NOCICEPTOR NEURONS CONTROL CANCER IMMUNOSURVEILLANCE

Abstract

The present disclosure provides methods of treating cancer by silencing tumor-innervating sensory neurons. The methods include treating cancer by genetic ablation of ion channels (e.g., TRPV1 or NaV1.8), local pharmacological silencing or blockade of neuropeptide release from tumor-innervating nociceptor (e.g., with QX-314 and BoNT/a), as well as the antagonism of the CGRP receptor RAMP1 (e.g., with BIBN 4096).

Description

BACKGROUND

Cytotoxic T cells express a variety of receptors, including PD-1 (Programmed Death-1), Tim-3 (T cell immunoglobulin and mucin domain-containing protein 3), and Lag-3 (Lymphocyte Activation Gene-3) (Dougan, M. et al. Annu Rev Immunol 27, 83-117 (2009); Chambers, C. A. et al. Annu Rev Immunol 19, 565-594 (2001); Topalian, S. L. et al. N Engl J Med 366, 2443-2454 (2012); Das, M. et al. Immunol Rev 276, 97-111 (2017)), that upon interaction with their cognate ligands, inhibit T cell function. These checkpoints ensure that immune responses to damage or infection are kept in check, preventing overly intense responses that might damage healthy cells (Baumeister, S. H. et al. Annu Rev Immunol 34, 539-573 (2016)). Tumor cells also express ligands for these immune checkpoints, which, once activated, block the cytolytic functions of T cells, thereby favoring survival of cancer cells (Baumeister, S. H., et al. Annu Rev Immunol 34, 539-573 (2016); Vesely, M. D. et al. Annu Rev Immunol 29, 235-271 (2011); Woo, S. R. et al. Annu Rev Immunol 33, 445-474 (2015)). Blocking the activity of immune checkpoint proteins releases this cancer cell-induced “brake” on the immune system, increasing the ability of the system to now eliminate tumor (Dougan, M. et al. Annu Rev Immunol 27, 83-117 (2009); Chambers, C. A. et al. Annu Rev Immunol 19, 565-594 (2001); Topalian, S. L. et al. N Engl J Med 366, 2443-2454 (2012); Baumeister, S. H. et al. Annu Rev Immunol 34, 539-573 (2016)). Several immune checkpoint inhibitors, including those targeting PD-L1, improve clinical outcomes for metastatic melanoma (Topalian, S. L. et al. N Engl J Med 366, 2443-2454 (2012); Wolchok, J. D. et al. N Engl J Med 369, 122-133, (2013); Long, G. V. et al. JAMA Oncol 3, 1511-1519 (2017)), but with varying degrees of effectiveness.

In addition to noxious stimuli detection leading to pain and prompting defensive reflexes, high threshold nociceptor neurons also promote host defenses through direct interaction with immune cells (Talbot, S. et al. Annu Rev Immunol 34, 421-447 (2016)). Neuro-immunity is mediated by the release of cytokines and neuropeptides and activation of their cognate receptors. While this dialogue helps to protect from danger, it may also contribute to disease. The somatosensory nervous system is positioned in epithelial and mucosal surfaces as well as primary and secondary lymphoid tissues to modulate immune responses (Talbot, S. et al. Neuron 87, 341-354, (2015); Rosas-Ballina, M. et al. Science 334, 98-101 (2011); Downing, J. E. et al. Immunol Today 21, 281-289 (2000); Veiga-Fernandes, H. et al. Cell 165, 801-811 (2016); McMahon, S. B. et al. Nat Rev Neurosci 16, 389-402 (2015); Foster, S. L. et al. Immunity 42, 403-405 (2015)). Nociceptors directly detect and respond to foreign antigens (Talbot, S. et al. Journal of neuroinflammation 6, 11 (2009)), immune cell-released cytokines (Talbot, S. et al. Neuron 87, 341-354), as well as toxins and surface receptors expressed by microbes and fungi (Kashem, S. W. et al. Trends Immunol 37, 440-450 (2016); Kashem, S. W. et al. Immunity 43, 515-526 (2015)). Nociceptor activation not only results in pain and itch but also local neuropeptide release from their peripheral terminals (Long, G. V. et al. JAMA Oncol 3, 1511-1519 (2017); Foster, S. L. et al. Front Immunol 8, 1463 (2017)). For example, Calcitonin Gene-Related Peptide (CGRP) increases T cell adhesion, blocks CD8 proliferation, reduces DC migration to lymph nodes, and suppresses FAS-L expression (Ding, W. et al. J Immunol 181, 6020-6026 (2008); Jimeno, R. et al. Immunol Cell Biol 90, 178-186 (2012); Mikami, N. et al. Journal of immunology 186, 6886-6893, (2011)). Overall, the peptides produced by and released from the peripheral terminals of nociceptors block the chemotaxis and polarization of lymphocytes, influencing the localization, duration, and type of inflammation (Talbot, S. et al. Neuron 87, 341-354, (2015); Goetzl, E. J. et al. Proc Natl Acad Sci USA 98, 13854-13859 (2001); Nussbaum, J. C. et al. Nature 502, 245-248 (2013); Cunin, P. et al. Journal of immunology 186, 4175-4182 (2011); Ganea, D. et al. Arch Immunol Ther Exp (Warsz) 49, 101-110 (2001)).

Cancer cells actively secrete growth factors promoting tumor hyper-innervation (neo-axonogenesis (Boilly, B. et al. Cancer Cell 31, 342-354 (2017); Saloman, J. L. et al. Proc Natl Acad Sci USA 113, 3078-3083 (2016); Zhao, C. M. et al. Sci Transl Med 6, 250ra115(2014); Isaacs, J. T. Science 341, 134-135 (2013); Magnon, C. et al. Science 341, 1236361 (2013)), and tumors are often painful or itchy (Walter, F. M. et al. BMC Fam Pract 11, 62 (2010); Yosipovitch, G. et al. Dermatol Ther 23, 590-596 (2010); Yosipovitch, G. et al. JAMA Dermatol 150, 1160-1166 (2014)). In a preclinical prostate cancer model, sensory neurons were found to promote tumor cell proliferation (Magnon, C. et al. Science 341, 1236361 (2013)), while nerve-derived noradrenaline activate an angiogenic switch that fuels exponential tumor growth (Zahalka, A. H. et al. Science 358, 321-326, (2017)). In addition, doublecortin⁺ neural progenitors were found to infiltrate prostate tumours, initiating neurogenesis, while their selective genetic depletion inhibits the early phases of tumour development (Mauffrey, P. et al. Nature 569, 672-678 (2019)). Experimental surgical denervation of a cancer-affected organ, such as gastric tumor, limits cancer growth (Boilly, B. et al. Cancer Cell 31, 342-354 (2017); Saloman, J. L. et al. Proc Natl Acad Sci USA 113, 3078-3083 (2016); Zhao, C. M. et al. Sci Transl Med 6, 250ra115(2014)), while vagotomised subjects have lower intestinal cancer mortality rates than their untreated counterparts (Boilly, B. et al. Cancer Cell 31, 342-354 (2017); Zhao, C. M. et al. Sci Transl Med 6, 250ra115(2014); Isaacs, J. T. Science 341, 134-135 (2013); Magnon, C. et al. Science 341, 1236361 (2013)). Similarly, blockade of synaptic vesicle release from neurons with botulinum toxin reduces growth in prostate cancer (Isaacs, J. T. Science 341, 134-135 (2013); Magnon, C. et al. Science 341, 1236361 (2013)). These findings led us to postulate that local release of neuropeptides from activated nociceptors suppresses immune surveillance and thereby increase metastasis (Isaacs, J. T. Science 341, 134-135 (2013); Magnon, C. et al. Science 341, 1236361 (2013)), Saloman, J. L. Trends Neurosci 39, 880-889 (2016)).

SUMMARY OF INVENTION

As provided herein, nociceptors play a regulatory role in the immune response to tumor growth, through the regulation of immune checkpoint receptor expression on cytotoxic CD8⁺ T-cells. Silencing tumor-innervating sensory neurons represents an innovative strategy for attenuating the immunomodulatory power of the nervous system and promoting anti-tumor activity. Genetic TRPV1 or Na_V1.8 lineage ablation, local pharmacological silencing or blockade of neuropeptide release from tumor-innervating nociceptors (e.g., with QX-314 or BoNT/a), and the antagonism of the CGRP receptor RAMP1 (e.g., with BIBN 4096) enhance tumor-infiltrating leukocyte (TIL) numbers and survival in mice, blunting tumor growth and TIL exhaustion.

In one aspect, provided herein are methods of treating cancer in a subject, the method comprising silencing tumor-innervating sensory neurons.

In another aspect, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a nociceptor modulating agent. In some embodiments, the nociceptor modulating agent is a nociceptor antagonist.

In a further aspect, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a neuropeptide modulating agent.

In one aspect, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that blocks the release or action of a neuropeptide from tumor-innervating neurons.

In another aspect, provided herein are methods of treating cancer in a subject, the method comprising administrating to the subject a therapeutically effective amount of an agent that blocks vesicle release from tumor-innervating nociceptors.

In a further aspect, provided herein are methods of treating cancer in a subject, the method comprising administering to a subject a therapeutically effective amount of a calcitonin gene-related peptide (CGRP) modulating agent. In some embodiments, the CGRP modulating agent is a CGRP receptor antagonist. In some embodiments, the CGRP receptor antagonist is a RAMP1 blocker.

In one aspect, provided herein are methods of treating cancer in a subject, the method comprising administering to a subject a therapeutically effective amount of QX-314, BoNT/a, and/or BIBN 4096.

In another aspect, provided herein are methods of treating cancer in a subject, the method comprising ablating an ion channel in a subject, wherein the ion channel is a sodium ion channel or TRPV ion channel. In some embodiments, the ion channel is Na_V1.8 and/or TRPV1.

In a further aspect, provided herein are compositions comprising (i) an anti-cancer agent, (ii) a nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor described herein, and (iii) optionally a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition described herein comprises (i) an anti-cancer agent, (ii) a nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor described herein, and (iii) a pharmaceutically acceptable carrier or excipient.

In another aspect, provided herein are kits comprising a pharmaceutical composition described herein or a nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor described herein and instructions for use (e.g., to treat cancer).

The details of certain embodiments of the disclosure are set forth in the Detailed Description of Certain Embodiments, as described below. Other features, objects, and advantages of the disclosure will be apparent from the Definitions, Figures, Examples, and Claims. It should be understood that the aspects described herein are not limited to specific embodiments, methods, apparati, or configurations, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1L: Melanoma sensitizes nociceptors. In comparison to control keratinocytes-injected skin (FIG. 1A), 14 days after B16F10-inoculation, abundant TRPV1⁺ nociceptors (red; TRPV1^Cre::Tdtomato^fl/wt) were found to innervate hyper-proliferating (green; BrdU⁺) cells (FIG. 1B). TRPV1⁺ nociceptors co-cultured with B16F10 have longer neurites (FIGS. 1C, 1E, 1F), reduced arborization (FIG. 1G), often form neuro-neoplastic contacts (FIG. 1C), then when cultured alone (FIGS. 1E-1G) or with non-tumorigenic keratinocytes (FIG. 1D). In co-culture, B16F0 or B16F10 cells sensitize the response of nociceptors to channel ligand (Capsaicin (100 nM), AITC (100 μM), ATP (1 μM)) used as control)) measured by calcium influx (FIG. 1H). Low concentration of the ligands induced minimal response in control neurons, while B16F10 show enhanced responses to ATP (FIG. 1H). L3-L5 DRG neurons harvested from mice 2-weeks post implantation with B16F10- or non-tumorigenic keratinocytes in the hindpaw were cultured and calcium flux generated by ligands tested. In comparison to keratinocytes, neurons from tumor-bearing mice were more sensitive to ATP (10 μM), and Capsaicin (1 μM; FIG. 1I). Neurons co-cultured with B16F10 cells release the neuropeptides CGRP (FIG. 1J). B16F10 cells alone do not releases neuropeptides (FIG. 1J). In comparison to DRG neurons(25) (FIG. 1K), B16F10 cells(26) (FIG. 1L) do not express the transcripts for Calca, Na_V1.8, Snap25, or TRPV1 as revealed by RNA sequencing. TRPV1⁺ nociceptors are labelled in red (FIGS. 1A-1D), BrdU proliferating cells are labelled in green (FIGS. 1A-1B), and nuclei are labelled in blue (FIGS. 1A, 1B, ID). B16F10 are labelled in green (FIG. 1C). Scale bar=100 μm. Mean±S.E.M; One-way ANOVA post-hoc Bonferroni (FIGS. 1E-1G) or unpaired Student's t-test (FIGS. 1H-1J); p values are showed in figure.

FIGS. 2A-2O: Nociceptor-released neuropeptides drive cytotoxic T-cell exhaustion. Cytotoxic CD8⁺ T-cells challenged with capsaicin-activated neurons conditioned media (CM) have increased proportion of PD1⁺Lag3⁺Tim3⁺ cells (FIG. 2A) and reduced INFγ⁺ (FIG. 2B) expressing cells. For cytotoxic CD8⁺ T-cells co-cultured with DRG neurons (FIGS. 2C-2D), capsaicin-stimulation increased the proportion of PD1⁺Lag3⁺Tim3⁺ cells (FIG. 2C), while it reduced proportion of INFγ⁺ (FIG. 2D) cells. The driving effects of neurons on CD8 T cells was null in absence of peptidergic neurons (TRPV1^creDTA^fl/wt neurons, FIGS. 2E-2F). The active crosstalk between DRG neurons and cytotoxic CD8⁺ T-cells when co-cultured for 48 h, leads to T-cells overexpression of the CGRP receptor Ramp1 (FIG. 2G). Cytotoxic CD8⁺ T-cells directly stimulated with CGRP (0.1 μM) increased the proportion of PD1⁺Lag3⁺Tim3⁺ cells (FIG. 2H), while it reduced proportion of INFγ⁺ (FIG. 21) cells. When co-cultured, T_c1-stimulated OT1-CD8⁺ T-cells leads to B16F10-OVA cells apoptosis (AnnexinV⁺7AAD⁺; FIGS. 2J-2L). When co-cultured, neurons and cytotoxic CD8⁺ T-cells actively crosstalk. Such interplay prompts nociceptors to release neuropeptides which, in turn, exhaust CD8⁺ T-cells preventing their elimination of B16F10-OVA (FIG. 2J). B16F10-OVA apoptosis decrease when CD8⁺ T-cells are challenged with capsaicin-stimulated nociceptor conditioned media (FIG. 2K) or CGRP (FIG. 2L). Representative image of DRG nociceptors (TRPV1^Cre::QuASR2-eGFP^fl/wt; green) cultured with B16F10-OVA-mCherry (red) cells (FIG. 2M). Representative image of cytotoxic CD8 T cells cultured with B16F10-OVA-mCherry cells without (FIG. 2N) or with nociceptors (FIG. 2O). B16F10-OVA-mCherry2 are labelled in red (FIGS. 2M-2O), TRPV1^Cre::QuASR2-eGFP^fl/wt nociceptors are labelled in green (FIGS. 2M, 2O), and OT1-CD8⁺ T-cells are labelled in blue (FIGS. 2N-2O). Scale bar 100 μm. Mean±S.E.M; One-way ANOVA post-hoc Bonferroni (FIGS. 2A-2L); p values are showed in figure.

FIGS. 3A-3I: Genetic elimination of nociceptors reduces TIL exhaustion. Orthotopic B16F10 tumor growth (FIG. 3A) was reduced in mice whose nociceptors are genetically ablated (TRPV1^Cre::DTA^fl/wt) while their median length of survival was increased by ˜250% (Mantel-Haenszel Hazard Ratio; FIG. 3B). Sensory neuron ablation also increased tumor infiltration of total (FIG. 3C) and INFγ⁺ CD8⁺ T-cells (FIG. 3D); while the proportion of PD1⁺Lag3⁺Tim3⁺ CD8⁺ T-cells was decreased (FIG. 3E). The reduction in B16F10 tumor growth observed in nociceptors ablated mice was absent following systemic CD3 depletion (FIG. 3F; αCD3, 200 μg/mice; i.p.; every 3 days). Nociceptor ablation also potentiated αPDL1 (6 mg/kg, i.p.; d7, d10, d13) mediated reduction in B16F10-OVA tumor growth (FIG. 3G). The daily optogenetic activation (3.5 ms, 10 Hz, 478 nm, 100 mW, giving approx. 2-6 mW/mm² with a 0.39 NA fiber placed 5-10 mm from the skin, for 20 min) of B16F10-inoculated skin in light sensitive Na_V1.8^Cre::ChR2^fl/wt mice enhanced tumor growth (FIG. 3H); while the genetic ablation of Na_V1.8⁺ lineage neurons (Na_V1.8^Cre::DTA^fl/wt) had the opposite effects (FIG. 3H). Finally, it was also found that sensory neuron ablation (TRPV1^Cre::DTA^fl/wt) decreases the growth of YUMMER1.7 cells; an immunogenic version of a Braf^V600ECdkn2a^−/−Pten^−/− melanoma cell line (FIG. 3I). Mean±S.E.M; Two-way ANOVA post-hoc Bonferroni (FIG. 3A, FIGS. 3F-3I); Mantel-Cox regression (FIG. 3B) or unpaired Student's t-test (FIG. 3C-3E); p values are showed in figure.

FIGS. 4A-4L: Silencing tumor-innervating nociceptors rescues anti-tumor immunity. B16F10 tumor growth (FIGS. 4A, 4C) and number of PD1⁺Lag3⁺Tim3⁺ CD8⁺ T-cells (FIGS. 4B, 4D) were reduced in mice whose tumor-innervating neuron are silenced by BoNT/a (FIGS. 4A-4B; 25 pg/μl; twice, i.d.) or QX-314 (FIGS. 4C-4D, 0.3%, i.d., q.d.). Tumor-innervating nociceptor silencing heighten αPDL1 (6 mg/kg, i.p.) decreased in B16F10-OVA tumor growth (FIG. 4E) and prolong the animals' median length of survival (FIG. 4F) by ˜270% (QX-314) and ˜800% (BoNT/a). CGRP levels are increased in B16F10 tumor surrounding skin explant in comparison to control skin; an effect further enhanced by capsaicin (1 μM; 3 h) but absent in skin pre-treated with BoNT/a (25 pg/μl) or QX-314 (0.3%; FIG. 4G). The RAMP1 antagonist BIBN4096 (5 mg/kg, i.p., day 6, 8, 10, 12, 14) decreased B16F10 tumor growth (FIG. 4H), and the proportion of intra-tumor PD1⁺Lag3⁺Tim3⁺ CD8⁺ T-cells (FIG. 4I), while it increased the numbers of INFγ⁺ CD8⁺ T-cells (FIG. 4J). In-silico analysis of single-cell RNA-sequencing of human melanoma (45) revealed that Ramp1⁺ CD8⁺ T-cells strongly overexpressed several immune checkpoint receptors (PD1, Tim3, Lag3, CTLA-4, CD28, ICOS, BTLA, CD27) in comparison to Ramp1⁻ CD8⁺ T-cells (FIG. 4K). Amongst other nociceptor markers, RNA sequencing data also showed that Calca, the gene encoding for CGRP, is overexpressed in melanoma skin biopsies in comparison to healthy skin (41) (FIG. 4L). Mean±S.E.M; Two-way ANOVA post-hoc Bonferroni (FIGS. 4A, 4C, 4E, 4H), one-way ANOVA post-hoc Bonferroni (FIG. 4G), Mantel-Cox regression (FIG. 4F), or unpaired Student's t-test (FIGS. 4B, 4D, 4I, 4J, 4L); p values are showed in figure.

FIGS. 5A-5C: Patient melanomas are innervated. Tubb3⁺ nerve fibers (IHC, brown; FIGS. 5A-5C) are present in three aggressive melanoma patient biopsies (FIGS. 5A-5C). Scale=100 μm.

FIG. 6: Immunocytes profiling. RNA sequencing of leukocyte subpopulations (47) revealed their basal expression of Cd45 and Ramp1. In contrast, immune cells do not express Snap25, Trpv1 and Na_V1.8.

FIGS. 7A-7C: Neurons-conditioned media drive CD8⁺ T-cells exhaustion. Cytotoxic CD8⁺ T-cells challenged with KCl-stimulated neuron conditioned media have increased proportion of PD1⁺Lag3⁺Tim3⁺ (FIG. 7A) and decreased level of INFγ⁺ (FIG. 7B) and TNFα⁺ (FIG. 7C) cells. Mean±S.E.M; unpaired Student's t-test; p values are showed in figure.

FIGS. 8A-8D: Nociceptors-conditioned media drive CD8⁺ T-cells exhaustion. Cytotoxic CD8⁺ T-cells challenged with KCl-stimulated neuron conditioned media have increased proportion of PD1⁺Lag3⁺Tim3⁺ cells (FIG. 8A) and decreased level of INFγ⁺ (FIG. 8B), TNFα⁺ (FIG. 8C) and IL2+(FIG. 8D) cells. These effects were absent when cytotoxic CD8⁺ T-cells were challenged with KCl-stimulated TRPV1^creDTA^fl/wt neuron conditioned media. Mean±S.E.M; One-way ANOVA post-hoc Bonferroni; p values are showed in figure.

FIGS. 9A-9G: Nociceptors drive CD8⁺ T-cells exhaustion. Capsaicin-challenge of DRG neurons co-cultured with cytotoxic CD8⁺ T-cells increases the proportion of PD1⁺ T cells (FIGS. 9A, 9D), while it decreased the level of TNFα⁺ (FIGS. 9B, 9E), IL2⁺ (FIGS. 9C, 9F) and INFγ⁺ (FIG. 9G) cells. Mean±S.E.M; unpaired Student's t-test; p values are showed in figure.

FIGS. 10A-10E: CGRP drives CD8⁺ T-cells exhaustion. CGRP increases cytotoxic CD8⁺ T-cells expression of PD1⁺ (FIG. 10A), Lag3⁺ (FIG. 10B), and Tim3⁺ (FIG. 10C) while it decreased level of INFγ⁺ (FIG. 10D) and TNFα⁺ (FIG. 10E). Mean±S.E.M; unpaired Student's t-test; p values are showed in figure.

FIGS. 11A-11G: Sensory neuron-released neuropeptides blunt cytotoxic T-cell anti-tumor immunity. Capsaicin-stimulated nociceptor conditioned media (FIGS. 11A-11C), neuron co-culture (FIGS. 11D-11F) or CGRP (FIG. 11G) reduces B16F10-OVA (Annexin V⁺) elimination by OVA-specific cytotoxic CD8⁺ T-cells (FIGS. 11A, 11D, 11G), while it increased mean fluorescence intensity of PD1⁺ (FIGS. 11B, 11E) and Lag3⁺ (FIGS. 11C, 11F) on the OT1 CD8⁺ T-cells. Mean±S.E.M; one-way ANOVA post-hoc Bonferroni (FIGS. 11A, 11D); or unpaired Student's t-test (FIGS. 11B, 11C, 11E-11G); p values are showed in figure.

FIGS. 12A-12I: Ablation of nociceptors decreases tumor growth. Orthotopic B16F10 tumor weight (FIG. 12A), size (FIG. 12B) and growth (FIGS. 12D-12I) were reduced in mice whose nociceptors are genetically ablated (TRPV1^Cre::DTA^fl/wt). The median length of survival of B16F10-inoculated sensory neuron ablated mice increased by 23%, while the one procured by αPDL1 blockade average 15.3% (FIG. 12C; meta-analysis of 16 published studies). Mean±S.E.M; unpaired Student's t-test (FIG. 12A); two-way ANOVA post-hoc Bonferroni (FIGS. 12D-12I); p values are showed in figure.

FIGS. 13A-13B: Nociceptors ablation prevent intra-tumoral CD8⁺ T-cells exhaustion. Sensory neuron ablation (TRPV1^Cre::DTA^fl/wt) increased B16F10 tumor infiltration of TNFα⁺ (FIG. 13A) and granzyme B⁺ (FIG. 13B) CD8⁺ T-cells. Mean±S.E.M; Unpaired Student's t-test; p values are showed in figure.

FIGS. 14A-14D: Nociceptors ablation prevent intra-tumoral CD4⁺ T-cells exhaustion. Sensory neuron ablation (TRPV1^Cre::DTA^fl/wt) increased B16F10 tumor infiltration of total (FIG. 14A), TNFα⁺ (FIG. 14C) and INFγ⁺ (FIG. 14D) CD4⁺ T-cells while the relative proportion of PD1⁺Lag3⁺Tim3⁺ CD4⁺ T-cells was decreased (FIG. 14B). Mean±S.E.M; Unpaired Student's t-test; p values are showed in figure.

FIGS. 15A-15F: Sensory neuron ablation decreases CD4⁺ and CD8⁺ T-cells exhaustion in tumor-draining lymph node. In comparison to B16F10-inoculated wildtype mice, nociceptor ablated animals have increased total (FIGS. 15A, 15B, 15D, 15E), INFγ⁺ (FIGS. 15C, 15F), CD8⁺ (FIGS. 15A-15C) and CD4⁺ (FIGS. 15D-15F) T-cells in tumor-draining lymph node. Mean±S.E.M; Unpaired Student's t-test; p values are showed in figure.

FIGS. 16A-16B: Nociceptors control CD3 anti-tumor immunity. Systemic CD3 depletion (FIG. 16A; αCD3, 200 μg/mice; i.p.; every 3 days) rescue tumor growth in nociceptor ablated mice (FIG. 16B). Mean±S.E.M.

FIGS. 17A-17D: Nociceptor neurons ablation potentiated αPDL1 reduction in tumor growth. Nociceptor ablation potentiated αPDL1 (6 mg/kg, i.p.; d7, d10, d13) mediated reduction in B16F10-OVA tumor growth (FIGS. 17A, 17B). The ablation also increased the infiltration of tumor specific CD8⁺ T-cells (FIG. 17C) which are less exhausted (FIG. 17D). These effects were further enhanced by αPDL1 treatment (FIGS. 17C-17D). Mean±S.E.M; One-way ANOVA post-hoc Bonferroni; p values are showed in figure.

FIGS. 18A-18C: Optogenetic activation of Na_V1.8⁺ nociceptors promotes TIL exhaustion. Daily optogenetic activation (3.5 ms, 10 Hz, 478 nm, 100 mW, giving approx. 2-6 mW/mm² with a 0.39 NA fiber placed 5-10 mm from the skin, for 20 min) of B16F10-inoculated skin in light sensitive Na_V1.8^Cre::ChR2^fl/wt mice decreased intra-tumor number of CD8 T cells (FIG. 18A), but increased the relative proportion of PD1⁺Tim3⁺ CD8⁺ T-cells (FIG. 18B) as well as secretion of CGRP in tumor surrounding skin explants (FIG. 18C). Genetic ablation of Na_V1.8⁺ lineage neurons (Na_V1.8^Cre::DTA^fl/wt) had the opposite effects (FIGS. 18A-18B). Mean±S.E.M; One-way ANOVA post-hoc Bonferroni; p values are showed in figure.

FIGS. 19A-19H: Sensory neuron ablation decreases CD8⁺ T-cells exhaustion in YUMMER1.7-inoculated mice. In comparison to YUMMER1.7-inoculated wildtype mice, nociceptor ablated animals have decreased tumor volume (FIG. 19A) and weight (FIG. 19B), as well as proportion of PD1⁺Lag3⁺Tim3⁺ CD8⁺ (FIG. 19E) and CD4⁺ (FIG. 19H) T-cells. Sensory neuron ablation also increased intra-tumor number of total (FIGS. 19C, 19D), INFγ⁺ (FIG. 19F), and TNFα⁺ (FIG. 19G) CD8⁺ (FIGS. 19C, 19E-19G) and CD4⁺ (FIGS. 19D, 19H) T-cells. Mean±S.E.M; Unpaired Student's t-test; p values are showed in figure.

FIG. 20A-20H: Botox silencing of B16F10-innervating neurons decrease tumor growth. When given prior to B16F10 inoculation, tumor volume (FIG. 20A), and weight (FIG. 20B) were reduced in mice treated with BoNT/a (i.d.; 25 pg/μl, day −3 and −1). In addition, silencing tumor-innervating neurons increased intra-tumoral number of total (FIG. 20C), INFγ⁺ (FIG. 20D) and Granzyme B⁺ (FIG. 20E) CD8⁺ T-cells. While t intra-tumoral CD4⁺ T-cells counts were not impacted (FIG. 20F), their exhaustion (FIG. 20G) was decreased in mice pre-treated with BoNT/A. Tumor-innervating nociceptor silencing heightens αPDL1 (6 mg/kg, i.p.) decreased B16F10-OVA tumor growth (FIG. 20H). Mean±S.E.M; Unpaired Student's t-test (FIGS. 20A-20G); p values are showed in figure.

FIGS. 21A-21E: Botox do not impact CD8⁺ T-cells function. When compared to vehicle exposed cells, BoNT/a (10-50 pg/ul; 24 h) do not impact the survival (FIG. 21A) of cultured cytotoxic CD8⁺ T-cells, nor their relative expression of PD1⁺Lag3⁺Tim3⁺ (FIG. 21B), TNFα⁺ (FIG. 21C), INFγ⁺ (FIG. 21D) and IL2⁺ (FIG. 21E). Mean±S.E.M.

FIGS. 22A-22I: Sensory neuron silencing prevents TILs exhaustion. Sensory neuron silencing with QX-314 decreased B16F10 tumor volume (FIG. 22A), and weight (FIG. 22B). QX-314 exposed B16F10-bearing mice show increased intra-tumoral number of total (FIG. 22C), INFγ⁺ (FIG. 22D) Granzyme B⁺ (FIG. 22E) and TNFα⁺ (FIG. 22F) CD8⁺ T-cells as well as total CD4⁺ T-cells (FIG. 22G). QX-314 decreased intra-tumoral proportion of PD1⁺Lag3⁺Tim3⁺ CD4⁺ T-cells (FIG. 22H). Tumor-innervating nociceptor silencing heighten αPDL1 (6 mg/kg, i.p.) decreased in B16F10-OVA tumor growth (FIG. 22I). Mean±S.E.M; Unpaired Student's t-test (FIGS. 22A-22H); p values are showed in figure.

FIGS. 23A-23E: QX-314 do not impact CD8⁺ T-cells function. When compared to vehicle exposed cells, QX-314 (50-150 μM; 24 h) do not impact the survival (FIG. 23A) of cultured cytotoxic CD8⁺ T-cells, nor their proportion of PD1⁺Lag3⁺Tim3⁺ (FIG. 23B), TNFα⁺ (FIG. 23C), INFγ⁺ (FIG. 23D) and IL2⁺ (FIG. 23E). Mean±S.E.M.

FIGS. 24A-24J: RAMP1 antagonism prevents TILs exhaustion. Blockade of the CGRP receptor RAMP1 with BIBN4096 (5 mg/kg, i.p.) decreased B16F10 tumor volume (FIG. 24A), and weight (FIG. 24B). RAMP1 blockade in B16F10-bearing mice increased intra-tumoral number of total (FIG. 24C), Granzyme B⁺ (FIG. 24D) and TNFα⁺ (FIG. 24E) CD8⁺ T-cells. Similar effects were found for total (FIG. 24F), TNFα⁺ (FIG. 24G), INFγ⁺ (FIG. 24H), and Granzyme B⁺ (FIG. 241) CD4⁺ T-cells. BIBN4096 decreased intra-tumoral proportion of PD1⁺ CD4⁺ T-cells (FIG. 24J). Mean±S.E.M; Unpaired Student's t-test; p values are showed in figure.

FIGS. 25A-25E: BIBN4096 do not impact CD8⁺ T-cells function. When compared to vehicle exposed cells, BIBN4096 (1-4 μM; 24 h) do not impact the survival (FIG. 25A) of cultured cytotoxic CD8⁺ T-cells, nor their proportion of PD1⁺Lag3⁺Tim3⁺ (FIG. 25B), TNFα⁺ (FIG. 25C), INFγ⁺ (FIG. 25D) and IL2⁺ (FIG. 25E). Mean±S.E.M.

FIGS. 26A-26D: Sensory neuron silencing prevents tumor-induced pain and itch. B16F10-hindpaw inoculation led to tumor induction (FIG. 26A), mechanical (FIG. 26B) and thermal pain hypersensitivities (FIG. 26C), while flank injection trigger occasional (˜30%) itch (FIG. 26D). These effects were stopped by sensory neuron silencing (QX-314 (0.3%, qd, i.d., FIGS. 26A-26D) and BoNT/a (25 pg/μl, FIGS. 26A-26C) or absent in mice whose sensory neuron are genetically ablated (TRPV1^cre::DTA^fl/wt, FIG. 26D). Mean±S.E.M; Unpaired Student's t-test (FIGS. 26B-26C) or one-way ANOVA post-hoc Bonferroni (FIG. 26D).

FIGS. 27A-27B: Sensory neuron silencing does not impact B16F10 survival. QX-314 (0.1-1%; 72 h; FIG. 27A) or BoNT/a (1.6-50 pg/μl; 24 h; FIG. 27B) did not impact the in vitro proliferation (FIG. 27A) or apoptosis (FIG. 27B) of cultured B16F10 cells.

FIG. 28: Single-cell RNA sequencing of human melanoma. In-silico analysis of single-cell RNA-sequencing of human melanoma revealed that Ramp1⁺ T-cells lost IL-2 expression but strongly overexpressed several immune checkpoint receptors (PD1, Tim3, Lag3, CTLA-4, CD28, ICOS, BTLA, CD27) in comparison to Ramp1⁻ T-cells. In-silico analysis of Tirosh et al (46).

FIG. 29: Single-cell RNA sequencing of human melanoma. Melanoma, cancer associated fibroblasts, macrophage, endothelial, natural killer, T, and B cells harvested from patients' melanoma do not express Calca. In addition, these cells do not express Snap25, the molecular target of BoNT/A. They also do not express Trp and Nav channels which are the molecular targets of QX-314. In-silico analysis of Tirosh et al (46).

