Patent Application
20240261364
This application contains an ST.26 compliant Sequence Listing, which is submitted in xml format via Patent Center and is hereby incorporated by reference in its entirety. The xml copy, created on Mar. 15, 2024, is named 138536-8001US4 Replacement Sequence Listing.xml and is 64,000 bytes in size.
Serine protease inhibitors (SERPINs) are a large family of proteins that are involved in diverse biological functions such as fibrinolysis, blood coagulation and inflammation. When SERPINs bind to their target serine proteases to inactivate the enzymatic activity, a conformational change occurs exposing a unique short peptide motif (5-11 amino acids).8,43 The protease-inhibitor complex binds to low-density lipoprotein receptor related protein (LRP1) at the newly exposed short peptide motif, a process which is conserved across the entire spectrum of serine protease inhibitors (SERPINs) such as alpha-1 antitrypsin (AAT) and antithrombin III (ATIII) (May 2013).22,25,43 Therefore, there is a need to develop novel SERPIN peptides and explore their prophylactic and therapeutic effects in various conditions and diseases.
In one aspect, this disclosure relates to various SERPIN peptides. In some embodiments, the SERPIN peptide comprises, consists essentially of, or consists of an amino acid sequence of X1-Z1-X2-Z2-X3-Z3-F-V-F-L-X4-Z4 (SEQ ID NO. 20), wherein X1 is V or L; X2 is R or F; X3 is R or K; X4 is M, Nle, or I; Z1 is any amino acid; Z2 is any amino acid; Z3 is any amino acid, and Z4 is a sequence of any five amino acids. In some embodiments, the SERPIN peptide comprises, consists essentially of, or consists of an amino acid sequence of X1-Z1-X2-Z2-X3-Z3-F-X4-F-L-Z4-X5 (SEQ ID NO. 21), wherein X1 is V or L; X2 is F or R; X3 is K or R; X4 is V, L, or M; X5 is a sequence any five amino acids; Z1 is any amino acid; Z2 is a sequence of any two amino acids; Z3 is any amino acid; and Z4 is M Nle, or I. In some embodiments, the SERPIN peptide comprises, consists essentially of, or consists of an amino acid sequence of X1-N-X2-P-F-X3-X4-X5-X6 (SEQ ID NO. 4), wherein X1 is R or F, X2 is Kor R, X3 is V or L, X4 is F, V or M, X5 is L, V or I, and X6 is M, I, or Nle. In some embodiments, the SERPIN peptide comprises, consists essentially of, or consists of an amino acid sequence of F-X3-X4-X5-X6 (SEQ ID NO. 15), wherein X3 is V or L, X4 is F, Vor M, X5 is L, Vor I, and X6 is M, I, or Nle. In some embodiments, the SERPIN peptide comprises, consists essentially of, or consists of an amino acid sequence of Z1-RFNRPFLVVIR-Z2 (SEQ ID NO. 17), Z1-RFNRPFLMIIR-Z2 (SEQ ID NO. 18), or Z1-RFNKPFVFL(NIe)R-Z2 (SEQ ID NO. 37), wherein Z1 and Z2 are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or between 1 and 3, between 1 and 5, between 1 and 6, between 1 and 7, between 1 and 8, between 1 and 9, or between 1 and 10 basic amino acids. In some embodiments, the SERPIN peptide comprises, consists essentially of, or consists of an amino acid sequence of VKFNKPFVFL(NIe)IEQNTK (SEQ ID NO. 35), VKFNKPFVFLM (SEQ ID NO. 38), LRFNRPFLVVI (SEQ ID NO. 39), VRFNRPFLMII (SEQ ID NO. 31), VKFNKPFVFL(Ne) (SEQ ID NO. 40), RFNRPFLVVIR (SEQ ID NO. 41), RFNRPFLMIIR (SEQ ID NO. 42), RFNKPFVFL(Nle)R (SEQ ID NO. 43), RRRFLVVIRRR (SEQ ID NO. 44), RRRFLMIIRRR (SEQ ID NO. 45), RRRFVFL(Nle)RRR (SEQ ID NO. 46), FVFLM (SEQ ID NO. 3), or FVFL(Nle) (SEQ ID NO. 10). In some embodiments, the SERPIN peptides disclosed herein has a size of between 5 and 30 amino acids, or less than 20 amino acids. In some embodiments, one or more amino acid residues of the SERPIN peptides disclosed herein are D-amino acids. In some embodiments, the SERPIN peptides disclosed herein is fused to an epitope tag, a half-life extender, or both.
In another aspect, disclosed herein is a pharmaceutical composition comprising an effective amount of a SERPIN peptide disclosed herein or a fusion thereof. In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, additive, preservative, or a combination thereof. In some embodiments, the pharmaceutical composition is formulated for oral administration, parenteral administration including subcutaneous administration, intramuscular administration, intravenous administration, and intrathecal administration, intradermal administration, transdermal administration, topical administration, intranasal administration, or inhalation.
In another aspect, disclosed herein is a method of treating a subject suffers from a disease or condition associated with TSLP. The method entails administering to the subject an effective amount of a SERPIN peptide, a fusion thereof, or a pharmaceutical composition disclosed herein. In some embodiments, the subject suffers from asthma, atopic dermatitis, allergic rhinoconjunctivitis, EoE, cancer, or rheumatoid arthritis. In some embodiment, the subject is a human. In some embodiments, the administration is by oral administration, parenteral administration including subcutaneous administration, intramuscular administration, intravenous administration, and intrathecal administration, intradermal administration, transdermal administration, topical administration, intranasal administration, or inhalation. In some embodiments, the SERPIN peptide is administered at a dose from about 0.001 mg/kg to about 4 mg/kg in human.
In another aspect, disclosed herein is a method of treating a subject suffers from a disease or condition associated with LRP1. The method entails administering to the subject an effective amount of a SERPIN peptide, a fusion thereof, or a pharmaceutical composition disclosed herein. In some embodiments, the subject suffers from peripheral neuropathy, neuropathic pain, neurodegenerative disease, sepsis, acute lung injury, acute respiratory distress, TH2 mediated allergic inflammation, or an infectious disease selected from the group consisting of COVID-19 infection, influenza, measles, alphavirus infection, rift-valley fever virus infection, dengue virus infection and Epstein Barr virus infection. In some embodiments, the subject suffers from Alzheimer's Disease or eosinophilic esophagitis. In some embodiments, the subject suffers from EoE, AMI, nerve injury, pain, gout, rheumatoid arthritis, or diabetes including type I and type II diabetes. In some embodiment, the subject is a human. In some embodiments, the administration is by oral administration, parenteral administration including subcutaneous administration, intramuscular administration, intravenous administration, and intrathecal administration, intradermal administration, transdermal administration, topical administration, intranasal administration, or inhalation. In some embodiments, the SERPIN peptide is administered at a dose from about 0.001 mg/kg to about 4 mg/kg in human.
This application contains at least one drawing executed in color. Copies of this application with color drawing(s) will be provided by the Office upon request and payment of the necessary fees.
Disclosed herein are C-terminal peptides derived from the SERPIN molecule and variants and derivatives thereof as well as their uses in prevention or treatment of various conditions by targeting LRP1. In certain embodiments, the SERPIN peptides are isolated peptides. In certain embodiments, the SERPIN peptides are synthetic peptides.
Precise coordination of the immune response is needed to promote inflammatory resolution and mitigate tissue damage, and targeting single cytokines or signaling pathways does not resolve all contributing factors in pathology of certain diseases such as those discussed here. A balanced inflammatory response plays a critical role in regeneration and repair and anti-inflammatory drugs have been associated with an opposing action on regeneration and tissue repair.3
Surprisingly, the peptides disclosed herein (1) exert neurotrophic effects, (2) have regenerative and healing properties, (3) show analgesic effects, (4) have anti-viral and anti-microbial properties, and/or (5) exert anti-allergic effects. This combination of activities provides a distinct mechanism in treating conditions associated with peripheral neuropathies such as diabetic peripheral neuropath, degenerative disorders, lung injury, allergic diseases and infectious disease. As disclosed in the examples herein, this surprising efficacy is contrasted with the merely anti-inflammatory activity of, e.g., an IL-1 antagonist. The peptides disclosed herein unexpectedly target a homeostatic and cell signaling receptor LRP1. The agonistic action of these peptides to LRP1 provides a unique mechanism beyond the anti-inflammatory effects previously described.
Accordingly, disclosed herein are SERPIN-derived peptides, pharmaceutical compositions comprising the SERPIN-derived peptides, and methods of using the same to treat a number of conditions where a dysregulated immune response or impaired endocytic function, or diseases in which LRP1 mediation could contribute to pathology, such as in conditions associated with peripheral nerve injury and resulting pain, lung injury, infectious disease and allergic inflammation. The unexpected regenerative and healing properties of these peptides allow use of the compositions comprising such peptides to new indications, and allow preventive intervention in conditions associated with, e.g., tissue injury.
Conditions Associated with LRP1
LRP1 functions as an endocytic and cell signal transduction receptor and has several ligands that induce specific cell signaling cascades that can contribute to cell survival and anti-inflammatory mechanisms.5,18,22,25 LRP1 is ubiquitously expressed on many different organs, abundantly in brain, lung, heart and immune cells. Because of these unique capabilities and wide expression on both tissues and immune cells, it plays a critical role in regulating inflammation, cellular metabolism, and maintaining homeostasis. For instance, LRP1 regulates inflammatory signaling pathways such as NFκB and JNK pathways that induce the conversion of pro-inflammatory (M1) macrophages to the anti-inflammatory (M2) macrophage phenotype, regulates the cytokine output, and contributes to effective migration and phagocytosis.22,26,51 In neutrophils, LRP1-dependent mechanisms lead to enhanced cell adhesion, chemotaxis, and antibacterial effects of these cells, thereby resisting immunosuppression25. During acute infection or injury, LRP1 also promotes inflammatory resolution through scavenging PAMPS and DAMPS from dying or injured tissue, to prevent the tissue injury cycle25. LRP1 was also shown to mediate autophagy during infection, an important metabolic process recently shown to play an important protective role in a variety of diseases. 4,10 Therefore, because of its multifunctional ability to regulate inflammation, targeting LRP1 has substantial potential to mitigate several aspects of the immune response that contributes to the pathology of several diseases including neurological disorders, infectious diseases, and allergic inflammatory disease.
