WO2023133562A1 - Methods of treating pain - Google Patents

Abstract

This disclosure features chemical entities and compositions containing the same that are useful for treating migraine (e.g., migraine pain) and neuralgia.

Description

Methods of Treating Pain CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of United States Provisional Application 63/297,589, filed on January 7, 2022. TECHNICAL FIELD This disclosure features chemical entities and compositions containing the same that are useful for treating migraine (e.g., migraine pain) and neuralgia. BACKGROUND Cutaneous tissue injury elicits a local vascular response, referred to as neurogenic inflammation, that is associated to a wider area of increased sensitivity to mechanical stimuli¹. A subset of C-fiber primary afferents, which mediate neurogenic inflammation, is the main source of the neuropeptides substance P (SP) and calcitonin gene related peptide (CGRP)^2,3. In rodents, noxious stimuli such as capsaicin, a pungent agonist of the transient receptor potential vanilloid 1 (TRPV1) channel⁴, evoke peripheral release of CGRP which induces arteriolar vasodilatation² and of SP which elicits plasma protein extravasation⁵, and produce sensory responses, which encompasses acute nociception and prolonged mechanical allodynia⁶. Capsaicin administration to the human skin elicits a similar pattern of responses, consisting of local cutaneous vasodilatation and focal and transient burning pain (min) associated with widespread, sustained mechanical hypersensitivity (hrs)⁷. While CGRP has been identified as the mediator of neurogenic vasodilatation in rodents² and humans⁸, the cellular and molecular mechanisms underlying mechanical allodynia associated with neurogenic inflammation are unknown. Mechanistic studies in animal models and humans have highlighted the role of CGRP in migraine pain⁹. Thus, small molecule antagonists of the CGRP receptor and monoclonal antibodies against CGRP or its receptor can relieve migraine pain¹⁰. The poor blood brain barrier penetration of some small molecule antagonists^11,12 and of monoclonal antibodies^13,14 suggests a peripheral contribution to CGRP-mediated migraine pain. However, little is known about the proalgesic actions of CGRP in the periphery. In mice, intraplantar injection of CGRP evokes mechanical allodynia¹⁵ and systemic CGRP causes facial grimace¹⁶. Periorbital CGRP injection, while failing to evoke spontaneous nociceptive behavior, produces sustained (~4 h) periorbital mechanical allodynia (PMA)¹⁷. CGRP released from trigeminal peripheral terminals mediates PMA in mice¹⁸ evoked by systemic (intraperitoneal) administration of the pro-headache agent glyceryl trinitrate (GTN)¹⁹. Facial cutaneous allodynia is one component of the migraine attack^20,21. Although the process that initiates migraine pain may originate in the central nervous system (CNS)^22,23, the cell type and signaling pathway by which CGRP acts in the periphery to cause pain are unknown. The CGRP receptor is a heterodimer of calcitonin receptor-like receptor (CLR), a G protein-coupled receptor (GPCR), and receptor activity-modifying protein 1 (RAMP1), a single transmembrane domain CLR chaperone²⁴. These two components coexist in cells that mediate the actions of CGRP, for example vascular myocytes². Satellite glial cells and Schwann cells express CLR/RAMP1 and are closely associated with peptidergic sensory neurons²⁵. While the extracellular space between the soma of trigeminal neurons and satellite glial cells is not a recognized locus for neurotransmission, the varicosities of C- fibers and the ensheathing Schwann cells are sites where neuropeptides, including CGRP²⁶, are normally released. Schwann cells from rat sciatic nerve respond to CGRP by increasing intracellular cAMP levels²⁷ and CLR/RAMP1 are expressed by Schwann cells that wrap CGRP+ve terminals of rat nociceptors^25,28,29. Schwann cells mediate mechanical allodynia in mouse models of neuropathic and cancer pain^30,31. Cutaneous Schwann cells can also directly activate sensory nerves to promote mechanical nociception³². Although GPCRs are usually considered to signal principally from the plasma membrane, GPCR kinases and E-arrestins (EARRs) rapidly terminate this signaling. Persistent endosomal signaling of GPCRs, including CLR/RAMP1, underlies sustained neuronal activation and nociception in the central nervous system (CNS)^33,34,35. Herein, it was hypothesized that mechanical allodynia associated with neurogenic inflammation is mediated by CGRP which targets CLR/RAMP1 in Schwann cells ensheathing peripheral endings of nociceptors. By selective RAMP1 gene deletion in Schwann cells, it was revealed that CGRP released from trigeminal terminals causes PMA by paracrine signaling to the surrounding Schwann cells. It was also hypothesized that persistent CGRP/CLR/RAMP1 signaling from endosomes in Schwann cells underlies sustained PMA. By using inhibitors of clathrin- and dynamin-mediated endocytosis and stimulus-responsive nanoparticles designed to release CLR/RAMP1 antagonists in acidified endosomes, it was found that CLR/RAMP1 endosomal signaling results in a cAMP-dependent release of nitric oxide (NO), which activates transient receptor potential ankyrin 1 (TRPA1), a proalgesic channel and sensor of oxidative stress³⁶. SUMMARY This disclosure features chemical entities and compositions containing the same that are useful for treating migraine (e.g., migraine pain) and neuralgia. In one aspect, this disclosure features methods for treating a condition selected from the group consisting of migraine (e.g., migraine pain) and neuralgia in a subject, the methods include administering to the subject an effective amount of an agent that selectively targets and inhibits endosomal CGRP receptor signaling in a Schwann cell in the subject. In one aspect, this disclosure features methods for treating a condition selected from the group consisting of migraine (e.g., migraine pain) and neuralgia in in a subject, the methods include contacting a Schwann cell in the subject with an effective amount of an agent that selectively targets and inhibits endosomal CGRP receptor signaling in the Schwann cell. In some embodiments, the condition is migraine (e.g., migraine pain). In certain embodiments, the migraine pain is CGRP-mediated migraine pain. In some embodiments, the condition is neuralgia (e.g., trigeminal neuralgia). The methods include treating one or more symptoms associated with migraine or neuralgia. In an aspect, the agent is an agent that inhibits endocytosis of the Schwann cell CGRP receptor. In certain embodiments, the agent is an inhibitor of a process selected from the group consisting of clathrin-mediated endocytosis; clathrin-independent endocytosis; caveolae-mediated endocytosis; micropinocytosis; dynamin-mediated endocytosis; dynamin-independent endocytosis; endosome maturation; and β-arrestin-mediated endocytosis. In certain embodiments, the agent is an inhibitor of a process selected from the group consisting of clathrin-mediated endocytosis; dynamin-mediated endocytosis; and β- arrestin-mediated endocytosis. In certain embodiments, the agent is an inhibitor of a process selected from the group consisting of dynamin 1-dependent endocytosis and dynamin 2-dependent endocytosis. In certain embodiments, the agent is an inhibitor of dynamin 2-mediated endocytosis. In some embodiments, the Schwann cell CGRP receptor is an activated Schwann cell CGRP receptor. In certain embodiments, the activated Schwann cell CGRP receptor is activated by interaction with CGRP. In certain embodiments, the CGRP is released from a TRPV1 nerve fiber surrounding the Schwann cell. The CGRP can be endogenous. The CGRP can be exogenous. In some embodiments, the agent achieves about 10%, or about 20% , or about 30%, or about 40%, or about 50%, about 60% or about 70% or about 80% or about 90% or about 95% or about 99%, or about 10% to about 50%, or about 50% to about 90% inhibition of endocytosis of the Schwann cell CGRP receptor. By way of example, in some implementations the agent can include an nucleic acid molecule targeting Dynamin 2, such as an antisense oligonucleotide targeting Dynamin-2 resulting in decreased mRNA or a siRNA targeting Dynamin-2. For the avoidance of doubt, as used herein and unless otherwise modified, the term “dynamin” is intended to include all isoforms, including but not limited to, dynamin 1, 2, and 3. The term “dynamin” includes all human isoforms. In an aspect, the agent is a CLR/RAMP1 antagonist. In some embodiments, the CLR/RAMP1 antagonist is encapsulated in a nanoparticle that is structurally predisposed to release the CLR/RAMP1 antagonist in the endosome. Examples of suitable nanoparticles can be found, e.g., in WO 2020/084471, which is incorporated herein by reference in its entirety. In some embodiments, the CLR/RAMP1 antagonist includes a lipid anchor that promotes insertion of the antagonist into a plasma membrane (optionally the antagonist and lipid anchor are connect by a linker, thereby forming a tripartite compound). Examples of suitable lipid anchors and linkers as well as methods of using the same to form a tripartite compound can be found, e.g., in WO 2017/112792, which is incorporated herein by reference in its entirety. In some embodiments, the agent includes a targeting moiety having an affinity for binding to the Schwann cell, e.g., the agent includes a targeting moiety having an affinity for binding to a plasma membrane-expressed moiety on the Schwann cell. In some embodiments, the targeting moiety selectively binds to a plasma membrane-expressed protein on the Schwann cell and triggers endocytosis of this protein. In certain embodiments, the targeting moiety selectively binds to a plasma membrane-expressed CLR/RAMP1 on the Schwann cell and triggers endocytosis of this protein. In certain embodiments, the targeting moiety selectively binds to plasma membrane-expressed CLR on the Schwann cell. In certain embodiments, the targeting moiety selectively binds to plasma membrane-expressed RAMP1 on the Schwann cell. In certain embodiments, the targeting moiety binds (e.g., selectively binds) to an interface created by the CLR/RAMP-1 heterodimer on the Schwann cell. Non-limiting examples of targeting moieties are described herein. In certain embodiments, the targeting moiety is or includes an antibody (e.g., monoclonal antibody. By way of example, the agent can be an antibody-nucleic acid molecule conjugate, such as an antibody-antisense oligonucleotide conjugate or an antibody-siRNA conjugate. In certain embodiments, the agent is a peripherally restricted agent. In some embodiments, upon administration of the agent to the patient, the level of reactive oxygen species released in the presence of the agent is less than the level of reactive oxygen species released in the absence of the agent. Methods for measuring the level of reactive oxygen species (directly or indirectly) are known to those skilled in the art. For example, the level of reactive oxygen species released in the absence and presence of the agent can be determined by measuring the activity of nitric oxide synthase, measuring the amount of reactive oxygen species released, and/or measuring the amount of biomarkers of oxidative stress present. In certain embodiments, the reactive oxygen species is nitric oxide (NO). In certain embodiments, the level of reactive oxygen species released in the presence of the agent is about 5 times less, about 10 times less, about 50 times less, or about 100 times less than the level of reactive oxygen species released in the absence of the agent. In some embodiments, upon administration of the agent to the subject, the level of interaction between Schwann cell TRPA1/NOX and a reactive oxygen species in the presence of the agent is less than the level of interaction between Schwann cell TRPA1/NOX and a reactive oxygen species in the absence of the agent. In some embodiments, the subject exhibits one or more of the following symptoms: medium to strong predominantly unilateral headache, light, noise and/or smell sensitivity, nausea or sickness, facial pain, sore eyes, balance disturbance, word finding difficulties, other neurological symptoms, such as sensory or motor disturbances, allodynia or any other of the features known to accompany, precede or follow a migraine attack, such as fatigue, nausea, cognitive difficulties, tiredness, ravenous hunger or thirst, reduced libido, depression, mania, mood swings, as well as changes in brain structure and function, such as white matter lesions or disturbances in functional connectivity. In certain embodiments, the subject exhibits one or more of the following symptoms: medium to strong predominantly unilateral headache, light, noise and/or smell sensitivity, nausea, facial pain, sore eyes, balance disturbance, and cognitive difficulties. In certain of these embodiments, the method further includes evaluating the severity of the one or more symptoms. Evaluating can include, for example, administering to the subject a clinician-administered instrument of evaluation (e.g., Patient Perception of Migraine Questionnaire (PPMQ-R); 6-item Headache Impact Test (HIT-6) disability score; 12-Item Short Form Health Survey (SF-12) score; Patient Global Impression of Change (PGIC) score; and/or Sport ConCuSSion ASSeSment tool 3 (SCAT-3) score). In certain embodiments evaluating includes providing the subject with a validated self-reporting instrument of evaluation (e.g., four-point pain scale (none, mild, moderate, severe) and/or the 11-point pain scale (0 = no pain, 10 = pain as bad as it could be). In certain embodiments, the severity and/or frequency of at least one of the symptoms is decreased after the first or subsequent administration of the agent. In some embodiments, the subject is suffering from 1 to 31 migraine days per month. In some embodiments, the subject is suffering from migraine with aura. In some embodiments, the subject is suffering from migraine without aura. In some embodiments, administration of the agent decreases migraine attack frequency experienced by the subject from a pre-administration level. For example, administration of the agent decreases the number of migraine days per month and/or headache hours per month experienced by the subject from a pre-administration level. As another example, administration of the agent decreases migraine attack severity experienced by the subject from a pre-administration level. In certain embodiments, the subject experiences a reduction of about 30% or greater in mean pain score and/or a reduction in the use of any acute headache medications from a pre-administration level. In certain embodiments, administration of the agent decreases the number and/or severity of one or more neurological symptoms associated with migraine experienced by the subject from a pre-administration level. Examples of the one or more neurological symptoms include those selected from the group consisting of phono-, photo-, and/or osmophobia, visual, sensory or motor disturbances, and allodynia. In certain embodiments, administration of the agent decreases the number and/or severity of one or more symptoms known to accompany, precede or follow a migraine attack from a pre-administration level. Examples of the one or more symptoms known to accompany, precede or follow a migraine attack include those selected from the group consisting of fatigue, nausea, cognitive difficulties, tiredness, ravenous hunger or thirst, muscle ache, reduced libido, depression, mania, and mood swings. In certain embodiments, administration of the agent reverses, slows the progression of, and/or prevents structural or functional nerve cell damage, such as white matter lesions or disturbances in functional connectivity, associated with migraine from a pre- administration level. In certain embodiments, administration of the agent reverses, slows the progression of, and/or prevents the transition of acute migraine to chronic migraine. In some embodiments, upon administration of the agent to the subject, the level of severity of allodynia in the presence of the agent is less than the level of severity of allodynia in the absence of the agent. I am unsure of how migraine pain is graded. In certain embodiments, in the allodynia is facial allodynia. In certain embodiments, the allodynia is periorbital mechanical allodynia. For example, the periorbital mechanical allodynia can be CGRP-mediated. As another example, the periorbital mechanical allodynia can be capsaicin-mediated. In certain embodiments, the agent is administered before onset of symptoms of a migraine attack. In other embodiments, the agent is administered concurrently with, or after, the onset of symptoms of a migraine attack. In certain embodiments, the agent is administered to the trigeminal region of the subject. For example, the agent can be administered locally to the trigeminal region of the subject. As a further example, the agent can be administered intranasally. In some embodiments, the migraine pain is refractory or resistant to other traditional or conventional therapies. In some embodiments, the agent is administered in combination with one or more additional therapies, e.g., one or more additional therapeutic agents. Non-limiting examples of additional therapeutic agents include one or more of: (i) an opioid analgesic, e.g., morphine, heroin, hydromorphone, oxymorphone, levorphanol, levallorphan, methadone, meperidine, fentanyl, cocaine, codeine, dihydrocodeine, oxycodone, hydrocodone, propoxyphene, nalmefene, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine or pentazocine; (ii) a nonsteroidal antiinflammatory drug (NSAID), e.g., aspirin, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tolmetin or zomepirac, cyclooxygenase-2 (COX-2) inhibitors, celecoxib; rofecoxib; meloxicam; JTE-522; L-745,337; NS398; or a pharmaceutically acceptable salt thereof; (iii) a barbiturate sedative, e.g., amobarbital, aprobarbital, butabarbital, butabital (including butalbital combinations, e.g., butalbital/aspirin/caffeine (Fiorinal.RTM., Actavis) or butalbital/paracetamol/caffeine (Fioricet.RTM., Cardinal Health)), mephobarbital, metharbital, methohexital, pentobarbital, phenobartital, secobarbital, talbutal, theamylal or thiopental; or a pharmaceutically acceptable salt thereof; (iv) a barbiturate analgesic, e.g., butalbital or a pharmaceutically acceptable salt thereof or a composition comprising butalbital. (v) a benzodiazepine having a sedative action, e.g., chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, or triazolam or a pharmaceutically acceptable salt thereof; (vi) an H1 antagonist having a sedative action, e.g., diphenhydramine, pyrilamine, promethazine, chlorpheniramine, or chlorcyclizine or a pharmaceutically acceptable salt thereof; (vii) a sedative such as glutethimide, meprobamate, methaqualone or dichloralphenazone or a pharmaceutically acceptable salt thereof; (viii) a skeletal muscle relaxant, e.g., baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, methocarbamol or orphrenadine or a pharmaceutically acceptable salt thereof; (ix) an NMDA receptor antagonist, e.g., dextromethorphan ((+)-3-hydroxy-N- methylmorphinan) or its metabolite dextrorphan ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine, pyrroloquinoline quinone or cis-4-(phosphonomethyl)-2- piperidinecarboxylic acid or a pharmaceutically acceptable salt thereof; (x) an alpha- adrenergic, e.g., doxazosin, tamsulosin, clonidine or 4-amino-6,7-dimethoxy-2-(5- methanesulfonamido-1,2,3,4-tetrahydroisoquinol- -2-yl)-5-(2-pyridyl) quinazoline; (xi) a COX-2 inhibitor, e.g., celecoxib, rofecoxib or valdecoxib; (xii) a coal-tar analgesic, in particular paracetamol; (xiii) a neuroleptic such as droperidol; (xiv) a vanilloid receptor agonist (e.g., resinferatoxin) or antagonist (e.g., capsazepine); (xv) a local anaesthetic, such as mexiletine; (xxii) a corticosteroid, such as dexamethasone; (xxiii) a serotonin receptor agonist or antagonist; (xxiv) a cholinergic (nicotinic) analgesic; (xxv) tramadol; (xxvi) a PDEV inhibitor, such as sildenafil, vardenafil or taladafil; (xxvii) an alpha-2-delta ligand such as gabapentin or pregabalin; and (xxviii) a cannabinoid. In some embodiments, the subject is a human. In some embodiments, the method further comprises identifying the subject in need of such treatment. This disclosure contemplates both in vitro and in vivo methods. In another aspect, this disclosure features methods of identifying a candidate agent for treating migraine pain, the method comprising: (i) providing a Schwann cell comprising a CGRP-activated CLR/RAMP1; (ii) contacting the Schwann cell comprising the CGRP-activated CLR/RAMP1 with the candidate agent; and (iii) measuring a parameter in the presence and absence of the agent. In certain embodiments, step (iii) includes (A) determining the level of reactive oxygen species released in the presence and absence of the candidate agent; and/or (B) determining the level of severity of allodynia in the presence of the candidate agent; and/or (C) determining the degree of light aversion. A candidate agent can be identified when the level of (A) and/or (B) and/or (C) in the presence of the candidate agent is less than the level of (A) and/or (B) and/or (C) in the absence of the candidate agent. In a further aspect, this disclosure features methods for inhibiting release of reactive oxygen species that activate pain-signaling nociceptors in a subject comprising administering to the subject an agent that selectively targets and inhibits endosomal CGRP receptor signaling in a Schwann cell. In still another aspect, this disclosure features methods for inhibiting release of reactive oxygen species that activate pain-signaling nociceptors in a subject comprising contacting a Schwann cell in the subject with an agent that selectively targets and inhibits endosomal CGRP receptor signaling in the Schwann cell. In one aspect, this disclosure provides chemical entities that include (i) a targeting moiety that selectively targets a Schwann cell; and (ii) a moiety that inhibits endosomal CGRP receptor signaling in the Schwann cell. In embodiments, (i) and (ii) are covalently connected. An "individual" or a "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. As used herein, the term "calcitonin gene-related peptide" and "CGRP" refers to any form of calcitonin gene-related peptide and variants thereof that retain at least part of the activity of CGRP. For example, CGRP may be .alpha.-CGRP or .beta.-CGRP. As used herein, CGRP includes all mammalian species of native sequence CGRP, e.g., human, canine, feline, equine, and bovine. As used herein, an "effective dosage" or "effective amount" of drug, compound, or pharmaceutical composition is an amount sufficient to effect beneficial or desired results. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing pain intensity, duration, or frequency of refractory migraine attack, and decreasing one or more symptoms resulting from refractory migraine (biochemical, histological and/or behavioral), including its complications and intermediate pathological phenotypes presenting during development of the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication, and/or delaying the progression of the disease of patients. An effective dosage can be administered in one or more administrations. For purposes of this disclosure, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. The terms “treat,” “treating,” and “treatment,” in the context of treating a disease or disorder, are meant to include alleviating or abrogating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition; or to slowing the progression, spread or worsening of a disease, disorder or condition or of one or more symptoms thereof. BRIEF DESCRIPTION OF DRAWINGS FIGs. 1a-1h show that Schwann cell RAMP1 mediates PMA evoked by CGRP. (Fig. 1a) Representative real-time PCR plot and cumulative data for GAPDH, S100, CLR and RAMP1 mRNA in HSCs and MSCs from trigeminal or sciatic nerve (n=3-4 independent experiments). (Fig. 1b) Representative images of DAPI and immunoreactive S100, RAMP1 and CLR in human and mouse cutaneous nerve bundles (scale, 10 μm human, 50 μm mouse) (n=3 subjects). (Fig. 1c) PMA induced by CGRP (1.5 nmol) or vehicle in C57BL/6J male and female mice (n=8 mice per group). (Fig. 1d) PMA induced by CGRP (1.5 nmol) or vehicle in male and female Plp1-Cre^ERT+/Ramp1^fl/fl and Control mice (n=8 mice per group) treated with periorbital 4-OHT. (Fig. 1e) Representative images and colocalization value (Rcoloc) of S100 and RAMP1 in periorbital nerve and sciatic nerve trunks from Plp1-Cre^ERT+;Ramp1^fl/fl and Control mice (scale: 20 μm) (n=3 replicates). TN (trigeminal nerve), SN (sciatic nerve). (Fig. 1f) PMA and paw mechanical allodynia induced by intraperitoneal (i.p.) CGRP (0.1 mg/kg) or vehicle in male and female C57BL/6J mice. (Fig. 1g) PMA and (Fig. 1h) paw mechanical allodynia induced by intraperitoneal (i.p.) CGRP (0.1 mg/kg) or vehicle in male and female Plp1- Cre^ERT+/Ramp1^fl/fl and Control mice (n=8 mice per group) treated with periorbital 4-OHT. Mean±SEM. *P<0.05, ***P<0.001 vs. Veh, Control-Veh, and TN-Control, ^§§§P<0.001. vs. Control-CGRP. 2-way (Fig. 1c, 1d, 1f, 1g, 1h) or 1-way (Fig. 1e) ANOVA, Bonferroni correction. FIGs. 2a-2o show that capsaicin induces PMA via CGRP and CLR/RAMP1 in Schwann cells. (Fig. 2a) Acute nociception and (Fig. 2b) PMA after periorbital injection of capsaicin (CPS, 50 pmol) or vehicle in C57BL/6J mice. (Fig. 2c) Acute nociception and (Fig. 2d) PMA after CPS (50 pmol) or vehicle in Trpv1^+/+ and Trpv1^–/– mice. (Fig. 2e) Acute nociception and (Fig. 2f) PMA after CPS (50 pmol) or vehicle in C57BL/6J mice pretreated with capsazepine (CPZ, 100 pmol) or vehicle. (Fig. 2g, 2h) PMA after periorbital SP (3.5 nmol), CPS (50 pmol) or vehicle in C57BL/6J mice pretreated (0.5 h) with L-733,060 (20 nmol) or vehicle. (Fig. 2i, 2j) PMA after CPS (50 pmol) or vehicle in C57BL/6J mice pretreated (0.5 h) with olcegepant (1 nmol) or CGRP8-37 (10 nmol) or vehicle. (Fig. 2k) PMA after CPS (50 pmol) or vehicle in Plp1-Cre^ERT+/Ramp1^fl/fl or Control mice treated with periorbital 4-OHT or vehicle. (Fig. 2l) PMA after periorbital CGRP (1.5 nmol) or vehicle and (Fig. 2m) acute nociceptive response and PMA after periorbital CPS (50 pmol) or vehicle in Adv-Cre^+/Ramp1^fl/fl or Control mice. (Fig. 2n) PMA and (Fig. 2o) paw mechanical allodynia induced by intraperitoneal (i.p.) CGRP (0.1 mg/kg) or vehicle in Adv-Cre^+/Ramp1^fl/fl or Control mice. Mean±SEM., n=8 mice per group. **P<0.01, ***P<0.001 vs. Veh/Veh, Trpv1^+/+-Veh and Control-Veh; ^§§P<0.01, ^§§§P<0.001 vs. Trpv1^+/+-CPS, CPS/Veh, SP/Veh, Control-CPS, Control-CGRP. 1-way (Fig. 2a, 2c, 2e and 2m left panel) or 2-way (Fig. 2b, 2d, 2f-2l, 2m right panel and 2n, 2o) ANOVA, Bonferroni correction. FIGs. 3a-3d show that GTN induces PMA via CGRP released from periorbital trigeminal terminals and, CLR/RAMP1 in Schwann cells. (Fig. 3a) PMA and (Fig. 3b) paw mechanical allodynia induced by intraperitoneal (i.p.) GTN (10 mg/kg) or vehicle in Adv- Cre^+/Ramp1^fl/fl or Control mice post-treated (1.5 hs after GTN) with olcegepant (1 nmol) or vehicle. (Fig. 3c) PMA and (Fig. 3d) paw mechanical allodynia induced by GTN (10 mg/kg, i.p.) or vehicle

