Treatment and prophylaxis of migraine

Abstract

A method of treatment or prevention of migraine is described which involves modulating the expression of alpha-calcitonin and beta-calcitonin gene-related peptides (α-CGRP and (β-CGRP, respectively), neuropeptides that are involved in migraine pathophysiology. Migraine is a common type of headache disorder that stands as the second highest cause of disability with estimated prevalence of about 15% globally. A cannabinoid formulation is described comprising primarily cannabichromene together with an excipient. A method of detecting the correlation between CALCA and CALCB genes with TRPM8 (Transient Receptor Potential Cation Channel Subfamily M Member 8) in spinal cord using RNA sequencing is described. An effective non-psychoactive therapy for migraine pain, migraine attacks and migraine prevention, based on the gene expression profile of patients, where patient response to cannabichromene treatment can be predicted by such biomarkers and benefit personalized therapy.

Description

TREATMENT AND PROPHYLAXIS OF MIGRAINECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 595,479, filed on November 2, 2023, entitled TREATMENT AND PROPHYLAXIS OF MIGRAINE, the content of which is hereby incorporated by reference.FIELD

[0002] The present disclosure relates generally to a formulation for prophylactic or medicinal use. More particularly, the present disclosure relates to cannabinoid formulations for use in prevention or treatment of migraine and / or management of migraine pain, and biomarkers for use in monitoring individuals.BACKGROUND

[0003] Individuals managing pain often turn to medicinal options that offer pain alleviation but are accompanied by unintended side-effects such as stomach upset, constipation, and risk of addiction and / or drug dependence. Alternative approaches are urgently needed.

[0004] Migraine is a complex and heterogeneous neurological disorder that leads to significant debilitation in millions of people worldwide.

[0005] The diagnosis of migraine is based on clinical criteria, and there is a need for reliable biomarkers that can enhance the diagnosis of the disease, monitor its progression, and predict the response to treatment. Biomarkers are measurable indicators of a biological process or condition that can be detected in the blood, saliva, urine, or cerebrospinal fluid. One of the most promising biomarkers for migraine is calcitonin gene-related peptide (CGRP), a neuropeptide that is involved in the activation of the trigeminovascular system, which mediates the pain and inflammation associated with migraine attacks. The most remarkable evidence of the involvement of CGRP in migraine pathogenesis is the elevated level of CGRP in physiological fluids in migraine patients during headache attacks and initiation of delayed migraine-like headaches after CGRP increase (Vandervorst et al., 2021 ; Demartini et al., 2023). CGRP release during a migraine attack leads to vasodilation of cerebral blood vessels, neuroinflammation and sensitization of pain pathways (Vandervorst et al., 2021). Studies have shown reduction of the CGRP levels after treatment with therapeutic CGRP antagonists or monoclonal antibodies (Demartini et al., 2023; Messoud et al., 2021).

[0006] In 2018, the U.S. Food and Drug Administration (FDA) updated the guidance for the acute migraine treatment trials to evaluate treatment efficacy based on pain freedom and freedom from the most bothersome symptoms, including photophobia, phonophobia or nausea at 2 hours as determined by patients (Ocheretyaner et al., 2022). Current FDA-approved drugs for migraine in adults exert their activity by blocking CGRP, the key neurotransmitter in the pathogenesis of migraines. Targeting CGRP is suggested to reduce the frequency and severity of migraine episodes. However, most of the approved drugs for migraine in adults are intended for migraine prevention with limited optionality for migraine treatment.