In terms of nerve injury and associated pain, injury to the peripheral nervous system induces an increase in the expression of LRP1. Previously, it was demonstrated that LRP1 agonist are capable of promoting axonal growth in the CNS and are capable of inducing regeneration after spinal cord injury.53 LRP1 is an endocytic receptor to a diverse number of ligands including tissue-type plasminogen activator (tPA), matrix metalloproteinase-9 (MMP-9), and activated a2-macroglobulin.14 These ligands are capable of inducing anti-inflammatory activity,39 activating the Schwann cell repair program21 and transactivation of cell signaling pathways in neurons associated with axonal regeneration. 38 LRP1 requires ligand-binding to activate cell-signaling, however, different ligands elicit distinct and sometimes opposing cell-signaling responses reflecting the ability of different ligands to assemble unique co-receptor complexes. Furthermore, many LRP1 ligands are multi-domain proteins with numerous effects on cell physiology that do not involve LRP1-binding. For example, tissue-type plasminogen activator (tPA) binds to LRP1 to promotes Schwann Cell (SC) survival and migration.23 Yet, by LRP1-independent activities, tPA elicits pain.1 El-tPA promotes survival of human iPSC-derived neural progenitor cells (iNPCs) and transplanted El-tPA activated iNPCs into rodents with severe spinal cord injury demonstrate improved motor functional recovery.40 Imbalances in the microenvironment following nerve injury may have severe consequences, including the development of chronic neuropathic pain states.12 In peripheral nervous system (PNS) injury, both inflammatory cytokines such as TNFα, IL-6 and IL-1β and anti-inflammatory cytokines such as IL-10 have been shown to play a central role in axon regeneration and repair.6
As demonstrated herein, the disclosed peptides robustly promote neuronal survival, axonal growth, and expression of regeneration associated genes, such as ATF3, in adult rat primary dorsal root ganglia (DRG) cultures. The disclosed peptides also act as a potent anti-inflammatory agent, inhibiting NFκB signaling in response to lipopolysaccharide (LPS). As an LRP1 agonist, the disclosed peptides target both SC and neurons, the dysfunction of which contributes to abnormal regeneration, poor functional recovery, and chronic neuropathic pain. Thus, the peptides disclosed herein can be used for treating PNIs associated with the development of chronic pain.
In terms of neurodegenerative disease such as Alzheimer's Disease (AD), LRP1's role has been extensively studied. AD is characterized by a progressive loss of cognitive abilities and formation of senile plaques, which are composed largely of amyloid β (Aβ), and tau protein aggregates called neurofibrillary tangles (NFTs) in the hippocampus and cortex of afflicted humans. The spread of protein aggregates during disease progression is a common theme underlying neurodegenerative disease pathology. As an endocytic receptor, LRP1 was shown to regulate brain and systemic clearance, degradation and production of amyloid β-peptide.54 As LRP1-mediated clearance of Aβ across the blood brain barrier (BBB) is the key event in the regulation of Aβ transcytosis from brain to periphery, targeting LRP1 with one or more peptides disclosed herein may serve as a novel treatment. Also, tau protein aggregates forming NTFs plays a central role in the pathogenesis of Alzheimer's disease. LRP1 functions to regulate tau protein endocytosis, accumulation and spread associated with worsened pathology.32 Therefore, the peptides disclosed herein may serve as an intervention that remediates both plaque and tangle pathologies through mediation of LRP1 associated protein aggregation of tau and amyloid aβ. The peptides disclosed herein also act on neuroinflammation, which plays a critical role in neurodegenerative disease. Microglial cells act as the resident immune cell of the brain, serving to maintain homeostasis in the environment. Reactive glia cells and associated neuroinflammation play a key role in both disease initiation and progression becoming activated through dysregulated clearance of beta amyloid and other damage associated molecular patterns (DAMPs).13 AB deposition and tau hyper-phosphorylation contribute to microglial activation, NFκB inflammatory pathway activation and associated pro-inflammatory cytokines such as TNFα, IL-6 and IL-1β, which contribute to neuronal damage and loss. Impaired autophagy, a homeostatic process that degrades and recycles proteins such as beta amyloid, has been associated with AD.7 LRP1 has been shown to mediate healthy lysosomal processing associated with autophagy. Therefore, through LRP1, the disclosed peptides can mediate several aspects of AD including healthy cell metabolism to reduce the spread of protein aggregation, alleviate neuroinflammation and improve neuronal dysfunction leading to survival and possibly regeneration of these cells.
Acute Lung Injury (ALI), leading to Acute Respiratory Distress Syndrome (ARDS), can be initiated by a variety of inflammatory insults such as pneumonia, traumatic injury, and/or infection. A key initiation step in ALI is the dysregulated innate immune response to damage associated or pathogen associated molecular patterns (DAMPs or PAMPs, respectively). Alveolar macrophages are activated by the infectious microbes in Toll-like and Nod-like Receptor signaling pathways that lead to further macrophage and circulating neutrophil recruitment. Neutrophils accumulate in the lungs and release proinflammatory cytokines and other cytotoxic substances causing exacerbation of the injury. The lung epithelium is damaged by these cells and their secreted products cause pulmonary edema and potential respiratory distress (ARDS). An increase in proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, IL-8, and IL-18 occurs resulting in a “cytokine storm” which is predictive of morbidity and mortality in sepsis. LRP1 was reported to be a critical player in sepsis and ARDS as it regulates lung inflammation and lung tissue repair.49 The peptides disclosed herein target LRP1, leading to the precise coordination of the immune response in order to restore homeostasis. Therefore, the disclosed peptides can have a significant impact to mitigate lung damage and improve survival through multiple mechanisms initiated by inducing specific LRP1-mediated signaling pathways. These mechanisms include rebalancing the cytokine output to promote inflammatory resolution, mediating autophagy to restore proper cell metabolic processes, restoring immune cell function and receptor scavenging to regulate the tissue injury cycle. These mechanisms help in infection clearance and allow the host to better fight infection. Based on these mechanisms, the disclosed peptides can be used as immunomodulatory therapeutic agents to prevent or treat ARDS by mediating LRP1 signaling.
Acute respiratory distress syndrome (ARDS) is a major complication in patients with severe COVID-19 illness. Clinical evaluation and retrospective studies out of Wuhan China show that all of the patients admitted to hospital presented with pneumonia, of those 29% developed ARDS and among critically ill patients who were non-survivors, a vast majority (81%) had developed ARDS.17,52 Therefore, patients who develop ARDS are at a substantially higher risk of death. Virally mediated activation of the innate immune response through the Toll-like Receptors (TLRs) initiates an inflammatory response that is necessary to eliminate the infection. In some cases, the initiating viral insults and triggers are highly amplified and uncontrolled, resulting in overstimulation of the immune cells and an aberrant cytokine release (known as a cytokine storm), resulting in tissue damage that can lead to organ failure and death. Cytokine regulation is a key factor in preventing the harmful effects of an overactive immune response and controlling the cytokine storm could have a significant impact on viral pneumonia progression to ARDS. In the case of SARS-COV-2, emerging evidence suggests that for a subpopulation of patients with severe illness, the cytokine storm is a contributing factor to mortality.27 Recently, clinical trials were launched for Kevzara (sarilumab), an interleukin-6 inhibitor (IL-6), for the treatment of coronavirus infected patients with lung complications. IL-6 is a biomarker associated with higher mortality rates in individuals with pneumonia. Those trials will provide critical insight into the effectiveness of single cytokine targeted therapeutics. However, historically, targeting a single cytokine or pathway in ARDS patients has not proved to be an effective approach.
LRP1 signaling mediated by the peptides disclosed herein may have therapeutic potential as an immunomodulatory strategy to improve COVID-19 patient outcomes through regulating several mechanisms of lung inflammation including curbing the cytokine storm, improving cell survival, regulating autophagy and cell metabolism to clear infection and controlling tissue repair signaling pathways to promote proper healing and prevent fibrosis. LRP1 is widely known to regulate protease/antiprotease activity and mediate viral entry.43 The novel coronavirus utilizes proteases (TMPRSS2) to regulate cell entry and infection and treatment with its corresponding protease inhibitor such as SERPIN blocked lung cell infection.16,24,28 SARS-COV-2 viral entry is dependent on a serine protease TMPRSS2 that primes the viral S protein involved in host cell entry, which is the first step in the viral replication cycle. Camostat mesylate is a natural SERPIN with protease inhibitor function of TMPRSS2 and was effective in reducing SARS-COV-2 viral entry, which can limit both infection and spread of the disease.16 An alpha-1 antitrypsin derivative disclosed herein such as SP163M is also capable of inhibiting TMPRSS2, potentially through LRP1 and may have effects in reducing SARS-COV-2 viral replication. As demonstrated herein, the disclosed peptides can reduce SARS-COV-2 replication in vitro, the mechanism of which may be LRP1 control of the host protease/viral protein interaction to inhibit viral entry. The disclosed peptides are also screened for viral activity against several other DNA and RNA viruses and found to inhibit replication of many, indicating a broad-spectrum mechanism through modifying host responses.
Arthropod-borne viruses are important causes of acute encephalitis and an emerging worldwide problem with an ever-growing risk for importation into new regions.11,31 The mosquito-borne encephalitic alphaviruses including Venezuelan (VEEV), eastern (EEEV) and western equine encephalitis viruses (WEEV) are endemic in the Americas and cause outbreaks of encephalomyelitis, which can spread into the United States. In humans VEEV causes a febrile illness typified by fever, malaise, and vomiting. In some cases, infection progresses to the central nervous system (CNS). Neurological cases have a mortality rate as high as 35% in children and 10% in adults, with long-term neurological deficits often seen in survivors.42 The overall estimate of VEEV survivors with neurological sequelae is 4-14% but may be even greater due to the misdiagnosis of arboviral encephalitis.33
VEEV infection is known to inhibit cellular transcription and translation in order to downregulate the innate immune response.45,48 In contrast, in the central nervous system (CNS), VEEV infection results in the upregulation of numerous genes in the inflammatory response and apoptotic pathway.45,48 Specifically, pro-inflammatory cytokines including interleukin-1β (IL-1β), IL-6, IL-12, and tumor necrosis factor-α (TNF-α) play a role in VEEV pathogenesis.9,20,30,34,35 Gene expression changes were analyzed in the brain tissue of VEEV infected mice and alterations in immune pathways involved in antigen presentation, inflammation, apoptosis and the traditional antiviral response were discovered.36 In addition, viral modulation of extracellular matrix and adhesion genes such as integrins, cadherin-1, cadheri-2, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1 (ICAM-1) in the brains of VEEV infected mice was observed.37 ICAM-1 knockout mice demonstrated reduced inflammation in the brain and a subsequent delay in the onset of clinical disease.37 These studies suggest that alphavirus-induced inflammation contributes substantially to neurological damage and that control of inflammation is a viable therapeutic strategy.
