Patent Application
20250115646
The contents of the text field submitted electronically herewith are incorporated herein by reference in their entirety: A computer readable format copy of the Sequence Listing (file name: SAMAR2017A_SL.txt, date recorded: Nov. 25, 2017, file size 289 kilobytes).
This invention relates generally to methods for optically modulating pain in animals and human. More specifically, the invention provides method for the use of opsin for modulating pain, wherein optical stimulation of specific neurons and/or other cells in targeted regions of the nervous system sensitized by opsin, using genetic technologies, leads to significant reduction of pain perception to noxious stimuli. Further, the invention relates to a method for inhibition of pain without use of exogenous opsin, wherein visual stimulation of eye (having endogenous opsin) is carried out. The invention also provides device(s) for controlled modulation neural and cellular activities in brain, eye or peripheral nervous system in order to treat different forms of chronic pain.
Modulation of neural activities by electrical and other means has enabled modulation of physiological functions. Opsins (light-sensitive ion-channel proteins) in combination with light have been used for modulation of neural activity.
Current pain relief approaches range from pharmaceutical to electrical or magnetic based deep brain stimulation and neuron ablation therapy, but there is no single treatment to alleviate different types of pain including sever migraine, phantom pain, chronic back pain and pain due to rheumatoid arthritis. In past, investigations of cellular mechanisms for physiological and pathological pain are focused mainly on the periphery and the spinal dorsal horn, considering potential less central side effects of drugs. However, despite progress achieved over many years, many forms of chronic pain are still resistant to conventional analgesics and drugs. Although there are a range of existing clinical options, a lot of patients don't respond to any of them, resulting in opioid dependence. Overprescription of opioids for chronic pain is severely costing many lives.
Different regions of central and peripheral nervous systems and specific cells are involved in pain perception, and its processing. For example, thalamus receives projections from multiple ascending pain pathways and is involved in processing of the nociceptive information before relaying the information to various cortical regions. The thalamic nuclei are involved in the sensory discriminative and affective motivational components of pain. The thalamic neurons project to the dorsal hom of spinal cord and modulates ascending nociceptive information. Changes in the gene expression, biochemistry, thalamic blood flow and responsiveness of thalamic neurons have been shown In the animal models for pain, which suggest role of the thalamus in modulating pain. Recent work has concentrated in testing the effect of electrical stimulation of the Thalamus, as a strategy for pain control. However, electrical pulses delivered via electrodes implanted in the deep brain (1, 2) lack the specificity in stimulating particular group of neurons, and thus the precise involvement of specific Thalamic neurons in pain modulation remains to be determined.
The advent of neuronal stimulation using optogenetics has enabled highly selective activation of specific as well as several types of neurons with millisecond temporal precision (3, 4) using light of different wavelengths. As compared to electrical stimulation, optogenetics is more specific and multiple types of neurons can be selectively targeted within the same region of the nervous device (5, 6). This light-assisted method of cellular stimulation eliminates the highly challenging requirement of placing electrodes in brain nuclei with relatively homogeneous group of neurons. This characteristic has also led to the emerging of optogenetics as a valuable expenmental tool and a promising approach for studying a variety of neurological disorders, such as blindness (7-10), drug-addiction (11, 12), conditioned fear (13), and Parkinsonian symptoms (14) in animal models, Channelrodopsin-2 (ChR2), a non-selective cation channel, is the most commonly used opsin for depolarization of neurons (4, 15).
Optogenetic modulation provides high cellular-specificity by introduction of light-activated molecular channels (opsins) by genetic targeting in a promoter-specific manner (4, 16, 17). In order to achieve optogenetic stimulation of specific neurons, the cells are typically transfected by a virus to express opsin, which gets activated, thus depolanzing the opsin-expressing cells when illuminated by light of specific band of light, characteristics of the opsin. For example, cells expressing ChR2 are sensitive to blue light.