or Control (treated with periorbital 4- OHT or vehicle) post-treated (1.5 hs after GTN) with olcegepant (1 nmol) or vehicle. Mean±SEM., n=8 mice per group. ***P<0.001 vs. Control-Veh/Veh; ^§§§P<0.001 vs. Control-GTN/Veh. 2-way ANOVA, Bonferroni correction. FIGs. 4a-4l show that functional CLR/RAMP1 is expressed by HSCs and undergoes clathrin- and dynamin-mediated endocytosis, which underlies nociception. (Fig. 4a, 4b) Effects of graded concentrations of CGRP on cAMP formation. (Fig. 4c, 4d) Effects of graded concentrations of olcegepant on CGRP (100 nM)-evoked cAMP formation. (Fig. 4e) Pharmacological targets. (Fig. 4f) Representative images of HSCs expressing Rab5a- GFP at 30 min after incubation with TAMRA-CGRP (100 nM). Arrows denote colocalization of TAMRA-CGRP and Rab5a-GFP. Arrowheads denote retention of a weak TAMRA-CGRP signal at the plasma membrane. Cells were preincubated with vehicle, Dyngo-4a (Dy4), Pitstop 2 (PS2), inactive analogs (PS2 and Dy4 inact) (all 30 μM) or sucrose (0.45 M). (n=4 independent experiments). Scale: 10 μm. (Fig. 4g, 4h) Quantification of localization of TAMRA-CGRP in endosomes (Fig. 4g) and of the number of TAMRA-CGRP+ve endosomes (Fig. 4h). (n=5-7 independent experiments, 5 cells imaged per experiment). (Fig. 4i, 4j) PMA induced by periorbital CGRP (1.5 nmol) or vehicle in C57BL/6J male and female mice pretreated (0.5 h) with PS2, Dy4, PS2 or Dy4 inact (all 500 pmol) (n=8 mice per group). (Fig. 4k, 4l) PMA induced by periorbital capsaicin (CPS, 50 pmol) or vehicle in C57BL/6J male mice pretreated (0.5 h) with PS2, Dy4, PS2 or Dy4 inact (all 500 pmol) (n=8 mice per group). Mean±SEM. **P<0.01, ***P<0.001 vs. Veh 0 min, and Veh/Veh; ^§§P<0.01, ^§§§P<0.001 vs. Veh 30 min, PS2 30 min, Dy4 30 min, CGRP/PS2 inact, CGRP/Dy4 inact, CPS/PS2 inact, CPS/Dy4 inact. 1- way (Fig. 4g, 4h) or 2-way (Fig. 4i-4l) ANOVA, Bonferroni correction. FIGs. 5a-5o show that CGRP leads to Gα protein activation and βARR2 recruitment at the plasma membrane and in early endosomes in HEK-hCLR/RAMP1 cells and HSCs- hCLR/RAMP1. Endosomal signaling generates sustained formation of cAMP in HSCs. (Fig. 5a-5d) CGRP (100 nM) increased EbBRET between Rluc8-mGα_s, Rluc8-mGα_sq, Rluc8-mGα_si, and Rluc2-βARR2 with RGFP-CAAX (Fig. 5a, 5b) and tdRGFP-Rab5a (Fig. 5c, 5d) in HEK-hCLR/RAMP1 cells. Fig. 5a, 5c time course. Fig. 5b, 5d area under curve (AUC). (Fig. 5e-5h) CGRP (100 nM) increased EbBRET between Rluc8-mGα_s, Rluc8-mGα_sq, Rluc8-mGα_si, and Rluc2-βARR2 with RGFP-CAAX (Fig. 5e, 5f) and tdRGFP-Rab5a (Fig. 5g, 5h) in HSC-hCLR/RAMP1 cells. Fig. 5e, 5g time course. Fig. 5f, 5h AUC. (Fig. 5i) Hypertonic sucrose (0.45 M) inhibited CGRP (100 nM)-stimulated EbBRET between hCLR-Rluc8 and tdRGFP-Rab5a in HEK-hCLR/RAMP1 cells. (Fig. 5j, 5k) Hypertonic sucrose (0.45 M) inhibited CGRP (100 nM)-stimulated EbBRET between Rluc8-mGα_s, Rluc8-mGα_sq, Rluc8-mGα_si and Rluc2-βARR2 with tdRGFP-Rab5a in HEK- hCLR/RAMP1 cells. (Fig. 5l, 5m) Sucrose (0.45 M) inhibited CGRP (100 nM)-stimulated EbBRET between Rluc8-mGαs, Rluc8-mGαsq, Rluc8-mGαsi, and Rluc2-βARR2 with tdRGFP-Rab5a in HSC-hCLR/RAMP1 cells. (Fig. 5n, 5o) Sucrose (0.45 M) inhibited CGRP (100 nM)-stimulated formation of cAMP in HSCs. Fig. 5n, time course. Fig. 5o, AUC. Mean±SEM. (n=5-10 independent experiments). *P<0.05, **P<0.01, ***P<0.001, vs. Veh. 1-way ANOVA, Dunnett’s correction (Fig. 5b, 5d, 5f, 5h) or parametric unpaired t test (Fig. 5i-5m, 5o). FIGs. 6a-6k show that endogenous and exogenous CGRP induces PMA via NO production. (Fig. 6a) Pharmacological targets. (Fig. 6b-6e) PMA after periorbital injection of CGRP (1.5 nmol), capsaicin (CPS, 50 pmol) or vehicle in C57BL/6J mice pretreated (0.5 h) with L-NAME (1 μmol) and cPTIO (200 nmol) or vehicle (n=8 mice per group). (Fig. 6f, 6g) Real-time PCR plot and cumulative data for GAPDH, S100, NOS1, NOS2 and NOS3 in primary HSCs and MSCs (n=3 independent experiments). (Fig. 6h) In-cell p- NOS3^s1177 ELISA in HSCs and IMS32 cells before (0) or after CGRP (1 μM) (n=4 independent experiments). cAMP assay in (Fig. 6i) HSCs, IMS32 and (Fig. 6j) MSCs from