The peptides disclosed herein can bind LRP1 and initiate the immune modulatory cascade. LRP1 expression increases during ischemia, tissue injury and viral infection.2,22,25,43 The binding of one or more disclosed peptides to LRP1 can inhibit the inflammatory response and induce pro-survival signaling through phosphorylation of protein kinase Akt. Therefore, targeting LRP1 has potential as a broad-spectrum therapeutic strategy for infectious disease.
Through LRP1 directed host mediated mechanisms, the SERPIN peptides can curb the harmful cytokine storm associated with severe COVID-19 disease, activate protective pathways to prevent lung damage, and/or clear infection faster and has direct impact on suppressing viral infection. Therefore, the dual anti-inflammatory and antiviral mechanism of the SERPIN peptides can have effects in improving survival in patients with
SARS-COV2 induced ARDS.
Eosinophilic Esophagitis
The peptides disclosed herein can serve as an adjunct treatment to mitigate the acute inflammatory response in patients with ST segment elevation myocardial infarction (STEMI). Additionally, SP163M has been shown to be safe and well tolerated in both Phase I and the ongoing Phase Ila clinical trials. The peptide therapy is unique in that it rebalances dysregulated immune responses and protects tissues from injury without any major immunosuppressive effects. Therefore, it is a safe anti-inflammatory drug that has broad-spectrum utility across a wide variety of immune-mediated diseases.
Eosinophilic Esophagitis (EoE) is a chronic, largely type-2 immune mediated allergic inflammatory response associated with esophageal dysfunction and disturbed epithelial barrier function. This food-allergen driven disease is characterized by eosinophil dominated inflammation and type-2 mediated immune responses that lead to esophageal damage. In the US, an estimated 150,000 patients (largely children) are currently suffering from this disorder that commonly causes esophageal pain, difficulty swallowing, food impaction, persistent heartburn, chest and abdominal pain, weight loss, nausea, vomiting and failure to thrive. Currently, there are no FDA approved therapeutics for the treatment of EoE, and management of the disease consists of diet restrictions, proton pump inhibitors, and corticosteroids. The current standard-of-care therapies do not adequately address the immune dysregulation that occurs in EoE and other allergic inflammatory diseases such as asthma or atopic dermatitis.
EoE is characterized by a high number of eosinophils, proteases, cathelicidin, serine proteases including the kallikreins (KLK5), as well thymic stromal lymphopoietin (TSLP)—a cytokine and master regulator of allergic type-2 inflammatory responses in the local environment.41 In esophageal epithelial cells, a loss of the function of a serine peptidase inhibitor, Kazal type 7-SPINK7, results in uncontrolled protease activity, release of pro-inflammatory cytokines such as TNFα, CCL2, GM-CSF, IL-8, and CXCL10 and inflammation. Recently, it was found that the serine protease KLK-5, which is an important mediator of epithelial barrier function, is a direct target of SPINK7 and loss of SPINK7 mediates EoE pathogenesis largely through uncontrolled KLK-5 protease activity. Interestingly, the SERPIN Alpha-1 antitrypsin, is capable of inhibiting KLK5 activity in vitro and allergen-induced esophageal eosinophilia in vivo. However, the mechanism of the activity is not yet clear and may involve LRP1, instead of or in addition to direct proteolytic inhibition.
Patients with eosinophilic asthma have lower levels of LRP1. In addition, LRP1 deletion specifically of CD11b and CD11c dendritic cells in mice results in heightened allergic inflammatory response in an allergic airway disease model.29 Mice with LRP1 deletion had increased antigen uptake and suffered increased eosinophilic inflammation, allergic sensitization, Th2 mediated cytokine production and a reduction in T-regulatory cells.29 Therefore, LRP1 could aid in maintaining homeostasis of proteases/inhibitors in the esophageal environment, mediating the TH2 responses and inhibiting inflammatory signaling pathways (NFκB, JNK) resulting in repair of esophageal dysfunction.
The anti-inflammatory and homeostatic effects of SP16 are mediated through targeting LRP1. Treatment by the peptides disclosed herein such as SP16 and SP163M effectively shuts down major inflammatory signaling pathways such as NFκB. During allergic inflammation, there is a crucial interplay of the innate and adaptive immune responses, and NFκB signaling can be described as a master transcriptional regulator of both responses. The peptides disclosed herein such as SP16 and SP163M effectively dampen pro-inflammatory cytokines IL-1β, IL-6 and TNFα, while increasing anti-inflammatory cytokine levels, such as IL-10 and TGFB. Signaling pathway analysis of SP16 also show inhibition of JAK/STAT signaling cascade, an important mediator of TH2 responses. Skewing of the hyper Th2 immune milieu through LRP1 dependent signaling and subsequent inhibition of Th2 cell mediated cytokines (IL-5, IL-4, IL-13) works to resolve allergic inflammation. Furthermore, SP16 has been shown to inhibit thymic stromal lymphopoietin (TSLP) in epithelial cells, and other important mediators (CCL11, CCL26) of eosinophilic inflammation. Another mechanism that SP16 controls inflammation is through LRP1 dependent endocytosis to clear dead cells/debris, proteases and other allergic triggers that lead to epithelial barrier damage. SP16 also works through LRP1 dependent PI3K/Akt pro-survival pathways to promote a favorable environment for epithelial barrier healing and repair. Therefore, a major benefit of SP16 and derivatives thereof is that although the peptides have strong anti-inflammatory properties, they are not a general immunosuppressive drug. Instead, the peptide works through LRP1 dependent pathways to alter the immune response and restore homeostasis.
The disclosed peptides such as SP 16 and SP163M demonstrated anti-inflammatory and pro-survival effects in a wide variety of inflammatory and auto-immune disease models. The peptides function as a potent LRP1 agonist with no protease inhibitor function and can reduce the cytokine TSLP release in SPINK7 knock out cells. In addition, the disclosed peptides can reduce TH2 mediated cytokines in human esophageal epithelial cells. The disclosed peptides can have significant implications in alleviating eosinophilic esophagitis by mediating control over LRP1.
Disclosed herein are SERPIN peptides including isolated, synthetic peptides and derivatives thereof that specifically bind to LRP1. LRP1 is an endocytic scavenger receptor for numerous ligands which exert biologically distinct functions. The LRP1 protein consists of a smaller (85 kD, β chain) intracellular fragment which spans the cell membrane, non-covalently attached to an extracellular fragment (515 kD, α Chain) which consists of ligand-binding-type repeats, responsible for the majority of ligand binding. In addition to its ability to mediate the endocytosis for various lipoproteins, protease/inhibitor complexes, viruses, matrix proteins and growth factors via its extracellular domain, LRP1 interacts with various scaffolding and signaling proteins via its intracellular domain to mediate cell signaling. Due to the multifunctional capability (both endocytic and cell signaling control) of LRP1, it is implicated in a variety of biological functions, including cell growth/survival, homeostasis, cell metabolism, cytokine regulation, and trafficking foreign antigens. Therefore, LRP1 is implicated to play a role in a variety of diseases.
In certain embodiments, the SERPIN peptide disclosed herein comprises, consists essentially of, or consists of a core sequence FNKPFVFLM (SEQ ID NO. 1) of the SP16 peptide which has a sequence of VKFNKPFVFLMIEQNTK (SEQ ID NO. 2). In certain embodiments, the SERPIN peptide disclosed herein is an analog or derivative of SP16 peptide, sharing at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SP16. The core sequence includes the LRP1 binding site having a sequence of FVFLM. Surprisingly, when Met of the core sequence is replaced by Nle, the activities of the SERPIN peptides increased significantly. Accordingly, in certain embodiments, the SERPIN peptide disclosed herein comprises, consists essentially of, or consists of a core binding motif having a sequence of X1-N-X2-P-F-X3-X4-X5-X6, wherein X1 is R or F, X2 is Kor R, X3 is V or L, X4 is F, V or M, X5 is L, V or I, and X6 is M, I, or Nle. In certain embodiments, the binding motif has a sequence of FNKPFVFLM (SEQ ID NO. 1), FNKPFVFL[Nle] (SEQ ID NO. 5), FNRPFLVVI (SEQ ID NO. 6), FNRPFLVV[Nle] (SEQ ID NO. 7), FNRPFLMII (SEQ ID NO. 8), or FNRPFLVI[Nle] (SEQ ID NO. 9). In certain embodiments, the SERPIN peptide disclosed herein comprises, consists essentially of, or consists of a LRP1 binding site having a sequence of F-X3-X4-X5-X6, wherein X3 is V or L, X4 is F, Vor M, X5 is L, Vor I, and X6 is M, I, or Nle. In certain embodiments, the LRP1 binding site has a sequence of FVFLM (SEQ. ID NO. 3), FVFL[Nle] (SEQ ID NO. 10), FLVVI (SEQ ID NO. 11), FLVV[Nle] (SEQ ID NO. 12), FLMII (SEQ ID NO. 13), or FLMI[Nle] (SEQ ID NO. 14).
In certain embodiments, the SERPIN peptides disclosed herein comprise, consist essentially of, or consist of a modified core binding motif by adding a flanking sequence comprising one or more basic amino acids, an arginine, or both of one or more basic amino acids and an arginine to either or both sides of the core binding motif. For example, the SERPIN peptide disclosed herein comprises, consists essentially of, or consists of an amino acid sequence of Z1-R-X1-N-X2-P-F-X3-X4-X5-X6-R-Z2 (SEQ ID NO. 16), wherein X1 is R or F, X2 is Kor R, X3 is Vor L, X4 is F, V or M, X5 is L, V or I, X6 is M, I, or Nle, and Z1 and Z2 are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or between 1 and 3, between 1 and 5, between 1 and 6, between 1 and 7, between 1 and 8, between 1 and 9, or between 1 and 10 basic amino acids. In certain embodiments, the SERPIN peptide disclosed herein comprises, consists essentially of, or consists of an amino acid sequence of Z1-RFNRPFLVVIR-Z2 (SEQ ID NO. 17), Z1-RFNRPFLMIIR-Z2 (SEQ ID NO. 18), or Z1-KFNKPFVFL(Nle)R-Z2 (SEQ ID NO. 19), wherein Z1 and Z2 are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or between 1 and 3, between 1 and 5, between 1 and 6, between 1 and 7, between 1 and 8, between 1 and 9, or between 1 and 10 basic amino acids.