To the extent that any specific disclosure in the aforementioned references or other literature may be considered to anticipate any genenc aspect of the present invention, the disclosure of the present invention should be understood to include a proviso or provisos that exclude of disclaim any such species that were previously disclosed. The aspects of the present invention, which are not anticipated by the disclosure of such literature, are also nonobvious from the disclosure of these publications, due at least in part to the unexpectedly superior results disclosed or alleged herein.
in order to meet the challenges, the inventor has created several molecules and methods for optically modulating pain in animals and human. The invention also provides device(s) for controlled modulation neural activities in brain and eye in order to treat different forms of chronic pain.
in one aspect, the disclosure provides Bioluminescent Bandwidth engineered Opsin-1 (B2EO-1, SEQ ID NO: 2) protein that, when expressed on cell membrane, excites the targeted celis upon activation by external (active) light illumination or by intrinsic bioluminescence emitted from the targeted cells themselves in presence of injected co-factors (e.g. furimazine, or analogs).
In another aspect, the disclosure provides Bioluminescent Bandwidth engineered Opsin (B2EO-2, SEQ ID NO: 3) that, when expressed on cell membrane, silences the targeted cells upon activation by external (active) light illumination or by intrinsic bioluminescence emitted from the targeted cells themselves in presence of injected co-factors.
The delivery of the B2EO opsin-genes to targeted region(s) of brain or peripheral nervous system is carried out by injection of virus carrying promoter-B2EO-1/2 or by other physical/chemical methods.
In one aspect, the disclosed invention provides method for the use of opsin for modulating pain, wherein optical stimulation of specific neurons in targeted regions of the nervous device sensitized by opsin, using genetic technologies, leads to significant reduction of pain perception to noxious stimuli.
According to another aspect of the invention, the invention includes a method for inhibition of pain without use of exogenous opsin, wherein visual stimulation of eye (having endogenous opsin) is carried out using specific wavelength and frequency of visible light.
Advantages of the present approach is the fact that it targets specific neurons and cells in targeted regions of the central and peripheral nervous systems, unlike global stimulation by existing electrical/magnetic approaches; thus generating better efficacy for modulating pain.
The present inventor investigated if optogenetic stimulation of excitatory neurons in different regions of Thalamus can modulate pain in animal model. The results presented herein show efficient pain inhibition in awakened mice subjected to optogenetic stimulation of ChR2-sensitized excitatory neurons in Thalamus. The results also demonstrated that the pain modulation is dependent on the frequency and intensity of optogenetic stimulation.
In another aspect, the present inventor demonstrated that use of different Bioluminescent Bandwidth engineered Opsins (B2EO-1, 2) leads to passive modulation of the cells by their activity-induced emission of bioluminescence, which when applied to neurons, fibroblasts, astrocytes/glia, immune cells, keratinocytes and/or vascular endothelial cells of central/peripheral nervous system leads to down-regulation of release of neurotransmitters, or release of pro-inflammatory cytokine(s); or up-regulation of endorphins and anti-inflammatory agents leading to pain inhibition.
According to yet another aspect of the invention, method of efficient pain inhibition uses eyes as optical windows to the pain perception circuits of the brain. In this method, suitable wavelength and mode of light stimulation of eye led to significant inhibition of pain in awakened animals.
In another aspect, the invention provides device(s) for controlled modulation of neural and other cellular activities in brain, eye and peripheral nervous systems in order to treat different forms of chronic pain.
In another embodiment, the present invention includes methods and uses of the B2EO-1, or B2EO-2 for treatment of pain: wherein the use comprises delivery of the B2EO-genes to different cells of different organs by either chemical, viral or physical transduction method; wherein activation of B2EO is achieved upon illumination of external light or intrinsic bioluminescence (in presence of co-factor); and wherein an effect is measured by an electrophysiology or other functional and behavioral analysis.