or Control mice treated with intraperitoneal 4-OHT (Fig. 6k) nitric oxide assay in HSCs and IMS32 cells treated with CGRP (1 μM) or vehicle. Some cells were treated with olcegepant (1 μM), CGRP8-37 (1 μM), SQ22536 (100 μM), L-NAME (100 μM) or vehicle (n=3 independent experiments). Mean±SEM. (-) represents the combination of different vehicles. *P<0.05, **P<0.01, ***P<0.001 vs. Veh/Veh, time 0 (min); ^§§§P<0.001 vs. CGRP/Veh, CGRP 1μM and 10 μM. 1-way (Fig. 6h-6k) or 2-way (Fig. 6b-6e) ANOVA, Bonferroni correction. FIGs. 7a-7l shows that CGRP induces ROS release via Schwann cell TRPA1 activation. (Fig. 7a) Representative images of localization of immunoreactive DAPI, S100, RAMP1 and TRPA1 in human abdominal and mouse periorbital cutaneous nerve bundles (Scale: 10 μm human, 50 μm mouse, inset 10 μm) (n=3 subjects). (Fig. 7b) Pharmacological targets. (Fig. 7c, 7d) PMA after periorbital injection of CGRP (1.5 nmol), capsaicin (CPS, 50 pmol) or vehicles in (Fig. 7c) Trpa1^+/+ and Trpa1^-/- mice and in (Fig. 7d) Adv- Cre⁺/Trpa1^fl/fl or Control mice (n=8 mice per group). (Fig. 7e, 7f) Ca²⁺ response in HSCs and IMS32 cells exposed to CGRP (10 μM) in the presence of olcegepant (100 nM), CGRP8-37 (100 nM), SQ22536 (100 μM), H89 (1 μM), L-NAME (10 μM), Ca²⁺-free medium, PBN (50 μM), ML171 (1 μM), A967079 (A96, 50 μM) or vehicle (n=4 independent experiments). (Fig. 7g) H₂O₂ release in HSCs and IMS32 cells exposed to CGRP (10 μM) in the presence of olcegepant (100 nM), CGRP8-37 (100 nM), SQ22536 (100 μM), L-NAME (10 μM), Ca²⁺-free medium, PBN (50 μM), A96 (50 μM), or vehicle (0.1 % DMSO) (n=3 independent experiments). (Fig. 7h) Nitric oxide release in HSCs and IMS32 cells exposed to CGRP (1 or 10 μM) in the presence of A96 (50 μM) or vehicle (n=3 independent experiments). (Fig. 7i-7k) PMA after CGRP (1.5 nmol) or vehicle in C57BL/6J male mice pre-treated (0.5 h, left) or post-treated (1h, right) with (Fig. 7i) A96 (300 nmol), (Fig. 7j) PBN (670 nmol) or (Fig. 7k) ML171 (250 nmol) or vehicle (n=8 mice per group). (Fig. 7l) PMA after CGRP (1.5 nmol), CPS (50 pmol) or vehicles in Plp1- Cre^ERT+/Trpa1^fl/fl or Control mice treated with 4-OHT (n=8 mice per group). Mean±SEM. (-) represents the combination of different vehicles. ***P<0.001 vs. Trpa1^+/+-Veh, Control-Veh, Veh and Veh/Veh; ^§§P<0.01, ^§§§P<0.001 vs. Trpa1^+/+-CGRP, Trpa1^+/+-CPS, Control-CGRP, Control-CPS, CGRP 1 μM and 1 μM, CGRP/Veh. 2-way (Fig. 7c, 7d, 7i- 7l) or 1-way (Fig. 7e-7h) ANOVA, Bonferroni correction. FIGs. 8a-8i show that DIPMA-MK-3207 nanoparticles target endosomal CLR/RAMP1 signaling and provide superior relief from CGRP-evoked PMA. (Fig. 8a) pH-responsive DIPMA-MK-3207. (Fig. 8b) Transmission electron micrograph image of DIPMA-MK- 3207 (Scale: 0.1 μm). (Fig. 8c) Physicochemical properties of DIPMA-MK-3207 and DIPMA-Ø. (Fig. 8d) Uptake of DIPMA-Cy5 into HSCs expressing EEA1-GFP. Cells were preincubated with DIPMA-Cy5 (40-60 ng/ml) for 30 min and were then incubated with TAMRA-CGRP (100 nM) for 30 min. Arrows denote accumulation of TAMRA-CGRP in early endosomes containing DIPMA-Cy5. Representative images from n=5 independent experiments (Scale: 10 μm). (Fig. 8e-8g). Effects of DIPMA-MK-3207, MK-3207, DIPMA-Ø or vehicle on CGRP- (100 nM) stimulated cAMP formation in HEK- rCLR/RAMP1 cells. (Fig. 8e) Time course and (Fig. 8f, 8g) integrated response (AUC, area under curve) before (1^st phase) and after (2^nd phase) washing to remove extracellular CGRP. (Fig. 8h) Concentration-response curves of the inhibition by DIPMA-MK-3207 or free MK-3207 on the Ca²⁺ response to CGRP in HSCs. Cells were preincubated with DIPMA-MK-3207 or MK-3207 (1-1000 nM) for 20 min at room temperature to allow nanoparticle accumulation in endosomes. Cells were washed, challenged with CGRP (1 μM), and Ca²⁺ responses were measured for 40 min. Maximal responses are shown. (Fig. 8i) PMA, expressed as area under the curve (AUC), after periorbital injection of CGRP (1.5 nmol), capsaicin (CPS, 50 pmol) or vehicles in C57BL/6J male mice pre-treated (0.5 h) with DIPMA-MK-3207, MK-3207 (0.1, 0.3, 1 pmol), DIPMA-Ø or vehicle (n=8 mice per group). Mean±SEM. ***P<0.001 vs. DIPMA-Ø/Veh, , ^###P<0.001 vs. MK-3207 0.3 pmol and MK-32071 pmol. (Fig. 8f, 8g, 8i) 1-way ANOVA, Bonferroni correction. FIG. 9 shows an exemplary schematic representation of the pathway that signal prolonged cutaneous allodynia elicited by CGRP released and associated with neurogenic inflammation. The pro-migraine neuropeptide, CGRP, released from trigeminal cutaneous afferents, activates CLR/RAMP1 on Schwann cells. CLR/RAMP1 traffics to endosomes, where sustained G protein signaling increases cAMP and stimulates PKA that results in nitric oxide synthase activation. The ensuing release of nitric oxide targets the oxidant- sensitive channel, TRPA1, in Schwann cells, which elicits persistent ROS generation. ROS triggers TRPA1 on adjacent C- (1) or AG-fiber (2) afferents resulting in periorbital allodynia, a hallmark of migraine pain. The inset shows several unmyelinated axons invaginated into a Schwann cell forming a Remak bundle. DETAILED DESCRIPTION This disclosure features chemical entities and compositions containing the same that are useful for treating migraine (e.g., migraine pain) and neuralgia. Methods of Treatment Migraine symptoms can be severely debilitating, with more than 90% of affected persons unable to work or to function normally during a migraine attack. Migraines can include severe throbbing and reoccurring headache pain (usually on only one side of the head, in contrast with stress headaches). Migraine attacks can last between about 4-72 hours, and typically include pain so severe that the patient is effectively disabled for the duration. In about 15-20% of migraines, patients can notice, for as much as 1-2 days before migraine onset (in a "prodome" portion of a migraine attack), other effects such as constipation, mood changes ranging from depression to euphoria, food cravings, neck stiffness, increased thirst or urination, and frequent yawning. Migraines can also include incapacitating neurological symptoms, including: visual disturbances, dizziness, nausea, vomiting, extreme sensitivity to senses (including sight, sound, and smell), and tingling or numbness in extremities or the face. About 25% of migraine attacks can include an "aura," a term for a set of nervous system symptoms, such as: visual and tactile hallucinations, loss of sensory acuity, and loss of physical bodily control (including limb weakness). Visual symptoms can include flashes of light, wavy zigzag vision, blind spots, and shimmering spots or "stars". Motor and tactile symptoms can include sensations of pins and needles, asymmetric weakness or numbness, difficulty speaking, tinnitus, and uncontrollable jerking or other movement. In certain embodiments, the symptoms of migraine include at least two of the following symptoms: medium to strong predominantly unilateral headache, light, noise and/or smell sensitivity, nausea or sickness, facial pain, sore eyes, balance disturbance, word finding difficulties, other neurological symptoms, such as sensory or motor disturbances, allodynia or any other of the features known to accompany, precede or follow a migraine attack, such as fatigue, nausea, cognitive difficulties, tiredness, ravenous hunger or thirst, reduced libido, depression, mania, mood swings, as well as changes in brain structure and function, such as white matter lesions or disturbances in functional connectivity. This disclosure features methods for treating, preventing, ameliorating, controlling, reducing incidence of, or delaying the progression of migraine. This disclosure features methods for treating, preventing, ameliorating, controlling, reducing incidence of, or delaying the progression of neuralgia. In some embodiments, administration of the agent achieves any one or more of the following: (i) decreasing migraine attack frequency; (ii) decreasing migraine attack severity; (iii) reducing any of the neurological symptoms associated with migraine, such as phono-, photo-, and/or osmophobia, visual, sensory or motor disturbances, allodynia; (iv) reducing any of the other features known to accompany, precede or follow a migraine attack, such as fatigue, nausea, cognitive difficulties, tiredness, ravenous hunger or thirst, muscle ache, reduced libido, depression, mania, mood swings; (v) reversing, retarding or preventing structural or functional nerve cell damage, such as white matter lesions or disturbances in functional connectivity, associated with migraine; and (vi) preventing, retarding or reversing the transition of acute migraine to chronic migraine. In certain embodiments, administration of the agent achieves any one or more of the following: decreasing migraine attack frequency; decreasing migraine attack severity; decreasing the severity of migraine symptoms; preventing disease progression and/or preventing disease chronification. The treating or reducing can comprise reducing the number of headache hours of any severity, reducing the number of monthly headache days of any severity, reducing the use of any acute headache medications (e.g., migraine-specific acute headache medications), reducing a 6-item Headache Impact Test (HIT-6) disability score, improving 12-Item Short Form Health Survey (SF-12) score (Ware et al., Med Care 4:220-233, 1996), reducing Patient Global Impression of Change (PGIC) score (Hurst et al., J Manipulative Physiol Ther 27:26-35, 2004), improving Sport ConCuSSion ASSeSment tool 3 (SCAT-3) score (McCrory et al. British Journal of Sports Medicine 47:263-266, 2013), or any combination thereof. In some embodiments, monthly headache hours experienced by the subject after said administering is reduced by 40 or more hours (e.g., 45, 50, 55, 60, 65, 70, 75, 80, or more) from a pre-administration level in the subject. Monthly headache hours may be reduced by more than 60 hours. In some embodiments, monthly headache hours experienced by the subject after said administering are reduced by 25% or more (e.g., 30%, 35%, 40%, 45%, 50%, or more) relative to a pre-administration level in the subject. Monthly headache hours may be reduced by 40% or more. In some embodiments, monthly headache days experienced by the subject after said administering is reduced by three or more days (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more days) from a pre-administration level in the subject. In some embodiments, the number of monthly headache days can be reduced by at least about 50% from a pre- administration level in the subject. Thus, in some aspects, the number of monthly headache days can be reduced by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 90%. Also disclosed are methods of decreasing a number of monthly headache hours experienced by a subject having refractory migraine. Also disclosed are methods of decreasing a number of monthly headache days experienced by a subject having refractory migraine. Also disclosed are methods of decreasing use of any acute headache medication in a subject having refractory migraine, e.g.,in an amount effective to decrease monthly use of the headache medication by the subject by at least 15% (e.g., 20%, 25%, 30%, 35%, 40%, or more). In some embodiments, the acute headache medication is selected from the group consisting of 5-HT1 agonists, triptans, opiates, ergot alkaloids, and non-steroidal anti-inflammatory drugs (NSAIDs). In some embodiments, the acute headache medication is selected from analgesics (e.g., acetylsalicylic acid, ibuprofen, naproxen, diclofenac, paracetamol, acetylsalicylic acid plus paracetamol plus caffeine, metamizol, phenazon, or tolfenamic acid); antiemetics (e.g., metoclopramide or domperidon); ergot alkaloids (e.g., ergotamine tartrate or dihydroergotamine); and triptans, i.e., 5-HT1 agonists (e.g., sumatriptan, zolmitriptan, naratriptan, rizatriptan, almotriptan, eletriptan, or frovatriptan). In some embodiments, the methods include selecting a subject who does not respond favorably to a migraine treatment selected from the group consisting of topiramate, carbamazepine, divalproex sodium, sodium valproate, valproic acid, flunarizine, candesartan, pizotifen, am itriptyline, venlafaxine, nortriptyline, duloxetine, atenolol, nadolol, metoprolol, propranolol, bisopropol, timolol, and onabotulinumtoxinA. In some embodiments, the methods include selecting a subject who does not respond favorably to a migraine treatment selected from the group consisting of topiramate, carbamazepine, divalproex sodium, sodium valproate, flunarizine, pizotifen, am itriptyline, venlafaxine, nortriptyline, duloxetine, atenolol, nadolol, metoprolol, propranolol, timolol, and onabotulinumtoxinA. In some embodiments, the methods include selecting a subject who does not respond favorably to a migraine treatment selected from the group consisting of propranolol, metoprolol, atenolol, bisopropol, topiramate, amitriptyline, flunarizine, candesartan, onabotulinumtoxinA, and valproic acid. In some embodiments, the methods include selecting a subject who does not respond favorably to a migraine treatment selected from propranolol/metoprolol, topiramate, flunarizine, valproate/divalproex, am itriptyline, venlafaxine, lisinopril, candesartan, and locally approved products (e.g. oxeterone or pizotifen). In other embodiments, the methods include selecting a subject who does not respond favorably to one or more migraine treatments of the following classes: beta-blockers, anticonvulsants, tricyclics, calcium channel blockers, angiotensin II receptor antagonists. For example, the subject may have documented inadequate response (in a medical chart or by treating physician's confirmation) to at least two preventive medications (from different clusters, as defined below). Or, the subject may have documented inadequate response (in a medical chart or by treating physician's confirmation) to two to four classes of prior preventive medications (from, e.g., different clusters, as defined below). As another example, the subject may have documented inadequate response (in a medical chart or by treating physician's confirmation) to two to three classes of prior preventive medications (from different clusters, as defined below) and a valrproate (e.g., divalproex sodium, sodium valproate, or valproic acid). Pharmaceutical Compositions and Administration In some embodiments, the chemical entities can be administered in combination with one or more conventional pharmaceutical excipients. Pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens, poloxamers or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, tris, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium-chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, and wool fat. Cyclodextrins such as α-, E, and γ-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3- hydroxypropyl-β-cyclodextrins, or other solubilized derivatives can also be used to enhance delivery of compounds described herein. Dosage forms or compositions containing a chemical entity as described herein in the range of 0.005% to 100% with the balance made up from non-toxic excipient may be prepared. The contemplated compositions may contain 0.001%-100% of a chemical entity provided herein, in one embodiment 0.1-95%, in another embodiment 75-85%, in a further embodiment 20-80%. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 22^nd Edition (Pharmaceutical Press, London, UK. 2012). In some embodiments, the chemical entities escribed herein or a pharmaceutical composition thereof can be administered to subject in need thereof by any accepted route of administration. Acceptable routes of administration include, but are not limited to, buccal, cutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, interstitial, intra-abdominal, intra-arterial, intrabronchial, intrabursal, intracerebral, intracisternal, intracoronary, intradermal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraovarian, intraperitoneal, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratesticular, intrathecal, intratubular, intratumoral, intrauterine, intravascular, intravenous, nasal, intranasal, nasogastric, oral, parenteral, percutaneous, peridural, rectal, respiratory (inhalation), subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transtracheal, ureteral, urethral and vaginal. In certain embodiments, a preferred route of administration is parenteral (e.g., intratumoral). Schwann cell As used herein, a “Schwann cell” is a cell of neural crest origin that forms a continuous envelope around each nerve fiber of peripheral nerves in situ. A Schwann cell can be identified as such by detecting the presence of one or more Schwann cell markers such as glial fibrillar acidic protein (GFAP), S100 protein, laminin, or nerve growth factor (NGF) receptor, e.g., using antibodies against these markers. Furthermore, Schwann cells have a characteristic morphology which can be detected by microscopic examination of cultures thereof. Schwann cells are known to play a key role in the study of neuropathic pain, wherein multiple receptors and channels are expressed in Schwann cells and regulated in different pain conditions. Although these molecules are not released, they activate intracellular signaling to release growth factors, cytokines, and chemokines that regulate pain states. In some embodiments, a Schwann cell can express a receptor or an active molecule that is regulated in different pain conditions. For example, a receptor in a Schwann cell can include, but is not limited to, a purigenic receptor (e.g., P2X4R, P2X2/3R, or P2X7R), a Toll-like receptor 2 (TLR2), a LDL receptor-related protein 1 (LRP1), a lysophophatidic acid 1 receptor (LPA1 R), a hydroxyl carboxylic acid receptor type 2 (HCAR2), a J- aminobutyric acid type B receptor (GABA-B), an epidermal growth factor receptor (ErbB), or calcitonin gene-related peptide (CGRP) receptor. In some embodiments, a Schwann cell expresses a CGRP receptor. In some embodiments, the CGRP receptor includes a calcitonin receptor-like receptor (CLR) or a calcitonin receptor (CTR). In some embodiments, the CGRP receptor further includes a receptor activity-modifying protein (RAMP). In some embodiments, a receptor activity-modifying protein (RAMP) can be a RAMP1, RAMP2, or RAMP3. In some embodiments, a Schwann cell CGRP receptor can include a CLR/RAMP1 heterodimer. Calcitonin gene-related peptide (CGRP) receptor As used herein, a “CGRP receptor” refers to a receptor expressed in both the peripheral and the central nervous system (CNS), including the trigeminovascular pathways. In some embodiments, in humans, CGRP is present in two isoforms, α-CGRP and β-CGRP, where α-CGRP is most abundantly found in primary spinal afferents from sensory ganglia, whereas β-CGRP is mainly found in the enteric nervous system. The CGRP receptor comprises three subunits: receptor activity-modifying protein 1 (RAMP1), calcitonin-like receptor (CLR) and receptor component protein (RCP). In some embodiments, as well as playing a role in cranial nociception, CGRP can be a potent general arterial vasodilator. Furthermore, at peripheral synapses, CGRP released from trigeminal terminals results in vasodilation via CGRP receptors on the smooth muscle cells of meningeal and cerebral blood vessels. In some embodiments, a CGRP receptor can include a receptor activity-modifying protein 1 (RAMP1) and a calcitonin-like receptor (CLR) at the cell surface to form a heterodimeric receptor complex. In some embodiments, the calcitonin-like receptor (CLR) is a human CLR. In some embodiments, the CLR includes the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 1 – human calcitonin receptor like receptor MEKKCTLNFLVLLPFFMILVTAELEESPEDSIQLGVTRNKIMTAQYECYQ KIMQDPIQQAEGVYCNRTWDGWLCWNDVAAGTESMQLCPDYFQDFDPSEK VTKICDQDGNWFRHPASNRTWTNYTQCNVNTHEKVKTALNLFYLTIIGHG LSIASLLISLGIFFYFKSLSCQRITLHKNLFFSFVCNSVVTIIHLTAVAN NQALVATNPVSCKVSQFIHLYLMGCNYFWMLCEGIYLHTLIVVAVFAEKQ HLMWYYFLGWGFPLIPACIHAIARSLYYNDNCWISSDTHLLYIIHGPICA ALLVNLFFLLNIVRVLITKLKVTHQAESNLYMKAVRATLILVPLLGIEFV LIPWRPEGKIAEEVYDYIMHILMHFQGLLVSTIFCFFNGEVQAILRRNWN QYKIQFGNSFSNSEALRSASYTVSTISDGPGYSHDCPSEHLNGKSIHDIE NVLLKPENLYN In some embodiments, the receptor activity-modifying protein 1 (RAMP1) is a human RAMP1. In some embodiments, the RAMP1 includes the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 2 – human Receptor activity-modifying protein 1 MARALCRLPRRGLWLLLAHHLFMTTACQEANYGALLRELCLTQFQVDMEA VGETLWCDWGRTIRSYRELADCTWHMAEKLGCFWPNAEVDRFFLAVHGRY FRSCPISGRAVRDPPGSILYPFIVVPITVTLLVTALVVWQSKRTEGIV CGRP evokes PMA by activating Schwann cell CLR/RAMP1 CLR and RAMP1 mRNA and immunoreactivity were detected in primary cultures of human Schwann cells (HSCs) or mouse Schwann cells (MSCs) taken from sciatic or trigeminal nerve (Fig. 1a). The S100+ve mouse Schwann cell line (IMS32) recapitulated features of primary MSCs, including expression of CLR and RAMP1 mRNA and immunoreactivity and TRPA1-dependent Ca²⁺ response to allyl isothiocyanate. Immunoreactive CLR and RAMP1 were also detected in S100+ve Schwann cells in nerve bundles in biopsies of human abdominal and mouse periorbital skin (Fig. 1b). In C57BL/6J male mice, periorbital CGRP elicited PMA (Fig. 1c). Periorbital CGRP also evoked PMA in female mice (Fig 1c). Periorbital CGRP did not elicit allodynia in the hind paw. Unless otherwise specified, all drugs were administered by subcutaneous periorbital injection. To assess the role of Schwann cell CLR/RAMP1 in CGRP-evoked PMA, Schwann cell-specific Cre mice (Plp-Cre^ERT) were crossed with RAMP1 floxed mice (Ramp1^fl/fl) to generate Plp-Cre^ERT+;Ramp1^fl/fl and