FIG. 30: Single-cell RNA sequencing of human melanoma. Melanoma, cancer associated fibroblasts, macrophage, endothelial, natural killer, T, and B cells harvested from patients' melanoma do not express Calca. In addition, these cells do not express Snap25, the molecular target of BoNT/A. They also do not express Trp and Nav channels which are the molecular targets of QX-314. In-silico analysis of Jerby-Arnon et al (45).

FIGS. 31A-31E: Higher neuronal-associated genes transcript expression worsened patient's survival. Across a population of 458 melanoma patients (40), enhanced RNA-sequencing gene expression (label as high; FIG. 31A) of Na_V1.7 (FIG. 31B), Piezo1 (FIG. 31C), Pgp9.5 (FIG. 31D), and Tubb3 (FIG. 31E) in biopsy correlate with decreased patient survival (p≤0.05). Relative gene expression across is shown in blue (FIG. 31A). Mantel-Cox regression (FIGS. 31B-31E).

FIG. 32: Increased transcript expression of neuronal-associated genes were found in melanoma biopsies. Melanoma skin biopsy show heighten expression (heatmap) of Calca, Ramp1, Pouf4f1, Eno2 and Tubb3 as well as other neuronal-enriched genes in comparison to benign skin nevi (41). Unpaired Student's t-test; p value showed in figure.

FIG. 33: Increased transcript expression of neuronal-associated genes were found in melanoma biopsies. Melanoma skin biopsy show heighten expression (heatmap) of Calca, Tubb3, Pouf4f1, and Eno2 as well as other neuronal-enriched genes encoding for neuropeptides and their receptors, ion channels receptors, and transcription factors in comparison to the skin of healthy patient (44). Unpaired Student's t-test; p value showed in figure.

FIG. 34: Increased transcript expression of neuronal-associated genes were found in melanoma biopsies. Melanoma skin biopsy show heighten expression (heatmap) of Ramp1, Calca, and Trpa1 as well as other neuronal-enriched genes encoding for neuropeptides and their receptors, ion channels receptors, and transcription factors in comparison to healthy patients' blood PBMC (42). Unpaired Student's t-test; p value showed in figure.

FIG. 35: PD1⁺ nociceptors decrease in tumor-innervating neurons. In comparison to TRPV1⁺ nociceptor neurons from keratinocytes-injected skin, FACS-sorted B16F10-infiltrating TRPV1⁺ nociceptor neurons express less Pd1 transcript. Mean±S.E.M.

FIG. 36: RNA-Sequencing signatures of human immune cell types. An in-silico analysis of Monaco et al. (49) revealed that immunocytes express Cd45; but not Na_V1.7, Na_V1.8 and Trpv1 which are relevant for the activity of QX-314. Similarly, immunocytes do not expressed Snap25 which is the molecular target of BoNT/A.

FIGS. 37A-37J: Melanoma sensitizes nociceptors. In comparison to control keratinocyte-injected skin (FIG. 37A), 14 days after B16F10-inoculation TRPV1⁺ nociceptor (red; TRPV1^Cre::Tdtomato^fl/wt) innervate hyper-proliferating (green; BrdU⁺) cells (FIG. 37B). TRPV1⁺ nociceptors co-cultured with B16F10 have longer neurites (FIGS. 37C, 37E, 37F), reduced arborization (FIG. 37G), often form neuro-neoplastic contacts (FIG. 37C), then when cultured with non-tumorigenic keratinocytes (FIG. 37D). In co-culture, B16F10 cells sensitize the response of nociceptors to channel ligand (Capsaicin (100 nM), AITC (100 uM), ATP (1 uM), (KCl (50 mM) used as control)) measured by calcium influx (FIG. 37H). Low concentration of the ligands induced minimal response in control neurons, while B16F10 show enhanced responses to ATP (FIG. 37H). L3-L5 DRG neurons harvested from mice 2-weeks post implantation with B16F10- or non-tumorigenic keratinocytes in the hindpaw were cultured and calcium flux generated by ligands tested. In comparison to keratinocytes, neurons from tumor-bearing mice were more sensitive to ATP (10 uM) and Capsaicin (1 uM; FIG. 37I). Neurons co-cultured with B16F10 cells release the neuropeptides VIP, SP and CGRP (FIG. 37J).

FIGS. 38A-38O: Nociceptors-released neuropeptide drives cytotoxic T cell exhaustion. Cytotoxic CD8 T cells challenged with conditioned media (CM) from naive and KCl-activated neurons have an increased proportion of PD1⁺Lag3⁺ cells (FIG. 38A), and reduced INFγ⁺ (FIG. 38B) and TNFα⁺ expressing cells (FIG. 38C). For co-cultured cytotoxic CD8 T cells and DRG neurons (FIGS. 38D-38F), capsaicin-stimulation increased the proportion of PD1⁺Lag3⁺Tim3⁺ cells (FIG. 38D) and reduced INFγ⁺ (FIG. 38E) and TNFα⁺ levels (FIG. 38F). Cytotoxic CD8 T cells directly stimulated with CGRP (0.1 uM) and VIP (1 uM) have an increased proportion of PD1⁺Lag3⁺ cells (FIG. 38G), while VIP enhances proportion of INFγ⁺ cells (FIG. 38H). CGRP (0.1 uM) and VIP (1 uM) both reduced the proportion of TNFα⁺ (FIG. 38I). OVA-specific cytotoxic CD8 T cells trigger apoptosis in B16F10-OVA (AnnexinV⁺7AAD⁺) cells (FIGS. 38J-380). B16F10-OVA apoptosis decreases when the cells are challenged with capsaicin-stimulated nociceptor conditioned media (FIG. 38J), co-cultured with DRG neurons (FIG. 38K) and stimulated with neuropeptides (FIG. 38L). Representative image of DRG nociceptors (TRPV1^cretd-tomato^fl/wt; green) cultured with B16F10-OVA-mCherry (red) cells (FIG. 38M). Representative image of cytotoxic CD8 T cells (blue) cultured with B16F10-OVA-mCherry cells (red) without (FIG. 38N) or with nociceptors (TRPV1^cretd-tomato^fl/wt; green; FIG. 38O).

FIGS. 39A-39J: Ablation of nociceptors decreases tumor growth. Orthotopic B16F10 tumor growth (FIG. 39A), weight (FIG. 39B), appearance (FIG. 39D), and the infiltration of PD1⁺Lag3⁺TIM3⁺ CD8 T cells (FIG. 39G) were reduced in mice whose nociceptors are genetically ablated (TRPV1^Cre::DTA^fl/wt) while their survival (FIG. 39C), and intra-tumor number of total (FIG. 39E) and INFγ⁺ CD8 T cells (FIG. 39F) were increased. The reduction in B16F10 tumor growth (FIG. 39H) and volume (FIG. 391) observed nociceptor in ablated mice was absent following systemic CD3 depletion in these mice (FIG. 39J; αCD3, 200 μg/mice; i.p.; every 3 days).

FIGS. 40A-40I: Activating nociceptors increases growth of solid tumors. αPDL1 (6 mg/kg, i.p.; d7, d10, d13) injections further reduced B16F10-OVA tumor growth when given to nociceptor ablated mice (FIG. 40A). In B16F10-OVA inoculated mice, sensory neuron ablation increased the infiltration of tumor-specific CD8 T cells (FIG. 40B) which are also less exhausted (FIG. 40C). Daily optogenetic activation of B16F10-inoculated skin in light sensitive Na_V1.8^Cre::ChR2^fl/wt mice enhanced tumor growth (FIG. 40D), intra-tumor number of CD8 T cells (FIG. 40E) and reduced number of intra-tumor PD1⁺Lag3⁺ CD8 T cells (FIG. 40F). Ablation of NaV1.8+ lineage neurons (Na_V1.8^Cre::DTA^fl/wt) had the opposite effects (FIG. 40D-40F). The tumor growth (FIG. 40G) of flank inoculated lung metastatic HPV⁺ oropharyngeal squamous cell carcinomas mEERL and MLM3 was decrease in sensory neuron ablated mice, while their survival (FIG. 40H) was increased. In contrast, the growth of EG7 lymphoma was enhanced in sensory neuron ablated mice (FIG. 40I).

FIGS. 41A-41P: Blocking vesicle release and silencing tumor-innervating nociceptor neurons rescues anti-tumor immunity. B16F10 tumor growth (FIG. 41A, 41E), and intra-tumor PD1⁺Lag3⁺TIM3⁺ CD8 T cells (FIG. 41C, 41G) were reduced in mice treated with BoNT/a (FIG. 41A-41D; 25 pg/μl; twice, i.d.), or QX-314 (FIG. 41E-41H, 100 μM, i.d., q.d.), while infiltration of total (FIG. 41B, 41F) and INFγ⁺ CD8 T cells (FIG. 41D, 41H) were increased. αPDL1 (6 mg/kg, i.p.) decreased in B16F10-OVA tumor growth (FIG. 411, 41K) and volume (FIG. 41J, 41L) was further enhanced in mice co-exposed to QX-314 (FIG. 41I-41J) or BoNT/a (FIG. 41K-41L). The Ramp1 antagonist BIBN (5 mg/kg) decreased B16F10 tumor growth (FIG. 41M), and intra-tumor number of PD1⁺ CD8 T cells (FIG. 41O), while numbers of total and INFγ⁺ CD8 T cells (FIGS. 41N, 41P, respectively) were enhanced.

FIGS. 42A-42B: Cancer cells control neuron sensitivity and growth. Calcium flux induced by nociceptor activating ligands (capsaicin (100 nM), AITC (100 uM), ATP (1 uM)) in lumbar neurons co-cultured with B16F0 or B16F10 cells (FIG. 42A). TRPV1⁺ nociceptors co-cultured with B16F0 or B16F10 have longer neurites, then when cultured with non-tumorigenic keratinocytes (FIG. 42B).

FIGS. 43A-43G: Neuropeptides drive CD8 exhaustion. In-silico analysis of various leukocyte population revealed their basal expression of various neuropeptide receptors, including the one for CGRP (FIG. 43A). Cytotoxic CD8 T cells either challenged with KCl-stimulated neuron conditioned media (FIGS. 43B-43C), or co-cultured with capsaicin-stimulated DRG neurons (FIGS. 43D-43G) have increased proportion of PD1⁺ (FIGS. 43B, 43D), Lag3⁺ (FIGS. 43C, 43E), Tim3⁺ (FIG. 43F), and decreased level of IL2⁺ (FIG. 43G).

FIGS. 44A-44L: Sensory neuron-released neuropeptides modify CD8 anti-tumor immunity. In comparison to TRPV1^cre::DTA^fl/wt neurons, cytotoxic CD8 T cells co-cultured with wildtype neurons have increased proportion of PD1⁺Lag3⁺ cells (FIG. 44A), and reduced levels of INFγ⁺ (FIG. 44B) TNFα⁺ (FIG. 44C) and IL2⁺ cells (FIG. 44D). Cytotoxic CD8 T cells co-cultured with naïve lumbar neurons showed increase transcripts expression of Ramp1 (FIG. 44E), Ramp3 (FIG. 44F), and Vpac2 (FIG. 44G). Neuropeptides (FIGS. 44H-44L) increased the proportion of PD1⁺ (FIG. 44H), Lag3⁺ (FIG. 44I), Tim3⁺ (FIG. 44J) cytotoxic CD8 T cells, while it decreased their expression of IL2 (FIG. 44K) and INFγ (FIG. 44L).

FIGS. 45A-45G: CD8 express neuropeptide receptors. Capsaicin-stimulated nociceptor conditioned media (FIGS. 45A-45C), neuron co-culture (FIGS. 45D-45F) or exogenous neuropeptide challenges (FIG. 45G) reduced B16F10-OVA (Annexin V⁺) elimination by OVA-specific cytotoxic CD8 T cells (FIGS. 45A, 45D, 45G), while it increased mean fluorescence intensity of PD1⁺ (FIGS. 45B, 45E) and Lag3⁺ (FIGS. 45C, 45F) on CD8 T cells.

FIGS. 46A-46Q: Sensory neuron ablation prevents intra-tumoral CD8 exhaustion. In comparison to B16F10-inoculated wildtype mice, nociceptor ablated animals have increased intra-tumoral number of total (FIG. 46A), TNFα⁺ (FIGS. 46C, 46H), granzyme B⁺ (FIGS. 46D, 46I), and INFγ⁺ (FIG. 46E) CD4 (FIGS. 46A-46E) and CD8 (FIGS. 46F-46I) T cells. Conversely, these mice display lower intra-tumoral number of PD1⁺Lag3⁺Tim3⁺ CD4 T cells (FIG. 46B) as well as PD1⁺ (FIG. 46F), Lag3⁺ (FIG. 46G) CD8 T cells. The tumor draining lymph node of B16F10-inoculated nociceptor ablated mice also have higher proportion (FIGS. 46J, 46N) and number (FIGS. 46K, 46O) of total (FIGS. 46J, 46K, 46N, 46O), INFγ⁺ (FIGS. 46L, 46P) and TNFα⁺ (FIGS. 46C, 46H, 46M, 46Q) CD4 (FIGS. 46J-46M) and CD8 (FIGS. 46N-46Q) T cells.

FIGS. 47A-47K: Sensory neuron ablation prevents intra-tumoral NK cell exhaustion. In comparison to B16F10-inoculated wildtype mice, nociceptor ablated animals have increased intra-tumoral proportion (FIG. 47A) and number (FIGS. 47B-47F) of total (FIGS. 47A, 47B), INFg+ (FIG. 47C), and granzyme B⁺ (FIG. 47D) NK (FIGS. 47A-47D). We found similar intra-tumoral number of total (FIG. 47G) NK T cells, but lower levels of PD1⁺ (FIG. 47H) and Lag3⁺ (FIG. 47I). Nociceptor ablated animals also showed reduced B16F10 tumor volume (FIG. 47J), an effect that was further enhanced by αPDL1 treatment (FIG. 47K; 6 mg/kg, i.p. day 7, 10 and 13).

FIGS. 48A-48Z: Sensory neuron silencing prevents CD8 exhaustion. Twenty-four hours BoNT/a (1.6-50 pg/μl) or 72 h QX-314 (0.1-1%) exposure had no impact on B16F10 tumor growth in vitro (FIGS. 48A, 48I). B16F10 tumor volume (FIGS. 48B, 48J, 48R), and weight (FIGS. 48C, 48K, 48S) were reduced in mice treated with BoNT/a (FIGS. 48B, 48C; 25 pg/μl), QX-314 (FIGS. 48J, 48K, 100 μM, i.d., q.d.) or BIBN (R-S, 5 mg/kg. i.d., q.d.). When given prior to (d-3, d-1), or immediately after (dl and d3), B16F10 inoculation, BoNT/a (i.d.; 1.6-50 pg/μl) increased intra-tumoral number of Granzyme B⁺ CD4 T cells (FIG. 48G), and reduced numbers of PD1⁺Lag3⁺TIM3⁺ CD4 T cells (FIG. 48H). BoNT/a treatment had no impact on B16F10 infiltration of Granzyme B⁺ CD8 T cells (FIG. 48D), as well as total (FIG. 48E), INFγ⁺ (FIG. 48F), and Granzyme B⁺ (FIG. 48G) CD4 T cells. QX-314 (100 μM, i.d., qd) increased tumor infiltration of Granzyme B+ (FIG. 48L) and TNFα+ (FIG. 48M) CD8 T cells as well as total number of CD4 T cells (FIG. 48N). QX-314 decrease tumor infiltration of Granzyme B+ (FIG. 48O), and PD1⁺Lag3⁺TIM3⁺ (FIG. 48P) CD4 T cells (FIG. 48H), but failed to impact levels of TNFα+ CD4 T cells (FIG. 48Q). In B16F10-bearing mice, BIBN (5 mg/kg, i.d.) increased tumor infiltration of Granzyme B+ CD8 T cells (FIG. 48T), TNFα+ CD8 T cells (FIG. 48U). as well as total (FIG. 48V), INFγ⁺ (FIG. 48W), Granzyme B⁺ (FIG. 48X) CD4 T cells. BIBN (5 mg/kg, i.d.) also decreased B16F10 infiltration of PD1+ (FIG. 48Y) and TNFα+ (FIG. 48Z) CD4 T cells.

FIGS. 49A-49E: Sensory neuron silencing prevents tumor growth. Intradermal inoculation of B16F10 to the mice hindpaw led to tumor growth (FIG. 49A), occasional (˜30%) itch (FIG. 49B), mechanical (FIG. 49C) and thermal pain hypersensitivities (FIG. 49D). These effects were absent in mice whose sensory neuron are genetically ablated (TRPV1^cre::DTA^fl/wt, FIG. 49D) or pharmacologically silenced (QX-314 (100 μM, qd, i.d., FIGS. 49A-49D) and BoNT/a (25 pg/μl, FIGS. 49A-49C). CGRP levels are shown in B16F10 tumor surrounding skin explant. Capsaicin-induced CGRP release from tumor-inoculated skin was absent in QX-314 and BoNT/A pre-treated skin (FIG. 49E).

DEFINITIONS

For convenience, certain terms employed herein, in the specification, examples, and claims are collected herein.

Unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.

The language “in some embodiments” and the language “in certain embodiments” are used interchangeably.

The singular terms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise.

Other than in the examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” “About” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, or more typically, within 5%, 4%, 3%, 2%, or 1% of a given value or range of values.

When a range of values (“range”) is listed, it encompasses each value and sub-range within the range. A range is inclusive of the values at the two ends of the range unless otherwise provided. For example “C_1-6 alkyl” encompasses, C₁, C₂, C₃, C₄, C₅, C₆, C_1-6, C_1-5, C_1-4, C_1-3, C_1-2, C_2-6, C_2-5, C_2-4, C_2-3, C_3-6, C_3-5, C_3-4, C_4-6, C_4-5, and C_5-6 alkyl.

The term “aliphatic” refers to alkyl, alkenyl, alkynyl, and carbocyclic groups. Likewise, the term “heteroaliphatic” refers to heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic groups.

The term “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 20 carbon atoms (“C_1-20 alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C_1-12 alkyl”). In some embodiments, an alkyl group has 1 to 10 carbon atoms (“C_1-10 alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms (“C_1-9 alkyl”). In some embodiments, an alkyl group has 1 to 8 carbon atoms (“C_1-8 alkyl”). In some embodiments, an alkyl group has 1 to 7 carbon atoms (“C_1-7 alkyl”). In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C_1-6 alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms (“C_1-5 alkyl”). In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C_1-4 alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms (“C_1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbon atoms (“C_1-2 alkyl”). In some embodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C_2-6 alkyl”). Examples of C_1-6 alkyl groups include methyl (C₁), ethyl (C₂), propyl (C₃) (e.g., n-propyl, isopropyl), butyl (C₄) (e.g., n-butyl, tert-butyl, sec-butyl, isobutyl), pentyl (C₅) (e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tert-amyl), and hexyl (C₆) (e.g., n-hexyl). Additional examples of alkyl groups include n-heptyl (C₇), n-octyl (C₈), n-dodecyl (C₁₂), and the like. Unless otherwise specified, each instance of an alkyl group is independently unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents (e.g., halogen, such as F). In certain embodiments, the alkyl group is an unsubstituted C_1-12 alkyl (such as unsubstituted C_1-6 alkyl, e.g., —CH₃ (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu or s-Bu), unsubstituted isobutyl (i-Bu)). In certain embodiments, the alkyl group is a substituted C_1-12 alkyl (such as substituted C_1-6 alkyl, e.g., —CH₂F, —CHF₂, —CF₃, —CH₂CH₂F, —CH₂CHF₂, —CH₂CF₃, or benzyl (Bn)).

The term “haloalkyl” is a substituted alkyl group, wherein one or more of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo. “Perhaloalkyl” is a subset of haloalkyl, and refers to an alkyl group wherein all of the hydrogen atoms are independently replaced by a halogen, e.g., fluoro, bromo, chloro, or iodo. In some embodiments, the haloalkyl moiety has 1 to 20 carbon atoms (“C_1-20 haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 10 carbon atoms (“C_1-10 haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 9 carbon atoms (“C_1-9 haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 8 carbon atoms (“C_1-8 haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 7 carbon atoms (“C_1-7 haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 6 carbon atoms (“C_1-6 haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 5 carbon atoms (“C_1-5 haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 4 carbon atoms (“C_1-4 haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 3 carbon atoms (“C_1-3 haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 2 carbon atoms (“C_1-2 haloalkyl”). In some embodiments, all of the haloalkyl hydrogen atoms are independently replaced with fluoro to provide a “perfluoroalkyl” group. In some embodiments, all of the haloalkyl hydrogen atoms are independently replaced with chloro to provide a “perchloroalkyl” group. Examples of haloalkyl groups include —CHF₂, —CH₂F, —CF₃, —CH₂CF₃, —CF₂CF₃, —CF₂CF₂CF₃, —CCl₃, —CFCl₂, —CF₂Cl, and the like.

The term “heteroalkyl” refers to an alkyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (e.g., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 20 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC_1-20 alkyl”). In certain embodiments, a heteroalkyl group refers to a saturated group having from 1 to 12 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC_1-12 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 11 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC_1-11 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 10 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC_1-10 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 9 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC_1-9 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 8 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC_1-8 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC_1-7 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 6 carbon atoms and 1 or more heteroatoms within the parent chain (“heteroC_1-6 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC_1-5 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC_1-4 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 3 carbon atoms and 1 heteroatom within the parent chain (“heteroC_1-3 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 to 2 carbon atoms and 1 heteroatom within the parent chain (“heteroC_1-2 alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 1 carbon atom and 1 heteroatom (“heteroC₁ alkyl”). In some embodiments, a heteroalkyl group is a saturated group having 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parent chain (“heteroC_2-6 alkyl”). Unless otherwise specified, each instance of a heteroalkyl group is independently unsubstituted (an “unsubstituted heteroalkyl”) or substituted (a “substituted heteroalkyl”) with one or more substituents. In certain embodiments, the heteroalkyl group is an unsubstituted heteroC_1-12 alkyl. In certain embodiments, the heteroalkyl group is a substituted heteroC_1-12 alkyl.

The term “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 1 to 20 carbon atoms and one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In some embodiments, an alkenyl group has 1 to 20 carbon atoms (“C_1-20 alkenyl”). In some embodiments, an alkenyl group has 1 to 12 carbon atoms (“C_1-12 alkenyl”). In some embodiments, an alkenyl group has 1 to 11 carbon atoms (“C_1-11 alkenyl”). In some embodiments, an alkenyl group has 1 to 10 carbon atoms (“C_1-10 alkenyl”). In some embodiments, an alkenyl group has 1 to 9 carbon atoms (“C_1-9 alkenyl”). In some embodiments, an alkenyl group has 1 to 8 carbon atoms (“C_1-8 alkenyl”). In some embodiments, an alkenyl group has 1 to 7 carbon atoms (“C_1-7 alkenyl”). In some embodiments, an alkenyl group has 1 to 6 carbon atoms (“C_1-6 alkenyl”). In some embodiments, an alkenyl group has 1 to 5 carbon atoms (“C_1-5 alkenyl”). In some embodiments, an alkenyl group has 1 to 4 carbon atoms (“C_1-4 alkenyl”). In some embodiments, an alkenyl group has 1 to 3 carbon atoms (“C_1-3 alkenyl”). In some embodiments, an alkenyl group has 1 to 2 carbon atoms (“C_1-2 alkenyl”). In some embodiments, an alkenyl group has 1 carbon atom (“C₁ alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C_1-4 alkenyl groups include methylidenyl (C₁), ethenyl (C₂), 1-propenyl (C₃), 2-propenyl (C₃), 1-butenyl (C₄), 2-butenyl (C₄), butadienyl (C₄), and the like. Examples of C_1-6 alkenyl groups include the aforementioned C_2-4 alkenyl groups as well as pentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and the like. Additional examples of alkenyl include heptenyl (C₇), octenyl (C₈), octatrienyl (C₈), and the like. Unless otherwise specified, each instance of an alkenyl group is independently unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents. In certain embodiments, the alkenyl group is an unsubstituted C_1-20 alkenyl. In certain embodiments, the alkenyl group is a substituted C_1-20 alkenyl. In an alkenyl group, a C═C double bond for which the stereochemistry is not specified (e.g., —CH═CHCH₃ or

may be in the (E)- or (Z)-configuration.

The term “heteroalkenyl” refers to an alkenyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (e.g., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 20 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC_1-20 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 12 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC_1-12 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 11 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC_1-11 alkenyl”). In certain embodiments, a heteroalkenyl group refers to a group having from 1 to 10 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC_1-10 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 9 carbon atoms at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC_1-9 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 8 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC_1-8 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 7 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC_1-7 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 6 carbon atoms, at least one double bond, and 1 or more heteroatoms within the parent chain (“heteroC_1-6 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 5 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC_1-5 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 4 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC_1-4 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 3 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (“heteroC_1-3 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 2 carbon atoms, at least one double bond, and 1 heteroatom within the parent chain (“heteroC_1-2 alkenyl”). In some embodiments, a heteroalkenyl group has 1 to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatoms within the parent chain (“heteroC_1-6 alkenyl”). Unless otherwise specified, each instance of a heteroalkenyl group is independently unsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a “substituted heteroalkenyl”) with one or more substituents. In certain embodiments, the heteroalkenyl group is an unsubstituted heteroC_1-20 alkenyl. In certain embodiments, the heteroalkenyl group is a substituted heteroC_1-20 alkenyl.

The term “alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 1 to 20 carbon atoms and one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C_1-20 alkynyl”). In some embodiments, an alkynyl group has 1 to 10 carbon atoms (“C_1-10 alkynyl”). In some embodiments, an alkynyl group has 1 to 9 carbon atoms (“C_1-9 alkynyl”). In some embodiments, an alkynyl group has 1 to 8 carbon atoms (“C_1-8 alkynyl”). In some embodiments, an alkynyl group has 1 to 7 carbon atoms (“C_1-7 alkynyl”). In some embodiments, an alkynyl group has 1 to 6 carbon atoms (“C_1-6 alkynyl”). In some embodiments, an alkynyl group has 1 to 5 carbon atoms (“C_1-5 alkynyl”). In some embodiments, an alkynyl group has 1 to 4 carbon atoms (“C_1-4 alkynyl”). In some embodiments, an alkynyl group has 1 to 3 carbon atoms (“C_1-3 alkynyl”). In some embodiments, an alkynyl group has 1 to 2 carbon atoms (“C_1-2 alkynyl”). In some embodiments, an alkynyl group has 1 carbon atom (“C₁ alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C_1-4 alkynyl groups include, without limitation, methylidynyl (C₁), ethynyl (C₂), 1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl (C₄), 2-butynyl (C₄), and the like. Examples of C_1-6 alkenyl groups include the aforementioned C_2-4 alkynyl groups as well as pentynyl (C₅), hexynyl (C₆), and the like. Additional examples of alkynyl include heptynyl (C₇), octynyl (C₈), and the like. Unless otherwise specified, each instance of an alkynyl group is independently unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents. In certain embodiments, the alkynyl group is an unsubstituted C_1-20 alkynyl. In certain embodiments, the alkynyl group is a substituted C_1-20 alkynyl.

The term “heteroalkynyl” refers to an alkynyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (e.g., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain. In certain embodiments, a heteroalkynyl group refers to a group having from 1 to 20 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC_1-20 alkynyl”). In certain embodiments, a heteroalkynyl group refers to a group having from 1 to 10 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC_1-10 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC_1-9 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 8 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC_1-8alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 7 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC_1-7 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 6 carbon atoms, at least one triple bond, and 1 or more heteroatoms within the parent chain (“heteroC_1-6 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 5 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC_1-5 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 4 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC_1-4 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 3 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (“heteroC_1-3 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 2 carbon atoms, at least one triple bond, and 1 heteroatom within the parent chain (“heteroC_1-2 alkynyl”). In some embodiments, a heteroalkynyl group has 1 to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatoms within the parent chain (“heteroC_1-6 alkynyl”). Unless otherwise specified, each instance of a heteroalkynyl group is independently unsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a “substituted heteroalkynyl”) with one or more substituents. In certain embodiments, the heteroalkynyl group is an unsubstituted heteroC_1-20 alkynyl. In certain embodiments, the heteroalkynyl group is a substituted heteroC_1-20 alkynyl.

The term “carbocyclyl” or “carbocyclic” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbon atoms (“C_3-14 carbocyclyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a carbocyclyl group has 3 to 14 ring carbon atoms (“C_3-14 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 13 ring carbon atoms (“C_3-13 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 12 ring carbon atoms (“C_3-12 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 11 ring carbon atoms (“C_3-11 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 10 ring carbon atoms (“C_3-10 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms (“C_3-8 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C_3-7 carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C_3-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 4 to 6 ring carbon atoms (“C_4-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C_5-6 carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C_5-10 carbocyclyl”). Exemplary C_3-6 carbocyclyl groups include cyclopropyl (C₃), cyclopropenyl (C₃), cyclobutyl (C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅), cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and the like. Exemplary C_3-8 carbocyclyl groups include the aforementioned C_3-6 carbocyclyl groups as well as cycloheptyl (C₇), cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇), cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇), bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C_3-10 carbocyclyl groups include the aforementioned C_3-8 carbocyclyl groups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀), cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl (C₁₀), spiro[4.5]decanyl (C₁₀), and the like. Exemplary C_3-8 carbocyclyl groups include the aforementioned C_3-10 carbocyclyl groups as well as cycloundecyl (C₁₁), spiro[5.5]undecanyl (C₁₁), cyclododecyl (C₁₂), cyclododecenyl (C₁₂), cyclotridecane (C₁₃), cyclotetradecane (C₁₄), and the like. As the foregoing examples illustrate, in certain embodiments, the carbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic carbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can be saturated or can contain one or more carbon-carbon double or triple bonds. “Carbocyclyl” also includes ring systems wherein the carbocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocyclyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system. Unless otherwise specified, each instance of a carbocyclyl group is independently unsubstituted (an “unsubstituted carbocyclyl”) or substituted (a “substituted carbocyclyl”) with one or more substituents. In certain embodiments, the carbocyclyl group is an unsubstituted C_3-14 carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted C_3-14 carbocyclyl.

In some embodiments, “carbocyclyl” or “cycloalkyl” is a monocyclic, saturated carbocyclyl group having from 3 to 14 ring carbon atoms (“C_3-14 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 10 ring carbon atoms (“C_3-10 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C_3-8 cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C_3-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms (“C_4-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C_5-6 cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C_5-10 cycloalkyl”). Examples of C_5-6 cycloalkyl groups include cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C_3-6 cycloalkyl groups include the aforementioned C_5-6 cycloalkyl groups as well as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C_3-8 cycloalkyl groups include the aforementioned C_3-6 cycloalkyl groups as well as cycloheptyl (C₇) and cyclooctyl (C₈). Unless otherwise specified, each instance of a cycloalkyl group is independently unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents. In certain embodiments, the cycloalkyl group is an unsubstituted C_3-14 cycloalkyl. In certain embodiments, the cycloalkyl group is a substituted C_3-14 cycloalkyl. In certain embodiments, the carbocyclyl includes 0, 1, or 2 C═C double bonds in the carbocyclic ring system, as valency permits.

The term “heterocyclyl” or “heterocyclic” or “heterocyclyl” refers to a radical of a 3-to 14-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or polycyclic (e.g., a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”) or tricyclic system (“tricyclic heterocyclyl”)), and can be saturated or can contain one or more carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more carbocyclyl groups wherein the point of attachment is either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. Unless otherwise specified, each instance of heterocyclyl is independently unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents. In certain embodiments, the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3-14 membered heterocyclyl. In certain embodiments, the heterocyclyl is substituted or unsubstituted, 3- to 7-membered, monocyclic heterocyclyl, wherein 1, 2, or 3 atoms in the heterocyclic ring system are independently oxygen, nitrogen, or sulfur, as valency permits.

In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatom include azirdinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclyl groups containing 1 heteroatom include azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing 1 heteroatom include tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing 2 heteroatoms include dioxolanyl, oxathiolanyl and dithiolanyl. Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms include triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1 heteroatom include piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing 2 heteroatoms include piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing 3 heteroatoms include triazinyl. Exemplary 7-membered heterocyclyl groups containing 1 heteroatom include azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1 heteroatom include azocanyl, oxecanyl and thiocanyl. Exemplary bicyclic heterocyclyl groups include indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, tetra-hydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl, naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl, 5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl, 5,7-dihydro-4H-thieno[2,3-c]pyranyl, 2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl, 4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl, 4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl, 4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl, 1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

The term “aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C_6-14 aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C₆ aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms (“C₁₄ aryl”; e.g., anthracyl). “Aryl” also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system. Unless otherwise specified, each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents. In certain embodiments, the aryl group is an unsubstituted C_6-14 aryl. In certain embodiments, the aryl group is a substituted C_6-14 aryl.

The term “heteroaryl” refers to a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-14 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl polycyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the point of attachment is on the heteroaryl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heteroaryl ring system. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, e.g., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). In certain embodiments, the heteroaryl is substituted or unsubstituted, 5- or 6-membered, monocyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur. In certain embodiments, the heteroaryl is substituted or unsubstituted, 9- or 10-membered, bicyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently oxygen, nitrogen, or sulfur.

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a heteroaryl group is independently unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents. In certain embodiments, the heteroaryl group is an unsubstituted 5-14 membered heteroaryl. In certain embodiments, the heteroaryl group is a substituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing 3 heteroatoms include triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4 heteroatoms include tetrazolyl. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include pyridinyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4 heteroatoms include triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing 1 heteroatom include azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary tricyclic heteroaryl groups include phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl, and phenazinyl.

The term “alkaryl” refers to an alkyl substituted by an aryl group (e.g., benzyl, phenethyl, or 3,4-dichlorophenethyl).

The term “alkcycloalkyl” refers to an alkyl substituted with a carbocyclic group (e.g., cyclopropylmethyl).

The term “alkheterocyclyl” refers to an alkyl substituted with a heterocyclic group (e.g., 3-furanylmethyl, 2-furanylmethyl, 3-tetrahydrofuranylmethyl, or 2-tetrahydrofuranylmethyl).

The term “unsaturated bond” refers to a double or triple bond.