In one aspect, the nucleic acid has at least one of 75%, 85%, 95% or 100% identity to SEQ ID NO: 2, or 3. In another embodiment, the invention includes a vector comprising the nucleic acid having 75%, 85%, 95% or 100% identity to at least one of SEQ ID NO: 2 or 3. In one aspect, the vector is selected from an adenovirus, adeno-associated virus or lentivirus vector. In another embodiment, the present invention includes a method of treating pain composing administering to a patient in need thereof a vector comprising the nucleic acid having 75%, 85%, 95% or 100% identity to at least one of SEQ ID NO: 2 or 3.
Details associated with the embodiments described above and others are described below.
The following drawings illustrate by way of example and not limitation. For the sake of brevity and clarity, every feature of a given structure is not always labeled in every figure in which that structure appears.
Tables 1-2 show Amino acid sequences of Bioluminescent Bandwidth engineered Opsins (B2EO): B2EO-1, and B2EO-2. Table-3 shows the DNA sequences of promoter (CAG) used upstream of B2EO-sequences for targeting specific cells as an example.
Formalin: post-formalin condition in absence of light stimulation.
Formalin+Light: post-formalin condition in presence of light stimulation of Thalamus.
Formalin: post-formalin condition in absence of light stimulation.
Formalin+Light: post-formalin condition in presence of light stimulation of Thalamus.
The present disclosure will now be described more fully hereinafter with reference to the accompanying drawings, in which some exemplary embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
Pain is a major world-wide health issue leading to severe impairment of normal psychological and physical conditions of the patients. While neuropathic pain (18, 19) is caused by damage to the nervous device, nociceptive pain (20) is caused by stimulation of peripheral nerve fibers that respond to severe harmful stimuli. Under persistent activation, nociceptive transmission to the dorsal horn (21, 22) may induce neuro-pathological changes that lower the threshold (allodynia) for pain signals to be transmitted to the sensory cortex, and thus enhances pain sensitivity. In addition, it may create non-nociceptive nerve fibers to respond to pain signals leading to enhanced pain sensation in response to noxious stimuli (hyperalgesia) (22). This process is difficult to reverse or eradicate in chronic pain, once established Chronic pain is estimated to affect 25 percent of Americans and account for more than 20 percent of all physician office visits (23). Sustained inhibition of many neuropathies and most idiopathic chronic pain is rarely achieved and management of chronic pain has emerged as a significant challenge. Therefore, there is an intense need for development of new therapeutic strategies for managing chronic pain, and improve quality of life.
Chronic pain can occur in response to long-term changes in plasticity along sensory pathways in peripheral nociceptors, spinal dorsal horn, sub-cortical areas including Thalamus as well as cortical areas that are involved in the processing of painful information (24). The thalamus receives projections from multiple ascending pain pathways and is involved in processing of the nociceptive information before relaying the information to various cortical regions. For example ventrobasal complex (VB) consisting of the Ventral posteromedial nucleus (VPM) and the Ventral posterolateral nucleus (VPL), is a relay nucleus of the thalamus for nociceptive stimuli received from nociceptive nerves in sensory discriminative component and the modulation of that stimuli to the primary somatosensory cortex. Similarly, the intralaminar nuclei is involved in affective-motivational component.
The thalamic neurons project to the dorsal hom of spinal cord via Periaqueductal gray (PAG) and modulates ascending nociceptive information. Changes in the gene expression, biochemistry, thalamic blood flow and responsiveness of thalamic neurons have been shown in the animal models for pain, which suggest role of the thalamus in modulating pain. Recent work has concentrated in testing the effect of electrical stimulation (
The advent (3, 4) of neuronal stimulation using optogenetics has enabled highly selective activation of specific neurons with millisecond-temporal precision. In contrast to electrical stimulation, optogenetics is more specific (by genetic targeting) and multiple types of neurons can be targeted at the same region of the nervous device (5, 6). Optogenetic stimulation provides high temporal precision (4, 15, 25-28) by introducing light-activatable molecular channels such as channelrhodopsin-2 (ChR2) into cells by genetic targeting. In addition to higher temporal resolution, optogenetics has several advantages over electrical stimulation such as cellular specificity and minimal invasiveness (29). Light-induced activation of ChR2, a non-selective cation channel, results in depolarization of only those cells that express ChR2. Selective activation of neurons by ms-pulsed blue light has been demonstrated in culture (27), brain slices, as well as in small animals (30-33). This optogenetic activation method is very promising for controlling cellular activities in-vitro as well as in-vivo as it only requires light of moderate intensity (˜1 mW/mm2) that can be delivered from a light emitting diode (LED) (34). Thus, optogenetics is emerging as a valuable experimental tool and a promising approach for intervening variety of neurological disorders such as blindness (7-9), drug-addiction (11, 12), and conditioned fear (13) in animal models.