(Control) mice. For selective deletion of RAMP1 in Schwann cells of the periorbital region, 4- hydroxytamoxifen (4-OHT) was administered daily for 3 days to Plp-Cre^ERT+;Ramp1^fl/fl and Control mice. CGRP elicited PMA in both male and female Control mice, that was similarly attenuated in Plp-Cre^ERT+;Ramp1^fl/fl mice (Fig. 1d). In Plp-Cre^ERT+;Ramp1^fl/fl mice 4-OHT treatment down-regulated RAMP1 immunoreactivity in S100+ve cells surrounding trigeminal but not sciatic nerve fibers (Fig. 1e) and did not prevent paw mechanical allodynia evoked by intraplantar CGRP, which was prevented by intraplantar 4-OHT. Intravenous CGRP provokes delayed headache attacks in patients³⁷. Intraperitoneal CGRP caused PMA and paw allodynia in male and female C57BL/6J mice without gender difference (Fig. 1f). In Plp-Cre^ERT+;Ramp1^fl/fl mice treated with periorbital 4-OHT PMA, but not paw allodynia, was similarly reduced in males and females in response to intraperitoneal CGRP (Fig. 1g, 1h). Systemic (intraperitoneal) 4-OHT reduced both PMA and paw allodynia by intraperitoneal CGRP. These results reveal an essential role for CLR/RAMP1 of Schwann cells surrounding periorbital trigeminal endings in PMA elicited by local and systemic CGRP. Stimulation of peptidergic trigeminal neurons evokes PMA by activating Schwann cell CLR/RAMP1 To explore the ability of endogenous CGRP to elicit PMA, capsaicin was administered, which activates TRPV1⁴ thus releasing CGRP and SP from peptidergic nociceptors³. Periorbital capsaicin elicited acute (~10 min) nociceptive behavior (Fig. 2a) and, like CGRP, caused prolonged (~4 h) PMA (Fig. 2b). Allodynia was detected in the periorbital area but not in the hind paw, indicating a local action. TRPV1 deletion (Trpv1^-/- mice) (Fig. 2c, 2d) or pretreatment with the TRPV1 antagonist capsazepine (Fig. 2e, 2f), prevented acute nociception and PMA. An antagonist (L-733,060) of the SP neurokinin 1 (NK₁) receptor, which prevented SP-evoked PMA (Fig. 2g), failed to diminish capsaicin- evoked acute nociception and PMA (Fig. 2h). The histamine H1 receptor antagonist, astemizole, inhibited SP-evoked PMA but did not affect CGRP- or capsaicin-evoked PMA. Pretreatment with the CLR/RAMP1 antagonists, CGRP8-37 or olcegepant, prevented PMA (Fig. 2i, 2j) but not acute nociception evoked by capsaicin. Importantly, 4- OHT markedly inhibited capsaicin-evoked PMA (Fig. 2k) but not acute nociception in Plp-Cre^ERT+;Ramp1^fl/fl mice observed in Control mice. CGRP-evoked PMA or capsaicin- evoked acute nociception and PMA were similar in mice with selective deletion of RAMP1 in nociceptors (Advillin-Cre⁺;Ramp1^fl/fl, Adv-Cre⁺;Ramp1^fl/fl) and Control mice (Fig. 2l, 2m). Both PMA and paw allodynia were similar in Adv-Cre⁺;Ramp1^fl/fl and Control mice after intraperitoneal CGRP (Fig. 2n, 2o). Thus, CGRP but not SP mediates allodynia resulting from excitation of TRPV1+ve peptidergic nociceptors, and PMA evoked by both endogenous and exogenous CGRP depends on CLR/RAMP1 of Schwann cells surrounding peripheral terminals of nociceptors, while the receptor of sensory nerve fibers is not implicated. Schwann cell CLR/RAMP1 mediates the CGRP-dependent PMA evoked by GTN Systemic GTN administration provokes sustained headaches in humans¹⁹. In mice intraperitoneal GTN elicits PMA¹⁸, that is in part mediated by CGRP release from periorbital trigeminal terminals¹⁸. Here, systemic GTN elicited PMA (Fig. 3a) and paw allodynia (Fig. 3b) that were similar in Control and