The term “unsaturated” or “partially unsaturated” refers to a moiety that includes at least one double or triple bond.

The term “saturated” or “fully saturated” refers to a moiety that does not contain a double or triple bond, e.g., the moiety only contains single bonds.

A group is optionally substituted unless expressly provided otherwise. The term “optionally substituted” refers to being substituted or unsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups are optionally substituted. “Optionally substituted” refers to a group which is substituted or unsubstituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” heteroalkyl, “substituted” or “unsubstituted” heteroalkenyl, “substituted” or “unsubstituted” heteroalkynyl, “substituted” or “unsubstituted” carbocyclyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group). In general, the term “substituted” means that at least one hydrogen present on a group is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds, and includes any of the substituents described herein that results in the formation of a stable compound. The present disclosure contemplates any and all such combinations in order to arrive at a stable compound. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety. The disclosure is not limited in any manner by the exemplary substituents described herein.

The term “halo” or “halogen” refers to fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

The term “quaternary amine” refers a cationic amine in which the nitrogen atom has four groups bonded to it and carries a positive charge. In some embodiments, the quaternary amines provided herein have a counterion. A “counterion” or “anionic counterion” or “pharmaceutically acceptable anion” (e.g., X⁻) is a negatively charged group associated with a positively charged group in order to maintain electronic neutrality. An anionic counterion may be monovalent (e.g., including one formal negative charge). An anionic counterion may also be multivalent (e.g., including more than one formal negative charge), such as divalent or trivalent. Exemplary counterions include halide ions (e.g., F⁻, Cl⁻, Br⁻, I⁻), NO₃⁻, ClO₄⁻, OH⁻, H₂PO₄⁻, HCO₃⁻, HSO₄⁻, sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonic acid-2-sulfonate, and the like), carboxylate ions (e.g., acetate, propanoate, benzoate, glycerate, lactate, tartrate, glycolate, gluconate, and the like), BF₄⁻, PF₄⁻, PF₆⁻, AsF₆⁻, SbF₆⁻, B[3,5-(CF₃)₂C₆H₃]₄]⁻, B(C₆F₅)₄⁻, BPh₄⁻, Al(OC(CF₃)₃)₄⁻, and carborane anions (e.g., CB₁₁H₁₂⁻ or (HCB₁₁Me₅Br₆)⁻). Exemplary counterions which may be multivalent include CO₃²⁻, HPO₄²⁻, PO₄³⁻, B₄O₇²⁻, SO₄²⁻, S₂O₃²⁻, carboxylate anions (e.g., tartrate, citrate, fumarate, maleate, malate, malonate, gluconate, succinate, glutarate, adipate, pimelate, suberate, azelate, sebacate, salicylate, phthalates, aspartate, glutamate, and the like), and carboranes. In some embodiments, the quaternary amine counterion is F⁻, Cl⁻, Br⁻, or I⁻. In some embodiments, the counterion is a non-coordinating anionic counterion. The term “non-coordinating anionic counterion” refers to an anion that interacts weakly with cations. Exemplary non-coordinating anions include, but are not limited to, ClO₄⁻, NO₃⁻, TfO⁻, BF₄⁻, PF₄⁻, PF₆⁻, and SbF₆⁻. Other examples of non-coordinating anions include, but are not limited to, B(C₆F₅)₄⁻, B[3,5-(CF₃)₂C₆H₃]₄]⁻, BPh₄⁻, Sb(OTeF₅)₆⁻, Al(OC(CF₃)₃)₄⁻, or a carborane anion (e.g., CB₁₁H₁₂⁻, CB₁₁(CF₃)₁₂⁻, or (HCB₁₁Me₅Br₆)⁻).

The term “nociceptor” refers to a sensory neuron. Nociceptors respond to damaging or potentially damaging stimuli by sending signals to the spinal cord and brain. Upon the brain recognizing a credible threat, the sensation of pain is created to direct attention to the originating body part in order to mitigate the threat. The action of nociceptors is called nociception.

A “sodium channel” is a membrane protein that form ion channels, conducting sodium ions (Na⁺) through a cell's plasma membrane. In neurons, sodium channels are responsible for the rising phase of action potentials. In some embodiments, the sodium channel is a Na_V1.6, Na_V1.7, Na_V1.8, and Na_V1.9 comprising sodium channel.

As used herein, the term “agent” means a molecule, group of molecules, complex, or substance. Agents include small molecules, peptides, proteins, nucleic acids, and the like.

A “neuropeptide modulating agent” is an agent modifies the activity of a neuropeptide. In some embodiments, the neuropeptide modulating agent is an agent that blocks the release of a neuropeptide. In some embodiments, the neuropeptide modulating agent is an agent that modifies the action of a neuropeptide.

The term “neuropeptide” refers to small proteins produced by neurons that act on G protein-coupled receptors and are responsible for slow-onset, long-lasting modulation of synaptic transmission.

The term “vesicle” refers to a structure within or outside a cell, consisting of liquid or cytoplasm enclosed by a lipid bilayer. Vesicles are involved in metabolism, transport, buoyancy, and temporary storage of food and enzymes.

A “nociceptor modulating agent” is an agent that modifies the activity of a nociceptor. In some embodiments, the nociceptor modulating agent inhibits the activity of a nociceptor (e.g., the agent is a nociceptor antagonist).

A “nociceptor antagonist” is an agent that inhibits the activity of a nociceptor. In some embodiments, the nociceptor antagonist is a sodium channel blocker. In some embodiments, the nociceptor antagonist is a calcium channel blocker. In some embodiments, the nociceptor antagonist is a sodium and calcium channel blocker.

A “sodium channel blocker” is an agent impairs the conduction of sodium ions (Na⁺) through sodium channels. In some embodiments, the sodium channel blocker is also a nociceptor antagonist. In some embodiments, the sodium channel blocker is Ranolazine, Phenytoin, Disopyramide, Lidocaine, Mexiletine, Triamterene, Lamotrigine, Amiloride, Moricizine, Oxcarbazepine, Quinidine, Procainamide, Tocainide, Amiodarone, Propafenone, Flecainide, Encainide, Ajmaline, Aprindine, Tetrodotoxin, Eslicarbazepine acetate, Pilsicainide, Eslicarbazepine, Carbamazepine, Ethotoin, Fosphenytoin, Rufinamide, or Lacosamide. In some embodiments, the sodium channel blocker is a compound comprising a quaternary amine (e.g., a quaternary amine of Formula (I), (IA), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV)).

A “calcium channel blocker” is an agent that disrupts the movement of calcium (Ca₂⁺) through calcium channels. In some embodiments, the calcium channel blocker is also a nociceptor antagonist. In some embodiments, the calcium channel blocker is ziconotide, amlodipine, clevidipine, diltiazem, felodipine, isradipine, nicardipine, nifedipine, nimodipine, nisoldipine, or verapamil. In some embodiments, the calcium channel blocker is ziconotide. In some embodiments, the calcium channel blocker is a compound comprising a quaternary amine (e.g., a quaternary amine of Formula (I), (IA), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV)).

A “sodium and calcium channel blocker” is an agent that is both a sodium channel blocker as well as a calcium channel blocker. In some embodiments the sodium and calcium channel blocker is CNCB-2 (Lee et al. Elife 2019 Nov. 25;8:e48118. doi: 10.7554/eLife.48118). In some embodiments, the sodium and calcium channel blocker is a compound comprising a quaternary amine (e.g., a quaternary amine of Formula (I), (IA), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV)).

An “ion channel blocker” or “channel blocker” refers to an agent that is a sodium channel blocker, calcium channel blocker, or sodium and calcium channel blocker.

An “antagonist” is a substance that interferes with or inhibits the physiological action of another.

The terms “calcitonin gene-related peptide modulating agent” and “CGRP modulating agent” are used interchangeably and refer to an agent that modifies the activity of calcitonin gene-related peptide. In some embodiments, the CGRP modulating agent acts through CGRP's receptor activity-modifying proteins (RAMPs). In some embodiments, the RAMP is RAMP1 or RAMP2.

The term “RAMP” or “receptor activity-modifying proteins” refers to a class of protein that interact with and modulate activity of numerous Class B G protein-coupled receptors including the receptors for secretin, calcitonin, glucagon, and vasoactive intestinal peptide. There are three types of RAMPs in mammals: RAMP1, RAMP2, and RAMP3. RAMP1 is a protein that in humans is encoded by the RAMP1 gene. In combination with the RAMP1 protein, calcitonin-receptor-like receptor functions as the CGRP receptor. RAMP2 is a protein which in humans is encoded by the RAMP2 gene. In the presence of RAMP2 protein, calcitonin-receptor-like receptor functions as an adrenomedullin receptor.

The terms “calcitonin gene-related peptide antagonist” and “CGRP antagonist” are used interchangeably to refer to agents that antagonize the CGRP. In some embodiments, the CGRP antagonist is BIBN 4096.

The term “ablate” as used herein refers to removal, especially the removal of a nociceptor. In some embodiments, the nociceptor is ablated via surgical, genetic, or chemical means.

The term “inhibition,” “inhibiting,” “inhibit,” or “inhibitor” refers to the ability of an agent to reduce, slow, halt or prevent activity of a particular biological process (e.g., activity of a nociceptor (e.g., nociception)) in a cell relative to vehicle.

The terms “silence” or “silencing” as used herein refers to preventing the normal activity of something. In certain embodiments, “silencing” means to partially prevent the normal activity of something. In certain embodiments, “silencing” means to completely prevent the normal activity of something (e.g., it is now inactive). In some embodiments, silencing a nociceptor means to prevent the normal activity of a nociceptor (e.g., via use of an agent that inhibits or antagonizes the nociceptor), or by removing the nociceptor (e.g., genetically or surgically ablating the nociceptor). In some embodiments, silencing a sensory neuron means to prevent the normal activity of a sensory neuron (e.g., via use of an agent that inhibits or antagonizes the sensory neuron), or by removing the sensory neuron (e.g., genetically or surgically ablating the sensory neuron).

The term “BIBN 4096”, “BIBN4096” and “BIBN” refer to a small molecule having the following structure:

The terms “BoNT” or “BONT” refer to botulinum neurotoxins. BoNTs are protein neurotoxins produced by neurotoxigenic strains of anaerobic and spore forming bacteria of the genus Clostridium (Clostridium botulinum, Clostridium butyrricum, Clostridium barati, and Clostridium argentinensis). “BoNT/a” and “BONT/a” refer to botulinum toxin type a.

“TeNT” refers to tetanus neurotoxin produced by Clostridium tetani.

The term “innervated”, “innervate”, or the like refers to nerves being supplied. In some embodiments, the tumors are innervated, such that the tumors are supplied with nerves. In some embodiments, innervated tumors are more aggressive than less innervated one.

A “subject” to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey)). In certain embodiments, the non-human animal is a fish, reptile, or amphibian. The non-human animal may be a male or female at any stage of development. The non-human animal may be a transgenic animal or genetically engineered animal. The term “patient” refers to a human subject in need of treatment of a disease.

The term “administer,” “administering,” or “administration” refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein, or a composition thereof, in or on a subject.

The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

An “effective amount” of a compound described herein refers to an amount sufficient to elicit the desired biological response. An effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, severity of side effects, disease, or disorder, the identity, pharmacokinetics, and pharmacodynamics of the particular compound, the condition being treated, the mode, route, and desired or required frequency of administration, the species, age and health or general condition of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is a prophylactic treatment. In certain embodiments, an effective amount is the amount of a compound described herein in a single dose. In certain embodiments, an effective amount is the combined amounts of a compound described herein in multiple doses. In certain embodiments, the desired dosage is delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage is delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). In certain embodiments, an effective amount of a compound for administration one or more times a day to a 70 kg adult human comprises about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of a compound per unit dosage form. In certain embodiments, the compounds provided herein may be administered orally or parenterally at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, preferably from about 0.1 mg/kg to about 40 mg/kg, preferably from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, and more preferably from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect. It will be appreciated that dose ranges as described herein provide guidance for the administration of compounds to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

A “therapeutically effective amount” of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount sufficient for silencing tumor-innervating sensory neurons. In certain embodiments, a therapeutically effective amount is an amount sufficient for blocking nociceptors. In certain embodiments, a therapeutically effective amount is an amount sufficient for treating cancer.

A “prophylactically effective amount” of a compound described herein is an amount sufficient to prevent a condition, or one or more symptoms associated with the condition or prevent its recurrence. A prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition. The term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent. In certain embodiments, a prophylactically effective amount is an amount sufficient for silencing tumor-innervating sensory neurons. In certain embodiments, a prophylactically effective amount is an amount sufficient for blocking nociceptors. In certain embodiments, a prophylactically effective amount is an amount sufficient for preventing cancer.

The term “prevent,” “preventing,” or “prevention” refers to a prophylactic treatment of a subject who is not and was not with a disease but is at risk of developing the disease or who was with a disease, is not with the disease, but is at risk of regression of the disease. In certain embodiments, the subject is at a higher risk of developing the disease or at a higher risk of regression of the disease than an average healthy member of a population.

The terms “neoplasm” and “tumor” are used herein interchangeably and refer to an abnormal mass of tissue wherein the growth of the mass surpasses and is not coordinated with the growth of a normal tissue. A neoplasm or tumor may be “benign” or “malignant,” depending on the following characteristics: degree of cellular differentiation (including morphology and functionality), rate of growth, local invasion, and metastasis. A “benign neoplasm” is generally well differentiated, has characteristically slower growth than a malignant neoplasm, and remains localized to the site of origin. In addition, a benign neoplasm does not have the capacity to infiltrate, invade, or metastasize to distant sites. Exemplary benign neoplasms include, but are not limited to, lipoma, chondroma, adenomas, acrochordon, senile angiomas, seborrheic keratoses, lentigos, and sebaceous hyperplasias. In some cases, certain “benign” tumors may later give rise to malignant neoplasms, which may result from additional genetic changes in a subpopulation of the tumor's neoplastic cells, and these tumors are referred to as “pre-malignant neoplasms.” An exemplary pre-malignant neoplasm is a teratoma. In contrast, a “malignant neoplasm” is generally poorly differentiated (anaplasia) and has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Furthermore, a malignant neoplasm generally has the capacity to metastasize to distant sites. The term “metastasis,” “metastatic,” or “metastasize” refers to the spread or migration of cancerous cells from a primary or original tumor to another organ or tissue and is typically identifiable by the presence of a “secondary tumor” or “secondary cell mass” of the tissue type of the primary or original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located. For example, a prostate cancer that has migrated to bone is said to be metastasized prostate cancer and includes cancerous prostate cancer cells growing in bone tissue. IN some embodiments, the tumor is innervated.

The term “cancer” refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See e.g., Stedman's Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990. Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing's sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenström's macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia/lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget's disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva). In some embodiments, cancer includes a benign and malignant tumors. In some embodiments, cancer includes a benign tumor. In some embodiments, the cancer is a tumor. In some embodiments, the cancer is a melanoma. In some embodiments, the melanoma is superficial spreading melanoma, nodular melanoma, acral-lentiginous melanoma, lentigo maligna melanoma, amelanotic melanoma, desmoplastic melanoma, ocular melanoma, or metastatic melanoma. In some embodiments, the cancer is metastatic melanoma.

These and other exemplary substituents are described in more detail in the Detailed Description, Examples, and Claims. The invention is not limited in any manner by the above exemplary listing of substituents. Additional terms may be defined in other sections of this disclosure.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Before the disclosed methods and uses are described in more detail, it should be understood that the aspects described herein are not limited to specific embodiments, methods, or uses, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and, unless specifically defined herein, is not intended to be limiting.

Described herein are methods of treating and preventing cancer. The data presented herein shows nociceptors play a regulatory role in the immune response to tumor growth, through the regulation of immune checkpoint receptor expression on cytotoxic CD8⁺ T-cells. Silencing tumor-innervating sensory neurons represents an innovative strategy for attenuating the immunomodulatory power of the nervous system and promoting anti-tumor activity.

Methods and Uses

In some embodiments, provided herein are methods of treating cancer in a subject comprising silencing tumor-innervating sensory neurons. In some embodiments, provided herein are methods of preventing cancer in a subject comprising silencing tumor-innervating sensory neurons.

In some embodiments, provided herein are methods of treating cancer in a subject comprising silencing tumor-innervating nociceptors. In some embodiments, provided herein are methods of preventing cancer in a subject comprising silencing tumor-innervating nociceptors.

In some embodiments, provided herein are methods of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a nociceptor modulating compound. In some embodiments, provided herein are methods of preventing cancer in a subject, the method comprising administering to the subject a prophylactically effective amount of a nociceptor modulating agent. In some embodiments, the nociceptor modulating agent is a nociceptor antagonist. In some embodiments, the nociceptor antagonist is a sodium channel blocker. In certain embodiments, the sodium channel is selected from Na_V1.6, Na_V1.7, Na_V1.8, and Na_V1.9. In some embodiments, the sodium channel is Na_V1.8. In some embodiments, the nociceptor antagonist is a calcium channel blocker. In some embodiments, the calcium channel is Ca_V1.1-1.4, Ca_V2.1, Ca_V2.2, Ca_V2.3, Ca_V3.1-3.3. In some embodiments, the calcium channel is Ca_V2.2.

In some embodiments, provided herein are methods of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a neuropeptide modulating agent. In some embodiments, provided herein are methods of preventing cancer in a subject comprising administering to the subject a prophylactically effective amount of a neuropeptide modulating agent. In some embodiments, the neuropeptide is calcitonin gene-related peptide (CGRP).

In some embodiments, provided herein are methods of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that blocks the release or action of neuropeptide from tumor-innervating neurons. In certain embodiments, provided herein are methods of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of an agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptors. In some embodiments, provided herein are methods of preventing cancer in a subject comprising administering to the subject a prophylactically effective amount of an agent that blocks the release or action of a neuropeptide from tumor-innervating neurons. In certain embodiments, provided herein are methods of preventing cancer in a subject comprising administering to the subject a prophylactically effective amount of an agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptors. In some embodiments, the neuropeptide is CGRP.

In some embodiments, the agent blocks the release of a neuropeptide. In some embodiments, the agent blocks the action of a neuropeptide. In some embodiments, the neuropeptide is CGRP.

In some embodiments, the nociceptor antagonist prevents release of a neuropeptide. In some embodiments, the neuropeptide is CGRP.

In certain embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is a compound comprising a quaternary amine. In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of the neuropeptide from tumor-innervating nociceptor is QX-314:

In certain embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is a compound selected from the group consisting of:

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is a quaternary amine of Formula (I):

wherein:

R^1F and R^1G together complete a heterocyclic or heteroaryl ring having at least one nitrogen atom;

each of R^1A, R^1B, and R^1C is independently selected from H, halogen, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, OR^1I, NR^1JR^1K, NR^1LC(O)R^1M, S(O)R^1N, SO₂R^1OR^1P, SO₂NR^1QR^1R, SO₃R^1S, CO₂R^1T, C(O)R^1U, and C(O)NR^1VR^1W;

each of R^1I, R^1J, R^1K, R^1L, R^1M, R^1N, R^1O, R^1P, R^1Q, R^1R, R^1S, R^1T, R^1U, R^1V, and R^1W is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

X¹ is selected from —CR^1XR^1Y—, —NR^1ZC(O)—, —OC(O)—, —SC(O)—, —C(O)NR^1AA—, —CO₂—, and —OC(S)—;

each of R^1X, R^1Y, R^1Z, and R^1AA is independently selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

each of R^1D and R^1E is independently selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, and C_3-6 carbocyclyl, wherein each R^1D and R^1E is optionally substituted with halogen, C_3-8 carbocyclyl, aryl, or heteroaryl; or R^1D and R^1E together form a 3-6-membered heterocyclic or carbocyclic ring; and

R^1H is selected from C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, and C_3-6 carbocyclyl, wherein R^1H is optionally substituted with halogen, C_3-8 carbocyclyl, aryl, or heteroaryl.

In some embodiments, R^1F and R^1G together complete a 4-8-membered heterocyclic ring. In some embodiments, R^1F and R^1G together complete a 5-, 6-, or 7-membered heterocyclic ring. In some embodiments, R^1F and R^1G together complete a pyrrolidine, piperidine, or azepane ring. In some embodiments, the heterocyclic ring formed by R^1F and R^1G is optionally substituted with C_1-4 alkyl, halogen, C_3-8 carbocyclyl, aryl, or heteroaryl.

In some embodiments, R^1A and R^1B are each independently H, halogen, C_1-4 alkyl, or CO₂R^1T. In some embodiments, R^1A and R^1B are each independently H, C_1-4 alkyl, or CO₂R^1T. In some embodiments, R^1A and R^1B are each independently C_1-4 alkyl or CO₂R^1T In some embodiments, R^1A and R^1B are each methyl.

In some embodiments, X¹ is —NHC(O)—.

In some embodiments, each of R^1D and R^1E is independently selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, and C_3-6 carbocyclyl, wherein each R^1D and R^1E is optionally substituted with halogen, C_3-8 carbocyclyl, aryl, or heteroaryl. In some embodiments, each of R^1D and R^1E is hydrogen. In some embodiments, each of R^1D and R^1E is independently C_1-4 alkyl, wherein each R^1D and R^1E is optionally substituted with halogen, C_3-8 carbocyclyl, aryl, or heteroaryl. In some embodiments, each of R^1D and R^1E is independently C_1-4 alkyl, wherein each R^1D and R^1E is substituted with halogen, C_3-8 carbocyclyl, aryl, or heteroaryl.

In some embodiments, R^1H is selected from C_1-4 alkyl, wherein R^1H is optionally substituted with halogen, C_3-8 carbocyclyl, aryl, or heteroaryl. In some embodiments, R^1H is selected from C_1-4 alkyl, wherein R^1H is substituted with halogen, C_3-8 carbocyclyl, aryl, or heteroaryl.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is a compound or agent described in (i) PCT publication WO2008/063603, WO2011/006073, WO2017/024037, WO2020/142657, WO2020/185830, WO2020/185928, WO2020/185915, or WO2020/185881, (ii) U.S. patent Ser. No. 10/780,083, 10927096, 10842798, 10828287, 10925865, or 10786485, or (iii) US patent publication US 2020/0290953, US 2020/0290965, or US 2020/0290979.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is a quaternary amine derivative or other permanently charged derivative of a compound selected from riluzole, mexilitine, phenytoin, carbamazepine, procaine, articaine, bupivicaine, mepivicaine, tocainide, prilocaine, diisopyramide, bencyclane, quinidine, bretylium, lifarizine, lamotrigine, flunarizine, and fluspirilene.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is QX-314, N-methyl-procaine, QX-222, N-octyl-guanidine, 9-aminoacridine, pancuronium, or another low molecular weight, charged molecule that inhibits voltage-gated sodium channels when present inside of said nociceptor. In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is D-890, CERM 11888, N-methyl-verapamil, N-methylgallopamil, N-methyl-devapamil, dodecyltrimethylammonium, or a quaternary amine derivative of verapamil, gallopamil, devapamil, diltiazem, fendiline, mibefradil, or farnesol amine.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is of Formula IA:

wherein:

each of R^1A′, R^1B′, and R^1C′ is, independently, selected from H, halogen, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, OR^1H′, NR^1I′R^1J′, NR^1K′C(O)R^1L′, S(O)R^1M′, SO₂R^1N′R^1O′, SO₂NR^1P′R^1Q′, SO₃R^1R′, CO₂R^1S′, C(O)R^1T′, and C(O)NR^1U′R^1V′;

each of R^1H′, R^1I′, R^1J′, R^1K′, R^1L′, R^1M′, R^1N′, R^1O′, R^1P′, R^1Q′, R^1R′, R^1S′, R^1T′, R^1U′, and R^1V′ is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl

X^1′ is selected from —CR^1W′R^1X′—, —NR^1Y′C(O)—, —OC(O)—, —SC(O)—, —C(O)NR^1Z′—, —CO₂—, and —OC(S)—;

each of R^1W′, R^1X′, R^1Y′, and R^1Z′ is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

R^1D′ is selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

each of R^1E′, R^1F′, and R^1G′ is, independently, selected from C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl; or

R^1D′ and R^1G′ together complete a heterocyclic ring having at least one nitrogen atom.

In a preferred embodiment, X^1′ is —NHC(O)—. Exemplary compounds of Formula IA include methylated quaternary ammonium derivatives of anesthetic drugs, such as N-methyl lidocaine, N,N-dimethyl prilocaine, N,N,N-trimethyl tocainide, N-methyl etidocaine, N-methyl ropivacaine, N-methyl bupivacaine, N-methyl levobupivacaine, N-methyl mepivacaine. These derivatives can be prepared using methods analogous to those described in Scheme 1. Compounds of Formula IA include

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is of Formula II:

wherein:

each of R^2A, R^2B, and R^2C is, independently, selected from H, halogen, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, OR^2I, NR^2JR^2K, NR^2LC(O)R^2M, S(O)R^2N, SO₂R^2OR^2P, SO₂NR^2QR^2R, SO₃R^2S, CO₂R^2T, C(O)R^2U, and C(O)NR^2VR^2W;

each of R^2I, R^2J, R^2K, R^2L, R^2M, R^2N, R^2O, R^2P, R^2Q, R^2R, R^2S, R^2T, R^2U, R^2V, R^2W is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

X² is selected from —CR^2XR^2Y—, —NR^2ZC(O)—, —OC(O)—, —SC(O)—, —C(O)NR^2AA—, —CO₂—, and —OC(S)—;

each of R^2X, R^2Y, R^2Z, and R^2AA is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

R^2D is selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

R^2E is H or C_1-4 alkyl; and

each of R^2F, R^2G, and R^2H is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

or R^2F and R^2G together complete a heterocyclic ring having two nitrogen atoms.

In some embodiments, when R^2F and R^2G form a heterocyclic ring having two nitrogen atoms, the resulting guanidine group is selected from

where R^2H is H or CH₃. In some embodiments, R^2F and R^2G combine to form an alkylene or alkenylene of from 2 to 4 carbon atoms, e.g., ring systems of 5, 6, and 7-membered rings. In a preferred embodiment, X² is —NHC(O)—. Exemplary compounds of formula II include N-guanidyl derivatives (e.g., —C(NH)NH₂ derivatives) of anesthetic drugs, such as desethyl-N-guanidyl lidocaine, N-guanidyl prilocaine, N-guanidyl tocainide, desethyl-N-guanidyl etidocaine, desbutyl-N-guanidyl ropivacaine, desbutyl-N-guanidyl bupivacaine, desbutyl-N-guanidyl levobupivacaine, desmethyl-N-guanidyl mepivacaine. These derivatives can be prepared using methods analogous to those described in Schemes 2-5.

The guanidyl derivatives described herein (e.g., the compounds of formula II) are presented in their uncharged base form. These compounds can be administered either as a salt (i.e., an acid addition salt) or in their uncharged base form, which undergoes protonation in situ to form a charged moiety.

The synthesis of parent drugs of formulas I and II are described in the literature. See, for example, U.S. Pat. No. 2,441,498 (synthesis of lidocaine), U.S. Pat. No. 3,160,662 (synthesis of prilocaine), DE Patent No. 2235745 (synthesis of tocainide), DE Patent No. 2162744 (synthesis of etidocaine), PCT Publication No. WO85/00599 (synthesis of ropivacaine), U.S. Pat. No. 2,955,111 (synthesis of bupivacaine and levobupivacaine), and U.S. Pat. No. 2,799,679 (synthesis of mepivacaine).

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is of Formula III:

wherein:

n=0-3;

m=0-3; wherein (n+m)=0-6;

each of R^3A, R^3B, and R^3C is, independently, selected from H, halogen, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, OR^3L, NR^3MR^3N, NR^3OC(O)R^3P, S(O)R^3Q, SO₂R^3RR^3S, SO₂NR^3TR^3U, SO₃R^3V, CO₂R^3W, C(O)R^3X, and C(O)NR^3YR^3Z;

each of R^3L, R^3M, R^3N, R^3O, R^3P, R^3Q, R^3R, R^3S, R^3T, R^3U, R^3V, R^3W, R^3X, R^3Y, R^3Z is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

Y³ is selected from —CR^3AAR^3AB—, NR^3ACC(O)—, —OC(O)—, —SC(O)—, —C(O)NR^3AD—, —CO₂—, and —OC(S)—;

each of R^3AA, R^3AB, R^3AC, and R^3AD is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

each of R^3D, R^3E, R^3F, and R^3G is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, C_2-6 heterocyclyl, C_6-12 aryl, C_7-14 alkaryl, and C_3-10 alkheterocyclyl; and

each of R^3H, R^3J, and R^3K is, independently, selected from C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl.

The quaternary nitrogen in formula III is identified herein as N′. Exemplary compounds of formula III include methylated quaternary ammonium derivatives of anesthetic drugs, such as N′-methyl procaine, N′-methyl proparacaine, N′-methyl allocain, N′-methyl encainide, N′-methyl procainamide, N′-methyl metoclopramide, N′-methyl stovaine, N′-methyl propoxycaine, N′-methyl chloroprocaine, N′,N′-dimethyl flecainide, and N′-methyl tetracaine. These derivatives can be prepared using methods analogous to those described in Scheme 1.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is of Formula IV:

wherein:

n=0-3;

m=0-3; wherein with (n+m)=0-6;

each of R^4A and R^4B is, independently, selected from H, halogen, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, OR^4L, NR^4MR^4N, NR^4O C(O)R^4P, S(O)R^4Q, SO₂R^4RR^4S, SO₂NR^4TR^4U, SO₃R^4V, CO₂R^4W, C(O)R^4X, and C(O)NR^4YR^4Z;

each of R^4L, R^4MR^4N, R^4O, R^4P, R^4Q, R^4R, R^4S, R^4T, R^4U, R^4V, R^4W, R^4X, R^4Y, and R^4Z is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

Y⁴ is selected from —CR^4AAR^4AB—, —NR^4ACC(O)—, —OC(O)—, —SC(O)—, —C(O)NR^4AD—, —CO₂—, and —OC(S)—;

each of R^4AA, R^4AB, R^4AC, and R^4AD is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

each of R^4C, R^4D, R^4E, and R^4F is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, C_2-6 heterocyclyl, C_6-12 aryl, C_7-14 alkaryl, and C_3-10 alkheterocyclyl;

X⁴ is selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and NR^4JR^4K;

each of R^4J and R^4K is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl; and

each of R^4G, R^4H, and R^4I is, independently, selected from C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl.

The quaternary nitrogen in formula IV is identified herein as N″. Exemplary compounds of formula III include methylated quaternary ammonium derivatives of anesthetic drugs, such as N″,N″,N″-trimethyl procaine, N″,N″,N″-trimethyl proparacaine, N″,N″,N″-trimethyl procainamide, N″,N″,N″-trimethyl metoclopramide, N″,N″,N″-trimethyl propoxycaine, N″,N″,N″-trimethyl chloroprocaine, N″,N″-dimethyl tetracaine, N″,N″,N″-trimethyl benzocaine, and N″,N″,N″-trimethyl butamben. These derivatives can be prepared using methods analogous to those described in Scheme 1.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is of Formula V:

wherein:

n=0-3;

m=0-3; with (n+m)=0-6;

each of R^5A, R^5B, and R^5C is, independently, selected from H, halogen, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, OR^5M, NR^5NR^5O, NR^5PC(O)R^5Q, S(O)R^5R, SO₂R^5SR^5T, SO₂NR^5UR^5V, SO₃R^5W, CO₂R^5X, C(O)R^5Y, and C(O)NR^5ZR^5AA;

each of R^5M, R^5N, R^5O, R^5P, R^5Q, R^5R, R^5S, R^5T, R^5U, R^5V, R^5W, R^5X, R^5Y, R^5Z, and R^5AA is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

Y⁵ is selected from —CR^5ABR^5AC, —NR^5ADC(O)—, —OC(O)—, —SC(O)—, —C(O)NR^5AE—, —CO₂—, and —OC(S)—;

each of R^5AB, R^5AC, R^5AD, and R^5AE is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

each of R^5D, R^5E, R^5F, and R^5G is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, C_2-6 heterocyclyl, C_6-12 aryl, C_7-14 alkaryl, and C_3-10 alkheterocyclyl;

R^5H is H or C_1-4 alkyl; and

each of R^5J, R^5K, and R^5L is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

or R^5J and R^5K together complete a heterocyclic ring having two nitrogen atoms.

In some embodiments, R^5J and R^5K form a heterocyclic ring having two nitrogen atoms, the resulting guanidine group is selected from:

where R^5L is H or CH₃. In certain embodiments, R^5J and R^5K combine to form an alkylene or alkenylene of from 2 to 4 carbon atoms, e.g., ring systems of 5, 6, and 7-membered rings.

The guanylated nitrogen in formula V is identified herein as N′. Exemplary compounds of formula V include N-guanidyl derivatives (e.g., —C(NH)NH₂ derivatives) of anesthetic drugs, such as such as desethyl-N′-guanidyl procaine, desethyl-N′-guanidyl proparacaine, desethyl-N′-guanidyl allocain, desmethyl-N′-guanidyl encainide, desethyl-N′-guanidyl procainamide, desethyl-N′-guanidyl metoclopramide, desmethyl-N′-guanidyl stovaine, desethyl-N′-guanidyl propoxycaine, desethyl-N′-guanidyl chloroprocaine, N′-guanidyl flecainide, and desethyl-N′-guanidyl tetracaine. These derivatives can be prepared using methods analogous to those described in Schemes 2-5.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is of Formula VI:

wherein:

n=0-3;

m=0-3; wherein with (n+m)=0-6;

each of R^6A and R^6B is, independently, selected from H, halogen, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, OR^6K, NR^6LR^6M, NR^6NC(O)R^6O, S(O)R^6P, SO₂R^6QR^6R, SO₂NR^6SR^6T, SO₃R^6U, CO₂R^6V, C(O)R^6W, and C(O)NR^6XR^6Y;

each of R^6K, R^6L, R^6M, R^6N, R^6O, R^6P, R^6Q, R^6R, R^6S, R^6T, R^6U, R^6V, R^6W, R^6X, and R^6Y is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

Y⁶ is selected from —CR^6ZR^6AA—, NR^6ABC(O)—, —OC(O)—, —SC(O)—, —C(O)NR^6AC—, —CO₂—, and —OC(S)—;

each of R^6Z, R^6AA, R^6AB, and R^6AC is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

each of R^6C, R^6D, R^6E, and R^6F is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, C_2-6 heterocyclyl, C_6-12 aryl, C_7-14 alkaryl, and C_3-10 alkheterocyclyl;

X⁶ is selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and NR^6ADR6^AE; each of R^6AD and R^6AE is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

R^6G is H or C_1-4 alkyl; and

each of R^6H, R^6I, and R^6J is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

or R^6H and R^6I together complete a heterocyclic ring having two nitrogen atoms.