In certain embodiments, the SERPIN peptide disclosed herein comprises, consists essentially of, or consists of an amino acid sequence of X1-Z1-X2-72-X3-Z3-F-V-F-L-X4-Z4 (SEQ ID NO. 20), wherein:
In some embodiments, this peptide comprises, consists essentially of, or consists of 20 or fewer amino acids.
In certain embodiments, the SERPIN peptide disclosed herein comprises, consists essentially of, or consists of an amino acid sequence of X1-Z1-X2-72-X3-Z3-F-X4-F-L-Z4-X5 (SEQ ID NO. 21), wherein:
In some embodiments, this peptide comprises, consists essentially of, or consists of 20 or fewer amino acids.
In certain embodiments, the SERPIN peptide disclosed herein comprises, consists essentially of, or consists of the sequence of VKFNKPFVFL(NIe)IEQNTK (SEQ ID NO. 35), VKFNKPFVFLM (SEQ ID NO. 25), LRFNRPFLVVI (SEQ ID NO.29), VRFNRPFLMII (SEQ ID NO.31), VKFNKPFVFL(Nle) (SEQ ID NO. 40), RFNRPFLVVIR (SEQ ID NO. 41), RFNRPFLMIIR (SEQ ID NO. 42), RFNKPFVFL(Nle)R (SEQ ID NO. 43), RRRFLVVIRRR (SEQ ID NO. 44), RRRFLMIIRRR (SEQ ID NO. 45), or RRRFVFL(Nle)RRR (SEQ ID NO. 46).
In certain embodiments, the SERPIN peptide disclosed herein has a size of between 5 and 30 amino acids, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, the SERPIN peptide disclosed herein has a size of 20 amino acids or less. A longer peptide may have a decreased solubility, whereas a shorter peptide may have decreased stability. As disclosed herein, various modifications can be made to improve stability such as adding poly R sequences or other flanking sequences and forming a fusion protein.
The SERPIN peptides include analogues or derivatives thereof. For example, the native sequence of the SERPIN peptides can be modified to enhance plasma stability and result in an increased binding affinity to the peptide's cognate receptor. In certain embodiments, the SERPIN peptides disclosed herein can be further modified to extend the shelf life and/or bioavailability using one or more non-natural peptide bonds or amino acids or by attaching to the peptide functional groups such as polyethylene glycol (PEG). In certain embodiments, the SERPIN peptides disclosed herein are modified by adding one or more amino acid residues such as arginine at either or both ends. In certain embodiments, the SERPIN peptides are modified by adding two, three, or four amino acid residues at both ends.
In certain embodiments, the SERPIN peptides disclosed herein are fused to one or more other peptides to form a fusion peptide or fusion protein. For example, one or more other peptides include an epitope tag such as ALFA-tag, V5-tag, Myc-tag, HA-tag, Spot-tag, T7-tag. NE-tag, a half-life extender such as poly(ethylene glycol) (PEG), Lipidation, FC fusion, or Albumin fusion, or both of an epitope tag and a half-life extender. In certain embodiments, the peptide comprises one or more D-amino acids, that is, one or more amino acids of the peptide have a D-configuration.
In another aspect, this disclosure relates to a pharmaceutical composition comprising, consisting essentially of, or consisting of an effective amount of one or more SERPIN peptides or fusion peptides disclosed herein. In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents, which are not the SERPIN peptides disclosed herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, additive, preservative, or a combination thereof. Examples of acceptable carriers include physiologically acceptable solutions, such as sterile saline and sterile buffered saline.
The term “an effective amount” as used herein refers to an amount of a composition that produces a desired effect. An effective amount of a composition may be used to produce a prophylactic or therapeutic effect in a subject, such as preventing or treating a target condition, alleviating symptoms associated with the condition, or producing a desired physiological effect. In such a case, the effective amount of a composition is a “therapeutically effective amount,” “therapeutically effective concentration” or “therapeutically effective dose.” The precise effective amount or therapeutically effective amount is an amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject or population of cells. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the composition (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication) or cells, the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. Further an effective or therapeutically effective amount may vary depending on whether the composition is administered alone or in combination with another composition, drug, therapy or other therapeutic method or modality. One skilled in the clinical and pharmacological arts will be able to determine an effective amount or therapeutically effective amount through routine experimentation, namely by monitoring a cell's or subject's response to administration of a composition and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy, 21st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.
In certain embodiments, the peptides or the pharmaceutical compositions disclosed herein may be formulated for oral administration, parenteral administration such as intravenous administration, intramuscular administration, subcutaneous administration (bolus injection or through a device such as an infusion pump), intradermal administration, transdermal administration, topical administration, and intranasal administration. In certain embodiments, a subcutaneous infusion pump can be used for delivery of the peptides or the pharmaceutical compositions disclosed herein. The peptides or the pharmaceutical compositions may be administered more than once. More specifically, after the initial administration, one or more additional doses may be given as a booster.
The SERPIN peptides or the pharmaceutical compositions disclosed herein have various functions. In certain embodiments, disclosed herein is a method of treating a subject in need thereof an effective amount of one or more SERPIN peptides, fusion peptides, or the pharmaceutical compositions disclose herein. In some embodiments, the subject suffers from a disease or condition in which LRP1 mediation contributes to pathology, such as in conditions associated with peripheral nerve injury and resulting pain, lung injury, infectious disease and allergic inflammation such as eosinophilic esophagitis. In some embodiments, the subject suffers from a disease associated with dysregulated immune response selected from the group consisting of peripheral neuropathies, neuropathic pain, COVID-19 infection, acute respiratory distress syndrome (ARDS), sepsis, SARS-COV-2, Influenza, alphavirus infection, and cytokine storm.
As used herein, “treating” or “treatment” of a condition may refer to preventing the condition, slowing the onset or rate of development of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or ending symptoms associated with the condition, generating a complete or partial regression of the condition, or some combination thereof. Treatment may also mean a prophylactic or preventative treatment of a condition.
As used herein, the term “subject” is a mammal. In some embodiments, the subject is human. In some embodiments, the subject has not received any prior treatment with serine protease inhibitors, such as alpha-1-antitrypsin treatment before the treatment with the peptides disclosed herein.
In some embodiments, the peptides can be used to reduce the serum TNF-a levels in human individuals who have pathologically increased TNF-a levels. The peptide causes a 75% decrease in serum TNF-α levels when administered in an effective amount to a human subject. In certain embodiments, the peptide results in a 50% or 75% decrease in serum TNF-α levels when administered in an effective amount to a human subject compared to the levels before administration of the peptide.
In some embodiments, the SERPIN peptides and fusions thereof or pharmaceutical compositions comprising the peptides or fusions can be used to treat a disease or condition associated with LRP1 such as EoE, AMI, nerve injury, pain, gout, rheumatoid arthritis, and diabetes including type I and type II diabetes. In some embodiments, the SERPIN peptides and fusions thereof or pharmaceutical compositions comprising the peptides or fusions can be used to treat a disease or condition associated with thymicomal lymphopoietin (TSLP), in particular, TSLP-associated allergic diseases such as allergic asthma, atopic dermatitis, allergic rhinoconjunctivitis, EoE, cancer, and rheumatoid arthritis.
In some embodiments, the SERPIN peptides and fusions thereof or pharmaceutical compositions comprising the peptides or fusions can be administered at a dose from about 0.001 mg/kg to about 4 mg/kg in human. In general, 3 μg in a mouse corresponds to 0.012 mg/kg in humans. Depending on the indication, severity, and administration route, a suitable dose can be selected accordingly. For example, for acute indications, fewer treatments with a higher dose in each treatment are administered; while for chronic indications requiring frequent and long-term treatment, a lower dose in each treatment is administered. In some indications where the inflamed tissue expresses high density of LRP1, a very low dose of the SERPIN peptides such as SP16 and SP163M is required. When a subject suffers from a nerve injury, neurons express very high density of LRP1. In vivo and in vitro studies demonstrated a significant effect with a low dose of 0.05 ug.
In some embodiments, the SERPIN peptides and fusions thereof or pharmaceutical compositions comprising the peptides or fusions is administered subcutaneously. In some embodiments, a SERPIN peptide such as SP16 or SP163M is administered subcutaneously to a human subject at a dose of between 0.05 mg/kg and 0.5 mg/kg, for example, at a dose of 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.45 mg/kg, or 0.5 mg/kg. In some embodiments, a SERPIN peptide such as SP16 or SP163M is administered subcutaneously to a human subject at a dose of 0.2 mg/kg or 0.4 mg/kg. In some embodiments, a SERPIN peptide such as SP16 or SP163M is administered orally to a human subject at a dose between 1 mg and 150 mg, for example, at a dose of 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, or 150 mg. In some embodiments, a SERPIN peptide such as SP16 or SP163M is administered orally to a human subject at a dose of 5 mg, 25 mg or 100 mg. In some embodiments, a SERPIN peptide such as SP16 or SP163M is administered locally, e.g., by a transdermal patch, optionally with pulsatile delivery, at a dose between 0.05 mg/kg and 0.5 mg/kg, for example, at a dose of 0.05 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg, 0.45 mg/kg, or 0.5 mg/kg.
In some embodiments, a single dose of the SERPIN peptides and fusions thereof or pharmaceutical compositions comprising the peptides or fusions is administered. In some embodiments, the SERPIN peptides and fusions thereof or pharmaceutical compositions comprising the peptides or fusions is administered as the sole therapeutic agent. In some embodiments, the SERPIN peptides and fusions thereof or pharmaceutical compositions comprising the peptides or fusions is administered in combination with a secondary therapeutic agent.
In some embodiments, the SERPIN peptides and fusions thereof or pharmaceutical compositions comprising the peptides or fusions is administered in a pulsatile mode or a continuous mode. In some embodiments, the SERPIN peptides and fusions thereof or pharmaceutical compositions comprising the peptides or fusions is administered via a transdermal patch, an inhaler, or an intranasal device.
The following examples are intended to illustrate various embodiments of the invention. As such, the specific embodiments discussed are not to be constructed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of invention, and it is understood that such equivalent embodiments are to be included herein. Further, all references cited in the disclosure are hereby incorporated by reference in their entirety, as if fully set forth herein.