Many forms of chronic pain are still resistant to conventional clinical approaches that have been targeting peripheral and spinal dorsal horn. Further, use of pharmaceutical drugs (agonists and antagonists) is not highly specific for targeted population. For example, bicuculine has major disadvantages: it is a competitive inhibitor (i.e., its efficacy will depend on the local GABAergic tone of the area) and unstable. To address this, promoter-driven expression of opsin (e.g. ChR2) was achieved in specific types of neurons (e.g. excitatory pyramidal neurons,
One of the examples where Opsin has been used in the past for pain modulation is by stimulation of excitatory neurons of the Anterior Cingulate Cortex (ACC). Optogenetic stimulation of inhibitory neurons in ACC led to decreased electrical activity in ACC, and significant reduction in pain response due to skin irritation and noxious stimulation in animals (35).
The disclosed invention includes methods for optically modulating pain in animals and human. The invention also provides device(s) for controlled modulation neural and cellular activities in brain, eye and peripheral nervous systems in order to treat different forms of chronic pain.
In one aspect, the disclosed invention provides method for the use of opsin for modulating pain, wherein optical stimulation of specific neurons in targeted regions of the Thalamus sensitized by opsin, using genetic technologies, leads to significant reduction of pain perception to noxious stimuli. Advantages of this invented approach is the fact that it targets specific neurons in targeted regions of the Thalamus, unlike global stimulation by existing electrical/magnetic approaches; thus generating better efficacy for modulating pain.
The present inventor investigated if optogenetic stimulation of excitatory neurons in different regions (VAL, VPL, VM, VPMpc) of Thalamus can modulate pain in animal model. The results presented herein show efficient pain inhibition in awakened mice subjected to optogenetic stimulation of ChR2-sensitized excitatory neurons in Thalamus. The results also demonstrated that the pain modulation is dependent on the frequency and intensity of optogenetic stimulation.
In another aspect, the present inventor demonstrated that use of different Bioluminescent Bandwidth engineered Opsins (B2EO-1, 2) leads to passive modulation of the cells by their activity-induced emission of bioluminescence, which when applied to neurons, fibroblasts, astrocytes/glia, immune cells, keratinocytes and/or vascular endothelial cells of central/peripheral nervous system leads to down-regulation of release of neurotransmitters, or release of pro-inflammatory cytokine(s); or up-regulation of endorphins and anti-inflammatory agents leading to pain inhibition.
For example, B2EO-1 when delivered to inhibitory pain neurons by use of promoters such as GAD65, SST, or NPY, upon light illumination, the release of GABA from inhibitory neurons will be enhanced, which will silence activities of pyramidal neurons involved in pain. Similarly, the vascular endothelial cells in central/peripheral nervous system, can be sensitized with the B2EO-1 (SEQ ID NO: 2) by use of promoters including but not limited to human VWF, or Tie1. The release of endorphins and anti-inflammatory agents such as opioid peptide can be enhanced upon light illumination, thus reducing pain.