In some embodiments, R^6H and R^6I form a heterocyclic ring having two nitrogen atoms, the resulting guanidine group is selected from:

where R^6J is H or CH₃. In some embodiments, R^6H and R^6I combine to form an alkylene or alkenylene of from 2 to 4 carbon atoms, e.g., ring systems of 5, 6, and 7-membered rings.

The guanylated nitrogen in formula V is identified herein as N″. Exemplary compounds of formula VI include N-guanidyl derivatives (e.g., —C(NH)NH₂ derivatives) of anesthetic drugs, such as such as N″-guanidyl procaine, N″-guanidyl proparacaine, N″-guanidyl procainamide, N″-guanidyl metoclopramide, N″-guanidyl propoxycaine, N″-guanidyl chloroprocaine, N″-guanidyl tetracaine, N″-guanidyl benzocaine, and N″-guanidyl butamben. These derivatives can be prepared using methods analogous to those described in Schemes 2-5.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is of Formula VII:

wherein:

n=0-3;

m=0-3; wherein with (n+m)=0-6;

each of R^7A, R^7B, and R^7C is, independently, selected from H, halogen, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, OR^7L, NR^7MR^7N, NR^7OC(O)R^7P, S(O)R^7Q, SO₂R^7RR^7S, SO₂NR^7TR^7U, SO₃R^7V, CO₂R^7W, C(O)R^7X, and C(O)NR^7YR^7Z;

each of R^7L, R^7M, R^7N, R^7O, R^7P, R^7Q, R^7R, R^7S, R^7T, R^7U, R^7V, R^7W, R^7X, R^7Y, and R^7Z is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

X⁷ is selected from —CR^7AAR^7AB—, —NR^7ACC(O)—, —OC(O)—, —SC(O)—, —C(O)NR^7AD—, —CO₂—, and —OC(S)—;

each of R^7AA, R^7AB, R^7AC, and R^7AD is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

each of R^7D, R^7E, R^7F, and R^7G is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, C_2-6 heterocyclyl, C_6-12 aryl, C_7-14 alkaryl, and C_3-10 alkheterocyclyl;

each of R^7H, R^7J, and R^7K is, independently, selected from C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl.

In a preferred embodiment, X⁷ is —C(O)NH—. Exemplary compounds of formula VII include methylated quaternary ammonium derivatives of anesthetic drugs, such as N′-methyl dibucaine. These derivatives can be prepared using methods analogous to those described in Scheme 1.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is of Formula VIII:

wherein:

n=0-3;

m=0-3; wherein (n+m)=0-6;

each of R^8A, R^8B, and R^8C is, independently, selected from H, halogen, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, OR^8L, NR^8MR^8N, NR^8OC(O)R^8P, S(O)R^8Q, SO₂R^8RR^8S, SO₂NR^8TR^8U, SO₃R^8V, CO₂R^8W, C(O)R^8X, and C(O)NR^8YR^8Z;

each of R^8L, R^8M, R^8N, R^8O, R^8P, R^8Q, R^8R, R^8S, R^8T, R^8U, R^8V, R^8W, R^8X, R^8Y, and R^8Z is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

X⁸ is selected from —CR^8AAR^8AB—, —NR^8ACC(O)—, —OC(O)—, —SC(O)—, —C(O)NR^8AD—, —CO₂—, and —OC(S)—;

each of R^8AA, R^8AB, R^8AC, and R^8AD is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

each of R^8D, R^8E, R^8F, and R^8G is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, C_2-6 heterocyclyl, C_6-12 aryl, C_7-14 alkaryl, and C_3-10 alkheterocyclyl;

R^8H is H or C_1-4 alkyl;

each of R^8I, R^8J, and R^8K is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl; or R^8I and R^8J together complete a heterocyclic ring having two nitrogen atoms.

In some embodiments, R^8I and R^8J form a heterocyclic ring having two nitrogen atoms, the resulting guanidine group is selected from

where R^8K is H or CH₃. In some embodiments, R^8I and R^8J combine to form an alkylene or alkenylene of from 2 to 4 carbon atoms, e.g., ring systems of 5, 6, and 7-membered rings.

The guanylated nitrogen in formula V is identified herein as N′. In a preferred embodiment, X⁸ is —C(O)NH—. Exemplary compounds of formula VIII include N-guanidyl derivatives (e.g., —C(NH)NH₂ derivatives) of anesthetic drugs, such as such as desethyl-N-guanidyl dibucaine. These derivatives can be prepared using methods analogous to those described in Schemes 2-5.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is of Formula IX:

wherein:

n=0-6;

each of R^9A, R^9B, R^9C, R^9D, and R^9E is, independently, selected from H, halogen, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, OR^9I, NR^9JR^9K, NR^9LC(O)R^9M, S(O)R^9N, SO₂R^9OR^9P, SO₂NR^9QR^9R, SO₃R^9S, CO₂R^9T, C(O)R^9U, and C(O)NR^9VR^9W;

each of R^9I, R^9J, R^9K, R^9L, R^9M, R^9N, R^9O, R^9P, R^9Q, R^9R, R^9S, R^9T, R^9U, R^9V, and R^9W is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

X⁹ is selected from —CR^9XR^9Y—, —O—, —S—, and —NR^9Z—; and each of R^9X, R^9Y, and R^9Z is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

Y⁹ is NR^9AANR^9ABNR^9AC or NR^9ADZ⁹;

each of R^9AA, R^9AB, and R^9AC is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, and C_2-4 alkynyl;

R^9AD is H or C_1-4 alkyl;

Z⁹ is

each of R^9F, R^9G, and R^9H is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, and C_2-4 alkynyl, or R^9F and R^9G together complete a heterocyclic ring having two nitrogen atoms.

In some embodiments, R^9F and R^9G form a heterocyclic ring having two nitrogen atoms, the resulting guanidine group is selected from

where R^9H is H or CH₃. In some embodiments, R^9F and R^9G combine to form an alkylene or alkenylene of from 2 to 4 carbon atoms, e.g., ring systems of 5, 6, and 7-membered rings. In a preferred embodiment, X⁹═—O—. Exemplary compounds of formula IX include N-guanidyl derivatives (e.g., —C(NH)NH₂ derivatives), such as N-guanidyl fluoxetine, and methylated quaternary ammonium derivatives, such as N,N-dimethyl fluoxetine. These derivatives can be prepared using methods analogous to those described in Schemes 1-5.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is of Formula X:

wherein:

W₃ is O, NH, NCH₂R^10J, NC(O)CH₂R^10J, CHCH₂R^10J, C═CHR^10J, or C═CHR^10K;

W₁-W₂ is S, O, OCHR^10K, SCHR^10K, N═CR^10K, CHR^10L—CHR^10K, or CR^10L═CR^10K;

each of R^10A, R^10B, R^10C, R^10D, R^10E, R^10F, R^10G, and R^10H is, independently, selected from H, OH, halide, C_1-4 alkyl, and C_2-4 heteroalkyl;

R^10J is CH₂CH₂X^10A or CH(CH₃)CH₂X^10A;

R^10L is H or OH; R^10K is H, OH, or the group:

X^10A is NR^10MR^10NR^10P, or NR^10QX^10C; X^10B is NR^10RR^10S or NX^10C;

each of R^10M, R^10N, R^10P, R^10R, and R^10S is, independently, selected from C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl, or R^10R, and R^10S together complete a heterocyclic ring having at least one nitrogen atom;

R^10Q is H or C_1-4 alkyl;

X^10C is

and

each of R^10T, R^10U, and R^10V is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, and C_2-4 alkynyl, or R^10T and R^10V together complete a heterocyclic ring having two nitrogen atoms.

In some embodiments, R^10T and R^10V form a heterocyclic ring having two nitrogen atoms, the resulting guanidine group is selected from

where R^10U is H or CH₃. In some embodiments, R^10T and R^10V combine to form an alkylene or alkenylene of from 2 to 4 carbon atoms, e.g., ring systems of 5, 6, and 7-membered rings.

Exemplary compounds of formula X include N-guanidyl derivatives (e.g., —C(NH)NH₂ derivatives) and methylated quaternary ammonium derivatives. N-guanidyl derivatives of formula X include, without limitation, N-guanidyl amoxapine, desmethyl-N-guanidyl trimipramine, desmethyl-N-guanidyl dothiepin, desmethyl-N-guanidyl doxepin, desmethyl-N-guanidyl amitriptyline, N-guanidyl protriptyline, N-guanidyl desipramine, desmethyl-N-guanidyl clomipramine, desmethyl-N-guanidyl clozapine, desmethyl-N-guanidyl loxapine, N-guanidyl nortriptyline, desmethyl-N-guanidyl cyclobenzaprine, desmethyl-N-guanidyl cyproheptadine, desmethyl-N-guanidyl olopatadine, desmethyl-N-guanidyl promethazine, desmethyl-N-guanidyl trimeprazine, desmethyl-N-guanidyl chlorprothixene, desmethyl-N-guanidyl chlorpromazine, desmethyl-N-guanidyl propiomazine, desmethyl-N-guanidyl prochlorperazine, desmethyl-N-guanidyl thiethylperazine, desmethyl-N-guanidyl trifluoperazine, desethyl-N-guanidyl ethacizine, and desmethyl-N-guanidyl imipramine. Methylated quaternary ammonium derivatives of formula X include, without limitation, N,N-dimethyl amoxapine, N-methyl trimipramine, N-methyl dothiepin, N-methyl doxepin, N-methyl amitriptyline, N,N-dimethyl protriptyline, N,N-dimethyl desipramine, N-methyl clomipramine, N-methyl clozapine, N-methyl loxapine, N,N-dimethyl nortriptyline, N-methyl cyclobenzaprine, N-methyl cyproheptadine, N-methyl olopatadine, N-methyl promethazine, N-methyl trimeprazine, N-methyl chlorprothixene, N-methyl chlorpromazine, N-methyl propiomazine, N-methyl moricizine, N-methyl prochlorperazine, N-methyl thiethylperazine, N-methyl fluphenazine, N-methyl perphenazine, N-methyl flupenthixol, N-methyl acetophenazine, N-methyl trifluoperazine, N-methyl ethacizine, and N-methyl imipramine. These derivatives can be prepared using methods analogous to those described in Schemes 1-5.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is a quaternary amine derived from orphenadrine, phenbenzamine, bepridil, pimozide, penfluridol, flunarizine, fluspirilene, propiverine, disopyramide, methadone, tolterodine, tridihexethyl salts, tripelennamine, mepyramine, brompheniramine, chlorpheniramine, dexchlorpheniramine, carbinoxamine, levomethadyl acetate, gallopamil, verapamil, devapamil, tiapamil, emopamil, dyclonine, pramoxine, lamotrigine, fendiline, mibefradil, gabapentin, amiloride, diltiazem, nifedipine, nimodipine, nitrendipine, cocaine, mexiletine, propafenone, quinidine, oxethazaine, articaine, riluzole, bencyclane, lifarizine, and strychnine. Still other compounds can be modified to incorporate a nitrogen atom suitable for quaternization or guanylation (e.g., fosphenytoin, ethotoin, phenytoin, carbamazepine, oxcarbazepine, topiramate, zonisamide, and salts of valproic acid).

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is a quaternized or guanylated derivative of a compound as described in Table 1.

TABLE 1
No.	Compound	Exemplary References
1	orphenadrine	U.S. Pat. No. 2,567,351 (see, e.g., the
		compounds of Examples 1-6 and the formula
		described at col.1, lines 10-24).
		U.S. Pat. No. 2,991,225 (see, e.g., the
		structure shown at col. 1, line 25).
2	phenbenzamine (RP-	Passalacqua et al., “Structure and
	2339; Antergan ®),	Classification of Hi-Antihistamines and
		Overview of Their Activities,” in Histamine
		and Hi -antihistamines in Allergic Disease,
		F.E.R. Simons, Ed., Informa Health Care
		(2002).
3	bepridil	U.S. Pat. No. 3,962,238 (see, e.g., Formulas
		I-V and compounds 1-6 of Table 1).
		U.S. RE30577
4	pimozide	See, e.g., Janssen et al., Arzneimittel-Forsch.
		18:261, 279, 282 (1968), and Journal of
		Neuroscience, 22(2):396-403 (2002)
5	penfluridol	U.S. Pat. No. 3,575,990 (see, e.g., the
		compounds of Formula (I), claims 1-7, and
		Examples I-XXXIII).
6	flunarizine	U.S. Pat. No. 3,773,939 (see, e.g., Formula
		(1) and the compound described at col. 5, line
		40).
7	fluspirilene	U.S. Pat. No. 3,238,216 (see, e.g., the
		compounds recited in any of claims 1-34).
8	propiverine	DD 106643
9	disopyramide	U.S. Pat. No. 3,225,054 (see, e.g., the
		compounds of Examples 1-15 and claims 1-3)
10	methadone	DE711069
		U.S. Pat. No. 2,983,757
11	tolterodine	U.S. Pat. No. 5,382,600 (see, e.g., Formula
		(I), the compounds described at col.3, lines
		20-39, in Table 1, and in claims 1-7)
12	tridihexethyl salts	U.S. Pat. No. 2,913,494 (see, e.g., col. 1,
		lines 15-22)
13	tripelennamine	U.S. Pat. No. 2,502,151 (see, e.g., Formula
		(1) and the compounds recited in claims 1-13)
14	mepyramine	U.S. Pat. No. 2,502,151
	(pyrilamine)
15	brompheniramine	U.S. Pat. No. 2,567,245 (see, e.g., the
		formula described at col. 1, lines 30-45, the
		compounds of Examples I-XXI, and the
		compounds recited in claims 1-15)
		U.S. Pat. No. 2,676,964 (see, e.g., the
		formula described at col.1, lines 5-28, the
		compounds of Examples I-XLIV, and the
		compounds recited in claims 1-14)
		U.S. Pat. No. 3,061,517 (see, e.g., the
		formula at col.1, lines 49-67, and the
		compounds described at col. 2, lines 17-19,
		col. 2, lines 40-43, col. 4, lines 2-7, and claims
		1-6)
16	chlorpheniramine
17	dexchlorphenir amine	U.S. Pat. No. 2,567,245 (see, e.g., the
		formula described at col. 1, lines 30-45, the
		compounds of Examples I-XXI, and the
		compounds recited in claims 1-15)
		U.S. Pat. No. 2,676,964 (see, e.g., the
		formula described at col.1, lines 5-28, the
		compounds of Examples I-XLIV, and the
		compounds recited in claims 1-14)
		U.S. Pat. No. 3,061,517 (see, e.g., the
		formula at col.1, lines 49-67, and the
		compounds described at col. 2, lines 17-19,
		col. 2, lines 40-43, col. 4, lines 2-7, and claims
		1-6)U.S. Pat. No. 2,766,174 (see, e.g., the
		formula described at col. 1, lines 41-72)
18	carbinoxamine	U.S. Pat. No. 2,606,195 (see, e.g., the
		formula described at col. 1, lines 7-24,
		Examples I-VIII, and in claims 1-3)
		U.S. Pat. No. 2,800,485
		GB 905993
19	levomethadyl acetate	Pohland et al., J. Am. Chem. Soc. 71:460
		(1949)
20	gallopamil	U.S. Pat. No. 3,261,859 (see, e.g., Formula
		(1), Examples 1-28, and claims 1-19)
		Theodore et al., J. Org. Chem. 52:1309 (1987)
21	verapamil	U.S. Pat. No. 3,261,859 (see, e.g., Formulas
		(I) and (IV), Examples 1-28, and claims 1-19)
22	devapamil	Godfraind, Calcium Channel Blockers,
23	tiapamil	Birkhauser Verlag (January 2004).
24	emopamil
25	dyclonine	Pofft, Chem. Tech. (Berlin) 4:241 (1952)
26	pramoxine	U.S. Pat. No. 2,870,151 (see, e.g., the
		formula described at col.1, lines 18-25, and
		the compounds of Examples I-XII and claims
		1-13).
27	lamotrigine	EP21121
		U.S. Pat. No. 4,602,017 (see, e.g., Formulas
		(I)-(III) and the compounds described at col.
		2, line 63-col. 3, line 12, Examples 1-5, and
		claims 1-2)
28	mibefradil	U.S. Pat. No. 4,808,605 (see, e.g., Formula 1
		described at col.1, lines 10-33 and the
		compounds described at col. 3, line 58-col. 7,
		line 6, Examples 1-41, and claims 1-15).
29	gabapentin	U.S. Pat. No. 4,024,175 (see, e.g., Formula
		(I) described at col.1, lines 5-17, Examples 1-
		12, and claims 1-11)
30	amiloride	U.S. Pat. No. 3,313,813 (see, e.g., the
		compounds described at col. 1, line 13-col.2,
		line 55, Examples 1-205, and claims 1-31)
31	diltiazem	U.S. Pat. No. 3,562,257 (see, e.g., Formula
		(1) described at col.1, lines 39-64, and the
		compounds described at col. 2, lines 15-30,
		Tables 1-3, and claims 1-43)
		U.S. Pat. No. 4,552,695 (see, e.g., the
		compound of Formula (I))
32	nifedipine	U.S. Pat. No. 3,485,847 (see, e.g., the
		Formula described at col. 1, line 40-col. 2, line
		6, the compounds of Examples 1-6, and claims
		1-27)
33	nimodipine	U.S. Pat. No. 3,799,934 (see, e.g., the
		Formula described at col. 1, lines 39-69, the
		compounds described at col. 4, line 50-col. 5,
		line 16, Examples 1-53, and claims 1-13)
34	nitrendipine
35	mexiletine	U.S. Pat. No. 3,954,872 (see, e.g., Formula
		(1) described at col.1, lines 14-35, and the
		compounds of Examples 1-6 and claims 1-4)
36	propafenone	DE2001431 (see, e.g., claims 1-4)
37	quinidine	Turner et al., The Alkaloids, Vol. 3, 1-63
		(1953)
		Mason et al., Ann. N.Y. Acad. Sci. 432:162-
		176(1984)
38	oxethazaine	U.S. Pat. No. 2,780,646 (see, e.g., the
		formula described at col. 1, lines 18-42, and
		the compounds of Examples 1-14 and claims
		1-8)
39	articaine	Becker et al., Anesth Prog. 53(3): 98-109
		(Fall 2006)
40	riluzole	U.S. Pat. No. 4,370,338 (see, e.g., the
		compound described at col. 1, line 15)
41	bencyclane	HU 151865
42	lifarizine	Grauert et al., J. Med. Chem. 45(17):3755-
		3764 (2002)
43	strychnine	Makarevich et al., “Quaternary salts of
		alkaloids,” Vol. 42, pages 473-476, Chemistry
		of Natural Compounds, Springer New York:
		2006.
44	fendiline	U.S. Pat. No. 3,262,977 (see, e.g., Formula
		(I), Examples 1-9, and the compounds of
		claims 1-9)

Exemplary calcium channel blockers include D-890, CERM 11888, N-methyl-verapamil, N-methylgallopamil, N-methyl-devapamil, and dodecyltrimethylammonium. Other exemplary compounds include any charged derivative, e.g., a quaternary amine derivative, of verapamil, gallopamil, devapamil, diltiazem, fendiline, mibefradil, terpene compounds (e.g., sesquiterpenes) such as those described in Norman et al. Agricultural and Biological Chemistry 49(10):2893-8 (1985), and other inhibitors of calcium channels (see, for example, Triggle, European Journal of Pharmacology, 375:311-325 (1999), Eller et al., British Journal of Pharmacology, 130:669-677 (2000), and Yamamoto et al., Current Topics in Medicinal Chemistry, 9:377-395 (2009), which can be prepared according to the methods described herein.

For example, Yamamoto et al. provides the following N-type calcium channel blockers (Table 2), which can be modified (e.g., quaternized or guanylated) according to the methods described herein.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is a quaternized or guanylated derivative of a compound as described in Table 2.

TABLE 2
No.	Compound	Exemplary References
45		Yamamoto et al., Bioorg. Med. Chem. 14:5333-5339 (2006).
46		Yamamoto et al., Bioorg. Med. Chem. Lett. 16:798-802 (2006).
47		Yamamoto et al., Bioorg. Med. Chem. Lett. 18:4813-4815 (2008).
48		See, e.g., WO08143263 and EP2149560 (e.g., Formula (I), the compounds of Tables 6-35, 43-110, 126-127, and the compounds of claims 1-6)
49		Miller et al., Soc. Neurosci. Abstr. 25(Part2):896.3 (1999)
50		WO0236567 (see, e.g., formulas I-IV, the compounds of Table 2 (Examples 1-111), and claims 1-5)
51		Zhang et al., Eur. J. Pharmacol. 587:24-47 (2008)
52		Baell et al., Bioorg. Med. Chem. 12:4025-4037 (2004)
53		Yamamoto et al., 22nd National Meeting of American Chemical Society, American Chemical Society: Washington, DC: Chicago, IL 2001; Kaneda et al, Soc. Neurosci. Abstr. 2T.332A5 (2001); Niidome et al., Soc. Neurosci.
		Abstr. 27:332.14 (2001); and
		Suzuki et al., Bioorg. Med.
		Chem. Eett. 13:919-922 (2003).
54	E-2051	Kaneda, Soc. Neurosci. Abstr.
		28:490.1 (2002)
55		WO07110449 (see, e.g., Formulas I-XIII, the compounds described at Paragraphs [0181]- [0183] and Examples 1-14, and claims 1-72)
56		WO06040181 (see, e.g., Formulas I-X, the compounds described at Paragraphs [0105]- [0109] and Examples 1-37, and in claims 1-56)
57		WO07118853 (see, e.g., Formulas I-XIII, the compounds described at Paragraph [0320] and Examples 1-19, and the compounds of claims 1-165)
58		WO07085357 (see, e.g., Formulas I-VII, the compounds described at Paragraphs [0065]- [0067], Examples 1-6, and claims 1-16)
59		WO07028638 (see, e.g., Formulas XXVI, the compounds described a Paragraphs [0119]-[0123], Examples 1-24, and claims 1-20)
60		WO07118854 (see, e.g., Formulas I-VII and the compounds of Examples 1-11 and claims 1-36)
61		WO08008398 (see, e.g., Formulas I, I′, I″, II, and II′; Examples 1-377, and claims 1-7)
62		WO08150447 (see, e.g., Formulas I, I′, I″, and the compounds of Examples 1-135 and claims 1-5
63		Knutsen et al., Bioorg. Med. Chem. Lett. 17: 662-667 (2007)
64		O’Neill, Brain Res. 888:138-149 (2001); Hicks et al., Eur. J. Pharmacol. 408:241-248 (2000)
65		WO07084394 (see, e.g., the compounds of Formulas I and Ia-Ig, and the compounds of Examples 1-11 and claims 1 and 2)
66		WO08066803 (see, e.g., Formulas I and II, the compound of Example 1, and claims 1-11)
67		WO07075524 (see, e.g., Formulas (I), (Ia)-(Ie), the compounds of Examples 1-184, and claims 1-16)
68		WO08133867 (see, e.g., Formulas (I) and (II), the compounds of Examples 1-163, and claims 1-16)
69		WO01045709 (see, e.g., Formula (1), the compounds of Example 4, and claims 24-38) WO06105670 (see, e.g., Formula (1), the compounds described at Paragraphs [0065] and [0066], and claims 1-13)
70		WO04089377 (see, e.g., Formula (1), Examples 1-5, original claims 1-13, and amended claims 1-17)
71		WO07071035 (see, e.g., Formula (1), the compounds of Examples 1-18, and claims 20-35)
72		WO08043183 (see, e.g., Formulas (1) and (2), the compounds of Examples 1-16, and claims 16-28)
73		WO04089922 (see, e.g., Formulas (l)-(4), the compounds of Examples 1-9, claims 1-17, and the compounds of Figure 1)
74		WO04105750 (see, e.g., Formulas (l)-(8), the compounds of Examples 1-10, claims 1-23, and Figure 1)
75		WO08031227 (see, e.g., Formulas (1) and (2), the compounds of Examples 1-20, and claims 21-37)
76		Tatsumi et al., Jpn. J. Pharmacol. 73:193 (1997);Aoki et al., Brain Res. 890:162-169 (2001); Katsumata et al., Brain Res. 969:168-174 (2003); Tamura et al., Brain Res. 890:170-176 (2001); Shi et al., J. Thorac. Cardiovasc Surg. 129:364-371 (2005); Small, IDrugs, 3:460-465 (2000); Suma et al., Jpn. J. Pharmacol. 73: 193 (1997); Shimidzu et al., Naunyn Schmiedebergs Arch. Pharamcol. 355:601-608 (1997); and Suma et al., Eur. J. Pharmacol.
		336:283-290(1997).
77		Seko et al, Bioorg. Med. Chem. Lett. 11:2067-2070(2001)
78		Seko et al., Bioorg. Med. Chem. 11:1901-1913 (2003). Seko et al., Bioorg. Med. Chem. Lett. 12:915-918 (2002)
79		Seko et al., Bioorg. Med. Chem. Lett. 12:2267-2269 (2002)
80		Menzler et al., Bioorg. Med. Chem. Lett. 10:345-347 (2000)
81		Malone et al., 217th National Meeting of the American Chemical Society, American Chemical Society: Washington DC: Anaheim CA 1999; Hu et al., J. Med. Chem. 42:4239-4249 (1999)
82		Hu et al., Bioorg. Med. Chem. Lett. 9:907-912 (1999)
83		Hu et al., Bioorg. Med. Chem. Lett. 9:2151-2156 (1999) Ryder et al., Bioorg. Med. Chem. Lett. 9:1813-1818 (1999)
84		Hu et al., Bioorg. Med. Chem. Lett. 9:1121-1126 (1999)
85		Bennett et al., Pain 33:87-107 (1988)
86		Hu et al., Bioorg. Med. Chem. 8:1203-1212 (2000)
87		Hu et al., Bioorg. Med. Chem. 8:1203-1212 (2000)
88		Hu et al., J. Med. Chem. 42:4239-4249 (1999)
89		Schelkun et al., Bioorg. Med. Chem. Lett. 9:2447-2452 (1999).
90		Yuen et al., Bioorg. Med. Chem. Lett. 8:2415-2418 (1998)
91		Song et al., J. Med. Chem. 43:3474-3477 (2000)
92		WO07125398 (see, e.g., Formula (1), the compounds of Examples 1-29, and claims 1-9)
93		WO08124118 (see, e.g., Formula I-VI, the compounds of Paragraphs [0129] and Examples 1-5, and claims 1-42)
94		Campbell et al., Eur. J. Pharmacol. 401:419-428 (2000)
95		Teodori et al., J. Med. Chem. 47:6070-6081 (2004)
96		Teodori et al., J. Med. Chem. 47:6070-6081 (2004)
97		Schroeder et al., Mol. Divers. 8:127-134 (2004).
98		WO06030211 (see, e.g., Formula (I), the compounds described at page 9, line 17-page 15, line 12, Examples 1-99, and claims 1-12)

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is of Formula XI:

wherein:

each R^11A, R^11B, and R^11C is selected, independently, from H or C_1-4 alkyl, and 0, 1, 2, or 3 of the dashed bonds () represents a carbon-carbon double bond (i.e., compounds of Formula (XI) can include 0, 1, 2, or 3 double bonds), provided that when 2 or 3 carbon-carbon double bonds are present, the double bonds are not adjacent to one another. Compounds that include 0, 1, or 2 double bonds can be prepared according to methods known in the literature, e.g., partial or total hydrogenation of the parent triene.

In some embodiments, compounds of Formula (XI) can be represented by the following formula (XI-A),

where each R^11A, R^11B, R^11C, and X is according to Formula (XI), and where each dashed bond represents an optional carbon-carbon double bond.

In some embodiments, compounds of Formula (XI) include those compounds that have a structure according to Formula (XI-B),

where each R^11A, R^11B, R^11C, and X is according to Formula (XI).

Exemplary compounds of Formula (XI) include

Amino acid derivatives, e.g., those described in U.S. Pat. No. 7,166,590 or in Seko et al., Bioorg. Med. Chem. Lett. 11(16):2067-2070 (2001), each of which is herein incorporated by reference, can also be used herein. For example, compounds having a structure according to Formula (XII) can be N-type calcium channel blockers.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is of Formula XII:

wherein:

each of R^12A, R^12B, R^12C, and R^12D is, independently, selected from C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, C_7-14 alkaryl, C_3-10 alkcycloalkyl, and C_3-10 alkheterocyclyl; or R^12A and R^12B together complete a heterocyclic ring having at least one nitrogen atom;

n is an integer between 1-5;

each of R^12E and R^12F is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, C_7-14 alkaryl, C_3-10 alkcycloalkyl, or C_3-10 alkheterocyclyl; and

X is any pharmaceutically acceptable anion.

Exemplary compounds of Formula (XII) include

Still other compounds that can be used herein are quaternary amine derivatives of flunarizine and related compounds (see, e.g., U.S. Pat. Nos. 2,883,271 and 3,773,939, as well as Zamponi et al., Bioorg. Med. Chem. Lett. 19: 6467 (2009)), each of which is hereby incorporated by reference. For example, compounds according to Formulas (XIII-A), (XIII-B), and (XIII-C) can be prepared according to, e.g., Zamponi et al., and used herein.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is of Formula XIII-A, XIII-B, or XIII-C:

wherein:

each R^13A-R^13J and R^13O-R^13T is selected, independently, from H, halogen, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, C_7-14 alkaryl, C_3-10 alkcycloalkyl, and C_3-10 alkheterocyclyl, OR^13AA, NR^13ABR^13AC, NR^13ADC(O)R^13AE, S(O)R^13AF, SO₂R^13AGR^13AH, SO₂NR^13AIR^13AJ, SO₃R^13AK, CO₂R^13AL, C(O)R^13AM, and C(O)NR^13ANR^13AO;

each of R^13AA-R^13AO is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl;

each R^13K, R^13L, R^13M and R^13N is, independently, H or C_1-4 alkyl, or R^13K and R^13L, or R^13M and R^13N, combine to form C═O, or R^13K and R^13M combine to form C═C;

R^13Y is H or C_1-4 alkyl;

R^13Z and R^13Z′ are, independently, selected from H, halogen, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, C_7-14 alkaryl, C_3-10 alkcycloalkyl, and C_3-10 alkheterocyclyl; and

X⁻ is any pharmaceutically acceptable anion.

Exemplary compounds according to Formulas (XIII-A)-(XIII-C) include

Derivatives of mibrefradil, such as those described in U.S. Pat. No. 4,808,605, hereby incorporated by reference can also be used. Exemplary mibrefadil derivatives include compounds of Formula (XIV). In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is of Formula XIV:

wherein:

n is an integer between 0-5;

R^14A is heterocyclyl (e.g., a heteroaryl such as benzimidazole),

each of R^14B, R^14C, R^14D, and R^14E is, independently, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, C_7-14 alkaryl, C_3-10 alkcycloalkyl, and C_3-10 alkheterocyclyl; and

R^14F is selected from H, halogen, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, C_2-4 heteroalkyl, C_7-14 alkaryl, C_3-10 alkcycloalkyl, and C_3-10 alkheterocyclyl, OR^14G, NR^14HR^14I, NR^14JC(O)R^14K, S(O)R^14L, SO₂R^14MR^14N, SO₂NR^14OR^14P, SO₃R^14Q, CO₂R^14R, C(O)R^14S, and C(O)NR^14TR^14V; and

each of R^14G-R^13AO is, independently, selected from H, C_1-4 alkyl, C_2-4 alkenyl, C_2-4 alkynyl, and C_2-4 heteroalkyl.

An exemplary compound of Formula (XIV) is

Charged derivatives of 4-piperidinylaniline compounds (e.g., Compounds (86)-(88) of Table 2) can be prepared according to methods known in the literature and described herein. For example, charged N-alkyl derivatives (e.g., N-methyl) of Compounds (86)-(88) can be prepared and used in the compositions, methods, and kits described herein.

Still other channel blockers that can be quaternized or guanylated according to the methods described herein are described, for example, in PCT Publication No. WO 2004/093813 (see, e.g., Tables 5, 6 and 8), which is herein incorporated by reference.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is a quaternized or guanylated derivative of a compound as described in Table 3.