As demonstrated herein, a small peptide fragment of the C-terminal end of alpha-1 antitrypsin (the prototypical SERPIN) is capable of binding to LRP1, exerting potent cell regenerative, tissue protective and immune-modulatory functions. Interestingly, the naturally occurring degradative C-terminal product of Alpha-1 Anti-trypsin (termed C-36) exhibits pro-inflammatory activity similar to lipopolysaccharide in both macrophages and neutrophils. 44 By excising a short fragment of the C-terminal end of Alpha-1 anti-trypsin, the anti-inflammatory sequences were identified. The amino acid sequences of the peptides tested are shown in Table 1 below. The core sequences containing the LRP1 binding site for each peptide are shown in bold and underlined.
The reporter cells (THP1-XBlue-MD2-CD14 cells) were treated with each peptide (50 μg/ml) before being insulted with LPS (5 ng/ml) and incubated overnight. The NFκB inducible (Secreted Embryonic Alkaline Phosphatase) SEAP was measured in the supernatant and read for absorbance. As shown in
In contrast, SP16 is capable of reducing NFκB activity, however, the short core peptide SP20 did not have an inhibitory effect. Although this truncated AAT derived peptide contains the core sequence and LRP1 binding site, it is unstable and therefore, exhibited no activity. When SP16 was flanked with triple arginine amino acids on both sides (termed “Poly-R”) to obtain SP21, the SP21 peptide's stability increased and NFκB inhibition increased as well relative to SP16. When the SP16 short core, SP20 peptide, was stabilized using triple arginine flanks to obtain SP22, the NFκB activity was significantly reduced. Similar effects were observed for the other two pairs of SERPIN peptides, SERPIN 2 short core (SP24) vs. SERPIN 2 short core poly-R (SP26), and SERPIN 3 short core (SP28) vs. SERPIN 3 short core Poly-R (SP29). Accordingly, this example demonstrates that shortening the peptide resulted in instability and lack of function, while stabilizing with the poly-R flanks resulted in heightened activity.
Moreover, these peptides all contain a LRP1 binding site, however, when the LRP1 binding site was truncated to obtain SP31, the anti-inflammatory activity of the peptide was lost and could not be restored with the Poly arginine flanks (SP32 peptide). This indicates that SERPINs contain an anti-inflammatory core motif that is dependent on LRP1.
To improve the bioavailability and stability of SP16, the peptide was modified by replacing the methionine with Norleucine (Nle) and acetylating and amidating the N and C terminus, respectively. The modified peptide is termed SP163M having the following sequence: VKFNKPFVFL[NIe]IEQNTK (SEQ ID NO. 35). Surprisingly, although one of the amino acids in the LRP1 binding site was mutated (M->Nle), SP163M significantly improved NFκB reduction in the reporter assay in comparison to the unmodified SP16 peptide, as shown in
The two peptides were compared for their ability to bind to LRP1. Binding assays confirmed SP163M′s improved binding capacity to LRP1 compared to unmodified SP16 peptide (
To confirm whether the proposed binding sequence FVFLM is where LRP1 interacts with the SERPIN peptides, SP16AALRP1 peptide having a sequence of VKFNKAAAAAAIEQNTK (SEQ ID NO. 36) was used. Compared to SP16 or SP163M, this peptide does not contain the LRP1 binding site and two other amino acids are replaced with alanine. In this assay, direct binding was assessed, as recombinant LRP1-FC fusion protein (Cluster II), was bound to protein G beads (dynabeads protein G). SP163M, SP16AALRP1, and scrambled control (SP34) were labeled with a fluorescent dye (Cy5.5) and added to the LRP1 bound beads (each peptide at 1 μg/ml), before excess or non-bound peptide was washed thoroughly away. Human IgG was used as a random protein control bound to the beads. The fluorescence of the beads was measured by flow cytometry to show the level of peptide binding to LRP1. As shown in
Next, whether blocking LRP1 signaling would have a negating effect on the peptides' ability to reduce NFκB activation was evaluated. The NFκB reporter assay was used and the cells were stimulated with LPS (5 ng/ml) to induce NFκB activity. SP163M (100 μg/ml) was added directly prior to each treatment. The cells were treated with an anti-LRP1 antibody that blocks LRP1 signaling (125 μg/ml), RAP (1 μM) (an LRP1 antagonist that blocks ligand binding), or an IgG antibody as a control 30 minutes prior to stimulation with LPS.
LRP1 has been shown to play a role in neuropathic pain. For instance, LRP1 agonists promote axonal regeneration (sprouting) and activates Schwann cell repair programs. In this example, adult dorsal root ganglion (DRG) were isolated from rats to assess the effects of SP163M on sprouting.
Sprouting in the DRG neuron cultures was further quantitatively assessed. Neuro-specific beta tubulin immunofluorescence was performed to measure the length of longest neurite in 300-400 cells per group.
In an effort to understand the molecular mechanisms underlying SP163M-induced neuronal sprouting, regenerative associated gene expression (ATF3, SPRRR1A, GAP43, and STMN2) changes at 72 hours were examined using qPCR. This test was to confirm that the observed sprouting effects were due to SP163M signaling via LRP1. RAP which prevents binding to LRP1 and inhibits LRP1-dependent signaling, was used to pre-treat the cultures 1 hour prior to SP163M treatment (500 ng). Increased expression of regeneration associated genes (RAGs) by SP163M appeared to be blocked by RAP, suggesting that SP163M induced these changes via LRP1 (
Next, a pain study was performed using the formalin pain test. This test is a tissue injury model that is widely used to screen potential pain therapeutics. In the model, formalin was injected subcutaneously into the hind paw, which induced a very reliable response consisting of a first phase and second phase. In the first phase, analgesic affects were tested as c-fiber activation due to peripheral stimulus (Substance P and Bradykinin) caused pain. During the second phase, there was an inflammatory reaction in peripheral tissue and central sensitization in spinal dorsal horn (histamine, serotonin, prostaglandin and bradykinin). SP163M was administered subcutaneously (50 μg) 1 hour prior to intraplantar formalin injection and then licking behavior was observed for 50 minutes. SP 163M significantly reduced the first phase, and thus appeared to act as an analgesic via modulation of C fiber activity. Moreover, SP163M significantly delayed the start of the second phase, suggesting SP163M may act as an analgesic and may also modulate inflammation and/or central sensitization (
The efficacy of SP163M was tested in a partial nerve ligation model by testing evoked pain measurements, both thermal and mechanical. SP163M was administered 1 hour prior to surgery (50 μg). Mice were then surgically ligated around part of the sciatic nerve. Mice were measured for evoked pain behaviors following both mechanical (Von Frey) and thermal (Hargreaves) on days 1, 3, 6, and 8. SP163M increased the pain threshold of mechanical allodynia and increases the tolerance to heat (
Given the effects of SP163M on improving both survivability and regeneration of neurons, SP163M could have potential as a treatment for certain CNS related neurodegenerative diseases such as Alzheimer's disease. Experiments were carried out to determine whether SP163M could cross the blood brain barrier (BBB), as LRP1 has been used to deliver drugs across the BBB via LRP-1 receptor mediated transcytosis. To assess the bioavailability of SP163M in the brain, mice were injected with LPS (2 mg/kg), and then 1 hour later injected with labeled SP163M (cy5.5) (50 μg) or vehicle. At 1 hour, 24 hours and 1 hour after the second dose of SP163M (25 hours), the mice were imaged for fluorescence in-vivo and then brain homogenate and serum were collected around 24 hours for measurement of fluorescence.
SERPINs have been found to have broad-spectrum anti-viral activity, for instance, in HIV. However, SERPINs have also been shown to play both positive and negative roles in the progression of chronic viral illnesses. Abnormally low alpha-1 anti-trypsin (AAT) levels have been observed in both HIV-1 and HCV infection and have been shown to correlate with progressive disease or the development of liver fibrosis, respectively. Conversely, there is mounting clinical evidence suggesting an association between increased levels of SERPIN expression and reduced incidence of HIV acquisition, or protracted disease progression. LRP1 plays an essential role in mediating anti-inflammatory cell signaling and modulation of several cytokines. Ligands to LRP1 have a similar anti-inflammatory activity. After ligand binding, the cytoplasmic fragment of LRP1 undergoes proteolysis by y-secretases and migrates to the nucleus, where it binds to the interferon regulatory factor-3 (IRF-3) and promotes its nuclear export and proteasomal degradation, thus limiting the expression of the pro-inflammatory genes in cultured fibroblasts and macrophages.55 The LRP1-agonist complex was also found to inhibit the interleukin-1 receptor associated kinase-1 (IRAK-1), leading to the down-regulation of the NF-κB pro-inflammatory signaling pathway in vascular smooth muscle cells.19 Therefore, SP163M's ability to inhibit HIV mediated pro-inflammatory cytokines and the transcytosis of SP163M in an in-vitro BBB model were tested. A 3D-BBB was constructed on a 3 μM trans-well insert. Microglia with or without HIV on a BBB insert were treated with increasing concentrations of SP163M (up to 100 μg/ml). Cytokines IL-6, MCP-1 and IL-8 were assessed after 24 hours.
LRP1 has been shown to be involved in the development of neurodegenerative diseases and in the regulation of the metabolism of amyloid- peptides (ABs) in the brain and periphery. Neuroinflammation plays a critical role in neurodegenerative disease, such as Alzheimer's Disease (AD). Reactive glia cells (such as microglial cells) play a key role in both disease initiation and progression becoming activated through dysregulated clearance of beta amyloid and other damage associated molecular patterns (DAMPs).13 AB deposition and tau hyper-phosphorylation contribute to microglial activation, NFκB inflammatory pathway activation and release of pro-inflammatory cytokines such as TNFα, IL-6 and IL-1β, which contribute to neuronal damage and loss. Therefore, the cytokine profile was explored in LPS induced microglial cells treated with SP163M. In microglial cells, SP163M reduced LPS mediated cytokines (largely TNF-alpha, p<0.02), while highly upregulating IL-10 (anti-inflammatory cytokine) in LPS treated cells (
In addition to neuroinflammation, impaired autophagy, a homeostatic process that degrades and recycles proteins such as beta amyloid, has been associated with AD.7 LRP1 has been shown to mediate healthy lysosomal processing associated with autophagy. Therefore, through LRP1, SP163M has potential to mediate several aspects of AD including healthy cell metabolism to reduce the spread of protein aggregation, alleviate neuroinflammation and improve neuronal dysfunction leading to survival and possibly regeneration of these cells. The effects of SP163M on autophagy markers were tested in microglial cells. IMG microglial cells were treated with SP163M (100 μg/ml) before addition of LPS (100 ng/ml) for 24 hours. Lysates were collected and western blot analysis of both LRP1 and microtubule-associated protein light chain 3 (LC3 I and II) was performed. The LC3II/I ratio, a commonly used marker for autophagy indicating autophagic flux, was determined.