B2EO-2 when expressed in excitatory neurons of targeted nervous system regions by use of promoter such as CaMKIIa, Thy1, or human synapsin 1, it will down regulate release of neurotransmitters ATP, Glutamate, and/or BONF upon light illumination. Similarly, upon expression of B2EO-2 in gial cells, their activities can be silenced by light and thus, pain. The dural fibroblasts in brain are known to release pro-inflammatory cytokine(s) such as IL-6 that stimulate dural afferents and enhance hyper-excitability leading to headache. Therefore, these fibroblasts can be specifically targeted by use of promoters, human MoMLV, or Col1α1; and upon sensitization with the B2EO-2 (SEQ ID NO: 3) light controlled suppression of release of pro-inflammatory cytokine(s) can be achieved, thus reducing migraine pain.
Dorsal root Ganglion (DRG) neurons of peripheral nervous system relay nerve injury-related primary afferent input to the spinal cord. The soma of DRG neurons are surrounded by small satellite glial cells (SGCs), which are connected by gap junctions and support DRG neurons by supplying nutrients and buffering extracellular ion and neurotransmitter levels. The interaction of SGCs and neurons vía paracrine signaling promote the pain-sensitization of peripheral nerves leading to chronic pain. The astrocytes, glia including SGCs in central/peripheral nervous system can be selectively sensitized with the B2EO-2 (SEQ ID NO: 3) by use of promoters such as GFAP, MBP,CMV, or U1snRNA; and controlled by light so as to down-regulate the release of neurotransmitters and ATP, thus reducing hyperexcitability of neurons toward pain.
immediately after nerve injury in diabetes or spinal stenosis, and other events leading to neuropathic pain, the resident immune cells, mast cells and macrophages are known to be activated which release pro-inflammatory cytokines, and chemokines. The immune cells, including macrophages and/or mast cells in central/peripheral nervous system, when specifically sensitized with the B2EO-2 (SEQ ID NO: 3) using promoters such as c-kit, ST2, or IL1RL1, their activities and thus release of pro-inflammatory cytokines can be controlled by light activation of B2EO-2. This leads to reduction of the release of histamine and pro-inflammatory reagents, thus reducing pain.
In another embodiment, the present invention includes methods and uses of the B2EO-1, or B2EO-2 for treatment of pain: wherein the use comprises delivery of the B2EO-genes to different cells of different organs by either chemical, viral or physical transduction method; wherein activation of B2EO is achieved upon intrinsic bioluminescence (in presence of co-factor such as furimazine or its analogs); and wherein an effect is measured by an electrophysiology or other functional and behavioral analysis.
According to yet another aspect of the invention, method of efficient pain inhibition uses eyes as optical windows to the pain perception circuits of the brain. In this method, suitable wavelength and mode of light stimulation of eye led to significant inhibition of pain in awakened animals.
In another aspect, the invention provides device(s) for controlled modulation of neural and other cellular activities in brain, eye and peripheral nervous systems in order to treat different forms of chronic pain.
According to another aspect of the invention, the invention includes a method for inhibition of pain without use of exogenous opsin, wherein visual stimulation of eye (having endogenous opsin) is carried out using specific wavelength and frequency of visible light. In this method, suitable wavelength and mode of light stimulation of eye led to significant inhibition of pain in awakened animals. The advantage of this aspect of the invention is the use of eyes as optical window to the pain perception circuits of the brain and use suitable wavelength(s) and mode of light stimulation of eye for pain modulation. Further, visual stimulation was observed to modulate sleep patterns in animals.
In another aspect, the invention provides device(s) for controlled modulation neural activities in brain and eye in order to treat different forms of chronic pain.
Below, the presently disclosed invention will be further described by way of examples, which are provided for illustrative purposes only and accordingly are not to be construed as limiting the scope of the invention.