mice. Olcegepant transiently and partially inhibited PMA (Fig. 3a), while did not affect GTN-evoked paw allodynia (Fig. 3b) in both mouse strains. RAMP1 deletion from trigeminal Schwann cells (Plp-Cre^ERT+;Ramp1^fl/fl mice) partially inhibited PMA (Fig. 3c), but not paw allodynia (Fig. 3d). Importantly, treatment with olcegepant reduced GTN-evoked PMA in Control mice, but failed to further inhibit the response in Plp-Cre^ERT+;Ramp1^fl/fl mice (Fig. 3c). Paw allodynia was unchanged by olcegepant (Fig. 3d). Together, the findings suggest that Schwann cell CLR/RAMP1 mediates the CGRP-dependent component of PMA in a mouse headache model. Clathrin- and dynamin-mediated endocytosis of CLR/RAMP1 in Schwann cells mediates PMA The CLR/RAMP1 signaling pathway in Schwann cells that mediates CGRP-evoked PMA was investigated. CGRP-stimulated cAMP formation was measured in HSCs using a virally-encoded cAMP cADDis reporter. CGRP stimulated a prompt concentration- dependent increase in cAMP formation in HSCs that was sustained for >300 s (Fig. 4a, 4b). The CLR/RAMP1 antagonist olcegepant caused a concentration-dependent inhibition of CGRP-stimulated cAMP formation (Fig. 4c-4e). Endosomal signaling of GPCRs, including CLR/RAMP1, controls nociception^33,34,35. To assess CLR/RAMP1 endocytosis in Schwann cells, HSCs expressing the early endosome marker Rab5a-GFP with TAMRA-CGRP were imcubated. In vehicle-treated cells, live cell imaging revealed uptake of TAMRA-CGRP into Rab5a-GFP+ve early endosomes within 10 min that continued for 30 min (Fig. 4f). Inhibitors of clathrin (PitStop2, PS2) or dynamin (Dyngo4a, Dy4) prevented the translocation of TAMRA- CGRP to endosomes causing retention of weak TAMRA-CGRP fluorescence at the cell surface (Fig. 4e, 4f). Quantification of TAMRA-CGRP fluorescence intensity in Rab5a- GFP+ve endosomes or the proportion of endosomes containing TAMRA-CGRP confirmed that PS2 and Dy4 inhibited endocytosis of TAMRA-CGRP (Fig. 4g, 4h). Inactive analogs had no effect. Hypertonic sucrose (0.45 M) inhibits clathrin-mediated endocytosis, including agonist-stimulated endocytosis of GPCRs³⁸. Hypertonic sucrose also inhibited the uptake of TAMRA-CGRP in HSCs (Fig. 4g, 4h). Injection of PS2 or Dy4, but not their inactive analogs, prevented CGRP-evoked PMA, both in male and female mice (Fig. 4i, 4j). PS2 or Dy4 also reversed capsaicin-evoked PMA (Fig. 4k, 4l). Thus, CGRP stimulates clathrin- and dynamin-mediated endocytosis of CLR/RAMP1 in Schwann cells, which sustains CGRP-evoked PMA. CLR activates Gα_s, Gα_q and Gα_i and recruits βARR2 to the plasma membrane and endosomes GPCRs, including CLR/RAMP1, can signal from endosomes by Gαs, Gαq and βARR-mediated mechanisms^33,34,35. Enhanced bystander bioluminescence resonance energy transfer (EbBRET) was used to study the activation of Gα and recruitment of βARR to the plasma membrane and early endosomes of HEK293T cells expressing human (h) CLR and RAMP1 (HEK-hCLR/RAMP1). CGRP-dependent activation of Gα_s, Gα_sq and Gαsi was assessed using an EbBRET assay that detects recruitment of mini (m) Gα coupled to Renilla (R)luc8 to the plasma membrane marker CAAX coupled to RGFP or the early endosome marker Rab5a coupled to tandem (td)RGFP. mGα proteins are N- terminally truncated Gα proteins that freely diffuse throughout the cytoplasm and bind to active conformations of GPCRs. Their translocation to GPCRs reflects Gα activation. mGα_sq and mGα_si were developed by mutating mGα_s residues to equivalent Gα_q and Gα_i residues. Recruitment of βARR was assessed by measuring EbBRET between Rluc2- βARR2 and RGFP-CAAX or tdRGFP-Rab5a. CGRP induced a rapid increase in EbBRET between Rluc8-mGα_s, Rluc8-mGα_sq, Rluc8-mGα_si and Rluc2-βARR2 with RGFP-CAAX, which was maximal at ~300 s and declined over 1000 s (Fig. 5a, 5b). CGRP increased EbBRET between Rluc8-mGαs, Rluc8-mGαsq, Rluc8-mGαsi and Rluc2-βARR2 with tdRGFP-Rab5a that was fully sustained for 1300 s (Fig. 5c, 5d). EbBRET was similarly used to study the activation of Gα and recruitment of βARR2 to the plasma membrane and endosomes of HSCs transfected with hCLR/RAMP1 (CLR/RAMP1 overexpression was required to amplify BRET signals). In HSCs, CGRP increased EbBRET between Rluc8- mGα_s, Rluc8-mGα_sq, Rluc8-mGα_si and Rluc2-βARR2 with RGFP-CAAX and tdRGFP- Rab5a (Fig. 5e-5h). EbBRET signals were sustained for 1300 s. To investigate the contribution of endocytosis to the activation of Gα proteins and βARRs in endosomes, cells were preincubated with hypertonic sucrose. CGRP increased EbBRET between hCLR-Rluc8 and tdRGFP-Rab5a in HEK-hCLR/RAMP1 cells, consistent with CLR endocytosis (Fig. 5i). Hypertonic sucrose inhibited these changes, which indicates an inhibition of endocytosis (Fig. 5i). Hypertonic sucrose caused a delayed yet more sustained activation of Rluc8-mGαs, Rluc8-mGαsq, Rluc8-mGαsi and Rluc2- βARR2 at the plasma membrane, and an almost complete inhibition of activation of Rluc8- mGα_s, Rluc8-mGα_sq, Rluc8-mGα_si and Rluc2-βARR2 in endosomes (Fig. 5j, 5k). Sucrose similarly delayed CGRP-induced recruitment of Rluc8-mGαs, Rluc8-mGαsq, Rluc8-mGαsi and Rluc2-βARR2 to the plasma membrane and almost completely inhibited activation of GD_s,sq,si and βARR2 in endosomes of HSCs expressing hCLR/RAMP1 (Fig. 5l, 5m). To examine the contribution of endosomal CLR/RAMP1 signaling to CGRP-induced cAMP formation, HSCs expressing the cADDis cAMP reporter with sucrose or vehicle were preincubated. In vehicle-treated cells, CGRP stimulated a rapid (1 min) increase in cAMP formation that was sustained for 30 min (Fig. 5n, 5o). Sucrose reduced but did not abolish the initial response, yet strongly inhibited the sustained phase of CGRP-stimulated cAMP formation (Fig. 5n, 5o). Thus, CGRP initially activates Gα and βARR at the plasma membrane, which is followed by sustained activation of Gα and βARR in early endosomes. Endocytosis is necessary for the recruitment of Gα and βARR to endosomes. GDs continues to signal in endosomes, leading to sustained cAMP formation. CLR/RAMP1 activation in Schwann cells releases NO, which initiates but does not sustain PMA The mechanisms that sustain PMA following CLR/RAMP1 activation and endocytosis in Schwann cells were investigated. Pre- but not post-treatment (60 min after CGRP or capsaicin) with CLR/RAMP1 antagonists, olcegepant or CGRP8-37, attenuated PMA evoked by capsaicin and in accordance with previous studies^17,18 PMA evoked by CGRP. Similarly, inhibitors of clathrin- and dynamin-mediated endocytosis had no effect when administered 60 min after CGRP or capsaicin. Thus, once induced by CGRP, CLR/RAMP1 antagonists or inhibitors of CRL/RAMP1 internalization are unable to attenuate PMA. Pre- but not post-treatment with the protein kinase A (PKA) inhibitor, H89, reduced PMA by CGRP and capsaicin. NO has been implicated in CGRP-mediated vascular responses². Although NO can release CGRP with proalgesic functions, the contribution of NO to CGRP-evoked allodynia is uncertain. Pretreatment with an NO synthase (NOS) inhibitor (L-NAME) or an NO scavenger (cPTIO) (Fig. 6a), abrogated CGRP-evoked PMA (Fig. 6b, 6c). L-NAME and cPTIO pretreatment also attenuated capsaicin-evoked PMA (Fig. 6d, 6e). However, L-NAME and cPTIO did not affect PMA when administered 60 min after CGRP or capsaicin. Thus, PKA-dependent NO release³⁹ is necessary to initiate, but is not sufficient to sustain, CGRP-evoked allodynia. In vitro findings recapitulated in vivo results. HSCs, MSCs and IMS32 cells predominantly expressed NOS3 (eNOS) mRNA, with little or no expression of NOS1 and NOS2 (nNOS and iNOS, respectively) mRNA (Fig. 6f, 6g). In both HSCs and IMS32 cells CGRP elicited a transient increase in NOS3 phosphorylation (i.e., activation), consistent with NO generation, which peaked at 5-10 min and declined within 30-60 min (Fig. 6h), and a cAMP increase that was prevented by olcegepant, CGRP8-37 and an adenylyl cyclase inhibitor (SQ22536), but not by L-NAME (Fig. 6i). The increase in cAMP evoked by CGRP but not that elicited by forskolin was reduced in cultured MSCs obtained from Plp-Cre^ERT+;Ramp1^fl/fl mice as compared to Control mice treated with intraperitoneal 4- OHT (Fig. 6j). In contrast, the CGRP-evoked increase in NO was attenuated by all these interventions, including NOS inhibition (Fig. 6k). The cAMP increase evoked by forskolin was unaffected by CLR/RAMP1 antagonism and NOS inhibition, and olcegepant failed to inhibit NO release by the NO donor NONOate, indicating selectivity. NO release evoked by CGRP, but not that evoked by NONOate was inhibited by PS2 and Dy4, but not their inactive analogs, further supporting selectivity. These results suggest that clathrin- and dynamin-dependent endocytosis and endosomal CLR/RAMP1 signaling evoke NOS activation and NO generation in Schwann cells. Schwann cell TRPA1 mediates CGRP-evoked PMA NO belongs to a series of reactive oxygen species (ROS) that target TRPA1⁴⁰. TRPA1 is coexpressed with TRPV1 and CGRP in a subpopulation of primary sensory neurons⁴¹. TRPA1 is expressed in Schwann cells of nerve bundles of human skin and mouse sciatic nerve, where it mediates mechanical allodynia in rodent models of pain^30,42. Immunoreactive TRPA1 was coexpressed with RAMP1 in S100+ve Schwann cells in human abdominal and mouse periorbital cutaneous nerves bundles (Fig. 7a). Thus, CLR/RAMP1 might engage signaling pathways that activate TRPA1 in trigeminal Schwann cells to initiate allodynia (Fig. 7b). This hypothesis was supported by the observation that both CGRP- and capsaicin-evoked PMA were reduced in Trpa1^-/- mice and in mice with sensory neuron-specific deletion of TRPA1 (Adv-Cre⁺;Trpa1^fl/fl) (Fig. 7c, 7d). Next, the signaling pathway by which the CLR/RAMP1 activates TRPA1 was investigated. In HSCs and IMS32 cells, CGRP stimulated a slowly developing yet sustained increase in Ca²⁺ response and increased H2O2 levels (Fig. 7e-7g). Olcegepant, CGRP8-37, SQ22536, H89, L-NAME, Ca²⁺-free medium, a ROS scavenger (PBN) or a NOX1 inhibitor (ML171) attenuated these responses (Fig. 7e-7g). A TRPA1 antagonist (A967079) inhibited CGRP-stimulated Ca²⁺ and H2O2 responses (Fig. 7e-7g) but did not affect CGRP-stimulated NO formation (Fig. 7h). CGRP-evoked Ca²⁺ responses were reduced in Schwann cells from Trpa1^-/- mice. These results support the hypothesis that CGRP liberates NO, which activates Schwann cell TRPA1; activated TRPA1 promotes a Ca²⁺-dependent H₂O₂ generation that sustains a feed-forward mechanism comprising TRPA1 channel engagement and ROS release. In vivo results corroborated this hypothesis. Whereas CLR/RAMP1 antagonists or NO inhibitors attenuated PMA only if given before CGRP or capsaicin, both pre- and post- treatment with a TRPA1 antagonist, a ROS scavenger and a NOX1 inhibitor reduced PMA (Fig. 7i-7k. Although pretreatment with TRPA1 or ROS inhibitors did not affect the acute nociception, they inhibited capsaicin-evoked PMA. Post-treatment also attenuated capsaicin-evoked PMA. These findings highlight the mechanistic differences between acute nociception and delayed PMA. After an initial and transient NO-dependent phase, PMA is sustained by persistent ROS liberation, which targets TRPA1 in Schwann cells. This hypothesis is robustly supported by the observation that PMA evoked by CGRP or capsaicin was markedly attenuated in mice with selective deletion of TRPA1 in Schwann cells (Plp-Cre^ERT+;Trpa1^fl/fl) (Fig. 7l). Targeting endosomal CGRP signaling provides superior relief of CGRP- and capsaicin-evoked PMA The finding that persistent GPCR signaling from endosomes mediates pain transmission suggests that GPCRs in endosomes rather than at the plasma membrane are a valid and perhaps superior target for the treatment of pain^33,34,35. Nanoparticles have been used to deliver chemotherapeutics to tumor, where endocytosis and endosomal escape are necessary for drug delivery to cytosolic and nuclear targets⁴³. The realization that GPCRs within endosomes are a therapeutic target, raises the possibility of exploiting the acid microenvironment of endosomes as a stimulus for nanoparticle disassembly and release of antagonist cargo³