[0007] Cannabinoids are a group of structurally similar compounds isolated from cannabis plants, which activate cannabinoid receptors in cells. Cannabinoids may be synthesized or may be isolated from cannabis plants or plant extracts (herein: a cannabinoid-containing plant extract). Cannabinoids can be isolated from plants or extracts to the extent that they are obtained in nearly pure, or essentially pure form, free of significant amounts of other naturally occurring compounds, such as other cannabinoids or plant-derived molecules such as terpenes. Known cannabinoids include but are not limited to tetrahydrocannabinol (THC); cannabidiol (CBD), cannabichromene (CBC); tetrahydrocannabidivarin (THCV); tetrahydrocannabinolic acid (THCA); cannabigerol (CBG); cannabidivarin (CBDV), cannabinol (CBN), and cannabidiolic acid (CBDA). Cannabis plants may be bred to have different amounts of a certain cannabinoid, as may be desirable for different purposes. THC and CBD have, to date, been considered as the predominant cannabinoids of interest.

[0008] CBD has been widely studied medicinal effects. CBD is regarded as having an effect on 5HT1 A receptor-mediated neurotransmission, as well as on anandamide metabolism and activation of TRPV1 receptor channels that facilitate CB1- and CB2-mediated responses (Crippa JS 2018).

[0009] A9-THC exerts partial agonistic activity on CB1 and CB2 receptors with high binding affinity with CB1 receptor leading to its psychoactive activity.

[0010] Cannabichromene (CBC) is a major non-psychotropic cannabinoid naturally found in the Cannabis sativa plant (ElSohly M 2014).

[0011] The proportion of each of these cannabinoids in the cannabis plant is, however, dependent on environmental growth conditions, geographical location, genetics, and chemotype (Lewis M A 2017).

[0012] CBC has moderate affinity (Ki ~ 100 nanomolar) only for CB2 receptors and binds to CB1 receptors only at concentrations higher than 1 micromolar (Shinjyo N 2013). The major CBC activity in brain has been suggested to be partly dependent onindirect activation of CB1 receptor by inhibition of cellular uptake of anandamide (De Petrocellis L 2011) and activation of TRPA1 (Transient Receptor potential A1) channels (Izzo and Capasso R 2012). In fact, CBC is found to be the most potent agonist of all the phytocannabinoids at TRPA1 channels (Maione S 2011). CBC has also shown antiinflammatory effects (Izzo and Capasso R 2012).

[0013] It has been demonstrated that CBD can act synergistically with A9-THC and contribute to the analgesic effect of medicinal-based cannabis extract (Russo 2011).

[0014] The agonistic activity of CBC with CB1 and CB2 receptors can offer a promising approach to potentiate the effect of other cannabinoids that exert their activities via binding and activation of CB1 an CB2 receptors.

[0015] Medicinal uses of cannabinoids are known, and formulations specifically to treat pain have been described. W02007 / 083098 A1 (GW Pharma Ltd) describes cannabinoid-containing plant extracts for treatment of neural degeneration. U.S. Patent Publication No. US2016 / 0106705 (United Cannabis Corp.) describes cannabis extracts having at least four cannabinoids and a terpene or flavonoid for use in relieving anxiety, pain, and related disorders. WO2016 / 044370 A1 (India Globalization Capital Inc.) teaches a topical pain-relieving formulation containing a combination of THC, CBD and cobalamin. WO2013 / 165251 A1 (ECHO Pharmaceuticals BV) describes a thin film evaporation method for obtaining THC-containing isolates, which may have trace only amounts of CBN or CBD. In WO2012 / 144892 A1 (Fytagoras BV), the use of acidic cannabinoids such as THC, CBD, and other cannabinoids for enhancing an animal’s natural cellular resistance to disease is described. Further, in WO2012 / 160358 A1 (GW Pharma Ltd.), the use of at least one of CBG, CBC, CBDV and THCV as a treatment of neuropathic pain is described.

[0016] WO2021 / 212209 A1 (Zyus Life Sciences Inc.) describes the use of a cannabichromene formulation for pain management is described.

[0017] W02020 / 223800 A1 (Zyus Life Sciences Inc.) describes the use of a cannabinoid formulation containing cannabichromene is described for use in pain management.