TABLE 3
No.	Compound	Exemplary References
105	Isradipine
106	Nickel Chloride
107	A-53930A	JP 08208690
108	AE-0047 Watanidipine	EP 00424901
	dihydrochloride
109	AGN-190604	Inflammation, 19(2):261-275
		(1995)
110	AGN-190744	EP372940
111	AH-1058	European Journal of
		Pharmacology, 398(1): 107-112
		(2000)
112	AHR 5360C	European Journal of
		Pharmacology 146(2-3): 215-22
		(1988)
113	AHR 12234	Archives Internationales de
		Pharamcodynamie et de Therapie
		301:131-50(1989)
114	AHR-12742	ZA 08604522
115	AHR-16303B	Journal of Cardiovascular
		Pharmacology 17(1): 134-44
		(1991)
116	AHR-16462B	Drug Development Research,
		22(3): 259-271 (1991)
117	AIT 110
118	AIT 111
119	AJ2615	WO 8601203 A1
120	AJ-3941	Arzneimittel Forschung
		46(6):567-71 (1996)
121	(+)-alismol	JP 04077420 A2
122	AM-336 (synthetic version of	WO9954350
	CVID marine cone snail venom)
123	AM 543
124	amlodipine	U.S. Pat. No. 4572902
125	S-(−)amlodipine	GB 2233974 A1
126	AN 132	EP 196648
127	animpamil LU 42668	EP 64158 A1
128	antioquine (alkaloid from stem	Journal of natural Products
	bark)	55(9):1281-6 (1992)
129	AP-1067	IDDB 268934
130	AQ-AH-208	CH 645628 A
131	AR 12456 (derivative of trapidil)	BE 902218 A1
		Cardiovascular Drug Reviews
		9(4):385-397 (1991)
132	aranidipine	U.S. Pat. No. 4446325
133	atosiban	EP 00112809
134	azenidipine CS 905	EP 88266922
135	B 84439	EP 240828
136	barnidipine (derivative of	U.S. Pat. No. 4220649
	nicardipine)	DE 02904552
137	BAY-E-6927	DE 2117571
138	BAY-K-9320	EP 9206
139	BAY-T-7207
140	BBR-2160	EP 28204 A2
141	BDF 8784	EP 25111
142	belfosdil/BMY 21891/SR7037	EP 173041 A1
143	Bencylcalne/EGYT-201	FR 151193
144	benipidine/KW3049/N akadipine	U.S. Pat. No. 4448964
145	bepridil	U.S. Pat. No. 3962238
146	bisaramil/RGH 2957	WO 9622096
147	BK 129	Methods and Findings in
		Experimental and Clinical
		Pharamcology 14(3): 175-81
		(1992)
148	BMS-181102	EP 559569
149	BMS-188107	U.S. Pat. No. 5070088
150	BMY 20014	DE 3512995 A1
151	BMY 20064	DE 3512995 A1
152	BMY-43011	Bioorganic and Medicinal
		Chemistry Letters, 3(12):2817-
		2820 (1993)
153	BN 50149	WO 9323082
154	BN 50175	WO 9323082
155	BN 50394	WO 9323082
156	BR 1022	Current Science 83(4):426-431
		(2002)
157	BRL 3287A	WO 9323082
158	BRL-32872	WO 09323024
159	buflomedil	U.S. Pat. No. 4326083
160	butoprozine	DE 2707048
161	CAF 603	Organic and Bioorganic
		Chemistry, 22:3349:52 (1994)
162	calciseptine (venom polypeptide)	WO 2000 069900
163	calcium antagonists	WO 9205165
164	calcium channel antagonists	WO 00236586
		WO 0236567
165	calcium channel blocker (L-type)	Journal of Medicinal Chemistry,
		39(15):2922-2938 (1996)
166	calcium channel blockers	EP 400665 A2
		U.S. Pat. No. 4965356
167	calcium channel blockers	WO 9526325
168	carvedilol	U.S. Pat. No. 4503067
169	caryachine	British Journal of Pharmacology,
		116(8):3211-8 (1995)
170	CD-349	EP 92936 A1
171	CD-832	EP 00370821
172	CER-2 metabolite of furnipidine	WO 9919302
173	cerebrocrast	DE 3534385
174	CERM 11956	EP 138684
175	CERM-12816	IDDB 283075
176	CGP 22442	WO 9323082
177	CGP 26797	WO 9323082
178	CGP 28727	WO 9323082
179	CGP 32413	WO 9323082
180	changrolin	Sci. Sin. (Engl. Ed.) 22(10): 1220-8
		(1979)
181	CHF-1521 (combination of
	delapril and manidipine)
182	cilnidipine	U.S. Pat. No. 4672068
183	cinnarizine	U.S. Pat. No. 3799934
184	civamide	WO 9640079
		U.S. Pat. No. 5840762
185	clentiazem/T A3090	EP 00127882
		U.S. Pat. No. 4567175
186	clevidipine	WO 9512578
187	CNS-1067	IDdb 211675
188	CNS-1237	Annals of the New York Academy
		of Sciences, 765 (Neuroprotective
		Agents):210-29 (1995)
189	CNS-2103 (from spider venom)	WO 9214709 A2
190	COR 28-22	WO 9323082
191	COR 2707C	WO 9323082
192	COR 3752C	WO 9323082
193	CP-060S	WO 9500471 A1
194	CPC-301	IDdb 231888
195	CPC 304	IDdb 185705
196	CPC-317	IDdb 185700
197	CPU 23	Yaoxue Xuebao, 25(11): 815-23
		(1990)
		CAN 114:143097
198	CPU-86017	EP 00538844
199	CRE 202	WO 9323082
200	CRE 204	WO 9323082
201	CRE 1005	WO 9323082
202	CRL-42752	WO 00003987
203	cronidipine (LF 2-0254)	EP 240398 A1
204	CV 159	FR 2511370 A1
205	D-2024 (verapamil(S))	WO 09509150
206	D2603	WO 9323082
207	dagapamil	WO 9323082
		EP 64158 A1
208	darodipine PY108068	EP 00000150
209	dauricine NSC 36413	Acta Pharmacologica Sinica 7(6):
		543-7(1986)
210	desmethyl verapamil
211	DHM9	WO 8604581 A1
212	DHP218/PAK9	EP 00121117
213	diclofurime	DE 79-29227999
214	dihydropyridine calcium channel	Journal of Medicinal Chemistry
	blockers	41(4):509-514 (1998)
215	diltiazem	U.S. Pat. No. 3562257
216	diperdipine	EP 00218996
217	dipfluzine	DE 3318577 A1
218	diproteverine BRL 40015	BE 866208
219	dopropidil	EP 00031771
220	dotarizine/FI 6026	U.S. Pat. No. 4883797
221	DTZ-323	Molecular Pharmacology,
		51(2):262-268 (1997)
222	E-2050	JP 2001199949 A2
223	E4080	EP 344577 A2
224	efonidipine hydrochloride	U.S. Pat. No. 4885284
225	EG 1088	EP 56637 A1
226	EGIS 3966	DE 4027052 A1
227	eglodipine	DE 3825962 A1
228	emopamil (racemic) SZ 45	DE 3344755 A1
229	(S)-emopamil	DE 3344755 A1
230	enalapril_nitrendipine, Vita-	EP 00884054
	Inveest
231	etafenonee LG 11457	DE 1265758
232	ethosuximide
233	eugenodilol	JP 11255719 A2
234	evodiamine	JP 52077098
235	F-0401	EP 00320984
236	falipamil AQA 39	Journal of Medicinal Chemistry,
		33(5): 1496-504 (1990)
237	fantofarone SR 33557	EP 235111 A1
		U.S. Pat. No. 4957925
238	fasudil (iv formulation), Asahi	U.S. Pat. No. 4678783
239	FCE-24265	EP 373645 A1
240	FCE-26262
241	FCE-27335
242	FCE-27892
243	FCE-28718	EP 00755931
244	fedopamil
245	felodipine	U.S. Pat. No. 4264611
246	felodipine + ramipril	WO 09607400
	(Astra/Aventis)
247	fendiline	U.S. Pat. No. 3262977
248	feniline
249	flezelastine, D 18024	EP 590551 A2
250	flordipine
251	fluodipine	U.S. Pat. No. 3773939
252	fluphenazine, S94	Journal of Medicinal Chemistry,
	SQ 4918	19(6):850-2 (1976)
	Triflumethazine
	Vespazine
253	fostedil KB 944	EP 10120
254	FPL 62129	EP 125803 A2
255	FR 46171
256	FR-172516	JP 09040647
257	FRC9411
258	FRG 8653
259	FRG-8701
260	furaldipine
261	furnidipine (CRE 319)	Journal of Medicinal Chemistry,
		38(15):2830-41 (1995)
262	GOE 5057
263	GOE 5584 A	EP 173933 A1
264	GOE 93007
265	GR 60139
266	GR 55234A (R-enantiomer of	Haemotalogica, 79(4):328-33
	telupidine)	(1994)
267	GR 55235A (L-enantiomer of	Haemotalogica, 79(4):328-33
	telupidine)	(1994)
268	GS-386
269	GYKI 46544
270	H32438
271	HA 22	U.S. Pat. No. 5240947
272	HA 23	U.S. Pat. No. 5240947
273	HA 1004
274	GA 1077
275	HE 30346
276	HNS 32	JP 08311007 A2
277	HOE 166	Molecular Pharmacology
		33(4):363-9(1988)
278	HOE 263
279	HP 406	U.S. Pat. No. 4521537
280	ICI206970	EP 293170 A1
		19881130
281	iganidipine	JP 63225355 A2
		19880920
282	IHC 72	Acta Pharmaceutica Sinica,
		27(6):407-11 (1992)
283	ipenoxazone
284	isradipine	U.S. Pat. No. 4466972
285	JTV-519	WO 09212148
286	KB 2796
287	KP-840	Yakubutsu, Seishin, Kodo,
		12(6):353 (1992)
288	KP873
289	KT-362	Archiv Der Pharmazie,
		328(4):313-6(1995)
290	KT 2230	General Pharmacology, 22(3):443-
		8(1991)
291	KW 3049 (see benipidine)
292	L-366682	EP 00444898
293	L-651582
294	L735821	WO 9514471 A1
		19950601
		British Journal of Pharmacology,
		132(1):101-110 (2001)
295	lacidipine GR 43659 Sn305	U.S. Pat. No. 4801599
		DE 03529997
296	LAS 30356
297	LAS 30398
298	LAS 30538	Journal of Pharmacy and
		Pharmacology, 44(10:830-5
		(1992)
299	LAS Z077
300	LCB-2514
301	lemildipine	P59152373A2
302	lercanidipine	U.S. Pat. No. 4705797
303	leualacin	EP 00358418
304	levosemotiadil SA 3212	WO 08700838
305	lidoflazine R7904	U.S. Pat. No. 3267104
306	lifarizine RS 87476	U.S. Pat. No. 0435417
307	LOE-908
308	lomerizine KB 2796	U.S. Pat. No. 4663325
		EP 00158566
309	LU 49700 (main metabolite of	DE 3642331 A1
	gallopamil)
310	LU 49938
311	LY-042826	European Journal of
		Pharmacology, 408(3):241-248
		(2000)
312	LY-393615	European Journal of
		Pharmacology, 408(3):241-248
		(2000)
313	manidipine/CV 4093/franidipine	U.S. Pat. No. 4892875
		EP 00094159
314	MCI 176 (MY7674)	EP 169537 A2
315	McN 5691 (see RWJ 26240)
316	McN-6186
317	MCN 6497
318	MD 260792
319	MDL 143
320	MDL 12330A
321	MDL 16582A	WO 9323082
322	MDL 72567	GB 2137622 A1
		19841010
		CAN 102:95548
323	MEM 1003/nimopidine
	analog/BAY Z 4406
324	mepirodipine
325	mesudipine
326	mibefradil	EP 00268148
		U.S. Pat. No. 4808605
327	minodipine
328	mioflazine
329	MJ 14712
330	monatepil maleate (AD 2615)	WO 08601203
		U.S. Pat. No. 4749703
331	MPC 1304
332	MPC 2101	FR 2514761 A1
333	MR-14134	Pharmacology, 51(2):84-95 (1995)
334	N-3601	EP 254322 A1
335	N 20776
336	N-allyl secoboldine
337	naltiazem Ro 23-6152	U.S. Pat. No. 4652561
338	NB 818
339	NC 1100
340	NC O 700
341	NCC 09-0026
342	nexopamil	EP 00271013
343	NH 2250
344	NH2716
345	nicainoprol RU 42924	DE 2934609
346	nicardipine (nifelan)	U.S. Pat. No. 3985847
347	nictiazem
348	nifedipine	U.S. Pat. No. 3485847
349	nigulipine	WO 8807525 A1
350	niludipine
351	nilvadipine FK 235	U.S. Pat. No. 4338322
		DE 02940833
352	nimodipine	U.S. Pat. No. 3842096
353	misoldipine Bay y 5552	U.S. Pat. No. 4154839
354	nitrendipine Bay k 5009	U.S. Pat. No. 3799934
355	NMDA/calcium channel	WO 09745115
	antagonists, Allelix
356	NKY 722
357	NMED 126 (MC-34D)	WO 0145709 A1
		U.S. Pat. No. 6387897
358	NMED 427	WO 0145709 A1
		U.S. Pat. No. 6387897
359	NMED 724	WO 0145709 A1
		U.S. Pat. No. 6387897
360	NMED 826	WO 0145709 A1
		U.S. Pat. No. 6387897
361	NMED JM-G-10	WO 0145709 A1
		U.S. Pat. No. 6387897
362	NMED 157 39-1B4	WO 0145709 A1
		U.S. Pat. No. 6387897
363	NMED 160 39-45-3	WO 0145709 A1
		U.S. Pat. No. 6387897
364	NNC-09-0026	WO 9201672
365	NP 252	Life Sciences, 48(2): 183-8 (1991)
366	NS 626
367	NS-638	U.S. Pat. No. 5314903
		EP 545845 A1
368	NS-649	EP 520200 A2
369	NS-696
370	NS-7	WO 09607641
371	NS 3034
372	NZ 105
373	olradipine S 11568	FR 2602231 A1
374	ONO-2921	WO 0000470 A1
375	OPC 13340
376	OPC 88117	EP 236140 A2
377	ORG 13020
378	Org-13061	Fundamental & Clinical
		Pharmacology, 11(5):416-426
		(1997)
379	OS AT (nifedipine)
380	osthole	JP 47000430
381	oxodipine IQB 837V	ES 531033 A1
382	P 0825
383	P 1268
384	palonidipine hydrochloride	Ep 128010 A2
385	PCA-50922
386	PCA-50938	Brain Research 772(1,2):57-62
		(1997)
387	PCA-50941
388	PCA 50982
389	PD-0204318	WO 9943658 A1
390	PD-029361	IDdb 300520
391	PD 122860	Ep 206747 A2
392	PD 151307	U.S. Pat. No. 6423689
		J. Med. Chern. 43:3472 (2000)
393	PD-157667	U.S. Pat. No. 5767129
394	PD-158143	WO 9705125 A1
395	PD 173212
396	PD 175069	WO 9854123 A1
397	PD 176078	WO 9955688
		J. Med. Chem. 43:3474 (2000)
398	PD 181283	Bioorganic & Medicinal Chemistry
		Letters, 9(16):2453-2458 (1999)
399	pelanserin
400	perhexiline	GB 1025578
401	petro synol	Tetrahedron, 49(45): 10435-8
		(1993)
402	PF 244
403	PFS 1144 (EO 122)	DE 2802208
404	pirmenol	U.S. Pat. No. 4112103
405	pirprofurol
406
407	PN 200110
408	PNU 156654E	WO 9705102 A1
409	pranidipine	EP 00145434
410	prenylamine
411	propiverine	DD 106643
412	ptilomycalin AM
413	QM 96233
414	QM 96159
415	QM 96127
416	QX-314	Biophysical Journal, 27(1):39-55
		(1979)
417	R56865	EP 184257 A1
418	R 59494	Ep 184257 A1
419	R71811
420	Rec 152288
421	Rec 152375, Rec 15/375
422	RGH-2716 (TDN 345)	EP 414421 A2
423	RGH 2970
424	riodipine
425	Ro-11-2933	EP 00523493
426	Ro 18-3981
427	Ro 40-5967
428	RO 445912 dithiane	Biochemical Pharmacology,
	derivatives of tiapamil	50(2): 187-96 (1995)
429	ronipamil
430	RS-5773	EP 00353032
431	RS 93007
432	RS 93522	U.S. Pat. No. 4595690
433	RU-43945	WO 9323082 Al
434	RWJ-22108	U.S. Pat. No. 04845225
435	RWJ-22726	U.S. Pat. No. 04845225
436	RWJ 26240 McN 5691	EP 146721 A2
437	RWJ 26899	EP 237191 A1
438	RJW-26902
439	RWJ-29009	EP 00493048
440	RWJ-37868	WO 0048584
441	ryanodine
442	S-(−)-amlodipine
443	S 11568
444	S 12967	ZA 9000231 A
445	S-12968	EP 00406502
446	S-2150	Ep 00615971
447	S-312-d	JP 03052890
448	S 830327
449	SA 2572	JP 63104969 A2
450	SA 2995
451	SA 3212
452	sabeluzole	Ep 184257 A1
453	safinamide	EP 400495 A1
454	sagandipine
455	salicylaldoxime	Clinical and Experimental
		Pharmacology and Physiology
		26(12):964-9 (1999)
456	SANK-71996
457	SB-201823A	WO 09202502
458	SB-206284A
459	SB 221420A	WO 9002494 A1
460	SB-237376	WO 0209761 A2
461	SB 262470	WO 0183546 A1
462	SC 30552
463	SDZ-249482
464	selodipine
465	semotiadil (SD 3211)	U.S. Pat. No. 4786635
		JP 09012576
466	SIM 6080	Ep 293925 A2
467	sipatrigine	EP 372934 A2
468	sinomenine (active from	WO 0269971 A1
	a Chinese medicinal plant)
469	siratiazem	WO 09117153
470	SKF-45675
471	SKF-96365	European Journal of
		Pharmacology 188(6):417-21
		(1990)
472	SKT-M-26
473	SL-34.0829	WO 0209761 A2
474	SL 651708
475	SL 851016
476	SL-870495
477	SM-6586	EP 00177965
478	S NX-124
479	SNX 185	WO 9310145 A1
480	S NX-236	WO 09313128
481	S NX-239	Pain, 60(1):83-90 (1995)
482	SNX-483 (peptides from tarantula	WO 9805780 A2
	venom)
483	sornidipine
484	SQ 31486	EP 205334 A2
485	SQ 31727
486	SQ 31765
487	SQ 32321
488	SQ 32324
489	SQ 32547	EP 400665 A2
490	SQ 32926	EP 400665 A2
491	SQ-33351	WO 09006118
492	SQ 33537
493	SQ 34399
494	SR-33805	EP 576347 A1
495	SUN 5647
496	SUN 6087
497	SUN-N8075	WO 9923072 A2
498	T-477	EP 00441539
499	TA-993	JP 01050872
500	taludipine
501	tamolarizine	EP 00354068
502	TDN-345
503	Teczem
504	temiverine	CAN 131:193592
505	terflavoxate	EP 72620 A1
506	terodiline TD 758	U.S. Pat. No. 3371014
507	tetrandrine	Clinical and Experimental
		Pharmacology and Physiology,
		23(8):715-753 (1996)
508	TH-1177
509	TH-9229	WO 09607415
510	thapsigargin	British Journal of Pharmacology,
		95(3):705-712 (1985)
511	tiapamil
512	tinctormine	Chemical & Pharmaceutical
		Bulletin 40(12):3355-7 (1992)
513	TJN 220 (O-ethylfangchinoline)	JP 63179878 A2
514	TMB 8	Journal of Cell Science 79:151-160
		(1985)
515	TN-871	European Journal of
		Pharmacology 342 (2/3): 167-175
		(1998)
516	TR 2957
517	trapidil
518	trimetazidine	U.S. Pat. No. 3262852
519	TY-10835	Pharmacometrics, 1998, 54:3
		(153)
520	U-88999	WO 9204338
521	U-92032	WO 09204338
522	U-92798	WO 9204338 A1
523	UK 1745	EP 653426 A1
524	UK-51656	EP 00089167
525	UK 52831	JP 59118782 A2
526	UK 55444	EP 00132375
527	UK 56593
528	UK-84149	EP 404359 A1
529	ULAH 99	European Journal of
		Pharmacology, 229(1):55-62
		(1992)
530	vantanipidine	EP 257616 A2
531	verapamil, verelan	U.S. Pat. No. 3261859
532	S-verapamil, D-2024,	WO 09509150
	levo verapamil
533	vexibinol	Chemical and Pharmaceutical
	Sophoraflavanone G	Bulletin 38(4): 1039-44 (1990)
534	vinigrol
535	vintoperol RGH 2981 RT 303	WO 9207851
536	vingrol
537	vintoperol/RGH 2981/RT 303	WO 9207851
538	VUF-8929	EP 467435 A2
539	VULM 993
540	vantanipidine	Ep 257616 A2
541	W787
542	WAS 4206
543	WK 269
544	WY 27569
545	WY 44644
546	WY 44705
547	WY 46622
548	WY 47324
549	xanthonolol	U.S. Pat. No. 5495005
550	Y19638
551	Y-22516	WO 9323082
552	Y208835
553	YC 114
554	YH-334	EP 00366548
555	YM 15430-1 (see YM 430)
556	YM-16151-4 (YM 151)	EP 00167371
557	YM-430 (YM 15430)	WO 0209761 A2
558	YS 035	BE 897244
559	YS 161
560	Z-6568	Journal of Mass Spectrometry,
		31(1):37-46 (1996)
561	ziconotiide omega	WO 9107980
	conotoxin/MVIIA/SNX-111
562	ZM-224832	EP 00343865
563	zonisamide	U.S. Pat. No. 4172896

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is a capsaicinoid. In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is capsaicin or resiniferatoxin.

In some embodiments, the nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is a neurotoxic protein. In some embodiments, the agent is a neurotoxic protein. In some embodiments, the neurotoxic protein is a clostridial neurotoxin. In some embodiments, the neurotoxic protein is produced by a clostridium. In certain embodiments, the neurotoxic protein is produced by Clostridium botulinum, C. argentinense, C. butyricum, C. baratii spp, or Clostridium tetani. In some embodiments, the neurotoxic protein is produced by Chryseobacterium piperi. In some embodiments, the neurotoxic protein is produced by Enterococcus faecium (BoNT/En). In some embodiments, the neurotoxic protein is produced by Weissella oryzae. In certain embodiments, the neurotoxic protein is a botulinum neurotoxin. In certain embodiments, the neurotoxic protein is a botulinum neurotoxin other than BoNT/a. In certain embodiments, the neurotoxic protein is a botulinum neurotoxin serotype selected from A, B, C, D, E, F, G, H, and, X, or variant or subtype thereof (e.g., BoNT/a subtype A1-A5 (i.e., BoNT/A1, BoNT/A2)). In certain embodiments, the neurotoxic protein is a botulinum neurotoxin serotype selected from A, B, C, D, E, F, and G, or variant or subtype thereof (e.g., BoNT/a subtype A1-A5 (i.e., BoNT/A1, BoNT/A2)). In certain embodiments, the neurotoxic protein is a botulinum neurotoxin serotype selected from B, C, D, E, F, G, H, and, X, or variant or subtype thereof. In certain embodiments, the neurotoxic protein is a botulinum neurotoxin serotype selected from A, B, E, and F. In some embodiments, the botulinum neurotoxin is a BoNT variant. In some embodiments, the BoNT variant is selected from BoNT/A1-A5, B1-B7, E1-E11, and F1-F7. In some embodiments, the botulinum neurotoxin is BoNT/a. In some embodiments, the neurotoxic protein is a BoNT-like toxin. In some embodiments, the neurotoxic protein is tetanus neurotoxin (TeNT). In some embodiments the agent is abobotulinumtoxinA, incobotulinumtoxinA, onabotulinumtoxinA, or rimabotulinumtoxinB.

In some embodiments, provided herein are methods of treating cancer in a subject comprising administrating to the subject a therapeutically effective amount of an agent that blocks vesicle release from tumor-innervating neurons. In some embodiments, provided herein are methods of treating cancer in a subject comprising administrating to the subject a therapeutically effective amount of an agent that blocks vesicle release from tumor-innervating nociceptors. In some embodiments, provided herein are methods of preventing cancer in a subject comprising administrating to the subject a prophylactically effective amount of an agent that blocks vesicle release from tumor-innervating neurons. In some embodiments, provided herein are methods of preventing cancer in a subject comprising administrating to the subject a prophylactically effective amount of an agent that blocks vesicle release from tumor-innervating nociceptors. In some embodiments, the agent is a neurotoxic protein. In some embodiments, the neurotoxic protein is a clostridial neurotoxin. In some embodiments, the neurotoxic protein is produced by a clostridium. In certain embodiments, the neurotoxic protein is produced by Clostridium botulinum, C. argentinense, C. butyricum, C. baratii spp, or Clostridium tetani. In some embodiments, the neurotoxic protein is produced by Chryseobacterium piperi. In some embodiments, the neurotoxic protein is produced by Enterococcus faecium (BoNT/En). In some embodiments, the neurotoxic protein is produced by Weissella oryzae. In certain embodiments, the neurotoxic protein is a botulinum neurotoxin. In certain embodiments, the neurotoxic protein is a botulinum neurotoxin other than BoNT/a. In certain embodiments, the neurotoxic protein is a botulinum neurotoxin serotype selected from A, B, C, D, E, F, G, H, and, X, or variant or subtype thereof (e.g., BoNT/a subtype A1-A5 (i.e., BoNT/A1, BoNT/A2)). In certain embodiments, the neurotoxic protein is a botulinum neurotoxin serotype selected from A, B, C, D, E, F, and G, or variant or subtype thereof (e.g., BoNT/a subtype A1-A5 (i.e., BoNT/A1, BoNT/A2)). In certain embodiments, the neurotoxic protein is a botulinum neurotoxin serotype selected from B, C, D, E, F, G, H, and, X, or variant or subtype thereof. In certain embodiments, the neurotoxic protein is a botulinum neurotoxin serotype selected from A, B, E, and F. In some embodiments, the botulinum neurotoxin is a BoNT variant. In some embodiments, the BoNT variant is selected from BoNT/A1-A5, B1-B7, E1-E11, and F1-F7. In some embodiments, the botulinum neurotoxin is BoNT/a. In some embodiments, the neurotoxic protein is a BoNT-like toxin. In some embodiments, the neurotoxic protein is tetanus neurotoxin (TeNT). In some embodiments the agent is abobotulinumtoxinA, incobotulinumtoxinA, onabotulinumtoxinA, or rimabotulinumtoxinB.

In certain embodiments, provided herein are methods of treating cancer in a subject comprising administering to a subject a therapeutically effective amount of a calcitonin gene-related peptide (CGRP) modulating agent. In certain embodiments, provided herein are methods of preventing cancer in a subject comprising administering to a subject a prophylactically effective amount of a calcitonin gene-related peptide (CGRP) modulating agent. In some embodiments, the CGRP modulating agent is a CGRP receptor antagonist. In certain embodiments the CGRP receptor antagonist is a RAMP1, RAMP3, or Vpac1 blocker. In some embodiments, the CGRP receptor antagonist is a RAMP1 blocker. In certain embodiments, the CGRP receptor antagonist is erenumab, fremanezumab, fremanezumab, eptinezumab, ubrogepant, or rimegepant. In certain embodiments, the CGRP receptor antagonist is BIBN 4096.

In one aspect, provided herein are methods of treating cancer in a subject comprising administering to a subject a therapeutically effective amount of one or more of QX-314, BoNT/a, and BIBN 4096. In one aspect, provided herein are methods of treating cancer in a subject comprising administering to a subject a therapeutically effective amount of QX-314 and BoNT/a. In one aspect, provided herein are methods of treating cancer in a subject comprising administering to a subject a therapeutically effective amount of QX-314. In one aspect, provided herein are methods of treating cancer in a subject, the method comprising administering to a subject a therapeutically effective amount of BIBN 4096.

In one aspect, provided herein are methods of preventing cancer in a subject comprising administering to a subject a prophylactically effective amount of one or more of QX-314, BoNT/a, and BIBN 4096. In one aspect, provided herein are methods of preventing cancer in a subject comprising administering to a subject a prophylactically effective amount of QX-314 and BoNT/a. In one aspect, provided herein are methods of preventing cancer in a subject comprising administering to a subject a prophylactically effective amount of QX-314. In one aspect, provided herein are methods of preventing cancer in a subject comprising administering to a subject a prophylactically effective amount of BIBN 4096.

In certain embodiments, provided herein are methods of treating cancer in a subject comprising ablating an ion channel in a subject, wherein the ion channel is a sodium ion channel or TRPV ion channel. In certain embodiments, provided herein are methods of preventing cancer in a subject comprising ablating an ion channel in a subject, wherein the ion channel is a sodium ion channel or TRPV ion channel. In certain embodiments, the sodium channel is selected from Na_V1.6, Na_V1.7, Na_V1.8, and Na_V1.9. In certain embodiments, the sodium channel is Na_V1.8. In some embodiments, the calcium channel is Ca_V2.2. In certain embodiments, the TRPV ion channel is TRPV1. In some embodiments, the ion channel is a calcium ion channel. In some embodiments, the calcium channel is Ca_V1.1-1.4, Ca_V2.1, Ca_V2.2, Ca_V2.3, Ca_V3.1-3.3. In some embodiments, the ion channel is genetically ablated. In some embodiments, the ion channel is ablated through genetic mutation. In some embodiments, the ion channel is ablated through genetic mutation during the development of the subject. In some embodiments, the ion channel is ablated through use of a denervating agent. In some embodiments, the denervating agent is capsaicin. In some embodiments, the denervating agent is resiniferatoxin.

In certain embodiments, provided herein are methods of treating cancer in a subject. In some embodiments, the cancer is skin cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, gastric cancer, or a tumor. In certain embodiments, the cancer is skin cancer. In some embodiments, the cancer is breast cancer. In certain embodiments, the cancer is prostate cancer. In some embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is a melanoma. In some embodiments, the melanoma is superficial spreading melanoma, nodular melanoma, acral-lentiginous melanoma, lentigo maligna melanoma, amelanotic melanoma, desmoplastic melanoma, ocular melanoma, or metastatic melanoma. In some embodiments, the cancer is metastatic melanoma. In certain embodiments, the cancer is a tumor. In some embodiments, the cancer is a benign tumor. In certain embodiments, the cancer is a malignant tumor. In certain embodiments, the is innervated.

In certain embodiments, the method decreases tumor growth, volume, and/or size.

In some embodiments, the method inhibits or decreases cancer cell proliferation.

In certain embodiments, the method increases subject survival.

In some embodiments, the method promotes anti-tumor activity.

In certain embodiments, the method increases lymphocyte numbers.

In some embodiments, the method improves response to chemotherapeutics.

In some embodiments, the method improves efficacy of immunotherapy.

In some embodiments, the method increases efficacy of αPDL1 treatment. In some embodiments, the method increases efficacy of PDL1 treatment. In some embodiments, when QX-314 or BoNT/a is used to treat cancer, the method increases efficacy of αPDL1 treatment. In some embodiments, when QX-314 or BoNT/a is used to treat cancer, the method increases efficacy of PDL1 treatment.

In some embodiments, the method leads to exhaustion of tumor-infiltrating lymphocytes.

In certain embodiments, the method decreases tumor comorbidities. In some embodiments, the comorbidity is pain or itch. In some embodiments, the comorbidity is pain. In certain embodiments, the comorbidity is itch.

In some embodiments, one or more additional therapies are administered to the subject. In some embodiments, an additional therapy is administered to the subject. In some embodiments, the additional therapy is chemotherapy, radioimmunotherapy, surgical therapy, immunotherapy, radiation therapy, or targeted therapy, or any combination thereof. In some embodiments, the additional therapy is chemotherapy. In some embodiments, the additional therapy is radioimmunotherapy. In some embodiments, the additional therapy is surgical therapy. In some embodiments, the additional therapy is immunotherapy. In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is targeted therapy. In some embodiments, the additional therapy is an anti-cancer agent.

In another aspect, provided herein is the use of a nociceptor modulating agent for treating or preventing cancer in a subject. In some embodiments, the nociceptor modulating agent is a nociceptor antagonist.

In a further aspect, provided herein is the use of a neuropeptide modulating agent for treating or preventing cancer in a subject.

In one aspect, provided herein is the use of an agent that blocks the release or action of a neuropeptide from tumor-innervating neurons for treating cancer in a subject.

In another aspect, provided herein is the use of an agent that blocks vesicle release from tumor-innervating nociceptors for treating cancer in a subject.

In a further aspect, provided herein is the use of a calcitonin gene-related peptide (CGRP) modulating agent for treating cancer in a subject. In some embodiments, the CGRP modulating agent is a CGRP receptor antagonist. In some embodiments, the CGRP receptor antagonist is a RAMP1 blocker.

In one aspect, provided herein is the use of QX-314, BoNT/a, and/or BIBN 4096 for treating cancer in a subject. In one aspect, provided herein is the use of QX-314 for treating cancer in a subject. In one aspect, provided herein is the use of BoNT/a for treating cancer in a subject. In one aspect, provided herein is the use of BIBN 4096 for treating cancer in a subject.

In another aspect, provided herein is the use of genetic mutation to ablate an ion channel in a subject, wherein the ion channel is a sodium ion channel or TRPV ion channel, for treating cancer. In some embodiments, the ion channel is Na_V1.8 and/or TRPV1.

Any of the methods or uses provided herein can be carried out employing a composition of the agent, blocker, protein, peptide, antagonist, or compound described herein (e.g., a composition comprising a neuropeptide modulating agent, an agent that blocks the release or action of a neuropeptide from tumor-innervating neurons, a nociceptor modulating agent such as a nociceptor antagonist, a sodium channel blocker, a calcium channel blocker, a sodium and calcium channel blocker, a compound comprising a quaternary amine, an agent that blocks vesicle release from tumor-innervating nociceptors, a neurotoxic protein, a calcitonin gene-related peptide (CGRP) modulating agent, a CGRP receptor antagonist, or RAMP1 blocker).

Compositions, Combinations, and Kits

In one aspect, also provided herein are compositions comprising (i) an anti-cancer agent, (ii) a nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor described herein, and (iii) optionally a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition described herein comprises (i) an anti-cancer agent, (ii) a nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor described herein, and (iii) a pharmaceutically acceptable carrier or excipient.

In certain embodiments, provided herein are compositions comprising an anti-cancer agent and a nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor which is as described herein. In certain embodiments, provided herein are compositions comprising an anti-cancer agent and a nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor which comprises a quaternary amine. In some embodiments, the composition comprises an anti-cancer agent and a nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor is selected from:

In some embodiments, the composition comprises an anti-cancer agent and a nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor of Formula (I), (IA), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII), or (XIV). In some embodiments, the composition comprises an anti-cancer agent and a nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor which is a quaternized or guanylated derivative of a compound as described in any of Tables 1-3. In some embodiments, the composition comprises an anti-cancer agent and a quaternary amine derivative or other permanently charged derivative of a compound selected from riluzole, mexilitine, phenytoin, carbamazepine, procaine, articaine, bupivicaine, mepivicaine, tocainide, prilocaine, diisopyramide, bencyclane, quinidine, bretylium, lifarizine, lamotrigine, flunarizine, and fluspirilene. In some embodiments, the composition comprises an anti-cancer agent and BIBN 4096. In some embodiments, the composition comprises an anti-cancer agent and a botulinum toxin. In some embodiments, the composition comprises an anti-cancer agent and BoNT/a.