SP163M works through the innate immune system to mitigate damaging inflammation and tissue injury to subsequently regulate the immune response and promote healing. A novel proteomic signaling pathway analysis was used to assess the impact of SP163M on the host dynamics following infection and treatment. SP163M was tested at various doses and time-points challenged with three distinct agents: an endotoxin (LPS) (bacterial agent), a highly inflammatory virus (Venezuelan Equine Encephalitic Virus), and a febrile virus known to cause mortality, particularly when host immune responses are dysregulated (Rift Valley Fever Virus). The heatmap of the top canonical pathways that were significantly changed in response to each insult shows that, regardless of the agent used, SP163M counteracted these changes, restoring the profile back to a normal state (canonical pathway heatmap for LPS shown in
Given the role of LRP1 in regulating the host responses, SP163M′s ability to restore homeostasis following acute lung injury was tested. LRP1 plays a key role in regulating lung inflammation during injury or infection. LRP1 regulates inflammatory signaling pathways (i.e. NFκB, JNK) to control the cytokine output, and contributes to effective migration and phagocytosis.22,26,51 In neutrophils, LRP1-dependent mechanisms lead to enhanced cell adhesion, chemotaxis, and antibacterial effects of these cells, thereby resisting immunosuppression25. During acute infection or injury, LRP1 also promotes inflammatory resolution through scavenging PAMPS and DAMPS (pathogen and damage associated molecular patterns) (from dying or injured tissue), to prevent tissue injury25. LRP1 was also shown to mediate autophagy during infection, an important metabolic process recently shown to play an important protective role in infectious lung disease.47 LRP1 also controls many signaling pathways important for appropriate tissue repair, particularly important in preventing lung fibrosis.49,50 Therefore, because of its multifunctional ability to regulate lung inflammation, targeting LRP1 has substantial potential to mitigate several aspects of the dysregulated immune response that contributes to acute respiratory distress syndrome (ARDS) progression.
SP163M was tested in an acute septic model of Klebsiella pneumonia (KP) infection, to determine whether SP163M is a viable treatment option for patients with severe lung infection. To assess the bioavailability of SP163M in the lung and tissues, C57BL/6 mice were treated with two doses of Cy5.5-SP163M (12 mg/kg) 24 hours apart. Plasma, liver, kidney and lung were collected and analyzed for SP163M (Fluorescence) at 1 hour after 1 dose (1 dose peak), 23 hours after 1 dose (1 dose trough) and 1 hour after second dose (2 dose peak).
Due to the profound effects of LRP1 in mitigating lung injury and regulating host mediated responses during viral infection, SP163M′s anti-viral effects in SARS-COV-2 infected cells were tested. Surprisingly, SP163M was able to significantly reduce viral replication. Vero cells were pretreated for one hour with SP163M or vehicle control, medium removed, infected with SARS-COV-2 (USA-WA1/2020 Strain) (MOI 0.1), inoculum removed, and post-treated with peptide. Virus in supernatant was measured by RT-qPCR at 48 hours infection. The dose response curve shows that at a dose of 100 μg/ml, SP163M inhibits SARS-COV-2 RNA significantly (
Next, the safety and tolerability of SP163M in subjects with pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) are to be tested. Reduction or change in plasma levels of inflammatory cytokines including interleukin-1 beta (IL-1), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8) is compared to baseline or placebo treated subject. Additionally, reduction or change of acute phase reactants (c-reactive protein (CRP), α-2 macroglobulin, fibrinogen, and D-dimer) is compared with baseline or placebo treated subject. The number or proportion of subjects requiring invasive and noninvasive mechanical ventilation over a 2-week period, the number or proportion of subjects showing improvement in oxygen saturation (SpO2) level as defined by both SpO2>95% and respiratory rate≤20/minute after 1 day or 1 dose of treatment, the time (days) on ventilator for subjects requiring invasive or noninvasive mechanical ventilation, the duration (days) of hospitalization, the time to clinical improvement (TTCI), which is defined as a National Early Warning Score 2 (NEWS2) of ≤2 to be maintained for 24 h (within a 2-week time frame), the number or proportion of subjects experiencing TTCI, which is defined as NEWS2 of ≤2 to be maintained for 24 h (within a 2-week time frame), all-causes mortality over study days 1-14, and/or change from baseline on Days 6 and 14 in Horowitz index or SpO2/FiO2 are assessed in SP163M-treated vs. placebo-treated subjects.
SP163M was also found to inhibit replication in other RNA viruses. Experiments were performed to analyze the impact of SP163M on alphavirus replication. Vero cells or primary astrocytes were pretreated for one hour with SP163M or water control, infected with VEEV TC83 luciferase virus (MOI 0.1), and post-treated with peptide. Luminescence was measured at 16 hours post infection. SP163M treatment reduced VEEV replication in Vero cells (
Because SP163M targets host responses, it could have broad spectrum utility with other viral agents. To better understand whether SP163M's action is alpha-virus specific, RNA or DNA specific or whether it has broad spectrum effects throughout many different viral families, a viral screen was performed with a panel of both RNA and DNA viruses. Of the viruses screened, only one DNA virus, Epstein Barr virus showed any anti-viral activity. Surprisingly, SP163M showed EC50 in many of the RNA viruses tested, with comparable activity to that of VEEV, among them influenza H1N1, yellow fever virus and Dengue fever virus (Table 2).
In terms of Eosinophilic Esophagitis (EoE), A1AT has been shown to attenuate experimental EoE in a murine model and in vitro. It is unknown if A1AT effects are mediated by inhibition of proteolytic activity or through activating LRP1 signaling. Although patients with eosinophilic asthma have lower LRP1 and in animal models, loss of LRP1 is associated with worsened allergic responses. SP163M does not contain any sequences for anti-protease activity, and is likely working through mediation of LRP1, not through proteolytic activity. Evidence shows that loss of esophageal expression of the serine peptidase inhibitor, kazal type 7 (SPINK7) is an upstream event in EoE pathogenesis. Therefore, the in-vitro effects of SP163M were tested in reducing thymic stromal lymphopoietin (TSLP), a key immune checkpoint cytokine involved in stimulating dendritic cells to polarize the generation of type 2 T cells (Th2), key cells involved in EoE pathogenesis, following loss of SPINK7.
Next, whether SP163M had any effects in mitigating a key chemoattractant for eosinophils, CCL26, was investigated. CCL26 is upregulated in the inflamed esophageal mucosa of EoE patients and the CCL26 gene is highly upregulated in the esophagus, according to genomic research. Molecular studies and animal models have implicated the importance of two key cytokines, IL-5 and IL-13 in the disease, which can stimulate release of CCL26 in EPC2 cells.
In an NFκB reporter cell line, SP163M caused a concentration-dependent reduction in LPS mediated NFκB activation as compared to a scrambled control peptide (
In terms of cell survival and regeneration, studies in primary dorsal root ganglion neurons show that SP163M (100 ng) robustly promoted neuronal survival, axonal growth, and expression of regenerative associated genes, such as ATF3, in ex-vivo cultures (
Studies in sepsis models demonstrate the potential therapeutic utility of SP16 to prolong survival and quench the pro-inflammatory cytokine response. Using a mouse model of lethal endotoxemia, in which mice were challenged with LPS (15 mg/kg), resulting in 100% mortality by 36 hours, the assessment brings focus to the potential of SP16 to regulate the cytokine storm underlying sepsis. In this model, all mice treated with SP16 survived (100% survival between 30 and 42 hours), whereas all control mice died (no survivors by 30 hours in the vehicle control group). SP16 treated animals displayed prolonged survival, 60% survival by 72-hrs (p<0.001 vs. vehicle) (
The effects of SP163M were evaluated in the formalin test, a pain model that has both neurogenic and inflammatory components. In this model, SP163M given as a single subcutaneous injection (50 μg per mouse) resulted in a reduction of the first phase (sensory nerve activation) indicating that SP163M has analgesic effects on the mice and a delay in the second phase (inflammatory reaction due to tissue injury), showing the anti-inflammatory effects of SP163M (
Accordingly, SERPIN peptides such as SP16 and SP163M have anti-inflammatory and immune-modulating properties, converging on a favorable environment for tissue regeneration and repair. Based on these mechanisms, SP16 and peptide derivatives thereof mediated LRP1 signaling may have therapeutic potential in the treatment of allergic inflammatory diseases such as atopic dermatitis and EoE.
Given that loss of SPINK7 is associated with profound cytokine activation, the effects of SP163M and AAT (A1AT) were tested for their ability to reduce thymic stromal lymphopoietin (TSLP), a key immune checkpoint cytokine involved in stimulating TH2 mediated immune responses, in SPINK7 KO cells.
Then the effects of SP163M and AAT were tested in a model of allergic inflammation. In the ovalbumin mouse model of allergic asthma, the mice were sensitized to ovalbumin by two intraperitoneal injections containing the adjuvant alum, a strong inducer of both innate and TH2 mediated immune responses and then challenged with ovalbumin given by intranasal instillation on five separate days, mice were treated with either SP163M (100 μg) or AAT (2 mg) daily during the challenge phase.
In this model, the bronchoalveolar lavage fluid (BALF) were measured for cytokines/chemokines, CCL24, IL-1α and IL-2 given that these innate immune mediators activate dendritic cells and contribute to T-cell responses.
These results suggest that SP163M has ameliorating effects on many of the pathophysiological mechanisms that drive allergic inflammatory diseases.
Studies conducted in rats were completed to assess the PK parameters of SP163M given systemically by intravenous injection. The half-life of the peptide was determined to be 1.9 hours. The Cmax (C0 μg/mL) was determined to be 2.5 μg/mL with an AUClast (μg-hr/mL) of 0.9 μg-hr/mL.
A preliminary pharmacokinetic study of patients given a single (0.2 mg/kg) subcutaneous injection of SP163M showed an average of 2.4 ng/ml (SEM±1.24 ng/L) at 90 minutes following injection. Six hours after injection, plasma concentrations of SP163M decreased to an average of 0.9 ng/ml (SEM±0.52 ng/ml) (
The 3 μg dose (0.15 mg/kg) of SP163M administered as a single injection to mice is equivalent to 0.012 mg/kg in humans. This is over 16-fold lower than the dose proposed in clinical protocols. Furthermore, the 0.15 mg/kg dose given to mice by subcutaneous injection demonstrated the same effective infarct size reduction as mice given 5 mg/kg (100 μg) by intraperitoneal injection, which is a 2-fold higher equivalent dose than the 0.2 mg/kg dose in humans. This provides evidence that a single subcutaneous administration of SP163M is very potent.