To target CLR in endosomes, self-assembling soft polymer nanoparticles containing a CLR antagonist were generated. Diblock copolymers were synthesized with a hydrophilic shell of P(PEGMA-co-DMAEMA) and a hydrophobic core of P(DIPMA-co- DEGMA) (Fig. 8a). Gel permeation chromatography and ¹H-nuclear magnetic resonance (¹H-NMR) confirmed the molecular weight and composition of nanoparticles. Nanoparticles were self-assembled with MK-3207, a potent hydrophobic antagonist of human CLR/RAMP1, forming DIPMA-MK-3207 (Fig. 8a). Empty nanoparticles (DIPMA-Ø) were used as a control. Nanoparticles were uniformly spherical, with similar diameter (30-35 nm) and ζ-potential (-0.4-1.3 mV) (Fig. 8b, 8c). DIPMA nanoparticles demonstrate a pH-dependent cargo release at pH<~6.5, consistent with the protonation of the DIPMA tertiary amine (pKa 6.1), charge repulsion and disassembly³⁴. DIPMA nanoparticles enter cells by clathrin- and dynamin-mediated endocytosis and disassemble in acidic early endosomes³⁴. To determine whether DIPMA nanoparticles target endosomes containing CLR/RAMP1, HSCs expressing early endosomal antigen-1-GFP (EEA1-GFP) were incubated with DIPMA-Cy5 for 30 min to allow accumulation in EEA1-GFP+ve endosomes (Fig. 8d). Cells were then incubated with TAMRA-CGRP, which was detected in endosomes containing Cy5-DIPMA within 5-10 min (Fig. 8d). Thus, DIPMA nanoparticles accumulate with CLR/RAMP1 in early endosomes of Schwann cells. To determine whether DIPMA-MK-3207 can antagonize CLR in endosomes, CGRP- stimulated cAMP formation was measured using the CAMYEL cAMP BRET sensor, which detects total cellular cAMP. HEK293 cells expressing rat CLR/RAMP1 (HEK- rCRL/RAMP1) were preincubated with graded concentrations of DIPMA-MK-3207 or free MK-3207, DIPMA-Ø or vehicle (control) for 30 min. Beginning at 0 min, baseline BRET was measured for 5 min, and cells were then challenged with CGRP. At 10 min, cells were washed to remove extracellular CGRP, and BRET was measured up to 35 min. In vehicle-treated cells, CGRP stimulated a prompt increase in cAMP formation (1^st phase, 6-10 min) that gradually declined after agonist removal from the extracellular fluid (2^nd phase, 11-35 min) (Fig. 8e). DIPMA-Ø did not affect this response. Free MK-3207 and DIPMA-MK-3207 (100, 316 nM) both inhibited CGRP-evoked cAMP in the 1^st phase to a similar extent (Fig. 8f). During the 2^nd phase, free MK-3207 was inactive at all concentrations whereas DIPMA-MK3207 (31.6, 100, 316 nM) strongly inhibited responses (Fig. 8g). The results suggest that DIPMA-MK3207 can antagonize the sustained phase of CGRP-stimulated formation of cAMP, which is attributable to endosomal CLR/RAMP1 signaling. To assess antagonism of the pain signaling pathway in HSCs, CGRP-evoked changes in Ca²⁺ response was measured, which depend on endosomal CGRP signaling and activation of TRPA1. HSCs were preincubated with graded concentrations of DIPMA- MK-3207 or MK-3207 for 20 min to allow accumulation in endosomes, and washed to remove extracellular compounds. At 10 min after washing, cells were challenged with CGRP and Ca²⁺ response was measured as an index of TRPA1 activity. DIPMA-MK-3207 inhibited CGRP-evoked increase in Ca²⁺ response (IC5015.4 nM, 95% confidence interval, 10.9 - 21.0 nM) more potently than free MK-3207 (IC₅₀2.9 μM, 95% confidence interval, 1.9 - 4.2 μM, P<0.0001) (Fig. 8h). To assess antinociception, DIPMA-MK-3207 or free MK-3207 (0.1, 0.3, 1.0 pmol) was injected into the periorbital region 30 min before periorbital injection of CGRP or capsaicin. DIPMA-MK-3207 (0.3, 1.0 pmol) more effectively inhibited PMA than the same doses of free MK-3207 (Fig. 8i). DIPMA-Ø had no effect. Thus, endosomal targeting enhances the efficacy of a CLR/RAMP1 antagonist in a preclinical model of migraine pain. Discussion The major findings of the present study are that CGRP causes PMA by activating CLR/RAMP1 of Schwann cells, CLR/RAMP1 signals from endosomes of Schwann cells to activate pain pathways, and endosomal CLR/RAMP1 can be targeted using nanoparticles and endocytosis inhibitors to relieve CGRP-evoked PMA. CLR/RAMP1 stimulation and trafficking to endosomes results in a persistent cAMP-dependent NOS activation and generation of NO, a mediator of migraine pain¹⁹. The role of NO in PMA is crucial, yet transient, as it is temporally limited to the engagement of TRPA1/NOX1, which releases ROS with a dual function. On one hand, ROS target TRPA1/NOX1 of Schwann cells to maintain ROS generation by a feed-forward mechanism. On the other hand, as suggested by experiments with selective TRPA1 deletion in primary sensory neurons, ROS target TRPA1 on nociceptors to signal allodynia to the CNS. Periorbital capsaicin injection elicited acute nociception mediated by TRPV1 excitation and ensuing afferent discharge, which signals pain to the CNS. In a larger cutaneous area, capsaicin evoked delayed and prolonged PMA. While the acute pain response is most likely dependent on ion influx associated with TRPV1 activation, the mechanism underlying mechanical hypersensitivity^7,44 has remained elusive. These findings support the existence of a paracrine mechanism that underlies PMA associated with neurogenic inflammation. It is suggested that capsaicin locally activates TRPV1+ve nerve fibers to generate action potentials that propagate antidromically into collateral fibers which release CGRP in a broader area, thus eliciting widespread PMA. PMA depends on the interaction between peptidergic nerve fibers, surrounding Schwann cells and nociceptive neurons that convey allodynic signals to the CNS. CGRP liberated from the varicosities of trigeminal TRPV1+ve nerve fibers binds to CLR/RAMP1 of adjacent Schwann cells. CNS perturbations may target the trigeminovascular system and initiate the migraine attack^22,23. These central mechanisms may underlie the delayed facial allodynia associated with migraine^20,21. However, the beneficial effect of anti-CGRP medicines that do not cross the blood brain barrier suggests that CGRP acts in the periphery to elicit pain. The peripheral site of the algesic action of CGRP released from peptidergic C-fibers has been proposed as the CLR/RAMP1 on adjacent non-peptidergic AG-fibers⁴⁵ and more precisely at the level of the node of Ranvier²⁸. The present results in Adv-Cre⁺;Ramp1^fl/fl mice suggest that CGRP does not act on trigeminal nociceptors to cause PMA in mice. This is consistent with failure of CGRP administration to elicit any itch, pain or axon reflex responses in humans⁴⁶. Instead, the results support the hypothesis that CGRP released from trigeminal nociceptors targets CLR/PAMP1 on Schwann cells that wrap their terminals to evoke PMA. Most Schwann cells in Remak bundles contain multiple unmyelinated axons from C- fiber nociceptors, including CGRP+ve fibers, which release the bulk of CGRP², and non- peptidergic isolectin B4+ve fibers⁴⁷. Thus, CGRP-evoked release of ROS from Schwann cells could induce allodynia by targeting TRPA1 on three neuronal subtypes, including the same AG- or C-fiber that releases CGRP, a different C-fiber of the same Remak bundle, or a different adjacent AG-fiber. The observation that both C-fiber and AG-fiber nociceptors contribute to capsaicin-evoked hypersensitivity in humans⁴⁸ supports the hypothesis that both types of neurons^28,45 are implicated in CGRP-mediated allodynia. CLR/RAMP1 signals from endosomes by G-protein-mediated mechanisms that activate a subset of compartmentalized signals, including cytosolic protein kinase C and nuclear extracellular signal regulated kinase; these kinases regulate excitation of spinal neurons and pain transmission³⁵. These results show that CLR/RAMP1 activates GD_s, GD_q and GDi and recruits EARRs in endosomes of Schwann cells, determined by EbBRET. Inhibitors of clathrin- and dynamin-mediated endocytosis blocked the recruitment of CLR/RAMP1, GD^and EARR to endosomes, which presumably requires CLR/RAMP1 endocytosis. GPCR/GD signaling complexes have also been detected in endosomes by using conformationally selective nanobodies⁴⁹. The observation that endocytosis inhibitors attenuated CGRP-stimulated cAMP formation and activation of NOS and TRPA1 reveals a central role for CLR/RAMP1 signaling in endosomes of Schwann cells in CGRP-evoked periorbital pain. Endocytosis of other Gs-coupled GPCRs is also necessary for the full repertoire of cAMP-mediated signaling outcomes, which entails endosomal recruitment of adenylyl cyclase 9⁵⁰ and assembly of metastable accumulations of PKA⁵¹. It was found that a nanoparticle-encapsulated CLR/RAMP1 antagonist, which targeted CLR/RAMP1 in endosomes and released cargo in the acidified endosomal microenvironment³⁴, also attenuated CGRP-stimulated cAMP formation and blunted TRPA1 activation. The observation that periorbital injection of inhibitors of clathrin and dynamin and of DIPMA-MK-3207 prevented CGRP- and capsaicin-evoked PMA provides the first evidence for a prominent role of endosomal CGRP signaling of pain from a peripheral site. The finding that nanoparticle encapsulation enhanced the potency of a CGRP antagonist for inhibition of endosomal signaling and resultant nociception supports the hypothesis that CLR/RAMP1 in endosomes mediates facial allodynia which contributes to migraine pain. Nanoparticle encapsulation similarly boosts the efficacy of an NK1 receptor antagonist in preclinical models of inflammatory and neuropathic pain³⁴. An antagonist of CLR/RAMP1 conjugated to a membrane lipid cholestanol also accumulates in endosomes and provides superior relief from pain³⁵, which reinforces the importance of CLR/RAMP1 endosomal signaling for pain transmission. Monoclonal antibodies to CGRP, although beneficial, are not effective in all patients¹⁰. While non-CGRP-dependent mechanisms might explain this failure⁵², monoclonal antibodies likely do not inhibit CGRP signaling in endosomes. The small molecule CLR/RAMP1 antagonist, rimegepant, was found to resolve migraine attacks in patients treated with the anti-CLR/RAMP1 monoclonal antibody, erenumab⁵³. This unexpected result was interpreted by the inherent membrane permeability of the lipophilic antagonist rimegepant⁵⁴ that might favor inhibition of CGRP signaling in endosomes⁵³, while neither receptor-targeted nor ligand-targeted monoclonal antibodies internalized with CLR/RAMP1 activated by CGRP⁵⁵. The results herein show a superior inhibition of CGRP signaling in Schwann cells and of PMA by DIPMA-MK-3207, which selectively targets receptor activity in endosomes, reveals a better approach to control allodynia. In 1936, Sir Thomas Lewis postulated¹ that in human skin action potentials are carried antidromically from the injured nerve terminal to collateral branches from where a chemical substance is released that produces the flare and increases the sensitivity of other fibers responsible for pain. CGRP has been previously identified as the mediator of neurogenic vasodilatation in rodents², and in humans⁸. Herein, it is proposed that CGRP is the ‘chemical substance’ that, via the essential role of endosomal CLR/RAMP1, TRPA1/NOX1 and oxidative stress of surrounding Schwann cells, sustains the enhanced sensitivity of primary sensory neurons associated with neurogenic inflammation (Fig. 9). The present results suggest that peripherally acting anti-CGRP medicines reduce migraine pain in part by targeting the facial allodynia that originates from CGRP-mediated endosomal signaling in Schwann cells. EXAMPLES Example 1 - Animals Male and female mice were use throughout (25-30 g, 5-8 weeks). The following strains of mice were used C57BL/6J mice (Charles River, RRID:IMSR_JAX:000664); wild-type (Trpa1^+/+) and TRPA1-deficient (Trpa1^-/-; B6129P-Trpa1^tm1Kykw/J; RRID:IMSR_JAX:006401, Jackson Laboratory) mice⁵⁶; wild-type (Trpv1^+/+) and TRPV1- deficient (Trpv1^-/-; B6129X1-Trpv1^tm1Jul/J, RRID:IMSR_JAX:003770, Jackson Laboratory) mice. Genetically modified mice were maintained as heterozygotes on a C57BL/6J background. To generate mice in which the Trpa1 and Ramp1 genes were conditionally silenced in Schwann cells/oligodendrocytes, homozygous 129S-Trpa1^tm2Kykw/J (floxed TRPA1, Trpa1^fl/fl, RRID:IMSR_JAX:008649 Jackson Laboratory) and C57BL/6N- Ramp1^{<tm1c(EUCOMM)Wtsi>/H} (floxed Ramp1, Ramp1^fl/fl Stock No: EM:07401, MRC HARWELL Mary Lion Center)⁵⁷ were crossed with hemizygous B6.Cg-Tg(Plp1- Cre^ERT)3Pop/J mice (Plp1-Cre^ERT, RRID:IMSR_JAX:005975 Jackson Laboratory), expressing a tamoxifen-inducible Cre in myelinating cells (Plp1, proteolipid protein myelin 1)³⁰. The progeny (Plp1-Cre^ERT;Trpa1^fl/fl and Ramp1-Cre^ERT;Trpa1^fl/fl) was genotyped by PCR for Trpa1, Ramp1 and Plp1-Cre^ERT. Mice negative for Plp1-Cre^ERT (Plp1-Cre^ERT-;Trpa1^fl/fl and Plp1-Cre^ERT-;Ramp1^fl/fl) were used as control. Both positive and negative mice to Cre^ERT and homozygous for floxed Trpa1 (Plp1-Cre^ERT;Trpa1^fl/fl and Plp1-Cre^ERT-;Trpa1^fl/fl, respectively) and floxed Ramp1 (Plp1-Cre^ERT;Ramp1^fl/fl and Plp1- Cre^ERT-;Ramp1^fl/fl) mice were treated with 4-hydroxytamoxifen (4-OHT) by subcutaneous periorbital (p.orb.) injection (0.02 mg/10 μl in corn oil once a day for 3 consecutive days). Some Plp1-Cre^ERT;Ramp1^fl/fl and Plp1-Cre^ERT-;Ramp1^fl/fl mice were treated with intraperitoneal (i.p.) or intraplantar (i.pl.) 4-OHT (1 mg/100 μl or 0.02 mg/10 μl in corn oil once a day for 3 consecutive days, respectively). Treatments resulted in Cre-mediated ablation of Trpa1 and Ramp1 in PLP- expressing Schwann cells/oligodendrocytes. To selectively delete the Trpa1 and Ramp1 gene in primary sensory neurons, Trpa1^fl/^fl and Ramp1^fl/fl mice were crossed with hemizygous Advillin-Cre mice (Adv-Cre)^30,58,59. Both positive and negative mice to Cre^ERT and homozygous for floxed Trpa1 (Adv-Cre⁺;Trpa1^fl/fl and Adv-Cre-;Trpa1^fl/fl, respectively) and floxed Ramp1 (Adv-Cre⁺;Ramp1^fl/fl and Adv-Cre-;Ramp1^fl/fl) were used. The group size of n = 8 animals for behavioral experiments was determined by sample size estimation using G*Power (v3.1)⁶⁰ to detect size effect in a post-hoc test with type 1 and 2 error rates of 5 and 20%, respectively. Mice were allocated to vehicle or treatment groups using a randomization procedure (http://www.randomizer.org/). Investigators were blinded to the identities (genetic background) and treatments, which were revealed only after data collection. No animals were excluded from experiments. All behavioral experiments were in accordance with European Union (EU) guidelines for animal care procedures and the Italian legislation (DLgs 26/2014) application of the EU Directive 2010/63/EU. Study was approved by the Italian Ministry of Health (research permits #383/2019-PR and #765/2019-PR). The behavioral studies followed the animal research reporting in vivo experiment (ARRIVE) guidelines⁶¹. Mice were housed in a temperature- and humidity-controlled vivarium (12 hr dark/light cycle, free access to food and water, 5 animals per cage). At least 1 hr before behavioral experiments, mice were acclimatized to the