In some embodiments, the additional pharmaceutical agent is an anti-cancer agent. Anti-cancer agents encompass biotherapeutic anti-cancer agents as well as chemotherapeutic agents.

Exemplary biotherapeutic anti-cancer agents include, but are not limited to, interferons, cytokines (e.g., tumor necrosis factor, interferon α, interferon γ), vaccines, hematopoietic growth factors, monoclonal serotherapy, immunostimulants and/or immunodulatory agents (e.g., IL-1, 2, 4, 6, or 12), immune cell growth factors (e.g., GM-CSF) and antibodies and fragments and variants thereof (e.g. HERCEPTIN (trastuzumab), T-DM1, AVASTIN (bevacizumab), ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN (rituximab), BEXXAR (tositumomab)).

Exemplary chemotherapeutic agents include, but are not limited to, anti-estrogens (e.g., tamoxifen, raloxifene, and megestrol), LHRH agonists (e.g., goscrclin and leuprolide), anti-androgens (e.g. flutamide and bicalutamide), photodynamic therapies (e.g. vertoporfin (BPD-MA), phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A (2BA-2-DMHA)), nitrogen mustards (e.g. cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas (e.g. carmustine (BCNU) and lomustine (CCNU)), alkylsulphonates (e.g. busulfan and treosulfan), triazenes (e.g., dacarbazine, temozolomide), platinum containing compounds (e.g., cisplatin, carboplatin, oxaliplatin), vinca alkaloids (e.g. vincristine, vinblastine, vindesine, and vinorelbine), taxoids (e.g., paclitaxel or a paclitaxel equivalent such as nanoparticle albumin-bound paclitaxel (ABRAXANE), docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin), polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 bound to three molecules of paclitaxel), paclitaxel-EC-1 (paclitaxel bound to the erbB2-recognizing peptide EC-1), and glucose-conjugated paclitaxel, e.g., 2′-paclitaxel methyl 2-glucopyranosyl succinate; docetaxel, taxol), epipodophyllins (e.g. etoposide, etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mytomycin C), anti-metabolites, DHFR inhibitors (e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (e.g., mycophenolic acid, tiazofurin, ribavirin, and EICAR), ribonuclotide reductase inhibitors (e.g. hydroxyurea and deferoxamine), uracil analogs (e.g. 5-fluorouracil (5-FU), floxuridine, doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosine analogs (e.g., cytarabine (ara C), cytosine arabinoside, and fludarabine), purine analogs (e.g. mercaptopurine and Thioguanine), Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylation inhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g. 1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g. staurosporine), actinomycin (e.g. actinomycin D, dactinomycin), bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline (e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDR inhibitors (e.g. verapamil), Ca²⁺ ATPase inhibitors (e.g. thapsigargin), imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g., axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™, AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®, SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474), vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), everolimus (AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumab ozogamicin (MYLOTARG®), temsirolimus (TORISEL®), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TK1258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and/or XL228), proteasome inhibitors (e.g., bortezomib (VELCADE)), mTOR inhibitors (e.g., rapamycin, temsirolimus (CCI-779), everolimus (RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)), oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbizine, prednisolone, dexamethasone, campathecin, plicamycin, asparaginase, aminopterin, methopterin, porfiromycin, melphalan, leurosidine, leurosine, chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin, aminopterin, and hexamethyl melamine.

In some embodiments, the anti-cancer agent is dacarbazine. In some embodiments, the anti-cancer agent is cisplatin.

In some embodiments, the composition comprises dacarbazine, QX-314, and optionally a pharmaceutically acceptable excipient. In some embodiments, the composition comprises dacarbazine, BoNT/a, and optionally a pharmaceutically acceptable excipient. In some embodiments, the composition comprises dacarbazine, BIBN4096, and optionally a pharmaceutically acceptable excipient.

In some embodiments, the composition comprises cisplatin, QX-314, and optionally a pharmaceutically acceptable excipient. In some embodiments, the composition comprises cisplatin, BoNT/a, and optionally a pharmaceutically acceptable excipient. In some embodiments, the composition comprises cisplatin, BIBN4096, and optionally a pharmaceutically acceptable excipient.

In some embodiments, the composition comprises dacarbazine, QX-314, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the composition comprises dacarbazine, BoNT/a, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the composition comprises dacarbazine, BIBN4096, and a pharmaceutically acceptable carrier or excipient.

In some embodiments, the composition comprises cisplatin, QX-314, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the composition comprises cisplatin, BoNT/a, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the composition comprises cisplatin, BIBN4096, and a pharmaceutically acceptable carrier or excipient.

In some embodiments, dacarbazine is used in combination with QX-314. In some embodiments, dacarbazine is used in combination with BoNT/a. In some embodiments, dacarbazine is used in combination with BIBN4096. In some embodiments, cisplatin is used in combination with QX-314. In some embodiments, cisplatin is used in combination with BoNT/a. In some embodiments, cisplatin is used in combination with BIBN4096.

Pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include bringing the compound described herein (i.e., the “active ingredient”) into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.

Pharmaceutical compositions can be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. A “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage, such as one-half or one-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w/w) active ingredient.

Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition.

Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate (Tween® 20), polyoxyethylene sorbitan (Tween® 60), polyoxyethylene sorbitan monooleate (Tween® 80), sorbitan monopalmitate (Span® 40), sorbitan monostearate (Span® 60), sorbitan tristearate (Span® 65), glyceryl monooleate, sorbitan monooleate (Span® 80), polyoxyethylene esters (e.g., polyoxyethylene monostearate (Myrj® 45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether (Brij® 30)), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic® F-68, poloxamer P-188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and/or mixtures thereof.

Exemplary binding agents include starch (e.g., cornstarch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and/or mixtures thereof.

Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoan preservatives, alcohol preservatives, acidic preservatives, and other preservatives. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant® Plus, Phenonip®, methylparaben, Germall® 115, Germaben® II, Neolone®, Kathon®, and Euxyl®.

Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates described herein are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle.

Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates described herein with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.

Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the art of pharmacology. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type can be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polethylene glycols and the like.

The active ingredient can be in a micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings, and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active ingredient can be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may comprise buffering agents. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating agents which can be used include polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of a compound described herein may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and/or patches. Generally, the active ingredient is admixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and/or any needed preservatives and/or buffers as can be required. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of an active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispensing the active ingredient in the proper medium. Alternatively or additionally, the rate can be controlled by either providing a rate controlling membrane and/or by dispersing the active ingredient in a polymer matrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices. Intradermal compositions can be administered by devices which limit the effective penetration length of a needle into the skin. Alternatively or additionally, conventional syringes can be used in the classical mantoux method of intradermal administration. Jet injection devices which deliver liquid formulations to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Ballistic powder/particle delivery devices which use compressed gas to accelerate the compound in powder form through the outer layers of the skin to the dermis are suitable.

Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments, and/or pastes, and/or solutions and/or suspensions. Topically administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient can be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, or from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant can be directed to disperse the powder and/or using a self-propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having a boiling point of below 65° F. at atmospheric pressure. Generally, the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).

Pharmaceutical compositions described herein formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and/or suspension. Such formulations can be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising the active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range from about 0.1 to about 200 nanometers.

Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition described herein. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 micrometers. Such a formulation is administered by rapid inhalation through the nasal passage from a container of the powder held close to the nares.

Formulations for nasal administration may, for example, comprise from about as little as 0.1% (w/w) to as much as 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may contain, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition described herein can be prepared, packaged, and/or sold in a formulation for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1-1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid carrier or excipient. Such drops may further comprise buffering agents, salts, and/or one or more other of the additional ingredients described herein. Other ophthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are also contemplated as being within the scope of this disclosure.

Although the descriptions of pharmaceutical compositions provided herein are directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with ordinary experimentation.

Compounds described herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions described herein will be decided by a physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disorder; the activity of the specific active ingredient employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

The compounds described herein and compositions provided herein can be administered by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. Specifically contemplated routes are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via blood and/or lymph supply, and/or direct administration to an affected site. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the agent (e.g., its stability in the environment of the gastrointestinal tract), and/or the condition of the subject (e.g., whether the subject is able to tolerate oral administration). In certain embodiments, the compound or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.

The exact amount of a compound required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of administration, and the like. An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, when multiple doses are administered to a subject, any two doses of the multiple doses include different or substantially the same amounts of a compound described herein. In certain embodiments, when multiple doses are administered to a subject, the frequency of administering the multiple doses to the subject is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject is three doses per day. In certain embodiments, when multiple doses are administered to a subject, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 μg and 1 μg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 10 mg and 30 mg, inclusive, of a compound described herein. In certain embodiments, a dose described herein includes independently between 30 mg and 100 mg, inclusive, of a compound described herein.

Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

A compound, as described herein, can be administered in combination with one or more additional pharmaceutical agents (e.g., therapeutically and/or prophylactically active agents). In some embodiments, a compound disclosed herein is administered with an anti-cancer agent. The compounds can be administered in combination with additional pharmaceutical agents that improve their activity (e.g., activity (e.g., potency and/or efficacy) in treating a disease in a subject in need thereof, in preventing a disease in a subject in need thereof, in reducing the risk to develop a disease in a subject in need thereof, improve bioavailability, improve safety, reduce drug resistance, reduce and/or modify metabolism, inhibit excretion, and/or modify distribution in a subject. It will also be appreciated that the therapy employed may achieve a desired effect for the same disorder, and/or it may achieve different effects. In some embodiments, the additional pharmaceutical agent achieves a desired effect for the same disorder. In some embodiments, the additional pharmaceutical agent achieves different effects.

The compound or composition can be administered concurrently with, prior to, or subsequent to one or more additional pharmaceutical agents, which may be useful as, e.g., combination therapies.

Pharmaceutical agents include therapeutically active agents. Pharmaceutical agents also include prophylactically active agents. Pharmaceutical agents include small organic molecules such as drug compounds (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins, and cells. In certain embodiments, the additional pharmaceutical agent is a pharmaceutical agent useful for treating and/or preventing a disease (e.g., proliferative disease, hematological disease, neurological disease, painful condition, psychiatric disorder, or metabolic disorder). Each additional pharmaceutical agent may be administered at a dose and/or on a time schedule determined for that pharmaceutical agent. The additional pharmaceutical agents may also be administered together with each other and/or with the compound or composition described herein in a single dose or composition or administered separately in different doses or compositions. The particular combination to employ in a regimen will take into account compatibility of the compound described herein with the additional pharmaceutical agent(s) and/or the desired therapeutic and/or prophylactic effect to be achieved. In general, it is expected that the additional pharmaceutical agent(s) in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.

Also encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a pharmaceutical composition or compound described herein and instructions for use. Also encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a pharmaceutical composition or compound described herein and instructions for administration of the composition or compound to a subject. The kits provided may comprise a pharmaceutical composition or compound described herein and instructions for administration of the composition or compound to a cancer patient.

Also encompassed by the disclosure are kits (e.g., pharmaceutical packs). The kits provided may comprise a pharmaceutical composition or compound described herein and a container (e.g., a vial, ampule, bottle, syringe, and/or dispenser package, or other suitable container). In some embodiments, provided kits may optionally further include a second container comprising a pharmaceutical excipient for dilution or suspension of a pharmaceutical composition or compound described herein. In some embodiments, the pharmaceutical composition or compound described herein provided in the first container and the second container are combined to form one unit dosage form.

Thus, in one aspect, provided are kits including a first container comprising a compound or pharmaceutical composition described herein. In certain embodiments, the kits are useful for treating a disease (e.g., cancer) in a subject in need thereof. In certain embodiments, the kits are useful for preventing a disease (e.g., cancer) in a subject in need thereof.

In certain embodiments, a kit described herein further includes instructions for using the kit. A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. In certain embodiments, the kits and instructions provide for treating a disease (e.g., cancer) in a subject in need thereof. In certain embodiments, the kits and instructions provide for preventing a disease (e.g., cancer) in a subject in need thereof. A kit described herein may include one or more additional pharmaceutical agents described herein as a separate composition.

EXAMPLES

In order that the present disclosure may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, pharmaceutical compositions, methods, and uses provided herein and are not to be construed in any way as limiting their scope.

Synthesis

The synthesis of compounds described herein may involve the selective protection and deprotection of alcohols, amines, ketones, sulfhydryls or carboxyl functional groups of the parent compound, the linker, the bulky group, and/or the charged group. For example, commonly used protecting groups for amines include carbamates, such as tert-butyl, benzyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 9-fluorenylmethyl, allyl, and m-nitrophenyl. Other commonly used protecting groups for amines include amides, such as formamides, acetamides, trifluoroacetamides, sulfonamides, trifluoromethanesulfonyl amides, trimethylsilylethanesulfonamides, and tert-butylsulfonyl amides. Examples of commonly used protecting groups for carboxyls include esters, such as methyl, ethyl, tert-butyl, 9-fluorenylmethyl, 2-(trimethylsilyl)ethoxy methyl, benzyl, diphenylmethyl, O-nitrobenzyl, ortho-esters, and halo-esters. Examples of commonly used protecting groups for alcohols include ethers, such as methyl, methoxymethyl, methoxyethoxymethyl, methylthiomethyl, benzyloxymethyl, tetrahydropyranyl, ethoxyethyl, benzyl, 2-napthylmethyl, O-nitrobenzyl, P-nitrobenzyl, P-methoxybenzyl, 9-phenylxanthyl, trityl (including methoxy-trityls), and silyl ethers. Examples of commonly used protecting groups for sulfhydryls include many of the same protecting groups used for hydroxyls. In addition, sulfhydryls can be protected in a reduced form (e.g., as disulfides) or an oxidized form (e.g., as sulfonic acids, sulfonic esters, or sulfonic amides). Protecting groups can be chosen such that selective conditions (e.g., acidic conditions, basic conditions, catalysis by a nucleophile, catalysis by a Lewis acid, or hydrogenation) are required to remove each, exclusive of other protecting groups in a molecule. The conditions required for the addition of protecting groups to amine, alcohol, sulfhydryl, and carboxyl functionalities and the conditions required for their removal are provided in detail in T. W. Green and P. G. M. Wuts, Protective Groups in Organic Synthesis (2^nd Ed.), John Wiley & Sons, 1991 and P. J. Kocienski, Protecting Groups, Georg Thieme Verlag, 1994.

Charged ion channel blockers (e.g., quaternary amines) can be prepared using techniques familiar to those skilled in the art. The modifications can be made, for example, by alkylation of the parent compound using the techniques described by J. March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, John Wiley & Sons, Inc., 1992, page 617. The conversion of amino groups to guanidine groups can be accomplished using standard synthetic protocols. For example, Mosher has described a general method for preparing mono-substituted guanidines by reaction of aminoiminomethanesulfonic acid with amines (Kim et al., Tetrahedron Lett. 29:3183 (1988)). A more convenient method for guanylation of primary and secondary amines was developed by Bernatowicz employing 1H-pyrazole-1-carboxamidine hydrochloride; 1-H-pyrazole-1-(N,N′-bis(tert-butoxycarbonyl) carboxamidine; or 1-H-pyrazole-1-(N,N′-bis(benzyloxycarbonyl)carboxamidine. These reagents react with amines to give mono-substituted guanidines (see Bernatowicz et al., J. Org. Chem. 57:2497 (1992); and Bernatowicz et al., Tetrahedron Lett. 34:3389 (1993)). In addition, thioureas and S-alkyl-isothioureas have been shown to be useful intermediates in the syntheses of substituted guanidines (Poss et al., Tetrahedron Lett. 33:5933 (1992)). In certain embodiments, the guanidine is part of a heterocyclic ring having two nitrogen atoms (see, for example, the structures below). The ring system can include an alkylene or

alkenylene of from 2 to 4 carbon atoms, e.g., ring systems of 5, 6, and 7-membered rings. Such ring systems can be prepared, for example, using the methods disclosed by Schlama et al., J. Org. Chem. 62:4200 (1997).

Compounds described herein (e.g., in Tables 1-3) can be prepared by alkylation of an amine nitrogen in the parent compound as shown in Scheme 1.

Alternatively, charged ion channel blockers can be prepared by introduction of a guanidine group. The parent compound can be reacted with a cynamide, e.g., methylcyanamide, as shown in Scheme 2 or pyrazole-1-carboxamidine derivatives as shown in Scheme 3 where Z is H or a suitable protecting group. Alternatively, the parent compound can be reacted with cyanogens bromide followed by reaction with methylchloroaluminum amide as shown in Scheme 4. Reagents such as 2-(methylthio)-2-imidazoline can also be used to prepare suitably functionalized derivatives (Scheme 5).

Any compounds containing an amine nitrogen atom (e.g., a compound selected from Compounds (1)-(563) or a compound according to Formulas (IA)-(XIV)) can be modified as shown in Schemes 1-5.

Example 1

Doublecortin-expressing neural progenitors initiate the neurogenesis found in prostate cancer (1). These autonomic neurons facilitate tumor development and dissemination (2) in part via nerve-derived noradrenaline which activates an angiogenic switch that fuels cancer growth (3, 4). While head and neck tumor-associated sensory nerves appear to transdifferentiate into adrenergic neurons following loss of TP53 (5), the overall impact of tumor neo-innervation by pain-initiating sensory neurons remains unclear. Here, it was found that malignant melanoma skin cancer cells directly interact with nociceptors by increasing neurite outgrowth, responsiveness to noxious ligands and neuropeptide release. In turn, CGRP, one such nociceptor-produced neuropeptide, directly increase exhaustion of cytotoxic T cells (PD1⁺Lag3⁺Tim3⁺INFγ⁻), limiting their capacity to eliminate melanoma cells. Genetic TRPV1 or Na_V1.8 lineage ablation, local pharmacological silencing or blockade of neuropeptide release from tumor-innervating nociceptors, as well as the antagonism of the CGRP receptor RAMP1, blunt tumor-infiltrating leukocyte exhaustion and tumor growth, nearly tripling B16F10-inoculated mouse survival. Single-cell RNA sequencing of human melanoma has revealed that Ramp1⁺ CD8⁺ T-cells are severely exhausted and increased in biopsy expression of Calca, the gene encoding for CGRP, correlated with worsened patient′ prognosis. It was concluded that reducing CGRP release from tumor-innervating nociceptors, by eliminating their immunomodulatory action on cytotoxic CD8⁺ T-cells, constitute an unexpected strategy to safeguard anti-tumor immunity.

Cytotoxic T-cells express a variety of receptors, including PD-1 (Programmed Death-1), Tim-3 (T cell immunoglobulin and mucin domain-containing protein 3), and Lag-3 (Lymphocyte Activation Gene-3)(6-9), which inhibit T-cell function after being activated by their cognate ligands. These checkpoint receptors ensure that immune responses to damage or infection are kept in check, preventing overly intense responses that might damage healthy cells(10). Tumor cells express ligands for these immune checkpoints, which, when activated, block the cytolytic functions of T-cells, favoring cancer cells survival (10-12). The posit role for neo-innervation in cancer as well as the known actions of neuropeptides on immune cells (13-22), led us to postulate that the local release of neuropeptides from activated nociceptors favors cancer growth by suppressing immune surveillance.

Breast cancer has been found to present increased sympathetic and decreased parasympathetic nerve densities (23), whereas prostate cancers are infiltrated with cholinergic fibers and are surrounded by adrenergic fibers (2). Here, it was sought to probe the extent of melanoma innervation and confirmed the presence of Tubb3 expressing fibers in each of the three melanoma patients' biopsies tested, whom were diagnosed with aggressive diseases (FIGS. 5A-5C). Next, CLARITY was used to obtain a 3-dimensional representation of the innervation present in 22d-B16F10 mouse tumors and confirmed the abundant presence of Tubb3⁺ neurons infiltrating the tumor and its border. To characterize the nature of this innervation, 10⁵ B16F10 cells or 10⁵ non-cancerous keratinocytes (MPEK-BL6) were inoculated (i.d.) into 8-week-old male and female nociceptor neurons reporter mice (TRPV1^Cre::Tdtomato^fl/wt) (24). Fourteen days post-inoculation, the B16F10 tumor showed nociceptor axonal innervation and outgrowth near the cancer cells (FIGS. 1A-1B).

When co-cultured with eGFP-B16F10 cells, fluorescently labeled (TRPV1^Cre::Tdtomato^fl/wt) DRG nociceptor neurites were found to largely extend toward the B16F10 cells, often forming physical contacts (FIGS. 1C-1D). In addition, the average neurite length of nociceptor neurons increased (number of intersecting radii; FIGS. 1E-1F) while the overall neuronal arborisation decreased (ramification index; FIG. 1G) when co-cultured with B16F10 cells. Cultured L3-L5 DRG nociceptors harvested from tumor-inoculated mice (on d14) extended longer neurites than their counterparts harvested from keratinocyte-injected mice (not shown). These findings indicate that nociceptors neurite outgrowth is enhanced when in proximity to melanoma cells, as opposed to keratinocytes.

Given that melanoma promotes axonogenesis, which leads to tumor innervation, it was next aimed to examine whether such physical proximity modulates nociceptor sensitivity. To test this, changes in calcium flux were measured, induced in response to sub-threshold concentrations of capsaicin (agonist of the heat sensing channel TRPV1 (Transient Receptor Potential Vanilloid-1)), mustard oil (agonist of the chemical sensing channel TRPA1 (Transient Receptor Potential Anykrin-1)), and ATP (agonist of the proton-sensing channel P2X3R). When nociceptors are cultured without melanoma cells, few DRG neurons responded to the ligands at the concentrations selected. However, the number of responsive neurons increased when co-cultured with B16F10 or B16F0 cancer cells (FIG. 1H). Similarly, the amplitude of calcium flux responses to the ligands was greater in ipsilateral L3-L5 DRG neurons harvested from tumor-inoculated mice (on d14) compared to those harvested from keratinocyte-injected mice (FIG. 1I). As opposed to B16F10 cells alone, DRG neurons co-cultured with B16F10 cells (5×10⁴ cells, 24 h) actively release neuropeptides in the media (e.g. CGRP; FIG. 1J). Unlike DRG neurons (25) (FIG. 1K), B16F10 cells (26) do not express transcripts for Calca, Tac1 or Vip (FIG. 1L); confirming their intrinsic incapacity to produce neuropeptides.

Cytotoxic CD8⁺ T cells express a wide variety (≥10) of neuropeptide receptors (FIG. 6). Given that nociceptors readily interact with CD8⁺ T cells in culture and that the neuropeptides they release block T_H1 immunity(27-30), it was tested whether these neuropeptides have direct effects on the expression of immune checkpoint receptors. First, splenocyte-isolated CD8⁺ T cells were cultured under type 1 CD8⁺ T-cell-stimulating conditions for 48 h and were then exposed to conditioned medium harvested from capsaicin-stimulated cultured DRG neurons (30 min). In comparison with naïve neuron media supplemented with capsaicin after harvesting, the conditioned medium containing nociceptor-produced neuropeptides increased the proportion of CD8⁺ T cells expressing PD1⁺Lag3⁺Tim3⁺ (FIG. 2A) and decreased the levels of INFγ⁺ (FIG. 2B) and TNFα⁺ (FIGS. 9A-9G) cells. The conditioned medium from potassium chloride (KCl)-stimulated neurons induced a similar phenotype (FIGS. 7A-7C).

To mimic their interactions within the tumor microenvironment, T_C1-stimulated CD8⁺ T-cells were co-cultured with DRG neurons (48 h) and the immunomodulatory role of peptidergic nociceptors were probed using gain-(capsaicin stimulation) and loss-of-function (TRPV1^Cre::DTA^fl/wt; genetically-engineered nociceptor ablated mice) approaches. Neuron stimulation with capsaicin (FIGS. 2C-2D; FIGS. 9A-9G) or KCl (FIGS. 7A-7C) increased the proportion of PD1⁺Lag3⁺Tim3⁺ (FIG. 2C) and decreased the levels of INFγ⁺ (FIG. 2D) and TNFα⁺ (FIGS. 9A-9G) CD8⁺ T-cells. These effects were absent when CD8⁺ T-cells were co-cultured in absence of TRPV1⁺ neurons (TRPV1^creDTA^fl/wt); confirming that peptidergic neurons drive CD8⁺ T-cells exhaustion (FIGS. 2E-2F; FIGS. 8A-8D). In contrast, capsaicin had no measurable impacts on CD8⁺ T-cells in the absence of neurons. These data suggest that neuron-secreted factors, rather than cell-cell contact, are responsible for the exhaustion of cytotoxic CD8 T cells.

When co-cultured, crosstalk occurs between neurons and cytotoxic CD8⁺ T-cells via cytokines and neuropeptides (22). Such prolong interactions (48 h culture) prompt CD8⁺ T-cells to overexpress the neuropeptide receptor Ramp1(FIG. 2G). Therefore, it was examined whether CGRP has direct immunomodulatory effects on cytotoxic CD8⁺ T-cells and found that it enhanced the proportion of PD1⁺Lag3⁺Tim3⁺ cells (FIG. 2H) and decreased the proportion of INFγ⁺ (FIG. 21) and TNFα⁺ (FIGS. 10A-10E) cells. Along with CGRP blocking CD8⁺ T-cells proliferation (31), the data highlight CGRP as a novel driver of CD8⁺ T-cells exhaustion.

As a litmus test for CD8⁺ T-cell exhaustion, it was probed whether nociceptive neuron-released neuropeptides can blunt the antitumor responses of cytotoxic CD8⁺ T-cells, by culturing B16F10-OVA and OT1 cytotoxic CD8⁺ T-cells, either with or without DRG neurons. OT1 cytotoxic T-cells drastically increased cancer cell apoptosis (annexinV⁺7AAD⁺ B16F10-OVA; FIGS. 2J-2K). B16F10-OVA apoptosis decreased when challenged with capsaicin-stimulated neuron conditioned medium (FIG. 2J), when co-cultured with nociceptors (FIG. 2K) or when stimulated with CGRP (FIG. 2L; FIGS. 11A-11G). Decreased tumor apoptosis correlated with OT1 cytotoxic T-cell exhaustion (FIGS. 11A-11G), which was also confirmed by live-cell imaging (FIGS. 2M-20).

Gastric tumor denervation limits growth, and vagotomised patients have lower mortality rates associated with intestinal cancer (2, 21, 32, 33). Nociceptor-produced neuropeptides have been shown to reduce immunity against bacteria (28) and fungi (34), and to promote the expression of immune checkpoint receptors on cytotoxic CD8⁺ T-cells (FIGS. 2A-2O) (13-18); therefore, it was sought to examine the interaction between cancer-nociceptor-CD8⁺ using a xenograft mouse model of triple-negative melanoma skin cancer, which is an established model of immunosurveillance (10). B16F10 cells were inoculated (i.d., 10⁵) into 8-week-old male and female nociceptor ablated (TRPV1^Cre::DTA^fl/wt) or intact mice (littermate controls). In both males (n=50) and females (n=68), tumor growth (˜350%) and weight (˜240%) were reduced in mice lacking nociceptors (FIG. 3A; FIGS. 12A-12I), whereas the median length of survival increased by 23% (p≤0.0001; FIG. 3B). This effect represents a 150% increase relative to the survival rate observed following αPDL1 blockade in B16F10 mice (meta-analysis of 16 publications; FIGS. 12A-12I). Consequently, intact mice succumb at a 2.5-fold higher rate than nociceptor ablated mice (0.4 Mantel-Haenszel hazard ratio; FIG. 3B).

Tumor-specific sympathetic denervation downregulates the expression of PDL1, PD1 and FOXP3 in tumor infiltrating lymphocytes (TIL), whereas parasympathetic innervation decreases the expression of PD1 and PDL1. TIL exhaustion was also correlated with relative distance from sympathetic terminals (23). In B16F10-bearing mice, the genetic ablation of nociceptor neurons increased the numbers of tumor-infiltrating CD8⁺ (FIG. 3C), which were characterized by reduced exhaustion (PD1⁺Lag3⁺Tim3⁺; FIG. 3D) and increased cytotoxic potential (INFγ⁺, TNFα⁺, Granzyme B⁺; FIG. 3D, FIGS. 13A-13B). Similar effects were observed for intra-tumoral CD4⁺ T-cells (FIGS. 14A-14D) as well as for T-cells found in tumor-draining lymph node (FIGS. 15A-15F). Therefore, it was next attempted to test whether the slower B16F10 growth observed in the TRPV1^Cre::DTA^fl/wt mice depended on lymphocytes. Upon the systemic depletion of CD3⁺ T cells (using αCD3), no differences in B16F10 tumor growth and volume were observed between TRPV1^Cre::DTA^fl/wt and littermate control mice (FIG. 3E; FIGS. 16A-16B), implying that slower tumor growth depended on nociceptors-induced lymphocyte exhaustion.

Blocking the activity of immune checkpoint proteins releases the cancer cell-induced “brake” on the immune system, increasing the ability of the system to eliminate tumor (6-8, 10). Immune checkpoint inhibitors, including those targeting PDL1, improve the clinical outcomes associated with metastatic melanoma (8, 35, 36). Given that nociceptor neurons drive CD8⁺ T cell exhaustion, the impacts of nociceptor absence on tumor elimination were assessed following PDL1 blockade. When used after tumor-establishment, αPDL1 (i.p; day 7, 10, 13) treatment resulted in the relative reduction of tumor growth which was enhanced by ˜2.5-fold in nociceptor ablated mice (FIG. 4G). B16F10-OVA tumor size was correlated with increased infiltration of tumor-specific CD8⁺ T-cells which displayed reduced exhaustion (PD1⁺Lag3⁺Tim3⁺), an effect exacerbated by αPDL1 treatment (FIGS. 17A-17D).

It was next sought to confirm the data using a second line of nociceptor ablated mice. Na_V1.8 is a sodium channel expressed by mechano- and thermos-sensitive neurons and ˜80% of nociceptors (28, 37). The ablation of Na_V1.8⁺ sensory neurons (Na_V1.8^Cre::DTA^fl/wt) slowed B16F10 tumor growth (FIG. 3H), reduced the proportion of PD1⁺Tim3⁺ CD8⁺ T cells within the tumor but increased the infiltration of total CD8⁺ T-cells (FIGS. 18A-18C). Note that unbiased RNA sequencing data unequivocally showed that TRPV1 and Na_V1.8 are not expressed by immune cells (FIG. 6).

Next, a gain-of-function approach was used, in which daily transdermal illumination (3.5 ms, 10 Hz, 478 nm, 100 mW, delivering ˜2-6 mW/mm² to a 0.39 NA fiber placed 5-10 mm from the skin, for 20 min) was used to stimulate optogenetically-controlled tumor-innervating Na_V1.8⁺ neurons. In comparison with light-insensitive littermate control mice, blue light-stimulated Na_V1.8^Cre::ChR2^fl/wt mice enhanced B16F10 tumor growth (FIG. 3H), increased proportion of PD1⁺Tim3⁺ CD8⁺ T-cells, and decreased infiltration of total CD8 T-cells (FIGS. 18A-18C). Tumor growth and CD8⁺ T-cells exhaustion correlated with secreted CGRP levels measured in the tumor-surrounding skin after optogenetic stimulation (FIG. 18C).

Sensory neuron depleted mice were then inoculated with the YUMMER1.7 cell line; which is a highly immunogenic form of the Braf^V600ECdkn2a^−/− Pten^−/− cell line that has been modified by UV exposure and represents a clinically relevant melanoma model. In comparison to mice whose sensory neurons are intact, slower tumor growth (˜3.3 fold; FIG. 3I) and reduced tumor weights (˜3.1 fold) were found in the absence of TRPV1⁺ neurons which correlated with a reduced (˜3.6-fold) proportion of PD1⁺Lag3⁺Tim3⁺ CD8⁺ T-cells (FIGS. 19A-19H). The ablation of sensory neurons also increased the infiltration of total (˜7.2-fold), INFγ⁺ (˜9.1-fold) and TNFα⁺ (˜9 fold) CD8 T-cells (FIGS. 19A-19H). Similar effects were observed for CD4⁺ T-cells (FIGS. 19A-19H).

The neonatal/embryonic ablation of neuronal subsets may lead to compensatory mechanisms. To circumvent this potential shortcoming, neurons were silenced using the FDA-approved cervical dystonia treatment Botulinum toxin A (BoNT/A), a neurotoxic protein produced by Clostridium botulinum, which acts by cleaving SNAP25 (38). This strategy caused the long-lasting (20 days) abolition of neurotransmitter′ release from skin-innervating autonomic fibers and somatosensory neurons (CGRP) and blocked the neuro-immune interplay occurring during skin infection (29). In addition, BoNT/a blockade of synaptic vesicle release from neurons reduce tumor growth in prostate cancer (2). BoNTA administration (25 pg/μl; 501; 5 i.d. sites) prior to tumor inoculation, reduced subsequent tumor growth (FIG. 4A) and the proportion of PD1⁺Lag3⁺Tim3⁺ CD8 T-cells (FIG. 4B), resulting in the survival of BoNT/A-treated B16F10-bearing mice (0.12 hazard ratio; FIG. 4F). BoNT/A treatment also increased the intra-tumor CD8⁺ and CD4⁺ T-cells counts and preserved their cytotoxic potential (INFγ, TNFα, Granzyme B; FIGS. 20A-20H).

A proven nociceptor-blocking strategy (39) was then used to silence tumor-innervating nociceptors. This protocol uses large pore ion channels (TRPV1) as cell-specific drug-entry ports to deliver QX-314, a charged and membrane-impermeable form of lidocaine, to block voltage-gated sodium (Na_V) channels. During inflammation, similar to that observed in tumor microenvironments, these ion channels open, allowing QX-314 to permeate into these neurons resulting in a long-lasting electrical blockade (37). Compared with vehicle, QX-314-mediated sensory neuron silencing (0.3%; daily i.d. surrounding the tumor) reduced melanoma growth (˜3.3-fold; FIG. 4C), tumor weight (˜5.2-fold; FIGS. 22A-22I) and the proportion of PD1⁺Lag3⁺Tim3⁺ CD8⁺ T-cells (˜15-fold; FIG. 4D). Sensory neuron silencing increased the intra-tumor numbers of CD8⁺ and CD4⁺ T-cells and preserved their cytotoxic potential (INFγ, TNFα, Granzyme B; FIGS. 22A-22I). Vehicle exposed B16F10-bearing mice succumb at a 2.7-fold higher rate (p≤0.0001) than QX-314-exposed mice (0.37 hazard ratio; FIG. 2F).