In an acute mouse model of rheumatoid arthritis (CAIA), it was shown that a single administration of SP163M given after paw swelling had started (Day 3) was capable of reducing paw swelling. The dose of 0.6 mg/kg in mice is equivalent to 0.048 mg/kg in humans. Furthermore, PBMCs collected from the arthritic mice treated daily with SP163M following subcutaneous administration (0.6 mg/kg) were exposed to LPS in an ex-vivo assay. This assay shows that cytokines IL-6 and TNF-α were significantly lower vs. vehicle.
In a model of acute lethal septicemia, SP163M was given at 3 doses (4 μg each) at −2, 0, and 0.5 hours of LPS administration. At this cumulative exposure of 0.6 mg/kg (0.048 mg/kg HED), SP163M significantly decreased mortality over a 72-hour period. Further, an acute model of endotoxemia in which TNFα was measured in the serum 1.5 hours following LPS (1 mg/kg) administration. SP163M at 0.2 mg/kg (0.016 mg/kg HED), 1 mg/kg (0.08 mg/kg HED) or 0.2 mg/kg (0.016 mg/kg) reduced this cytokine in the plasma. Given that all the doses exhibited anti-inflammatory effects, a lower dose is likely needed to achieve a dose dependent response.
Finding reliable biomarkers to assess the pharmacodynamic effects of SP163M is an important aspect of clinical trials. The epithelium derived cytokine thymic stromal lymphopoietin (TSLP) and other immune cell derived cytokines/chemokines are important mediators of the allergic reaction which are modified by SP163M in preclinical studies. Furthermore, LRP1 plays a role as a negative regulator of adaptive immune responses mediated by dendritic cells in allergic airway inflammation. Therefore, the ex-vivo effects of SP163M on patient PBMCs induced with IL-13/Poly I:C can be tested. These PBMCs will be analyzed for the cytokine/chemokine expression profile (either by PCR or multiplex). Another assay employs the use of a co-culture model of airway epithelial cells in air liquid interface (ALI) and patient derived dendritic cells and PBMCs to explore the effects of SP163M on TSLP, IL-10 and Tregs. The peptides disclosed herein such as SP16 and SP 163M will reduce TSLP mediated effects, and induce favorable dendritic cell mediated T-cell responses, consisting of high IL-10 and Treg cells that are important in the suppression of allergen specific Th2 responses.
Mechanistic studies of tolerance in EoE: Esophageal biopsy specimen is used for scoring for pathological features such as epithelial inflammation and eosinophil infiltration; single cell mRNA, patient reported outcomes (PROs), scoring of esophageal endoscopic features, confocal microscopy analysis of epithelial junctional proteins, and detection of SP163M by Mass Spectrometry or analysis of LRP1 expression. Biomarkers include peripheral blood absolute eosinophil counts (AEC), CRP, TSLP, IL-5, IL-33, IL-6, IL-8, and chemokines such as CCL-26, CCL-11, MCT, CCL27, MIP-3B, and CXCL12. PBMC is used for ex vivo priming of patient cells and cytokine profile. Flow cytometry analysis of biopsies and PBMCs is performed to analyze populations of Th2, Tregs, eosinophils, MCs and ILC2 cells.
Mechanistic studies of tolerance in atopic dermatitis: Skin biopsy is performed to score for pathological features such as epithelial inflammation and eosinophil infiltration. Additional studies include single cell mRNA, patient reported outcomes (PROs), confocal microscopy analysis of epithelial junctional proteins, detection of SP163M by Mass Spectrometry or analysis of LRP1 expression. Biomarkers include peripheral blood absolute eosinophil counts (AEC), CRP, TSLP, IL-5, IL-33, IL-6, IL-8 and chemokines such as CCL-26, CCL-11, MCT, CCL27, MIP-38, and CXCL12. PBMC is used for ex vivo priming of patient cells and cytokine profile. Flow cytometry analysis of biopsies and PBMCs is performed to analyze populations of Th2, Tregs, eosinophils, MCs and ILC2 cells.
Mechanistic studies of tolerance in asthma: Cells extracted from BAL fluids (lung lavage) are used for scoring for pathological features such immune cells associated with inflammation. Additional studies include single cell mRNA, patient reported outcomes (PROs), confocal microscopy analysis of BAL cells, detection of SP163M by Mass Spectrometry or analysis of LRP1 expression. Biomarkers include peripheral blood absolute eosinophil counts (AEC), CRP, TSLP, IL-5, IL-33, IL-6, IL-8, and chemokines such as CCL-26, CCL-11, MCT, CCL27, MIP-3B, and CXCL12. PBMC is used for ex vivo priming of patient cells and cytokine profile. Flow cytometry analysis of biopsies and PBMCs is performed to analyze populations of Th2, Tregs, eosinophils, MCs and ILC2 cells.
The references, patents and published patent applications listed below, and all references cited in the specification above are hereby incorporated by reference in their entirety, as if fully set forth herein
1. Berta, T., Y. C. Liu, Z. Z. Xu and R. R. Ji (2013). “Tissue plasminogen activator contributes to morphine tolerance and induces mechanical allodynia via astrocytic IL-1beta and ERK signaling in the spinal cord of mice.” Neuroscience 247: 376-385.
2. Christia, P., M. Bujak, C. Gonzalez-Quesada, W. Chen, M. Dobaczewski, A. Reddy and N. G. Frangogiannis (2013). “Systematic characterization of myocardial inflammation, repair, and remodeling in a mouse model of reperfused myocardial infarction.” J Histochem Cytochem 61(8): 555-570.
3. Cooke, J. P. (2019). “Inflammation and Its Role in Regeneration and Repair.” Circ Res 124(8): 1166-1168.
4. Feng, Y., B. Liu, X. Zheng, L. Chen, W. Chen and Z. Fang (2019). “The protective role of autophagy in sepsis.” Microb Pathog 131: 106-111.
5. Franchini, M. and M. Montagnana (2011). “Low-density lipoprotein receptor-related protein 1: new functions for an old molecule.” Clin Chem Lab Med 49(6): 967-970.
6. Fregnan, F., L. Muratori, A. R. Simões, M. G. Giacobini-Robecchi and S. Raimondo (2012). “Role of inflammatory cytokines in peripheral nerve injury.” Neural regeneration research 7(29): 2259-2266.
7. Gali, C. C., E. Fanaee-Danesh, M. Zandl-Lang, N. M. Albrecher, C. Tam-Amersdorfer, A. Stracke, V. Sachdev, F. Reichmann, Y. Sun, A. Avdili, M. Reiter, D. Kratky, P. Holzer, A. Lass, K. K. Kandimalla and U. Panzenboeck (2019). “Amyloid-beta impairs insulin signaling by accelerating autophagy-lysosomal degradation of LRP-1 and IR-β in blood-brain barrier endothelial cells in vitro and in 3XTg-AD mice.” Molecular and Cellular Neuroscience 99: 103390.
8. Gettins, P. G. W. and S. T. Olson (2016). “Inhibitory serpins. New insights into their folding, polymerization, regulation and clearance.” The Biochemical journal 473(15): 2273-2293.
9. Griffin, D., B. Levine, W. Tyor, S. Ubol and P. Despres (1997). “The role of antibody in recovery from alphavirus encephalitis.” Immunol Rev 159: 155-161.
10. Grosso, R. A., P. V. Subirada Caldarone, M. C. Sanchez, G. A. Chiabrando, M. I. Colombo and C. M. Fader (2018). “Hemin induces autophagy in a leukemic erythroblast cell line through the LRP1 receptor.” Biosci Rep.
11. Gubler, D. J. (2002). “The global emergence/resurgence of arboviral diseases as public health problems.” Archives of Medical Research 33(4): 330-342.
12. Hashizume, H., J. A. DeLeo, R. W. Colburn and J. N. Weinstein (2000). “Spinal glial activation and cytokine expression after lumbar root injury in the rat.” Spine (Phila Pa 1976) 25(10): 1206-1217.
13. Hemonnot, A .- L., J. Hua, L. Ulmann and H. Hirbec (2019). “Microglia in Alzheimer Disease: Well-Known Targets and New Opportunities.” Frontiers in Aging Neuroscience 11(233).
14. Herz, J. and D. K. Strickland (2001). “LRP: a multifunctional scavenger and signaling receptor.” J Clin Invest 108(6): 779-784.
15. Hoffmann, M., H. Kleine-Weber, S. Schroeder, N. Krüger, T. Herrler, S. Erichsen, T. S. Schiergens, G. Herrler, N .- H. Wu, A. Nitsche, M. A. Müller, C. Drosten and S. Pöhlmann (2020). “SARS-COV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.” Cell.
16. Hoffmann, M., H. Kleine-Weber, S. Schroeder, N. Krüger, T. Herrler, S. Erichsen, T. S. Schiergens, G. Herrler, N. H. Wu, A. Nitsche, M. A. Müller, C. Drosten and S. Pohlmann (2020). “SARS-COV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor.” Cell 181(2): 271-280.e278.
17. Huang, C., Y. Wang, X. Li, L. Ren, J. Zhao, Y. Hu, L. Zhang, G. Fan, J. Xu, X. Gu, Z. Cheng, T. Yu, J. Xia, Y. Wei, W. Wu, X. Xie, W. Yin, H. Li, M. Liu, Y. Xiao, H. Gao, L. Guo, J. Xie, G. Wang, R. Jiang, Z. Gao, Q. Jin, J. Wang and B. Cao (2020). “Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.” The Lancet 395(10223): 497-506.
18. Joslin, G., R. J. Fallon, J. Bullock, S. P. Adams and D. H. Perlmutter (1991). “The SEC receptor recognizes a pentapeptide neodomain of alpha 1-antitrypsin-protease complexes.” J Biol Chem 266(17): 11282-11288.
19. Kawamura, A., D. Baitsch, R. Telgmann, R. Feuerborn, G. Weissen-Plenz, C. Hagedorn, K. Saku, S. M. Brand-Herrmann, A. von Eckardstein, G. Assmann and J. R. Nofer (2007). “Apolipoprotein E interrupts interleukin-1beta signaling in vascular smooth muscle cells.” Arterioscler Thromb Vasc Biol 27(7): 1610-1617.