experimental room and behavior was evaluated between 9:00 am and 5:00 pm. All the procedures were conducted following the current guidelines for laboratory animal care and the ethical guidelines for investigations of experimental pain in conscious animals set by the International Association for the Study of Pain⁶². Animals were anesthetized with a mixture of ketamine and xylazine (90 mg/kg and 3 mg/kg, respectively, i.p.) and euthanized with inhaled CO2 plus 10-50% O2. Example 2 - Cell lines Primary cultures of human Schwann cells (HSCs, #1700, ScienCell Research Laboratories) were grown and maintained in Schwann cell medium (#1701, ScienCell Research Laboratories) at 37 °C in 5% CO₂ and 95% O₂. Cells were passaged at 90% confluency and discarded after 12 passages. HEK293T (#CRL-3216™, American Type Culture Collection) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with heat inactivated fetal bovine serum (FBS, 10%), L-glutamine (2 mM), penicillin (100 U/ml) and streptomycin (100 mg/ml) at 37 °C in 5% CO2 and 95% O2. The mouse Schwann cell line (IMS32 cells, #PMC-SWN-IMS32-COS, Cosmo Bio USA) was grown and maintained in Schwann cell medium (#PMC-SWN-MM-COS, Cosmo Bio USA) at 37 °C in 5% CO2 and 95% O2^63,64. All cells were used when received without further authentication. Example 3 - Behavioral experiments Treatment protocol. Subcutaneous injections were made in the periorbital area 2-3 mm from the external eyelid corner¹⁷. Briefly, the mouse was lifted by the base of the tail and placed on a solid surface with one hand and the tail was pulled back. Then, it was quickly and firmly picked up by the scruff of the neck with the thumb and index finger of the other hand. Injection was made rapidly by a single operator with minimal animal restraint. Mice received unilateral (right side) injections (10 μl/site) of CGRP (1.5 nmol in 0.9% NaCl), SP (3.5 nmol in 0.9% NaCl), capsaicin (10, 50, 100 pmol in 0.1% dimethyl sulfoxide, DMSO), or vehicles (control). Mice received bilateral injections (10 μl/site, right side same site as stimulus, left side symmetrical to right side) of antagonists and inhibitors. CGRP (1.5 nmol in 0.9% NaCl) or vehicle was also administered by intraplantar (i.pl., 20 μl/site) or systemic (0.1 mg/kg, i.p.) injection. GTN was administered at 10 mg/kg, i.p. injection. Acute nociception. Immediately after the p.orb. injection, mice were placed inside a plexiglass chamber and spontaneous nociception was assessed for 10 min by measuring the time (s) that the animal spent rubbing the injected area of the face with its paws^17,65. Periorbital mechanical allodynia. PMA was assessed using the up-down paradigm^66,67 in the same mice in which acute nociceptive responses were monitored. Briefly, mice were placed in a restraint apparatus designed for the evaluation of periorbital mechanical thresholds¹⁷. One day before the first behavioral observation, mice were habituated to the apparatus. PMA was evaluated in the periorbital region over the rostral portion of the eye (i.e., the area of the periorbital region facing the sphenoidal rostrum)⁶⁸ before (basal threshold) and after (0.5, 1, 2, 4, 6, 8 hr) treatments. On the day of the experiment, after 20 min of adaptation inside the chamber, a series of 7 von Frey filaments in logarithmic increments of force (0.02, 0.04, 0.07, 0.16, 0.4, 0.6 and 1.0 g) were applied to the periorbital area perpendicular to the skin, with sufficient force to cause slight buckling, and held for approximately 5 s to elicit a positive response. Mechanical stimuli were applied homolaterally outside the periorbital area at a distance of 6-8 mm from the site where stimuli were injected. The response was considered positive by the following criteria: mouse vigorously stroked its face with the forepaw, head withdrawal from the stimulus, or head shaking. Mechanical stimulation started with the 0.16 g filament. Absence of response after 5 s led to the use of a filament with increased force, whereas a positive response led to the use of a weaker (i.e. lighter) filament. Six measurements were collected for each mouse or until four consecutive positive or negative responses occurred. The 50% mechanical withdrawal threshold (expressed in g) was then calculated from these scores by using a δ value of 0.205, previously determined. Paw mechanical allodynia. Paw mechanical allodynia was evaluated by measuring the paw withdrawal threshold by using the up-down paradigm^66,67. Mice were acclimatized (1 hr) in individual clear plexiglass boxes on an elevated wire mesh platform, to allow for access to the plantar surfaces of the hind paws. von Frey filaments of increasing stiffness (0.07, 0.16, 0.4, 0.6 and 1.0, 1.4 and 2 g) were applied to the hind paw plantar surfaces of mice with enough pressure to bend the filament. The absence of a paw being lifted after 5 s led to the use of the next filament with an increased force, whereas a lifted paw indicated a positive response, leading to the use of a subsequently weaker filament. Six measurements were collected for each mouse or until four consecutive positive or negative responses occurred. The 50% mechanical withdrawal threshold (expressed in g) was then calculated. Example 4 - Primary culture of mouse Schwann cells Mouse Schwann cells (MSC) were isolated from sciatic or trigeminal nerves of C57BL/6J, and from sciatic nerve of Trpa1^+/+ and Trpa1^-/-, Plp1-Cre^ERT+;Ramp1^fl/fl and Plp1-Cre^ERT-;Ramp1^fl/fl mice^30,69. The epineurium was removed, and nerve explants were divided into 1 mm segments and dissociated enzymatically using collagenase (0.05%) and hyaluronidase (0.1%) in Hank's Balanced Salt Solution (HBSS, 2 hr, 37 °C). Cells were collected by centrifugation (800 rpm, 10 min, room temperature) and the pellet was resuspended and cultured in DMEM containing fetal calf serum (10%), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ml), neuregulin (10 nM) and forskolin (2 μM). Three days later, cytosine arabinoside (Ara-C, 10 mM) was added to remove fibroblasts. Cells were cultured at 37 °C in 5% CO2 and 95% O2. The culture medium was replaced every 3 days and cells were used after 15 days of culture. Example 5 - Calcium imaging HSCs, IMS32 and sciatic nerve MSCs cells were plated on poly-L-lysine-coated (8.3 μM) 35 mm glass coverslips and maintained at 37 °C in 5% CO2 and 95% O2 for 24 hr. Cells were loaded (40 min) with Fura-2 AM-ester (5 μM) added to the buffer solution (37 °C) containing (in mM) 2 CaCl₂; 5.4 KCl; 0.4 MgSO₄; 135 NaCl; 10 D-glucose; 10 HEPES and bovine serum albumin (BSA, 0.1%) at pH 7.4. Cells were exposed to CGRP (0.01-10 μM) or vehicle (0.9% NaCl) and the Ca²⁺ response was monitored for approximately 40 min. In another set of experiments, IMS32 cells were exposed to AITC (30 μM) or vehicle (0.03% DMSO). The Ca²⁺ response to CGRP was monitored in the presence of CGRP8-37 (100 nM), olcegepant (100 nM), SQ22536 (100 μM), L-NAME (10 μM), A967079 (50 μM), PBN (50 μM), H89 (1 μM), ML171 (1 μM) or vehicle (0.1 % DMSO), and in the presence of DIPMA-MK-3207 (1-1000 nM) and MK-3207 free drug (0.01-1000 μM) or DIPMA-empty. Some experiments used Ca²⁺-free buffer solution containing EDTA (1 mM). Results were expressed as percent increase in ratio_340/380 over baseline normalized to the maximum effect induced by ionomycin (5 μM) added at the end of each experiment. Example 6 - In-cell ELISA assay HSCs or IMS32 cells were plated in 96-well black wall clear bottom plates (Corning Life Sciences) (5 × 10⁵ cells/well) and maintained at 37 °C in 5% CO₂ and 95% O₂ for 24 hr. HSCs and IMS32 cells were exposed to CGRP (1 and 10 μM, respectively) or its vehicle (phosphate buffered saline, PBS) for 5, 10, 15, 30 and 60 min, at 37 °C, then washed with DMEM pH 2.5 and fixed in 4% paraformaldehyde for 30 min. Cells were then washed with TBST (0.05%) and blocked with donkey serum (5%) for 4 hr at room temperature and incubated overnight 4 °C with eNOS^pS1177 (#ab184154, rabbit polyclonal, 1:100, Abcam). Cells were then washed and incubated with donkey anti-rabbit IgG conjugated with horseradish peroxidase (HRPO, 1:2000, Bethyl Laboratories Inc.) for 2 hr at room temperature. Cells were then washed and stained using SIGMAFAST OPD for 30 min protected from light. After the incubation period, the absorbance was measured at 450 nm. Change in NOS3 phosphorylation was calculated as a percentage of the signal in vehicle-treated cells. Example 7 - cAMP ELISA assay cAMP level was determined by the CatchPoint™ cyclic-AMP fluorescent assay kit (#R8088, Molecular Device) according to the manufacturer’s protocol. Briefly, HSCs or IMS32 cells were plated in 96-well black wall clear bottom plates (Corning Life Sciences) (5 × 10⁵ cells/well) and maintained in 5% CO₂ and 95% O₂ (24 hr, 37 °C). The cultured medium was replaced with HBSS added with olcegepant (100 nM), CGRP8-37 (100 nM), SQ22536 (100 μM), L-NAME (10 μM) or vehicle (0.1% DMSO in HBSS) for 20 min at room temperature. HSCs or IMS32 cells were then stimulated with CGRP (1 and 10 μM, respectively), forskolin (1 μM, positive control) or their vehicles (HBSS) and maintained for 40 min at room temperature protected from light. Signal was detected 60 min after exposure to the stimuli. cAMP level was calculated using cAMP standards and expressed as nmol/1. Example 8 - Nitric oxide assay Nitric oxide was determined by using the fluorometric-orange assay kit (#ab219932, Abcam) according to the manufacturer’s protocol. HSCs or IMS32 cells were plated 96- well black wall clear bottom plates (Corning Life Sciences) (5 × 10⁵ cells/well) and maintained in 5% CO₂ and 95% O₂ (24 hr, 37 °C). The cultured medium was replaced with Hanks' balanced salt solution (HBSS) added with olcegepant (100 nM), CGRP8-37 (100 nM), SQ22536 (100 μM), L-NAME (10 μM), A967079 (50 μM), PS2 or Dy4 (both 30 μM), PS2 inact or Dy4 inact (30 μM) or vehicle (0.1% DMSO in HBSS) for 20 min at room temperature. HSCs or IMS32 cells were then stimulated with CGRP (1 and 10 μM, respectively), diethylamine NONOate (1 mM, positive control) or their vehicles (HBSS) and maintained for 40 min at room temperature protected from light. Signal was detected 60 min after exposure to the stimuli. Change in nitric oxide level was calculated as percentage of the vehicle. Example 9 - Immunofluorescence Anesthetized mice were transcardially perfused with PBS and 4% paraformaldehyde. Trigeminal and sciatic nerves were removed, postfixed for 24 hr, and paraffin-embedded. Human and mouse formalin fixed paraffin embedded (FFPE) sections (5 μm) were incubated with primary antibodies: TRPA1 (#ab58844, rabbit polyclonal, 1:400, Abcam), S100 (#ab14849, mouse monoclonal [4B3], 1:300, Abcam), CLR (#NLS6731, rabbit polyclonal, 1:30, Novus Biologicals), RAMP1 (#ab241335, rabbit polyclonal, 1:200, Abcam), diluted in fresh blocking solution (PBS, pH 7.4, 2.5% normal goat serum, [NGS]). Sections were then incubated with the fluorescent polyclonal secondary antibodies Alexa Fluor 488 and 594 (#A32731, #A327271:600; Invitrogen), and coverslipped using mounting medium with DAPI (Abcam). The Pearson correlation (Rcoloc) value for RAMP1 and S100 in the colocalization studies were calculated using the colocalization Plugin of the ImageJ software (ImageJ 1.32J, National Institutes of Health). The use of FFPE sections of human abdominal cutaneous tissues was approved by the Local Ethics Committee of the Florence University Hospital (Area Vasta Toscana Centro) (18271_bio/2020), according to the Helsinki Declaration, and informed consent was obtained. Example 10 - cAMP cADDIS assay HSCs were plated on poly-D-lysine-coated 96-well black wall clear bottom plates (Corning Life Sciences) (25 × 10³ cells/well) and incubated in 5% CO₂ and 95% O₂ for 4-6 hr. HSCs were transduced with the baculovirus mediated Green Upward cADDIS cAMP reporter (25 μl/well, Montana Molecular) following manufacturer’s instructions, and cells were incubated in 5% CO₂ and 95% O₂ (48 hr, 37 °C). HSCs were washed twice in HBSS plus HEPES (10 mM) pH 7.4. Cells were incubated in HBSS/HEPES with the CLR/RAMP1 antagonists olcegepant (100 pM-100 μM) or vehicle (control) for 30 min. Plates were mounted in a FlexStation3 plate reader (Molecular Devices) and fluorescence (485-500 excitation, 515-530 emission with cutoff at 510) was monitored. Baseline was measured for 1 min, and cells were stimulated with human CGRPD (100 pM-10 μM) or forskolin (10 μM, positive control). For single cell imaging, HSCs were plated on poly-D- lysine-coated 35 mm glass bottom dishes (MatTek, Ashland) (40 × 10³ cells/dish) and incubated in 5% CO2 and 95% O2 (overnight, 37 °C). HSCs cells were transduced with Green Upward cADDIS and incubated for 48 hr. HSCs were washed twice in HBSS/HBS and mounted on a Leica DMI8 microscope (Wetzlar, Germany). Fluorescence (470/40 excitation, 527/30 emission) was measured every 5 s. Baseline was measured for 30 s, and HSCs were challenged with human CGRPD (100 nM). Images were analyzed with ImageJ (NIH). To inhibit endocytosis, cells were incubated in HBSS containing 0.45 M sucrose or normal HBSS (control) for 30 min at 37 °C before cAMP assays. Example 11 - CAMYEL BRET cAMP assay HEK293 cells stably expressing the CAMYEL BRET sensor (~2x10⁶) were seeded into 90 mm Petri dish (Corning™, USA) in DMEM/FBS/Geneticin and incubated in 5% CO2 and 95% O2 (24 hr, 37 °C). Prior to the transfection, the medium was changed to fresh DMEM/FBS/Geneticin and rat CLR/RAMP1 was transfected (2.5 μg CLR/RAMP1 DNA/dish) using JetPEI (Polyplus Transfection, France) at a 1:6 ratio. After 24 hr, cells were plated in poly-L-lysine coated black 96 well CulturPlate (Perkin Elmer, USA) and incubated in 5% CO₂ and 95% O₂ (24 hr, 37 °C). BRET was assessed using a LUMIstar (BMG LABTECH, Germany). On the day of the assay, cells were equilibrated in HBSS for 30 min, supplemented with 12 mM HEPES at 37 °C in CO2-free incubator. DIPMA- MK-3207 or free MK-3207 was incubated for 25 min, followed by the addition of coelentrazine-h (50 μM) for 5 min. Baseline was then measured for 5 min, followed by stimulation with CGRP (100 nM, ~EC50), vehicle (HBSS) or forskolin (1 μM, positive control), and further measurements for 5 min. Buffer was then replaced by HBSS with coelentrazine-h (50 μM) and measurements were resumed for further 25 min. Example 12 - Statistical analysis Results are expressed as mean ± standard error of the mean (SEM). For multiple comparisons, a one-way analysis of variance (ANOVA) followed by the post-hoc Bonferroni’s test or Dunnett’s test was used. Two groups were compared using Student’s t- test. For behavioral experiments with repeated measures, the two-way mixed model ANOVA followed by the post-hoc Bonferroni’s test was used. Statistical analyses were performed on raw data using Graph Pad Prism 8 (GraphPad Software Inc.). IC₅₀ values and confidence intervals were determined from non-linear regression models using Graph Pad Prism 8 (GraphPad Software Inc.). 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Claims