Example 1—
Example 2—
Example 3—
Example 4—Pain assay using skin-irritant (Formalin) was carried out to evaluate the effect of light stimulation of thalamus. The formalin test is a widely used tonic model of continuous pain involving neurogenic, inflammatory, and central mechanisms of nociception. Both transgenic and wild type mice were randomly assigned to receive either sham stimulation or laser stimulation. Mice generated from breeding of Thy1-ChR2 male mouse with two female wild type mice was used for the assay. Behavioral testing was performed by an observer unaware of the expression of ChR2 in the mice. Each mouse was randomly assigned to receive light stimulation or sham stimulation. Therefore the mice were allocated to one of two groups (1: mice without light treatment; 2: mice with light stimulation). Approximately 7 days after implanting the fiber stub, the fiber coupled to LED (emission wavelength: 465 nm) was aligned to couple to the fiber stub. A pre-formalin baseline test was performed in which all mice received sham stimulation (no light) for 60 min. The time the mice spent on lifting and licking the paw was calculated and the pain sensation was scored as follows: paw lifting as 1 and paw licking as 2. Then, another baseline (with light alone, but no formalin) pain scoring was carried out. After these two pre-formalin baselines, the fiber coupled to LED was removed and mice returned to their cages.
After more than 2 days in the cages, the mice were subjected to the formalin test (40) where each mouse tested individually in an observation chamber for the 60 min test period. As with the pre-formalin test, the fiber coupled to LED was aligned to couple to the fiber stub. Animals were then administered a 20 μL subcutaneous injection of 1% formalin into the dorsal hind paw of the mouse using a micro-syringe with a 26-gauge needle. The light stimulation (or sham stimulation in interval of more than 2 days) was then applied and the amount of time the animal spent lifting or licking the injected paw was recorded during the 60 minute test period. The power of light delivered from the fiber was set at 10 mW. The light flash continued with repetition rate of 10 Hz and pulse width of 10 ms (i.e., 10% duty cycle during each stimulation cycle).
Example 5—To evaluate whether or not optogenetic stimulation of neurons in the Thalamus would modulate pain behavior, the formalin test assay of acute inflammatory pain (40) was conducted 1 wk after implantation of the fiber stub.
As expected, the pain scores following the formalin injection (
Example 6—Following behavioral measurements, mice were sacrificed and brains were removed carefully and post-fixed in 4% PFA for 24 to 48 hrs, and subsequently transferred to PBS. The brain tissues were sliced at 500 μm sections using Bain slicer (Zivic Instruments). Slices were then mounted on slides and the location of the fiber stub in each slice was examined “blind” to behavioral outcome and group designation for all mice using a upright laser scanning confocal microscope (Olympus Fluoview 1000) with a 2.5× objective.
Optogenetic stimulation of specific Thalamic and other deep brain regions created tremor and paralysis of awakened animals. The strength of tremor and paralysis was dependent on intensity and frequency of optogenetic stimulation of brain. Further, optogenetic stimulation of specific Thalamic and other deep brain regions created roll and yaw motion of the head.
Example 7—Here, histological (
Example 8—In order to translate optogenetic stimulation for pain inhibition, an implantable optical neural stimulator device is proposed which comprises of an implantable light source and/or waveguide carrying stimulation light designed to be permanently inserted in to targeted region(s) so as to deliver light to the targeted nervous system region(s) that has been genetically modified to express an opsin.
An implantable power supply (in the skull, below the skin), drives the implantable light source to generate pulses of light when triggered by the patient or health care provider until switched off manually or by a pre-set program. The implantable power supply is coupled to one or more implantable inductive coils configured to receive magnetic flux from a transcutaneous magnetic flux source configured to recharge the implantable power supply The controller coupled to the light source, is also implanted and configured to result in controlled light intensity sufficient to elicit or inhibit activities in specific neurons or other cell types pre-sensitized with an opsin in targeted region(s) of the nervous system(s). The external controller (8060) is configured to wirelessly communicate with the implantable unit(s). The controller also communicates with an implantable photo-sensor that can measure intensity of delivered light inside the tissue and thus determine undesired loss that may occur during transmission from light source(s) via the waveguide(s). The implantable photo-sensor can be either a photodiode, a photovoltaic cell, a pyroelectric sensor, a photoresistor, a phototransistor, or a photoconductor; and is positioned with respect to the light-delivering waveguide (8050) so as to get a small fraction of incident light. The controller (8060) is designed to act in response to an undesired light loss level above a preset threshold by flagging the event in a software log file, and in case of an undesired light loss level above a preset threshold, the controller is programmed to stop the power supplied to the light source(s). Furthermore, cellular activity sensor (impedance sensor, a capacitance sensor, an electroneurogram sensor, or an electroencephalogram sensor) is placed near the targeted tissue to measure the light-activated cellular signal from the targeted nervous system region(s). The controller (8060) is designed to act in response to a preset out-of-range signal detected by the cellular activity sensor by stopping the power supplied to the light source (8040).