20. Kehn-Hall, K., A. Narayanan, L. Lundberg, G. Sampey, C. Pinkham, I. Guendel, R. Van Duyne, S. Senina, K. L. Schultz, E. Stavale, M. J. Aman, C. Bailey and F. Kashanchi (2012). “Modulation of GSK-3beta activity in Venezuelan equine encephalitis virus infection.” PLOS One 7(4): e34761.
21. Landowski, L. M., M. Pavez, L. S. Brown, R. Gasperini, B. V. Taylor, A. K. West and L. Foa (2016). “Low-density Lipoprotein Receptor-related Proteins in a Novel Mechanism of Axon Guidance and Peripheral Nerve Regeneration.” The Journal of biological chemistry 291(3): 1092-1102.
22. Lillis, A. P., L. B. Van Duyn, J. E. Murphy-Ullrich and D. K. Strickland (2008). “LDL receptor-related protein 1: unique tissue-specific functions revealed by selective gene knockout studies.” Physiol Rev 88(3): 887-918.
23. Mantuano, E., M. S. Lam, M. Shibayama, W. M. Campana and S. L. Gonias (2015). “The NMDA receptor functions independently and as an LRP1 co-receptor to promote Schwann cell survival and migration.” Journal of Cell Science 128(18): 3478-3488.
24. Matsuyama, S., N. Nao, K. Shirato, M. Kawase, S. Saito, I. Takayama, N. Nagata, T. Sekizuka, H. Katoh, F. Kato, M. Sakata, M. Tahara, S. Kutsuna, N. Ohmagari, M. Kuroda, T. Suzuki, T. Kageyama and M. Takeda (2020). “Enhanced isolation of SARS-COV-2 by TMPRSS2-expressing cells.” Proceedings of the National Academy of Sciences 117(13): 7001.
25. May, P. (2013). “The low-density lipoprotein receptor-related protein 1 in inflammation.” Curr Opin Lipidol 24(2): 134-137.
26. May, P., H. H. Bock and J. R. Nofer (2013). “Low density receptor-related protein 1 (LRP1) promotes anti-inflammatory phenotype in murine macrophages.” Cell Tissue Res 354(3): 887-889.
27. Mehta, P., D. F. McAuley, M. Brown, E. Sanchez, R. S. Tattersall and J. J. Manson (2020). “COVID-19: consider cytokine storm syndromes and immunosuppression.” The Lancet 395(10229): 1033-1034.
28. Meyer, M. and I. Jaspers (2015). “Respiratory protease/antiprotease balance determines susceptibility to viral infection and can be modified by nutritional antioxidants.” American journal of physiology. Lung cellular and molecular physiology 308(12): L1189-L1201.
29. Mishra, A., X. Yao, A. Saxena, E. M. Gordon, M. Kaler, R. A. Cuento, A. V. Barochia, P. K. Dagur, J. P. McCoy, K. J. Keeran, K. R. Jeffries, X. Qu, Z .- X. Yu and S. J. Levine (2018). “Low-density lipoprotein receptor– related protein 1 attenuates house dust mite– induced eosinophilic airway inflammation by suppressing dendritic cell– mediated adaptive immune responses.” Journal of Allergy and Clinical Immunology 142(4): 1066-1079.e1066.
30. Muehlenbein, M. P., F. B. Cogswell, M. A. James, J. Koterski and G. V. Ludwig (2006). “Testosterone correlates with Venezuelan equine encephalitis virus infection in macaques.” Virol J 3: 19.
31. Olival, K. J. and P. Daszak (2005). “The ecology of emerging neurotropic viruses.” Journal of Neurovirology 11(5): 441-446.
32. Rauch, J. N., G. Luna, E. Guzman, M. Audouard, C. Challis, Y. E. Sibih, C. Leshuk, I. Hernandez, S. Wegmann, B. T. Hyman, V. Gradinaru, M. Kampmann and K. S. Kosik (2020). “LRP1 is a master regulator of tau uptake and spread.” Nature 580(7803): 381-385.
33. Ronca, S. E., K. T. Dineley and S. Paessler (2016). “Neurological Sequelae Resulting from Encephalitic Alphavirus Infection.” Front Microbiol 7: 959.
34. Schoneboom, B. A., K. M. Catlin, A. M. Marty and F. B. Grieder (2000). “Inflammation is a component of neurodegeneration in response to Venezuelan equine encephalitis virus infection in mice.” J Neuroimmunol 109(2): 132-146.
35. Schoneboom, B. A., J. S. Lee and F. B. Grieder (2000). “Early expression of IFN-alpha/beta and iNOS in the brains of Venezuelan equine encephalitis virus-infected mice.” J Interferon Cytokine Res 20(2): 205-215.
36. Sharma, A., B. Bhattacharya, R. K. Puri and R. K. Maheshwari (2008). “Venezuelan equine encephalitis virus infection causes modulation of inflammatory and immune response genes in mouse brain.” BMC Genomics 9: 289.
37. Sharma, A., M. Bhomia, S. P. Honnold and R. K. Maheshwari (2011). “Role of adhesion molecules and inflammation in Venezuelan equine encephalitis virus infected mouse brain.” Virology Journal 8: 197-197.
38. Shi, Y., E. Mantuano, G. Inoue, W. M. Campana and S. L. Gonias (2009). “Ligand binding to LRP1 transactivates Trk receptors by a Src family kinase-dependent pathway.” Sci Signal 2(68): ra18.
39. Shi, Y., T. Yamauchi, A. Gaultier, S. Takimoto, W. M. Campana and S. L. Gonias (2011). “Regulation of cytokine expression by Schwann cells in response to alpha2-macroglobulin binding to LRP1.” J Neurosci Res 89(4): 544-551.
40. Shiga, Y., A. Shiga, P. Mesci, H. Kwon, C. Brifault, J. H. Kim, J. J. Jeziorski, C. Nasamran, S. Ohtori, A. R. Muotri, S. L. Gonias and W. M. Campana (2019). “Tissue-type plasminogen activator-primed human iPSC-derived neural progenitor cells promote motor recovery after severe spinal cord injury.” Scientific Reports 9(1): 19291.
41. Simon, D., B. Page, M. Vogel, C. Bussmann, C. Blanchard, A. Straumann and H.-U. Simon (2018). “Evidence of an abnormal epithelial barrier in active, untreated and corticosteroid-treated eosinophilic esophagitis.” Allergy 73(1): 239-247.
42. Steele, K. E. and N. A. Twenhafel (2010). “REVIEW PAPER: pathology of animal models of alphavirus encephalitis.” Vet Pathol 47(5): 790-805.
43. Strickland, D. K., S. C. Muratoglu and T. M. Antalis (2011). “Serpin-Enzyme Receptors LDL Receptor-Related Protein 1.” Methods Enzymol 499: 17-31.
44. Subramaniyam, D., P. Glader, K. von Wachenfeldt, J. Burneckiene, T. Stevens and S. Janciauskiene (2006). “C-36 peptide, a degradation product of alpha1-antitrypsin, modulates human monocyte activation through LPS signaling pathways.” Int J Biochem Cell Biol 38(4): 563-575.
45. Taylor, K. G. and S. Paessler (2013). “Pathogenesis of Venezuelan equine encephalitis.” Vet Microbiol 167(1-2): 145-150.
46. Toldo, S., D. Austin, A. G. Mauro, E. Mezzaroma, B. W. Van Tassell, C. Marchetti, S. Carbone, S. Mogelsvang, C. Gelber and A. Abbate (2017). “Low-Density Lipoprotein Receptor-Related Protein-1 Is a Therapeutic Target in Acute Myocardial Infarction.” JACC Basic Transl Sci 2(5): 561-574.
47. Wang, Y., K. Jiang, Q. Zhang, S. Meng and C. Ding (2018). “Autophagy in Negative-Strand RNA Virus Infection.” Frontiers in Microbiology 9(206).
48. Weaver, S. C., C. Ferro, R. Barrera, J. Boshell and J. C. Navarro (2004). “Venezuelan equine encephalitis.” Annu Rev Entomol 49: 141-174.
49. Wujak, L., P. Markart and M. Wygrecka (2016). “The low density lipoprotein receptor-related protein (LRP) 1 and its function in lung diseases.” Histol Histopathol 31(7): 733-745.
50. Wujak, L., J. Schnieder, L. Schaefer and M. Wygrecka (2018). “LRP1: A chameleon receptor of lung inflammation and repair.” Matrix Biology 68-69: 366-381.
51. Yang, L., C.-C. Liu, H. Zheng, T. Kanekiyo, Y. Atagi, L. Jia, D. Wang, A. N'songo, D. Can, H. Xu, X.-F. Chen and G. Bu (2016). “LRP1 modulates the microglial immune response via regulation of JNK and NF-κB signaling pathways.” Journal of Neuroinflammation 13(1): 304.
52. Yang, X., Y. Yu, J. Xu, H. Shu, J. a. Xia, H. Liu, Y. Wu, L. Zhang, Z. Yu, M. Fang, T. Yu, Y. Wang, S. Pan, X. Zou, S. Yuan and Y. Shang (2020). “Clinical course and outcomes of critically ill patients with SARS-COV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study.” The Lancet Respiratory Medicine.
53. Yoon, C., E. A. Van Niekerk, K. Henry, T. Ishikawa, S. Orita, M. H. Tuszynski and W. M. Campana (2013). “Low-density Lipoprotein Receptor-related Protein 1 (LRP1)-dependent Cell Signaling Promotes Axonal Regeneration.” Journal of Biological Chemistry 288(37): 26557-26568.
54. Zlokovic, B. V., R. Deane, A. P. Sagare, R. D. Bell and E. A. Winkler (2010). “Low-density lipoprotein receptor-related protein-1: a serial clearance homeostatic mechanism controlling Alzheimer's amyloid β-peptide elimination from the brain.” Journal of neurochemistry 115(5): 1077-1089.
55. Zurhove, K., C. Nakajima, J. Herz, H. H. Bock and P. May (2008). “Gamma-secretase limits the inflammatory response through the processing of LRP1.” Science signaling 1(47): ra15-ra15.
This application is a continuation of U.S. patent application Ser. No. 17/753,013, filed Feb. 15, 2022, which is the 371 national stage application of PCT/US2021/040572, filed Jul. 6, 2021, which claims priority to U.S. Provisional Patent Application No. 62/705,591, filed Jul. 6, 2020, the content of which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62705591 | Jul 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17753013 | Feb 2022 | US |
| Child | 18454013 | US |