WHAT IS CLAIMED IS: 1. A method for treating a condition selected from the group consisting of migraine pain and neuralgia in a subject in need thereof, the method comprising administering to the subject an effective amount of an agent that selectively targets and inhibits endosomal CGRP receptor signaling in a Schwann cell in the subject.

2. A method for treating a condition selected from the group consisting of migraine pain and neuralgia in in a subject in need thereof, the method comprising contacting a Schwann cell in the subject with an effective amount of an agent that selectively targets and inhibits endosomal CGRP receptor signaling in the Schwann cell.

3. The method of claim 1 or 2, wherein the condition is migraine pain.

4. The method of claim 1 or 2, wherein the condition is neuralgia.

5. The method of claim 5, wherein the condition is trigeminal neuralgia.

6. The method of any one of claims 1-5, wherein the migraine pain is CGRP-mediated migraine pain.

7. The method of any one of claims 1-6, wherein the agent is an agent that inhibits endocytosis of the Schwann cell CGRP receptor.

8. The method of any one of claims 7, wherein the agent is an inhibitor of a process selected from the group consisting of clathrin-mediated endocytosis; clathrin-independent endocytosis; caveolae-mediated endocytosis; micropinocytosis; dynamin-mediated endocytosis; dynamin-independent endocytosis; endosome maturation; and β-arrestin- mediated endocytosis.

9. The method of claim 7 or 8, wherein the agent is an inhibitor of a process selected from the group consisting of clathrin-mediated endocytosis; dynamin-mediated endocytosis; and β-arrestin-mediated endocytosis.

10. The method of any one of claims 3-9, wherein the agent is an inhibitor of a process selected from the group consisting of dynamin 1-dependent endocytosis and dynamin 2- dependent endocytosis.

11. The method of any one of claims 3-12, wherein the agent is an inhibitor of dynamin 2-mediated endocytosis.

12. The method of any one of claims 7-11, wherein the Schwann cell CGRP receptor is an activated Schwann cell CGRP receptor.

13. The method of claim 12, wherein the activated Schwann cell CGRP receptor is activated by interaction with CGRP.

14. The method of claim 13, wherein the agent comprises an anti-sense molecule targeting Dynamin 2, such as an oligonucleotide targeting Dynamin-2 resulting in decreased mRNA, such as an siRNA targeting Dynamin-2.

15. The method of claim 1 or 2, wherein the agent is a CLR/RAMP1 antagonist.

16. The method of claim 153, wherein the CLR/RAMP1 antagonist is encapsulated in a nanoparticle that is structurally predisposed to release the CLR/RAMP1 antagonist in the endosome.

17. The method of claim 13, wherein the CLR/RAMP1 antagonist comprises a lipid anchor that promotes insertion of the antagonist into a plasma membrane.

18. The method of any one of claims 1-15, wherein the agent comprises a targeting moiety having an affinity for binding to a plasma membrane-expressed moiety on the Schwann cell.

19. The method of claim 16, wherein the targeting moiety selectively binds to a plasma membrane-expressed protein on the Schwann cell and triggers endocytosis of this protein..

20. The method of claim 18, wherein the targeting moiety selectively binds to plasma membrane-expressed CLR on the Schwann cell.

21. The method of claim 18, wherein the targeting moiety selectively binds to plasma membrane-expressed RAMP1 on the Schwann cell.

22. The method of any one of claims 18-21, wherein the targeting moiety is an antibody.

23. The method of claim 22, wherein the agent is an antibody- nucleic acid molecule conjugate, such as an antibody-antisense oligonucleotide conjugate or an antibody-siRNA conjugate.

24. The method of any one of claims 1-23, wherein the agent is a peripherally restricted agent.

25. The method of any one of claims 1-24, wherein, upon administration of the agent to the patient, the level of reactive oxygen species released in the presence of the agent is less than the level of reactive oxygen species released in the absence of the agent.

26. The method of claim 25, wherein the level of reactive oxygen species released in the absence and presence of the agent is determined by measuring the activity of nitric oxide synthase, measuring the amount of reactive oxygen species released, and/or measuring the amount of biomarkers of oxidative stress present.

27. The method of claim 25 or 26, wherein the reactive oxygen species is nitric oxide (NO).

28. The method of any one of claims 1-27, wherein the subject exhibits one or more of the following symptoms: medium to strong predominantly unilateral headache, light, noise and/or smell sensitivity, nausea, facial pain, sore eyes, balance disturbance, and cognitive difficulties.

29. The method of claim 28, wherein the method further comprises evaluating the severity of the one or more symptoms.

30. The method of claim 29, wherein evaluating comprises administering to the subject a clinician-administered instrument of evaluation (e.g., Patient Perception of Migraine Questionnaire (PPMQ-R); 6-item Headache Impact Test (HIT-6) disability score; 12-Item Short Form Health Survey (SF-12) score; Patient Global Impression of Change (PGIC) score; and/or Sport ConCuSSion ASSeSment tool 3 (SCAT-3) score).

31. The method of claim 30, wherein evaluating comprises providing the subject with a validated self-reporting instrument of evaluation (e.g., four-point pain scale (none, mild, moderate, severe) and/or the 11-point pain scale (0 = no pain, 10 = pain as bad as it could be).

32. The method of any one of claims 28-31, wherein the severity and/or frequency of at least one of the symptoms is decreased after the first or subsequent administration of the agent.

33. The method of any one of claims 1-32, wherein the subject is suffering from 1 to 31 migraine days per month.

34. The method of any one of claims 1-33, wherein the subject is suffering from migraine with aura.

35. The method of any one of claims 1-33, wherein the subject is suffering from migraine without aura.

36. The method of any one of claims 1-35, wherein administration of the agent decreases migraine attack frequency experienced by the subject from a pre-administration level.

37. The method of claim 36, wherein administration of the agent decreases the number of migraine days per month and/or headache hours per month experienced by the subject from a pre-administration level.

38. The method of any one of claims 1-37, wherein administration of the agent decreases migraine attack severity experienced by the subject from a pre-administration level.

39. The method of claim 38, wherein the subject experiences a reduction of about 30% or greater in mean pain score and/or a reduction in the use of any acute headache medications from a pre-administration level.

40. The method of any one of claims 1-39, wherein administration of the agent decreases the number and/or severity of one or more neurological symptoms associated with migraine experienced by the subject from a pre-administration level.

41. The method of claim 40, wherein the one or more neurological symptoms are selected from the group consisting of phono-, photo-, and/or osmophobia, visual, sensory or motor disturbances, and allodynia.

42. The method of any one of claims 1-41, wherein administration of the agent decreases the number and/or severity of one or more symptoms known to accompany, precede or follow a migraine attack from a pre-administration level.

43. The method of claim 42, wherein the one or more symptoms known to accompany, precede or follow a migraine attack are selected from the group consisting of fatigue, nausea, cognitive difficulties, tiredness, ravenous hunger or thirst, muscle ache, reduced libido, depression, mania, and mood swings.

44. The method of any one of claims 1-43, wherein administration of the agent reverses, slows the progression of, and/or prevents structural or functional nerve cell damage, such as white matter lesions or disturbances in functional connectivity, associated with migraine from a pre-administration level.

45. The method of any one of claims 1-44, wherein administration of the agent reverses, slows the progression of, and/or prevents the transition of acute migraine to chronic migraine.

46. The method of any one of claims 1-45, wherein the agent is administered before onset of symptoms of a migraine attack.

47. The method of any one of claims 1-45, wherein the agent is administered concurrently with, or after, the onset of symptoms of a migraine attack.

48. The method of any one of claims 1-47, wherein the agent is administered to the trigeminal region of the subject.

49. The method of any one of claims 1-48, wherein the agent is administered locally to the trigeminal region of the subject.

50. The method of any one of claims 1-49, wherein the agent is administered intranasally.

51. The method of any one of claims 1-50, wherein the migraine pain is refractory or resistant to other traditional or conventional therapies including CLR or CGRP antibodies and sumatriptan.

52. The method of any one of claims 1-51, wherein the agent is administered in combination with one or more additional therapies.

53. The method of any one of claims 1-52, wherein the subject is a human.

54. The method of any one of claims 1-53, wherein the method further comprises identifying the subject in need of such treatment.

55. A method of identifying a candidate agent for treating migraine pain, the method comprising: (i) providing a Schwann cell comprising a CGRP-activated CLR/RAMP1; (ii) contacting the Schwann cell comprising the CGRP-activated CLR/RAMP1 with the candidate agent; and (iii) (A) determining the level of reactive oxygen species released in the presence and absence of the candidate agent; and/or (B) determining the level of severity of allodynia in the presence of the candidate agent; (C) determining the degree of light aversion wherein a candidate agent is identified when the level of (A) and/or (B) in the presence of the candidate agent is less than the level of (A) and/or (B) in the absence of the candidate agent..

56. A chemical entity that includes (i) a targeting moiety that selectively targets a Schwann cell; and (ii) a moiety that inhibits endosomal CGRP receptor signaling in the Schwann cell.