Example 9—
Dorsal root Ganglion (DRG) neurons of peripheral nervous system relay nerve injury-related primary afferent input to the spinal cord. The soma of DRG neurons are surrounded by small satellite glial cells (SGCs), which are connected by gap junctions and support DRG neurons by supplying nutrients and buffering extracellular ion and neurotransmitter levels. The interaction of SGCs and neurons via paracrine signaling promote the pain-sensitization of peripheral nerves leading to chronic pain. The astrocytes, glia including SGCs in central/peripheral nervous system can be selectively sensitized with the B2EO-2 (SEQ ID: 3) by use of promoters such as GFAP, MBP,CMV, or U1snRNA: and controlled by self-generated bioluminescent light so as to down-regulate the release of neurotransmitters and ATP, thus reducing hyperexcitability of neurons toward pain. Similarly, the dural fibroblasts in brain are known to release pro-inflammatory cytokine(s) such as IL-6 that stimulate dural afferents and enhance hyper-excitability leading to headache. Therefore, these fibroblasts are specifically targeted by use of promoters, human MoMLV, or Col1α1; and upon sensitization with the B2EO-2 (SEQ ID: 3), self-generated bioluminescent light controlled suppression of release of pro-inflammatory cytokine(s) is achieved, thus reducing migraine pain.
Example 10—
B2EO-1 when delivered to inhibitory pain neurons by use of promoters such as GAD65, SST, or NPY, upon intrinsic bioluminescence generation, the release of GABA from inhibitory neurons is enhanced, which silences the activities of pyramidal neurons involved in pain. Similarly, the vascular endothelial cells in central/peripheral nervous system, can be sensitized with the B2EO-1 (SEQ ID: 2) by use of promoters including but not limited to human VWF, or Tie1. The release of endorphins and anti-inflammatory agents such as opioid peptide is enhanced upon bioluminescence light generation, thus reducing pain.
Example 11—
Immediately after nerve injury in diabetes or spinal stenosis, and other events leading to neuropathic pain, the resident immune cells, mast cells and macrophages are known to be activated which release pro-inflammatory cytokines, and chemokines. The immune cells, including macrophages and/or mast cells in central/peripheral nervous system, when specifically sensitized with the B2EO-2 (SEQ ID: 3) using promoters such as c-kit, ST2, or IL1RL1, their activities and thus release of pro-inflammatory cytokines is controlled by bioluminescence light activation of B2EO-2. This leads to reduction of the release of histamine and pro-inflammatory reagents, thus reducing pain.
Example 12—In order to evaluate the whether pain can be inhibited by visual stimulation (using eyes as optical windows to the pain perception circuits of the brain), different wavelength bands and mode of light stimulation (e.g. frequency) was examined. The mice were individually placed inside optically-transparent cages (with water and food) between two counter-propagating light beams.
In the present invention, use of blue light (400-500 nm) was avoided to minimize damage to retina via heating and photo-chemical toxicity. Further, the band 550-600 nm was excluded to minimize absorption by blood (and thus avoid any potential damage to eye). The spectrum of the optimized strobe light used for modulating pain is shown in
Example 13—To evaluate whether or not optical stimulation of photoreceptors in the retina would modulate pain behavior, the formalin test assay of acute inflammatory pain (40) was conducted.