Nociceptor neurons express PD1, a level found to be reduced in tumor-innervating neuron (FIG. 35). Nevertheless, cell-cell interplay between PDL1⁺ TILs and PD1⁺ neurons may result in immune exhaustion. Here, it was found that αPDL1-mediated tumor reductions were increased by ˜2.5-fold in nociceptor ablated mice (FIG. 3G; FIGS. 17A-17D), ˜10 fold when used in BoNT/A pre-treated mice (FIG. 4E; FIGS. 20A-20H) or ˜5 fold when used in combination with QX-314 (FIG. 4E; FIGS. 22A-22I). Given that αPDL1 induces allodynia by increasing nociceptor activity, it is therefore likely to promote neuropeptide release and subsequent TIL exhaustion. The silencing of nociceptors may augment αPDL1 efficacy by limiting its pro-nociceptive effects, safeguarding the anti-tumor immunity of the host (FIGS. 17A-17D). Overall, these data indicated that silencing nociceptors (QX-314 or BoNT/A) may be a potent adjuvant treatment for immune checkpoint blockers.

In support of the neuronal specificity of BoNT/A and QX-314, unbiased RNA-sequencing analysis has shown that in contrast to nociceptors (FIG. 1K), B16F10 (FIG. 1L) and immune cells (FIG. 6) do not express Snap25, Trpv1, Na_V1.7 or Na_V1.8. In addition, BoNT/A and QX-314 have no impacts on B16F10 survival (FIGS. 27A-27B) or CD8⁺ T-cells function (FIGS. 21A-21E and FIGS. 23A-23E). Tumor growth, mechanical and thermal pain-related hypersensitivity measured in hindpaw-inoculated B16F10 mice, and itching (observed in ˜30% of mice with flank inoculated tumor) were absent in mice with genetically ablated or pharmacologically silenced nociceptors (FIGS. 26A-26D). Capsaicin-induced CGRP release from tumor-inoculated skin was also blunted in QX-314 and BoNT/A pre-treated skin (FIG. 4G). These data support the capacity of QX-314 and BoNT/a to i) silence tumor-innervating neurons and ii) prevent the release of CGRP and its subsequent immunomodulation of TILs.

Adrenergic and cholinergic nerve fiber densities in tumors have been correlated with poor clinical outcomes (2). Whether nociceptor innervation of melanomas has a similar effect remains unclear. Here, it was found that patient′ survival (oncolnc database; 459 patients (40)) was negatively correlated (p≤0.05) with increased tumor expression of NaV1.7, Piezo1, Pgp9.5 and Tubb3 (FIGS. 31A-31E). Patient′ biopsies also showed an increased expression (p≤0.05) of Calca, Ramp1 and Na_V1.8 expression compared with healthy skin (FIG. 4L; FIG. 33), PBMCs (FIG. 34) or benign nevi (FIG. 32) (41-44).

In support of the neuronal origin of these genes and in contrast with doublecortin (1), it was found that Calca, Na_V1.7, Piezo1, Pgp9.5 as well as Snap25, Trpv1 and Na_V1.8, are not expressed by TILs, cancer-associated fibroblasts, endothelial cells or melanoma cells according to the in-silico analysis of two independent single-cell RNA-sequencing performed on melanoma biopsies (45, 46) (FIGS. 29, 30) as well as the Immgen (47) (FIG. 6) and human protein atlas (48, 49) (FIG. 36) databases. Overall, these data, in addition to the presence of Tubb3⁺ neurons in biopsy samples (FIGS. 5A-5C), support a link between melanoma innervation, the heightened expression of neuropeptides or their cognate receptors and the worsening of clinical prognosis. These data are once more indicative of the translational potential of BoNT/A and QX-314, to silence tumor-innervating nociceptors.

CD8⁺ T-cells overexpress Ramp1when co-cultured with sensory neurons (FIG. 2G), and CGRP increases CD8⁺ T-cells expression of immune checkpoint receptors (FIG. 2H), reduces their production of cytotoxic granules (FIG. 2I), and blunts the OT1-CD8⁺ T-cells capacity to eliminate B16F10-OVA melanomas (FIG. 2L; FIGS. 10A-10E; FIGS. 11A-11G). CGRP receptor antagonism using BIBN4096 blocked the deleterious neuro-immune interplay during microbe infections and rescued host anti-bacterial activity (27). Here, it was found that BIBN4096, when administered systemically once every two days, reduced B16F10 growth (˜1.5-fold; FIG. 2H), tumor weight (˜1.9-fold; FIGS. 24A-24J) and the proportion (˜2.5-fold) of PD1⁺Lag3⁺Tim3⁺ CD8 T cells (FIG. 2I). RAMP1 blockade also increased the intra-tumor number of total (˜3-fold; FIGS. 24A-24J) and INFγ⁺ (˜3-fold; FIG. 2J) CD8⁺ T-cells. Similar effects were observed for intra-tumor CD4⁺ T-cells (FIGS. 24A-24J). It is worth noting that BIBN4096 (1-4 μM) have no impact on cultured B16F10 survival and do not prevent the effects of αCD3/αCD28 stimulation on CD8⁺ T-cells (FIGS. 25A-25E).

Single-cell RNA-sequencing of human melanomas revealed that ˜1% of tumor-infiltrating CD8⁺ T-cells expressed Ramp1 (45). When clustered, these Ramp1⁺ CD8⁺ T-cells revealed their drastic overexpression of the immune checkpoint receptors PD1, Tim3, Lag3, CTLA4, CD27 coupled with a loss of the pro-proliferative cytokine IL-2 (FIG. 4K). Similar findings were obtained from an independent single cell RNA-sequencing analysis of melanoma-infiltrating T cells (46) (FIG. 28). Reminiscent of severe exhaustion (45, 46) (FIG. 4K; FIG. 28), the expression profile of Ramp1⁺ tumor-infiltrating CD8⁺ T-cells support the concept that nociceptor-released CGRP promotes CD8⁺ T-cells exhaustion (FIG. 2H, 2L) and the use of BIBN4096 as a targeted therapy to safeguard anti-tumor immunity (FIG. 4H-4I; FIGS. 24A-24J).

In summary, the data support a regulatory role for nociceptors in the immune responses to tumor growth, through the regulation of immune checkpoint receptors expression on cytotoxic CD8⁺ T-cells. Silencing tumor-innervating sensory neurons represents an innovative strategy for attenuating the immunomodulatory power of the nervous system and promoting anti-tumor activity.

Methods for Example 1

Animals

The Institutional Animal Care and Use Committees of Boston Children's Hospital, and the Université de Montréal (CDEA #19027; #19028) approved animal procedures. Mice were housed in standard environmental conditions (12 h light/dark cycle; 23° C.; food and water ad libitum) at facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. 8-week old C57BL6 (Jax, #000664); OT1 (Jax, #003831) (50), TRPV1^cre (Jax, #017769) (51), ChR2^fl/fl (Jax, #012567) (52), td-tomato^fl/fl (Jax, #007908) (53), DTA^fl/fl (Jax, #009669) (54), and QuASR2^fl/fl (Jax, #028678) (55) mice were purchased from Jackson Laboratory. Na_V1.8^Cre mice (56) were generously supplied by Professor Rohini Kuner (Heidelberg University). The cre/lox toolbox was used to genetically-engineered the various mice lines used (TRPV1^cre::DTA^fl/wt, TRPV1^cre:: QuASR2^fl/wt, TRPV1^cre::Tdtomato^fl/wt, NaV1.8^cre::DTA^fl/wt, NaV1.8^cre::ChR2^fl/wt and littermate control) by crossing male heterozygote Cre mice to female homozygous loxP mice. Cre driver lines used are viable and fertile and abnormal phenotypes were not detected. Offspring were tail clipped; tissue was used to assess the presence of transgene by standard PCR, as described by Jackson Laboratory. Offspring were used at 8 weeks of age.

Cell Line

B16F0 (#CRL-6322) (57), B16F10 (#CRL-6475) (58), YUMMER1.7 (Marcus Bosenberg, Yale University)(59), EG7-OVA (#CRL-2113) (60) were purchased from ATCC. B16F10-OVA (61) and B16F10-OVA-mCherry2 (62) were kindly supplied by Dr. Matthew F. Krummel (University of California San Francisco) while mEERL (63) and MLM3 (63) cell lines were generated and used by Dr. Paola Vermeer (University of South Dakota). These cell lines were cultured in complete Dulbecco's Modified Eagle's Medium high glucose (DMEM, Gibco #D5796) supplemented with 10% fetal bovine serum (Biochrom, #12483020) and 1% penicillin/sterptomycin (Gibco, #15140163), and maintained at 37° C. in a humidified incubator with 5% CO₂.

Cancer Inoculation

Cancer cells (1×10⁵) were resuspended in PBS and injected (i.d., 100 μl) to the mice right flank. Growth was daily assessed using a handheld caliper. Mice were euthanized when tumor reached 1000-1500 mm (57, 58, 62). Tumor and their draining lymph node (tdLN) were harvested. Tumors were enzymatically digested in DMEM (Gibco, #D5796)+2 mg/ml collagenase D (Sigma, #C5138)+0.03 mg/ml DNAse I (Sigma, #10104159001) under constant shaking (30 min, 37° C.). Tumor draining lymph nodes were dissected in PBS, mechanically dissociated using a plunger, strained (70 μm), washed with PBS. The cell suspensions were then strained (70 μm), washed and RBC were lysed (Life Technologies, #A1049201; 2 min).

Drugs

QX-314 (64) (Tocris, #2313; 0.3%) was injected (i.d.) daily in 5 points around the tumor (treatment began once tumor was visible). BIBN4096 (Tocris, #4561; 5 mg/kg) was injected (i.p.) on day 6, 8, 10, 12 and 14. Botulinum neurotoxin A (65) (List biological labs, #130B; 25 pg/μl) was injected (i.d.) three and one day prior to, or one and three days after, tumor inoculation. αPD-L1 (66) (Bioxcell, #BE0101, 6 mg/kg) was injected (i.p.) on day 7, 10 and 13.

In Vivo Depletion of CD3

200 μg/mice of anti-mouse CD3 (67) (Bioexcell, #BE0001) was injected (i.p.) 3 days prior to B16F10 inoculation (1×10⁵; i.d.) and continued every 3 days. Blood samples were taken twice weekly to confirm depletion and tumor growth measured daily, as previously described.

Immunophenotyping

Single cells were resuspended in FACS buffer (PBS, 2% FCS, EDTA), Fc blocked (0.5 mg/ml, 10 min; BD Biosciences, #553141) and stained (30 min, 4° C.) with ZombieAqua (Biolegend, #423102) and monoclonal antibodies (anti-CD45-BV421 (1:100, Biolegend, #103134), anti-CD11b-APC/Cy7 (1:100, Biolegend, #101226), anti-CD8-AF700 (1:100, Biolegend, #100730), anti-CD4-PerCP/Cyanine5.5 (1:100, Biolegend, #100540), anti-NK-FITC (1:100, Biolegend, #108706), anti-PD-1-PE-Cy7 (1:100, Biolegend, #109110), anti-Lag3-PE (1:100, Biolegend, #125208), anti-Tim-3-APC (1:100, Biolegend, #119706).

Intracellular Cytokine Staining

Cells were stimulated (4 h) with Brefeldin A (Biolegend, #423303), washed, fixed/permeabilized (BD Biosciences; #554714) and stained with anti-IFN-7-BV650 (Biolegend, #505832), anti-TNFα-FITC (Biolegend, #506304), anti-Granzyme B-APC (Biolegend, #396407).

Skin Explant

3 h post-exposure to vehicle (100 al), QX-314 (0.3%, 100 μl) or BoNT/a (25 pg/μL, 100 μl), tumor-surrounding skin was harvested using 10 mm punch biopsies. The biopsies were transferred into 24-well plates and cultured into DMEM containing 1 μl/ml of protease inhibitor (Sigma, #P1860) and capsaicin (1 μM. Sigma, #M2028). After 30 min incubation (37° C.), the supernatant was collected and CGRP release analyzed (65) using a commercial ELISA (Cayman Chemical, #589001).

Neuron Culture

Mice were sacrificed and dorsal root ganglia (DRG) were dissected out into DMEM medium (Corning, #10-013-CV), completed with 50 U/mL penicillin and 50 μg/ml streptomycin (Fisher, #MT-3001-C1), and 10% FBS (Seradigm, #3100). Cells were then dissociated in HEPES buffered saline (Sigma, #51558) completed with 1 mg/mL collagenase IV (Sigma, #C0130)+2.4 U/mL dispase II (Sigma, #04942078001) and incubated for 80 minutes at 37° C. Ganglia were triturated with glass Pasteur pipettes of decreasing size in supplemented DMEM medium, then centrifuged over a 10% BSA gradient, plated on laminin (Sigma, #L2020) coated cell culture dishes. Cells were cultured with Neurobasal-A medium (Gibco, #21103-049) completed with 0.05 ng/μL NGF (Life Technologies, #13257-019), 0.002 ng/μL GDNF (Peprotech, #450-51-10), 0.01 mM AraC (Sigma, #C6645) and 200 mM L-Glutamine (VWR, #02-0131) (68).

Calcium Imaging

L3-L5 DRG neurons were harvested and co-cultured with B16F10, B16F0 or MPEK-BL6 for 24-48 h. The cells were then loaded with 5 mM Fura-2 AM (BioVision, #2243) in complete Neurobasal-A medium for 30 min at 37° C., washed into Standard Extracellular Solution (SES, 145 mM NaCl, 5 mM KCl, 2 mM CaCl₂), 1 mM MgCl₂, 10 mm glucose, 10 mM HEPES, pH 7.5), and response to noxious ligands (100 nM capsaicin; 100 μM AITC; 1 μM ATP) analyzed at room temperature. Ligands were flowed (15 s) directly onto neurons using perfusion barrels followed by buffer washout (105-sec minimum) (68). Cells were illuminated by a UV light source (Xenon lamp, 75 watts, Nikon), 340 nm and 380 nm excitation alternated by an LEP MAC 5000 filter wheel (Spectra services), and fluorescence emission captured by Cool SNAP ES camera (Princeton Instruments). 340/380 ratiometric images were processed, background corrected and analyzed (IPLab software; Scientific Analytics) and Microsoft Excel used for post-hoc analyses.

Immunofluorescence

2×10³ DRG neurons were co-cultured with 2×10⁴ B16F10-mCherry-OVA for 24-48 h. The cells were fixed (4% paraformaldehyde; 30 min), permeabilized (0.1% Triton X-100, 20 min), and blocked (PBS, 0.1% Triton X-100, 5% BSA, 30 min). The cells were rinsed (PBS), stained and mounted with vectashield containing DAPI (Vector Laboratories, #H-1000). Images were acquired using a Ti2 Nikon fluorescent microscope.

Clarity

Passive CLARITY protocol (69) was used to cleared 14d-inoculated B16F10 tumors. Peripheral nerves were stained with an A647 conjugated anti-tubulin β3 (1:100, Biolegend, #801209) polyclonal antibody. Samples were then submerged in PROTOS index-matching imaging medium (LifeCanvas, #SHIELF), imaged (5×) with lightsheet microscope (Zeiss Z.1) and data showed as 2 mm-thick maximum projection (scale=500 μm).

CD8 Isolation

6-8 weeks-old male and female mice were euthanized, spleen harvested in ice-cold PBS (5% FBS), and mechanically dissociated. The cells were strained (70 μm), RBC lysed (Life Technologies, #A1049201; 2 min), and counted using a hemocytometer. Naïve CD8⁺ T cells were magnet sorted (Stem cell, #19853A) and cultured (DMEM+FBS 10%, Pen/Strep +non-essential amino acid (Corning, #25-025-C1)+vitamin+β-mercaptoethanol (Gibco, #21985-023)+L-Glutamine (VWR, #02-0131)+sodium pyruvate (Corning, #25-000-C1)). Cell purity was confirmed after magnet sorting by labeling cells against CD62L and the numbers of CD8⁺CD62^hi immunophenotyped by flow cytometry. To generate cytotoxic T lymphocytes, 1×10⁶ naïve CD8⁺ T cells were seeded and stimulated for 48 h under Tc1 inflammatory condition (2 μg/ml plate bounded αCD3/αCD28 (Bioxcell, #BE00011, #BE00151)+10 ng/ml rIL-12 (Biolegend, #577008)+10 μg/ml of anti-IL-4 (Bioxcell, #BE0045).

In Vitro Cytotoxic CD8 T Cell Stimulation with Neuropeptides

CD8 T cells were isolated and stimulated for 48 h in 96 wells plate. In the presence of peptidase inhibitor, the cells were treated with either of CGRP (0.1 uM), VIP (1 uM) and SST (0.1 uM), or a combination of these. Expression of PD-1, Lag-3 and Tim-3 as well as INFγ, TNFα and IL-2 were immunophenotyped by flow cytometry.

Co-Culture

Naïve DRG neurons (1×10⁴) were seeded in T cell media (supplemented with 0.05 ng/μL NGF (Life Technologies, #13257-019), 0.002 ng/μL GDNF (Peprotech, #450-51-10), and co-cultured with Tc1 CD8 T cells (1×10⁵) in presence of IL-2 (575408). In some instances, co-cultures were stimulated with either of capsaicin (300 nM, twice/day) or KCl (40 mM). After 48 h, the cells were collected by centrifugation (5 min at 1300 rpm), stained and immunophenotyped by flow cytometry.

In Vitro Cytotoxic CD8 T Cell Stimulation with Neurons' Conditioned Media

DRG neurons were cultured (24 h) in complete T cell media supplemented with 1 μl/ml protease inhibitor (Sigma, #P1860), 0.05 ng/μL NGF (Life Technologies, #13257-019), and 0.002 ng/μL GDNF (Peprotech, #450-51-10) and stimulated with KCl (40 mM). The conditioned media or vehicle were collected after 10 min and added to CD8 T cells for 48 h. The CD8 T cells expression of exhaustion markers (PD1, Lag3 and Tim3) and cytokine (INFγ, TNFα, IL-2) were analyzed by flow cytometry.

Apoptosis

1×10⁴ naïve TRPV1^Cre::QuASR2^fl/wt DRG neurons were co-cultured (24-72 h) with 1×10⁵ OVA-specific cytotoxic CD8 T cells harvested from OT1 mice and 1×10⁵ B16F10-mCherry-OVA in T cell media (supplemented with 0.05 ng/μL NGF (Life Technologies, #13257-019), 0.002 ng/μL GDNF (Peprotech, #450-51-10). After 48 h co-culture, the cells were collected by centrifugation (5 min at 1300 rpm), stained using anti-Annexin V-APC, and 7-AAD, (Biolegend, #640930) and immunophenotyped by flow cytometry. Alternatively, co-cultures were imaged every 6 h using a fluorescent microscope (Nikon, Ti2).

Pain Behavioral

10⁵ B16F10 cells or 10⁵ non-cancerous keratinocytes (MPEK-BL6) were inoculated intradermally in the mice left hindpaw. Once every two-day, the thermal and mechanical sensitivity was evaluated using a plantar test (Hargreaves' method) analgesia meter (70) (IITC Life Science, Model 390G,) and von Frey monofilaments (70) (UgoBasile, #52-37450-275), respectively.

B16F10 Survival

2×10⁵ B16F10 cells were cultured in 6-well-plate and challenged with BoNT/a (0-50 pg/μl) for 24 h, QX-314 (0-1%) for 72 h or vehicle. B16F10 cells survival was assessed using anti-annexin V-APC and 7-AAD (Biolegend #640930) using flow cytometry.

qPCR

After 48 h in co-culture with DRG neurons, live CD8 T cells were purified by flow cytometry (FACSAria), RNA extracted in Trizol (Sigma-Aldrich, #93289), cDNA generated (SuperScript® VILO™ cDNA Synthesis Kit) and transcript expression (Ramp1, Ramp3, Vpac2, GAPDH) quantified (Power SYBR Green PCR Master mix) by qPCR (BMS; Mic thermocycler), as instructed by the manufacturer (70).

Neurite Outgrowth Assessment

2×10³ DRG neurons from TRPV1^Cre::Tdtomato^fl/wt mice were harvested and co-cultured (2×10⁴) with non-tumorigenic keratinocytes or B16F10-GFP. The cells were fixed, imaged (Nikon, Ti2 fluorescent microscope) and neurite outgrowth analyzed using ImageJ.

In-Silico Analysis

The OncoLnc database (www.oncolnc.org) was used to assess transcript expression of 333 neuronal-enriched genes (neuronal membrane proteins, neural stem cell markers, transcription factors, ion channel receptors, and neuropeptides) in 459 skin cancer (SKCM) tumor biopsies from the Cancer Genome Atlas (TCGA) database. 206 of these genes were expressed, and 108 selected based on their negative Cox coefficient value, indicating a link between lower gene expression and improved patient survival. The cBioPortal (www.cbioportal.org) allowed us to link gain and loss-of-function mutations to 333 neuronal-enriched genes and survival of 1517 skin cancer patient. The Oncomine database (www.oncomine.org), was used to browse previously published sequencing datasets (71-74), to compare the expression of 30 nociceptor-enriched genes in normal skin or benign nevus with melanoma biopsies.

Patients Biopsy

Melanoma biopsies were collected at Sanford Health and classified by a board-certified pathologist. Given that patient samples were de-identified and no information other than tumor type was provided, the Sanford Health IRB company established that these samples were not considered human subjects research and no IRB number was assigned. DERM103 patient sample classified as a malignant melanoma from the left posterior shoulder, MART-1 and HMB45 positive, melanocytic in nature (Elastic positive) and MIB-1 positive (increased proliferation). DERM107 patient sample classified as malignant melanoma, high mitotic index, invasive, stage pT2a, MART-1, SOX-10, P16, and HMB45 positive. DERM110 patient sample classified as malignant melanoma; high mitotic index, Clark's level IV, stage pT1b, the tumor appears to be invading the epidermis with a fusion of malignant sheets of cells, strongly positive for SOX-10, MART-1, HMB45 with a moderate increase in proliferative index.

Statistics

Data expressed as mean±S.E.M. Statistical significance determined by one-way or two-way ANOVA for multiple comparisons and two-tail unpaired Student's t-test for single variable comparison. P values less than 0.05 were considered significant. Numbers of animals are defined in figure legends.

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Example 2

Tumor denervation limits cancer growth, but the mechanisms behind this are unknown. Provided herein is the finding that malignant melanoma cells directly interact with pain-initiating nociceptor neurons by increasing neuron outgrowth, responsiveness to noxious ligands and neuropeptide release. In turn, nociceptor neuropeptides increase exhaustion of cytotoxic T cells (PD1⁺Lag3⁺Tim3⁺INFγ⁻), limiting their capacity to eliminate melanoma cells. Genetic ablation (TRPV1^cre::DTA^fl/wt; Na_V1.8^cre::DTA^fl/wt), local pharmacological silencing (QX-314) as well as blockade of vesicle release (BoNT/a) from tumor-innervating nociceptors enhance tumor-infiltrating leukocyte (TIL) numbers and survival in mice subject to orthotropic melanoma inoculation, blunting tumor growth and TIL exhaustion. Similar findings were obtained in mice inoculated with a Braf^V600ECdkn2a^−/− Pten^−/− melanoma as well as in a model of lung metastatic HPV⁺ oropharyngeal squamous cell carcinoma. These effects are absent upon CD3⁺ depletion and are phenocopied by local administration of a calcitonin gene-related peptide (CGRP) receptor antagonist. Silencing or ablating sensory neurons also increases the response to systemic PDL1 blockade. Further support for involvement of nociceptors in cancer comes from the increased tumor growth and greater TIL exhaustion produced by optogenetic activation (Na_V1.8^cre:ChR2^fl/wt) of tumor-innervating nociceptors and Tubb3⁺ innervation of metastatic melanoma patients' biopsies. Provided herein is the conclusion reducing neuropeptide release from tumor-innervating nociceptors, by eliminating their immunomodulatory action on cytotoxic CD8 T cell, may be a useful therapeutic intervention to boost immune surveillance.

Shown herein is nociceptor neurons stimulated by B16F10-melanoma cells release neuropeptides that modulate cytotoxic CD8 T cell activity, including an overexpression of immune checkpoint receptors. Targeted genetic ablation or local temporary pharmacological silencing of tumor-innervating sensory neurons decreases the growth of either triple-negative B16F10 melanoma, Braf^V600ECdkn2a^−/− Pten^−/− melanoma or lung metastatic HPV⁺ oropharyngeal squamous cell carcinomas, in a CD3-dependant manner. These treatments also prevent TIL exhaustion and increase the response to anti-PD-L1 blockade. Optogenetic activation of tumor-innervating sensory neurons, furthermore, drives increased cancer growth and TIL exhaustion. Therefore, it was concluded that nociceptor neurons dampen CD8 T cell activity by expressing ligands for⁵² and controlling the expression of immune checkpoint receptors. In consequence, nociceptor sensory neurons emerge as a therapeutic driver of immune regulation in a cancer setting.

It was first probed whether melanoma modulate the growth and sensitivity of nociceptors. To do this, 8-week-old male and female mice whose sensory neurons were fluorescently labeled (TRPV1^Cre::Tdtomato^fl/wt; a lineage reporter virtually labelled all nociceptors⁷²) were inoculated intradermally with 10⁵ B16F10 cells or non-cancerous keratinocytes (MPEK-BL6). Fourteen days post-inoculation, the B16F10 tumor showed nociceptor axonal innervation of the BrDu⁺ cells (FIGS. 37A-37B). In support of this, IHC was used and found Tubb3 expressing fibers in three aggressive melanomas biopsies.

It was then sought to probe the nature of the interplay between melanoma cancer cells and nociceptors. To do this, a double reporter co-culture assay was devised that included fluorescently labeled (TRPV1^Cre::Tdtomato^fl/wt) DRG nociceptor neurons and eGFP-expressing B16F10 cells, or non-tumorigenic keratinocytes. When co-cultured with B16F10 cells, nociceptor neurites typically extend toward B16F10 cells, forming contacts (FIGS. 37C-37D). In addition, the average neurite length of nociceptor neurons increased (number of intersecting radi; FIGS. 37E, 37F, 42B) while the overall neuronal arborisation decreased (ramification index; FIG. 37G) when co-cultured with B16F10 cells. Ipsilateral L3-L5 DRG nociceptors harvested from tumor-inoculated mice (on d14) put in culture for 72 h extend longer neurites than their counterparts harvested from keratinocyte-injected mice (not shown). These findings indicate that nociceptors grow differently when in proximity to melanoma cells as opposed to 1 keratinocytes.

Given that melanoma promotes axonogenesis, which leads to innervation it was next aimed to test whether this would physical proximity modulates the sensitivity of nociceptor neurons. To test this, the number and the amplitude of calcium flux induced in response to sub-threshold concentration of capsaicin (an exogenous agonist of the heat sensing channel TRPV1 (Transient Receptor Potential Vanilloid-1))-, mustard oil-(an exogenous agonist of the chemical sensing channel TRPA1 (Transient Receptor Potential Anykrin-1)), and ATP-(an exogenous agonist of the proton-sensing channel P2X3R) was measured. It was found that when nociceptors are cultured without melanoma cells the ligands, at the concentration selected activated a few DRG neurons. However, the numbers of responsive neurons increased when co-cultured with B16F10 cells (FIG. 37H; FIG. 42A). Similarly, when tested for the amplitude of calcium flux responses to the ligands, ipsilateral L3-L5 DRG neurons harvested from tumor-inoculated mice (on d14) had heightened sensitivity in comparison to their counterparts harvested from keratinocyte-injected mice (FIG. 37I). In addition, it was found that neurons co-cultured with B16F10 cells (5×10⁴ cells, 24 h) increase release of SP, CGRP, and VIP (FIG. 37J).

In-silico analysis of the Immgen database (⁷³) revealed that cytotoxic CD8⁺ T cells express a wide variety of (≥10) neuropeptide receptors (FIG. 43A). Given that nociceptors readily interact with CD8 T cells when put in co-culture and that the neuropeptides they release block T_H1 immunity^74-77, it was tested if these neuropeptides have direct effects on cytotoxic CD8⁺ T cell′ expression of immune checkpoint receptors. It was first sought to test the immunomodulatory effect of KCl-stimulated (30 min) cultured DRG neuron conditioned media (CM), which contains nociceptor-produced neuropeptides. Splenocyte-isolated CD8⁺ T cells were cultured under T_C1-stimulating conditions for 48 h and then exposed to CM. In comparison to control media (with KCl), CM increased the proportion of CD8⁺ T cells expressing PD1⁺Lag3⁺ (FIG. 38A), PD1⁺ (FIG. 43B), Lag3⁺ (FIG. 43C), and decreased levels of INFγ⁺ (FIG. 38B), and TNFα⁺ (FIG. 38C) cells. CM from capsaicin-stimulated neuron displayed a similar phenotype (not shown). Next, T_C1-stimulated CD8 T cells with neurons were co-cultured to mimic their local interaction. An increased proportion of PD1⁺Lag3⁺Tim3⁺ (FIG. 38D), PD1⁺ (FIG. 43D), Lag3⁺ (FIG. 43E), Tim3⁺ (FIG. 43F), and decreased levels of INFγ⁺ (FIG. 38E), TNFα⁺ (FIG. 38F), and IL2⁺ (FIG. 43G) was noted when CD8⁺ T cells were with DRG neurons.

Based on these data, the focus was on investigating the immunomodulatory role of TRPV1⁺ nociceptors, the major NP producers, using pharmacological gain (capsaicin) and loss-of-function (TRPV1^Cre::DTA^fl/wt) approaches. Lymphocyte′ exhaustion was reduced when CD8 cells were co-cultured with DRG neurons from TRPV1^Cre::DTA^fl/wt (a mouse genetically-engineered to ablate nociceptors), while capsaicin stimulation of wildtype DRG neurons cultured with CD8 T cells had the opposite effects (FIGS. 44A-44D). Of note, capsaicin had no measurable impact in the absence of neurons. It was also found that CD8 T cells increase the expression of neuropeptide receptors Ramp1, Ramp3, and Vpac1 when co-cultured with sensory neurons (FIGS. 44E-44G), indicating a possible neuropeptide-mediated inhibitory loop between the two populations of cells.

Given the breadth of mediators (SP, CGRP, and VIP) released by nociceptors when exposed to melanoma cells (FIG. 37J), it was sought to define the immunomodulatory effects of the individual peptides. In the presence of a peptidase inhibitor, individual neuropeptides (CGRP, VIP, SST) enhanced the proportion of PD1⁺Lag3⁺Tim3⁺ (FIG. 38G), INFγ⁺ (FIG. 38H), PD1⁺ (FIG. 44H), Lag3⁺ (FIG. 44I), Tim3⁺ (FIG. 44J) and IL2⁺ CD8 T cells (FIG. 44K) while it had no impact on TNFα⁺ expression (FIG. 38I). Overall, the various immunomodulatory effects of the neuron CM, or co-culture, to one NP could not ascribed, it was found that combining neuropeptides prompted a decreased proportion of INFγ⁺ CD8 T cells (FIG. 44L). Taken together, these data support a role for nociceptor-released NP as modulators of CD8 T cell exhaustion.

Then in order to test whether neuropeptides released from sensory neuron impact CD8 T cells anti-tumor responses, DRG neurons were co-cultured with B16F10-OVA cancer cells, and OT1 cytotoxic T cells. OT1 cells were harvested from naïve mouse splenocyte CD8 T cells primed under T_C1-stimulating conditions for 48 h. As expected, the presence of OT1 cytotoxic T cells drastically increased AnnexinV⁺7AAD⁺ B16F10-OVA cells mediated apoptosis in the cancer cells (FIGS. 38J-38K). It was also observed a decrease in tumor apoptosis when B16F10-OVA and OT1 cytotoxic T cells were stimulated with CM from neurons (induced by capsaicin; FIGS. 38J, 45A). Apoptosis was similarly induced in the cancer cells by co-culture with nociceptors (FIGS. 38K, 45D) or after NP stimulation (FIGS. 38L, 45G). These phenotypes correlate with an enhanced ratio of PD1⁺ and Lag3⁺ cytotoxic T cells (FIGS. 45B, 45C, 45E, 45F). These effects were confirmed using live-cell imaging, which showed that nociceptors (TRPV1^Cre::QuASR2-eGFP^fl/wt) enhanced B16F10-OVA-mCherry survival by diminishing the activity of OT1 cytotoxic T cells (FIGS. 38M-380).

Given that secreted neuropeptides influence the activation of immune cells^26,74,75,77, antigen trafficking to skin draining lymph node and drive the expression of immune checkpoint receptors on cytotoxic CD8⁺ T cells (FIGS. 38A-380)^78-83, it was tested whether these neurons modulate the progression of B16F10 tumors in vivo. B16F10 (i.d. 1×10⁵) cells were inoculated in 8-week-old male and female nociceptor ablated (TRPV1^Cre::DTA^fl/wt) or intact mice. In both male and female, tumor growth, volume, and weight were reduced in mice whose nociceptor were ablated and overall survival was enhanced (FIGS. 39A-39D; 47J). While NKT cells were not impacted (FIG. 47G), the numbers of tumor-infiltrating CD8⁺ (FIG. 39E), CD4⁺ (FIG. 46A) and NK (FIGS. 47A-47B) cells were enhanced in sensory neuron ablated mice, and MDSCs were reduced (not shown).