Example 14—
Example 15—1410: in absence of intact photosensitivity of retina (in case of no light perception due to retinal degenerative diseases), the optical retinal stimulator may not realize the goal of pain inhibition. In order to evaluate the effect of optical retinal stimulator in inhibiting pain of photo-sensitized retina, mice with ChR2-opsin expression in retinal ganglion cells are used.
The terms “a” and “an” are defined as one or more unless this disclosure explicitly requires otherwise. The term “substantially” is defined as largely but not necessarily wholly what is specified (and includes what is specified; e.g., substantially 90 degrees includes 90 degrees and substantially parallel includes parallel), as understood by a person of ordinary skill in the art. In any disclosed embodiment, the terms “substantially,” “approximately,” and “about” may be substituted with “within [a percentage] of” what is specified, where the percentage includes 0.1, 1, 5, and 10 percent.
Further, a molecule or method that is configured in a certain way is configured in at least that way, but it can also be configured in other ways than those specifically described.
The terms “comprise” (and any form of comprise, such as “comprises” and “comprising”), “have” (and any form of have, such as “has” and “having”), “include” (and any form of include, such as “includes” and “including”) and “contain” (and any form of contain, such as “contains” and “containing”) are open-ended linking verbs. As a result, an apparatus that “comprises,” “has,” “includes” or “contains” one or more elements possesses those one or more elements, but is not limited to possessing only those elements. Likewise, a method that “comprises,” “has,” “includes” or “contains” one or more steps possesses those one or more steps, but is not limited to possessing only those one or more steps.
Any embodiment of any of the apparatuses, systems, and methods can consist of or consist essentially of—rather than comprise/include/contain/have—any of the described steps, elements, and/or features. Thus, in any of the claims, the term “consisting of” or “consisting essentially of” can be substituted for any of the open-ended linking verbs recited above, in order to change the scope of a given claim from what it would otherwise be using the open-ended linking verb.
The feature or features of one embodiment may be applied to other embodiments, even though not described or illustrated, unless expressly prohibited by this disclosure or the nature of the embodiments.
Some references, which may include publications, patents, and patent applications, are cited and discussed in the description of this invention. The citation and/or discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any such reference is “prior art” to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference were individually incorporated by reference.
The specification and examples herein provide a complete description of the structure and use of illustrative embodiments Although certain embodiments have been described with a certain degree of particularity, or with reference to one or more individual embodiments, those skilled in the art could make numerous alterations to the disclosed embodiments without departing from the scope of this invention. As such, the various illustrative embodiments of the devices are not intended to be limited to the particular forms disclosed. Rather, they include all modifications and alternatives falling within the scope of the claims, and embodiments other than the one shown may include some or all of the features of the depicted embodiment. For example, components may be omitted or combined as a unitary structure, and/or connections may be substituted. Further, where appropriate, aspects of any of the examples described above may be combined with aspects of any of the other examples described to form further examples having comparable or different properties and addressing the same or different problems. Similarly, it will be understood that the benefits and advantages described above may relate to one embodiment or may relate to several embodiments.
Furthermore, the claims are not intended to include, and should not be interpreted to include, means-plus- or step-plus-function limitations, unless such a limitation is explicitly recited in a given claim using the phrase(s) “means for” or “step for,” respectively.
To the extent that any specific disclosure in the references or other literature may be considered to anticipate any generic aspect of the present invention, the disclosure of the present invention should be understood to include a proviso or provisos that exclude of disclaim any such species that were previously disclosed. The aspects of the present invention, which are not anticipated by the disclosure of such literature, are also nonobvious from the disclosure of these publications, due at least in part to the unexpectedly superior results disclosed herein.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth above, are specifically incorporated by reference.
This application claims the benefit of U.S. provisional application No. 62/426,402 filed Nov. 25, 2016, which application is incorporated herein by reference.
This invention was made with private funding by NanoScope Technologies, LLC. The government has no rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62426402 | Nov 2016 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15822185 | Nov 2017 | US |
| Child | 18887005 | US |