The genetic ablation of nociceptors preserved the cytotoxic potential of CD8 T cells (increased number of Granzyme B⁺, INFγ⁺, TNFα⁺, INFγ⁺TNFα⁺), while preventing their exhaustion (reduced ratio of PD1⁺Lag3⁺Tim3⁺, PD1⁺Lag3⁺, PD1⁺, Tim3⁺, Lag3⁺) (FIG. 39F-39G, FIG. 46F-46I). TRPV1^Cre::DTA^fl/wt also preserved CD4+(FIG. 46B-46E), NK (FIGS. 47C-47F) and NKT (FIGS. 47H-47I) cells activation. An absence of sensory neurons had a similar impact on number and activation of lymphocytes within the tumor-draining lymph node (FIG. 46J-46Q). Next, it was probed whether the decrease tumor growth in the TRPV1^Cre::DTA^fl/wt mice depends on lymphocytes. To test this, a monoclonal antibody was used to systemically deplete CD3⁺ T cells and found after this treatment there was no difference in B16F10 tumor growth and volume between TRPV1^Cre::DTA^fl/wt and littermate control mice (FIGS. 39H-39J), implying an action of nociceptors on lymphocytes.

Given that nociceptor neurons drive CD8⁺ T cells exhaustion and that melanoma is highly responsive to immune checkpoint blockers¹⁶, an experiment was devised using a B16F10-OVA cancer cell line to test whether genetic ablation of nociceptor sensory neurons increases tumor elimination upon PDL1 blockade. After tumor-establishment (≥1 cm³ tumor), TRPV1^Cre::DTA^fl/wt and littermate control mice were treated (on day 7, 10 and 13) with a rat anti-mouse PDL1 (6 mg/kg; clone 29F.1A12; i.p.), and effects assessed on day 14. PDL1 blockade in naïve mice reduced tumor growth and size and increased infiltration of tumor-specific cytotoxic T cells, effects that were enhanced in TRPV1^Cre::DTA^fl/wt mice (FIGS. 40A-40C). Sensory neuron ablated mice have reduced tumor growth (FIG. 40A), and volume (FIG. 47K) which is coupled with increased infiltration of total (FIG. 40B) and H2 KB-OVA⁺ (FIG. 40C) CD8 T cells.

While not supported by the Immgen database⁸⁴, immune cells have been reported to express TRPV1⁸⁵. As such, it was decided to confirm the findings in Na_V1.8^Cre mice, since this sodium channel is expressed by mechano- and thermos-sensitive neurons and represents 80% of nociceptors^86,87 but not in immune cells. Sensory neuron ablation using the Na_V1.8^Cre::DTA^fl/wt line also slowed B16F10 tumor growth and reduced the proportion of PD1⁺Lag3⁺ CD8⁺ T cells within the tumor, while it increases infiltration of total CD8 T cells (FIGS. 40D-40F). Next, these data were confirmed using a nociceptor gain-of-function approach by stimulating (transdermal illumination; 2 mW/mm², 1 Hz, 20 min, BID for 14 d) optogenetically-controlled tumor-innervating neurons. In comparison to light-insensitive littermate control mice, blue light-stimulated Na_V1.8^Cre::ChR2^fl/wt enhanced B16F10 tumor growth, proportion of PD1⁺Lag3⁺ CD8⁺ T cells while it increased the infiltration of total CD8 T cells (FIGS. 40D-40F).

Next, the effect of nociceptors on Braf^V600ECdkn2a^−/−Pten^−/− melanoma was probed and found reduced tumor growth in the absence of TRPV1⁺ lineage neurons. To determine whether the contribution of nociceptors to tumor growth is a general phenomenon (rather than limited to melanoma), their contribution to tumor growth was tested in an aggressive and innervated model of metastatic head and neck squamous cell carcinoma (MLM3) as well as the parental line from which it was derived (mEERL)⁸⁸. When intradermally implanted in control or TRPV1 nerve ablated mice, slower mEERL and MLM3 growth in absence of sensory neurons was found (FIG. 40G), while their overall survival increased (FIG. 40H). Finally, the impact of nociceptor ablation in a lymphoma model was tested. As opposed to the solid tumor models, ectopic EG7 lymphoma growth (FIG. 401) and spreading (not shown) were enhanced in TRPV1^Cre::DTA^fl/wt mice.

Neonatal ablation of neuronal subsets using diphtheria toxin may lead to compensatory mechanisms. To confirm the pro-tumorigenic effects of nociceptors in adult, pharmacological approaches were used to block vesicle releases from, or silence, tumor-innervating neurons.

First, the effect of BoNT/a, a neurotoxic protein produced by Clostridium Botulinum was tested. BoNT/a acts by cleaving SNAP-25⁸⁹, a component of the neuronal SNARE complex, triggering a long-lasting (20 days) blockade of neurotransmitter′ release by preventing the vesicle docking with the membrane⁸⁹. This strategy targets all neurons¹², which included skin-innervating autonomic fibers (acetylcholine, noradrenaline) as well as nociceptors (CGRP, SP), and was successfully used to block neuro-immune interplay in skin infection⁷⁶. Thus, BoNT/a was injected (25 pg/μl; 50 ul; 5 injection point) 1 and 3 day prior, or 1 and 3 days after, B16F10 cells inoculation. In both case, BoNT/a reduced tumor growth (FIG. 41A), volume (FIG. 48B), and weight (FIG. 48C) when compared to vehicle-injected skin. BoNT/a treatment after tumor implantation increased CD8 infiltration (FIG. 41B;FIG. 48E), and preserved their cytotoxic potential (increased number of Granzyme B⁺; FIGS. 48D, 48G), and prevented their exhaustion (reduced ratio of PD1⁺Lag3⁺Tim3⁺; FIG. 41C, 48H).

Next, an efficient pain^26,90,91 and itch⁹² nociceptor-blocking strategy was modified to silence tumor-innervating nociceptors. The protocol uses non-selective large pore ion channels (TRPA1 and TRPV1) as cell-specific drug-entry ports that deliver a charged and membrane-impermeable form of lidocaine (QX-314) into sensory fibers to block sodium currents. During inflammation, as found in tumor micro-environments, these ion channels on the surface of nociceptors open, allowing the small-sized (263 Da) QX-314 to permeate into these neurons resulting in a long-lasting electrical blockade (21457220). Here, it was tested whether skin surrounding tumor-injected with QX-314 amplifies the host anti-tumor activity. It was found that QX-314 mediated sensory neuron silencing (100 μM) reduces tumor growth (FIG. 41E), volume (FIG. 48J) and weight (FIG. 48K) as compared to vehicle-injection. As opposed to BoNT/a, QX-314 enhances the infiltration of CD8+(FIG. 41F) and CD4+(FIG. 48N) T cells, preserved their cytotoxic potential (increased number of INFγ⁺, TNFα⁺, Granzyme B⁺; FIG. 41H, FIGS. 48L, 48M, 480, 48Q), and prevented their exhaustion (decrease ratio of PD1⁺Lag3⁺Tim3⁺; FIG. 41G, FIG. 48P).

QX-314 (0.1-1%) and BoNT/a (1.6-50 pg/ml) failed to impact B16F10 survival (FIGS. 48A, 48I) or lymphocyte′ function (not shown) when applied directly in cell culture. Intradermal inoculation of B16F10 to the mouse hindpaw led to tumor growth (FIG. 49A), occasional (˜30%) itch (FIG. 49B), mechanical (FIG. 49C) and thermal pain-related hypersensitivity (FIG. 49D). These effects were absent in mice whose sensory neuron are genetically ablated (FIG. 49D) or pharmacologically silenced with QX-314 (100 μM, qd, i.d., FIG. 49A-49D) or treated with BoNT/a (25 pg/μl, FIGS. 49A-49C). In a bath-organ culture of tumor-inoculated skin, it was found that capsaicin-induced CGRP release was absent in samples pre-treated with QX-314 or BoNT/a (FIG. 49E). These data support the capacity of QX-314 or BoNT/a to block neuropeptide release from tumor-innervating neurons, preventing their subsequent immunomodulation of TILs. Finally, it was tested whether silencing tumor-innervating neurons would modulate the response to PDL1 blockade and found that QX-314 or BoNT/a enhanced αPDL1-mediated tumor shrinkage (FIGS. 41I-41L).

CGRP administration increased CD8 T cell exhaustion and reduced their production of cytotoxic granules when co-cultured with melanoma while TILs expressed higher levels of CGRP receptor RAMP1 and RAMP3 when co-cultured with sensory neurons (FIGS. 38A-380, FIGS. 43A-43G, FIGS. 44A-44L, FIGS. 45A-45G). Given that the selective CGRP receptor antagonist BIBN blocks deleterious neuro-immune interplay during microbe infection and rescues host anti-bacterial activity⁷⁴, studies were carried out to test its effect within the tumor microenvironment. Similar to QX-314, CGRP blockade with BIBN reduced tumor growth (FIG. 41M), volume (FIG. 48R), and weight (FIG. 48S). While it did not impact CD8⁺ infiltration (FIG. 41N), BIBN preserved these cells cytotoxic potential (increasing relative infiltration of INFγ⁺, TNFα⁺, Granzyme B⁺; FIG. 41P, FIGS. 48T-48U), and prevented their exhaustion (decrease ratio of PD1⁺; FIG. 41O). BIBN also enhanced the influx of total and cytotoxic CD4 T cells, and stopped their exhaustion (FIGS. 48V-48Z).

In contrast to tumor neo-angiogenesis, the impact of cancer neo-innervation on clinical prognosis remains unclear. While recent data highlight the importance of parasympathetic and sympathetic neurons on tumor growth^{12,14,15,57,58,93-97}, the role for nociceptor neurons in skin and other cancers remain unclear, even though the skin is densely innervated with nociceptor^11,15,94.

Given that nociceptor-produced neuropeptides alter immune responses⁹⁸, shutting down T_H1 immunity against bacteria 75 and fungi 34 the nature of cancer-nociceptor-CD8⁺ interplay was probed using a xenograft mouse model of triple-negative melanoma skin cancer, a robust and established model of immune surveillance^16,21,23 It was found that nociceptor neurons are activated by cancer cells, responding with local release of neuropeptides from their peripheral terminal, which, then through an action on cytotoxic T lymphocytes leads to a failure of immune surveillance and tumor growth. Overall, by expressing ligand for¹⁰, and controlling the expression of, immune checkpoint receptors, sensory neurons emerge as a driver of regulatory immunity. Since silencing tumor-innervating neurons rescues the host anti-tumor immunity and improves response to checkpoint blockade, this may constitute an adjuvant therapy for the treatment of skin cancers.

The discovery of axonal guidance molecules demonstrated that nerves are directed to their targets by attractive and repulsive cues^99,100 Similar signals help blood vessels reach their targets^101-103 Such parallelism emerged between the actions of growth factors that direct angiogenic sprouting and those that regulate axon terminal arborisation^104,105. Here, it was found that melanoma, both in vitro and in vivo, drives axonogenesis in nociceptor neurons, which might be the driver for the innervation of these tumors. It likely involves melanoma-released growth factors¹⁰⁶, exosome or other modulators that act on the neurons. Thus, given the high exosome cargo produced by melanoma¹⁰⁷ and its negative clinical impact¹⁰⁸ as well as response to checkpoint blockers¹⁰⁹, may also contribute to melanoma hyper-innervation.

Clinically, vagal denervation increases the risk of some cancer^6,124,125 while, higher densities of adrenergic and cholinergic nerve fibers correlated with poor clinical outcome¹⁴. Human breast, melanoma, and prostate cancer patients taking β-blockers have lower recurrence and mortality^122,126,127. Breast cancer biopsies showed increased sympathetic and decreased parasympathetic nerve density¹¹⁷, while prostate cancers are infiltrated with cholinergic fibers and surrounded by adrenergic fibers¹⁴. Consistent with adrenergic signaling as cancer-promoting, norepinephrine upregulates VEGF, IL-6, and IL-8, in turn, increasing melanoma aggressiveness¹²⁸.

Cancer cells produced mediators may sensitize nociceptors by increasing the expression and lowering the activation threshold of ion channel transducer (Julius, 2013). Here, it was found that hindpaw inoculation of B16F10 cells led to mechanical allodynia and thermal hyperalgesia, while flank injection led to spontaneous itching (in thirty percent of the tested animals). Both the pain hypersensitivity and pruritus were relieved by sensory neuron silencing (QX-314) or by blocking neuropeptide release (BoNT/a) and absent in mice whose nociceptor neurons are genetically ablated (TRPV1^creDTA^fl/wt). Sensitizing effects of B16F10 cells was observed in vitro when DRG nociceptor neurons were co-cultured with melanoma, as well as in neurons harvested from tumor-inoculated mice. Such drivers may include melanoma produced TSLP, IL-1β, INFγ, all known nociceptor sensitizers in other contexts²⁵, or melanoma-produced growth factors such as platelet-derived growth factor or transforming growth factor¹⁰⁶ sensitize nociceptors in the context of painful neuropathy¹¹⁰. Finally, the alarmin HMGB1 which is expressed by melanoma¹¹¹ and correlate with disease severity¹¹² can also initiate pain¹¹³.

Lumbar nociceptor neurons express PD-1, whose activation by PD-L1 blocks pain hypersensitivity in melanoma-bearing mice¹⁰. Such data contrast with the clinical neuropathies experienced by melanoma patients and with experimental findings provided herein. The absence of T cells or other PD-L1 bearing cells in this in vitro co-culture system could explain such discrepancy. However, it is unclear why PD-L1 expressing TIL do not readily silence tumor-innervating neurons in vivo. Given that TILs are drawn to fight the cancer cells it may limit their crosstalk with neurons to secreted signals.

Noxious stimuli activate membrane transducers in the peripheral terminals of nociceptors initiate reflexes, sensations, and local release of neuropeptides²⁵. Here, it was found that the B16F10-nociceptor co-culture lead to the release of SP, VIP, and CGRP. Such neuropeptides may have multiple roles; driving i) tumor progression, ii) secretion of tumor-associated cytokines, iii) neo-vascularization, iv) metastasis or v) controlling immune responses^{3,7-9,11,12,114-116} Neuropeptides promote antigen trafficking in the LN and the chemotaxis and polarization of lymphocytes; influencing the localization, extent, and type of inflammation (Cunin et al., 2011; Ganea and Delgado, 2001; Goetzl et al., 2001; Nussbaum et al., 2013; Talbot et al., 2015). Here, it was found that nociceptor-produced neuropeptides induced single (PD1⁺), double (PD1⁺Lag3⁺) and triple (PD1⁺Lag3⁺Tim3⁺) expression of immune checkpoint receptors on cytotoxic CD8 T cells. It was also found that CD8 T cells exposed to sensory neurons upregulate the expression of CGRP receptor RAMP1 and RAMP3 as well as the VIP receptor VPAC1. CGRP decreases the proliferation (IL-2⁺), and cytotoxic capacity (TNFα⁺, INFγ⁺, Granzyme B⁺) of these cytotoxic CD8 T cells, while local treatment with BIBN, a RAMP1 blocker, prevents cytotoxic CD8 T cells exhaustion and rescues anti-tumor immunity.

The elimination of B16F10-OVA cancer cells by OT1 cytotoxic CD8 T cells decreases when they are co-cultured with nociceptors or exposed to nociceptor-produced neuropeptides. Such as in the case of pancreatic ductal cancer¹¹, B16F10 cancer cell growth slowed down in nociceptor ablated mice. Furthermore, these tumors now have a higher content of cytotoxic CD8 T cells, which bear less immune checkpoint receptor expression and have a lower content of cytotoxic granules. This decrease in tumor growth was prevented by systemic αCD3 depletion, confirming a nociceptor-T cell-mediated effect. These are the first to show that nociceptor neuropeptide (CGRP) drives immune checkpoint receptor expression on TILs.

Tumor-specific sympathetic denervation downregulates the expression of PDL1, PD1 as well as FOXP3 while parasympathetic innervation decreased TIL expression of PD-1 and PD-L1¹¹⁷. The authors also discovered that exhaustion of TILs correlates with their distance from sympathetic nerves¹¹⁷. It is unclear how sympathetic nerves drive these effects, and whether there is a contribution of sensory neurons, but the data suggests that nociceptors help control the expression of immune checkpoint receptors, and promote regulatory immunity within the tumor microenvironment.

At neuro-neoplastic contact, cancer cells produce mediators (e.g. alarmin, TSLP or IFNγ, growth factors, or sEVs) that may prompt nociceptor axonogenesis increasing innervation of the tumor, sensitization which will produce pain or itch and neuropeptide release which alters the immune system. Does this constitute a host warning response to danger or more plausibly a maladaptive maladaptive neuro-immune crosstalk similar to that occurring in diseased contexts²⁵, but which here contributes to tumor growth? Ablation of skin neurons enhanced the local immune infiltration, in a manner similar to microbes which hijack the nociceptor interplay with the immune systems to modify regulatory-mediated immunity^74,77 and facilitates the survival of the cancer cells. Supporting this, it was found that while DRG neurons do not upregulate the proliferation of melanoma cells in vitro, their presence increased tumor growth by enhancing the migration of myeloid-derived suppressor cells supporting the onset of an immunosuppressive and pro-tumorigenic microenvironment¹¹⁵.

The neuropeptides expressed by and released from nociceptors also depend on the type of bacteria activating them, indicating that different microbes tweak nociceptor activity in a way that promotes their survival⁷⁷. It is conceivable that different cancer types may act similarly, either blocking or increasing neuropeptide release from different sensory neurons. This could explain why tumor denervation increases breast cancer growth¹¹⁷ while it suppresses prostate, melanoma or ovarian cancer development^14,52,58,94.

Despite tremendous strides, the success of immune therapy in cancer is limited by its variability amongst patients and cancer types, and the adverse effects it produces¹⁶. Here, it was found that targeting nociceptors with QX-314 or BoNT/A increased the response to immune checkpoint ligands, and may, therefore, be a potential adjuvant therapy to PD-L1 and other blockers.

QX-314 and BoNT/A abolished the local neuro-immune interplay^26,76, and by decreasing immune checkpoint receptor expression, safeguard host anti-tumor immune activity resulting in a decrease in tumor growth and increased survival. Silencing tumor-innervating sensory neurons is, therefore, an innovative strategy to attenuate the immunomodulatory power of the nervous system to promote anti-tumor activity. Local administration of a CGRP receptor blocker⁷⁶ also prevents TIL exhaustion and may be worth exploring as adjuvant therapy for tumor types responsive to checkpoint blockade and where tumor innervation by nociceptors is a feature.

QX-314 also reversed melanoma-induced pain and itch and its activity support the presence of a sufficient levels of inflammation in the tumor micro-environment to allow its uptake into tumor-innervating nociceptors through large pore ion channels. This symptom suppression and immune surveillance enhancing strategy offers three potential advantages: (1) high specificity (the effect is limited to sensory neurons that express activated large pore channels), (2) long-lasting activity, and (3) limited side-effects, the charge on QX-314 would limit diffusion through lipid membranes and redistribution²⁶.

In summary, the data supports a regulatory role for nociceptor over immune response to tumor growth through the regulation of the expression of immune checkpoint receptors on cytotoxic CD8⁺ T cells. Silencing cancer-innervating nociceptors or blocking the release or action of neuropeptides prevents cytotoxic T cell exhaustion and represents an innovative strategy to safeguard host anti-tumor immune responses.

Methods for Example 2

Experimental Procedures

The Institutional Animal Care and Use Committees of Boston Children's Hospital, and the Université de Montréal (CDEA #19027; #19028) approved animal procedures. Either of MPEK-BL6 (non-tumorigenic keratinocytes), B16F10¹³³ Braf^V600ECdkn2a^−/− Pten^{−/− 134}, mEERL¹³⁵ or MLM3¹³⁵ cancer cells were implanted (10⁵ cells; i.d., left flank) to 8-weeks old male and female wildtype, littermate control or to genetically-engineered mice whose sensory neurons are fluorescent, ablated or controlled by light. Tumor growth and survival was monitored daily, and animals were euthanized when the tumor reached 1 cm³. Tumors and sdLN were harvested, and TILs number and innervation were respectively analyzed by flow cytometry or immunofluorescence. QX-314 (100 μM, 100 μL, i.d)⁹² and BIBN (5 mg/kg, 100 μL, i.d)⁷⁶ were administered once daily after tumors become visible (˜day 5), while BoNT/a was administered (25 pg/μl, 100 μL, i.d)⁷⁶ prior to or right after tumor inoculation.

Statistics

Data expressed as mean±S.E.M. Statistical significance determined by one-way or two-way ANOVA for multiple comparisons and two-tail unpaired Student's t-test for single variable comparison. P values less than 0.05 were considered significant. Numbers of animals are defined in figure legends.

Animals

Mice were housed in standard environmental conditions (12 h light/dark cycle; 23° C.; food and water ad libitum) at facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. 8-week old C57BL6 (Jax, #000664); BALB/c (Jax, #001026), OT1 (Jax, #003831), TRPV1^cre (Jax, #017769), ChR2^fl/fl(Jax, #012567), td-tomato^fl/fl (Jax, #007908), DTA^fl/fl (Jax, #009669),^{136 136 13 135 136 138 138 142 142 142 142 142 142 142 142 142 141 140 141 141} (Voehringer et al., 2008) GCaMP6^fl/fl (Jax, #024105) mice were purchased from Jackson Laboratory. Na_V1.8^cre mice were generously supplied by Professor Rohini Kuner (Heidelberg University). The cre/lox toolbox was used to genetically-engineered the various mice lines used (TRPV1^cre::DTA^fl/wt, TRPV1^cre::GCaMP6^fl/wt, TRPV1^cre::Tdtomato^fl/wt, NaV1.8^cre::DTA^fl/wt, NaV1.8^cre::ChR2^fl/wt and littermate control) by crossing male heterozygote Cre mice to female homozygous loxP mice. All Cre driver lines used are viable and fertile and abnormal phenotypes were not detected. Offspring were tail clipped; tissue was used to assess the presence of transgene by standard PCR, as described by Jackson Laboratory. Offspring were used at 8 weeks of age.

Cell Line

B16F0 (ATCC), B16-F10 (ATCC), B16F10-OVA (ATCC), B16F10-mCherry2 (ATCC), mEERL (ATCC), MLM3 (ATCC), EG7 (ATCC) were cultured in complete Dulbecco's Modified Eagle's Medium high glucose (DMEM, Gibco) supplemented with 10% fetal bovine serum (Biochrom) and 1% penicillin/steptomycin (Gibco), and maintained at 37° C. in a humidified incubator with 5% CO₂.

Cancer Inoculation

Cancer cells (1×10⁵) were resuspended in PBS and injected (i.d., 100 l) to the mice right flank. Growth was daily assessed using a handheld caliper. Mice were euthanized when cancer reach 1000-1500 mm³, tumors and tumor draining lymph node (tdLN) harvested and immunophenotype by flow cytometry.

Tumor Immonophenotyping

Tumor were enzymatically digested (DMEM+2 mg/ml collagenase D (Sigma)+0.03 mg/ml DNAse I (Sigma) under constant shaking (30 min, 37° C.). The cell suspension was then strained (70 um), washed and RBC lysed (Life Technologies, ACK lysis buffer, 2 min). Single cells were then resuspended in FACS buffer (PBS, 2% FCS, EDTA), Fc blocked (0.5 mg/ml, 10 min; BD Biosciences) and stained (45 min, 4° C.) with monoclonal antibodies (anti-CD45-BV421 (1:100, Biolegend), anti-CD11b-APC-cy7 (1:100, Biolegend), anti-CD8-PercP (1:100, Biolegend), anti-CD4-FITC (1:100, Biolegend), anti-PD-1-PE-cy7 (1:100, Biolegend), anti-Lag3-PE (1:100, Biolegend), anti-Tim-3-APC (1:100, Biolegend).

Intracellular Cytokine Staining

Cells were stimulated (4 h) with Brefeldin A (Biolegend, #423304), washed, fixed/permeabilized (BD Biosciences; #554714) and stained (anti-IFN-γ FITC (Biolegend), anti-TNF-α PE (Biolegend), anti-Granzyme B APC (Biolegend, #504118).

Drugs

QX-314 (Tocris, #2313; 100 μM) was injected (i.d.) daily in 5 points around the tumor (treatment began once tumor was visible). BIBN4096 (Tocris, #4561; 5 mg/kg) was injected (i.d.) on day 6, 8, 10, 12 and 14. Botulinum neurotoxin A (List biological labs, #130B; 25 pg/μl) was injected (i.d.) three and one day prior to, or one and three days after, tumor inoculation. In either of vehicle, QX-314, BoNT/a, littermate control or TRPV1^cre::DTA^fl/wt mice, αPD-L1 (Bioxcell, 6 mg/kg) was injected (i.p.) on day 5, 8 and 11.

Skin Explant

1 h post exposure to vehicle (100 μL), QX-314 (100 μM, 100 μL) or BoNT/a (25 pg/μL, 50 L), tumor-surrounding skin was harvested using 10 mm punch biopsies and transferred to a 24-well plates (DMEM+protease inhibitor (1 ul/ml). After 30 min incubation (37° C.), the supernatant was collected and CGRP analyzed by commercial ELISA (Cayman Chemical).

Neuron Culture

Mice were sacrificed and dorsal root ganglia (DRG) were dissected out into DMEM medium (Corning, #10-013-CV), completed with 50 U/mL penicillin and 50 μg/mL streptomycin (Fisher, #MT-3001-C1), and 10% FBS (Seradigm, #3100). Cells were then dissociated in HEPES buffered saline (Sigma) completed with 1 mg/mL collagenase IV (Sigma, #C0130)+2.4 U/mL dispase II (Sigma, #04942078001) and incubated for 80 minutes at 37° C. Ganglia were triturated with glass Pasteur pipettes of decreasing size in supplemented DMEM medium, then centrifuged over a 10% BSA gradient, plated on Laminin (Sigma, #L2020) coated cell culture dishes. Cells were cultured with Neurobasal-A medium (Gibco, #21103-049) completed with 0.05 ng/μL NGF (Life Technologies, #13257-019), 0.002 ng/μL GDNF (Peprotech, #450-51-10), 0.01 mM AraC (Sigma, #C6645) and 200 nM L-Glutamin (VWR, #02-0131).

CD8 Isolation

Adult mice (8-10 weeks-old) were euthanized, spleen harvested (ice-cold PBS, 5% FBS), and mechanically dissociated. The cells were strained (70-μm), RBC lysed (ACK lysing buffer) and counted using a hemocytometer. Naïve CD8⁺ T cells were magnet sorted (Stem cell, #19853A) or FACS sorted and cultured (DMEM+FBS 10%, Pen/Stp, non-essential amino acid, vitamin, β-mercaptoethanol, L-glutamine, and sodium pyruvate). To generate cytotoxic T lymphocytes, 1×106 naïve CD8⁺ T cells will be seeded and stimulated for 48 h under TC1 inflammatory condition (2 μg/ml αCD3/αCD28 (Bioxcell)+10 ng/ml rIL-12 (Biolegend)+10 μg/ml of anti-IL-4 (Bioxcell).

Co-Culture

Naïve DRG neurons (1×10⁴) were seeded in T cell media (supplemented with 0.05 ng/μL NGF (Life Technologies, #13257-019), 0.002 ng/μL GDNF (Peprotech, #450-51-10), IL-2 and co-cultured with Tc1 CD8 T cells (1×10⁵). After 48 h co-culture, Tc1 cells were collected by centrifugation (5 min at 1300 rpm), stained and immunophenotyped by flow cytometry.

Apoptosis

Naïve TRPV1^Cre::Tdtomato^fl/wt DRG neurons (1×10⁴) were co-cultured (24-72 h) with OT1 T_C1 CD8 T cells (1×10⁵) and B16F10-mCherry2-OVA (1×10⁵). in T cell media (supplemented with 0.05 ng/μL NGF (Life Technologies, #13257-019), 0.002 ng/μL GDNF (Peprotech, #450-51-10), IL-2 After 48 h co-culture, Tc1 cells were collected by centrifugation (5 min at 1300 rpm), stained and immunophenotyped by flow cytometry. Anti-annexin V APC (Biolegend), 7-AAD

Immunofluorescence

2×10³ DRG neurons were co-cultured with 2×10⁴ B16F10-mCherry2 for 24-48 h. The cells were then fixed (4% paraformaldehyde; 30 min), permeabilized (0.1% Triton X-100, 20 min), and blocked (PBS, 0.1% Triton X-100, 5% BSA, 30 min). The cells were rinsed (PBS), and stained with mounted with Vectashield (Vector Laboratories, #H-1000) containing DAPI (Tocris, #5748). Images were acquired using a Ti2 Nikon fluorescent microscope.

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INCORPORATION BY REFERENCE

The present application refers to various issued patents, published patent applications, scientific journal articles, and other publications, all of which are incorporated herein by reference. The details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the Detailed Description, the Figures, the Examples, and the Claims.

EQUIVALENTS AND SCOPE

In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

Claims

1. A method of treating cancer in a subject, the method comprising silencing tumor-innervating sensory neurons.

2. A method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a neuropeptide modulating agent.

3. A method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that blocks the release or action of a neuropeptide from tumor-innervating neurons.

4. A method of treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a nociceptor modulating agent.

5. The method of claim 4, wherein the nociceptor modulating agent is a nociceptor antagonist.

6. The method of claim 5, wherein the nociceptor antagonist is a sodium channel blocker, calcium channel blocker, or sodium and calcium channel blocker.

7. The method of claim 6, wherein the sodium channel is NaV1.8.

8. The method of any one of claims 2-7, wherein the neuropeptide modulating agent, agent that blocks the release or action of a neuropeptide, nociceptor modulating agent, or nociceptor antagonist is a compound comprising a quaternary amine.

9. The method of any one of claims 2-8, wherein the neuropeptide modulating agent, agent that blocks the release or action of a neuropeptide, nociceptor modulating agent, or nociceptor antagonist is QX-314:

10. The method of any one of claims 2-8, wherein the neuropeptide modulating agent, agent that blocks the release or action of a neuropeptide, nociceptor modulating agent, or nociceptor antagonist is a compound selected from the group consisting of:

11. The method of any one of claims 2-8, wherein the neuropeptide modulating agent, agent that blocks the release or action of a neuropeptide, nociceptor modulating agent, or nociceptor antagonist is a quaternary amine derivative or other permanently charged derivative of a compound selected from riluzole, mexilitine, phenytoin, carbamazepine, procaine, articaine, bupivicaine, mepivicaine, tocainide, prilocaine, diisopyramide, bencyclane, quinidine, bretylium, lifarizine, lamotrigine, flunarizine, and fluspirilene.

12. A method of treating cancer in a subject, the method comprising administrating to the subject a therapeutically effective amount of an agent that blocks vesicle release from tumor-innervating nociceptors.

13. The method of any one of claims 2-7 and 12, wherein the neuropeptide modulating agent, agent that blocks the release or action of a neuropeptide, an agent that blocks vesicle release, nociceptor modulating agent, or nociceptor antagonist is a neurotoxic protein.

14. The method of claim 13, wherein the neurotoxic protein is a botulinum neurotoxin or tetanus toxin (TeNT).

15. The method of claim 14, wherein the botulinum neurotoxin is BoNT/a.

16. A method of treating cancer in a subject, the method comprising administering to a subject a therapeutically effective amount of a calcitonin gene-related peptide (CGRP) modulating agent.

17. The method of any one of the preceding claims, wherein the CGRP modulating agent is a CGRP receptor antagonist.

18. The method of claim 17, wherein the CGRP receptor antagonist is a RAMP1, RAMP3, or Vpac1 blocker.

19. The method of claim 17 or 18, wherein the CGRP receptor antagonist is a RAMP1 blocker.

20. The method of any one of claims 17-19, wherein the CGRP receptor antagonist is erenumab, fremanezumab, fremanezumab, eptinezumab, ubrogepant, or rimegepant.

21. The method of any one of claims 17-19, wherein the CGRP receptor antagonist is BIBN 4096.

22. A method of treating cancer in a subject, the method comprising ablating an ion channel in a subject, wherein the ion channel is a sodium ion channel or TRPV ion channel.

23. The method of claim 22, wherein the sodium ion channel is NaV1.8.

24. The method of claim 22, wherein the TRPV ion channel is TRPV1.

25. The method of any one of claims 22-24, wherein the ion channel is genetically ablated.

26. The method of any one of claims 1-25, wherein the cancer is skin cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, gastric cancer, or a tumor.

27. The method of any one of claims 1-26, wherein the cancer is melanoma.

28. The method of any one of claims 1-27, wherein the cancer is a tumor.

29. The method of any one of claims 1-28, wherein the method decreases tumor growth, volume, and/or size.

30. The method of any one of claims 1-29, wherein the method inhibits or decreases cancer cell proliferation.

31. The method of any one of claims 1-30, wherein the method increases subject survival.

32. The method of any one of claims 1-31, wherein the method promotes anti-tumor activity.

33. The method of any one of claims 1-32, wherein the method increases lymphocyte numbers.

34. The method of any one of claims 1-33, the method improves response to chemotherapeutics.

35. The method of any one of claims 1-34, wherein the method decreases tumor comorbidities.

36. The method of claim 35, wherein the comorbidity is pain or itch.

37. The method of any one of claims 1-36 further comprising administering to the subject an additional therapy.

38. The method of claim 37, wherein the additional therapy is chemotherapy, radioimmunotherapy, surgical therapy, immunotherapy, radiation therapy, or targeted therapy, or any combination thereof.

39. The method of claim 38, wherein the method increases efficacy of the immunotherapy.

40. The method of any one of claims 1-39, where the method leads to exhaustion of tumor-infiltrating lymphocytes.

41. A method of treating cancer in a subject, the method comprising administering to a subject a therapeutically effective amount of (i) an anti-cancer agent and (ii) QX-314, BoNT/a, or BIBN 4096.

42. The method of claim 41, wherein the anti-cancer agent is a biotherapeutic anti-cancer agent or a chemotherapeutic agent.

43. The method of claim 41 or 42, wherein the anti-cancer agent or chemotherapeutic agent is dacarbazine or cisplatin.

44. A composition comprising (i) an anti-cancer agent, (ii) a nociceptor modulating agent, nociceptor antagonist, neuropeptide modulating agent, an agent that blocks vesicle release, or agent that blocks the release or action of a neuropeptide from tumor-innervating nociceptor described herein, and (iii) optionally a pharmaceutically acceptable excipient.

45. The composition of claim 44, wherein the composition comprises (i) dacarbazine or cisplatin, (ii) QX-314, BoNT/a, or BIBN 4096, and (iii) optionally a pharmaceutically acceptable excipient.