[0018] The potential of certain individual cannabinoids to have primary medicinal effects for treatment of migraine, has not been fully explored. It is desirable to provide a cannabinoid formulation with beneficial properties for use in treatment of migraine, and to utilize strategies for prevention and monitoring of migraine with accurate biomarkers.SUMMARY

[0019] It is an object of the present disclosure to obviate or mitigate at least one disadvantage of previous formulations for the treatment or prevention of migraine.

[0020] Cannabichromene (CBC) has not previously been established as able to act as a primary medicinal ingredient for treatment of migraine, and it has not heretofore been shown that CBC would have impact on biomarkers of migraine.

[0021] The formulation described herein is for use in treatment or prevention of migraine.

[0022] There is provided herein a formulation for use in a method of treatment or prophylaxis of migraine in a subject in need thereof, said formulation comprising a primary cannabinoid and one or more excipient, diluent or carrier, wherein said primary cannabinoid is cannabichromene (CBC).

[0023] The formulation may be essentially free of tetrahydrocannabinol (THC). The formulation may comprise one or more secondary cannabinoids, preferably cannabidiol (CBD). When present, the one or more secondary cannabinoids may be present in an amount of up to 15% by weight of the primary cannabinoid. The formulation may be prepared in a dosage form selected from the group consisting of a pill, tablet, gel capsule, syrup, oil-based spray, topical or nasal formulation, and liquid oil form. The formulation may provide a total amount of from about 10 mg to about 200 mg of primary cannabinoid per dose, preferably from about 10 mg to about 100 mg per dose, or from about 20 mg to about 50 mg per dose.

[0024] A method is described herein for treatment or prophylaxis of migraine in a subject in need thereof, comprising administering to said subject an effective amount of a formulation comprising a primary cannabinoid and an excipient, diluent or carrier, wherein said primary cannabinoid is cannabichromene (CBC).

[0025] In this method, the formulation used may be essentially free of tetrahydrocannabinol (THC). Further, the formulation may additionally comprise one or more secondary cannabinoids, preferably cannabidiol (CBD). When present, the one or more secondary cannabinoids may be present in the formulation in an amount of up to 15% by weight of the primary cannabinoid. The formulation administered in this method may be in a dosage form selected from the group consisting of a pill, tablet, gel capsule, syrup, oil-based spray, topical or nasal formulation, and liquid oil form. The formulation may provide the subject with a total amount of from about 10 mg to about 200 mg of primary cannabinoid per dose, preferably from about 10 mg to about 100 mg per dose, or from about 20 mg to about 50 mg per dose. This method for treatment or prophylaxis ofmigraine may further comprise the step of evaluating CALC or TRPM8 gene expression following administration to determine migraine status.

[0026] Use of a formulation is described herein for treating migraine in a subject in need thereof, in which the formulation comprises an effective amount of a primary cannabinoid and an excipient, diluent or carrier, and the primary cannabinoid consists of cannabichromene (CBC). A use of a formulation for preparation of a medicament for treating migraine in a subject in need thereof is also described, in which the formulation comprises an effective amount of a primary cannabinoid and an excipient diluent or carrier, and in which the primary cannabinoid consists of cannabichromene (CBC). In such uses, the formulation may be essentially free of tetrahydrocannabinol (THC). Further, the formulation may additionally comprising one or more secondary cannabinoids, such as preferably cannabidiol (CBD). When the one or more secondary cannabinoids is present, it may be used in an amount of up to 15% by weight of the primary cannabinoid. The formulation may be prepared in a dosage form selected from the group consisting of a pill, tablet, gel capsule, syrup, oil-based spray, topical or nasal formulation, and liquid oil form. The formulation may provide a total amount of from about 10 mg to about 200 mg of primary cannabinoid per dose, preferably from about 10 mg to about 100 mg per dose, or from about 20 mg to about 50 mg per dose.

[0027] A method is described herein for identifying a subject responsive to CBC for treatment or prophylaxis of migraine, the method comprising evaluation of CALC and TRPM8 gene expression, upon treatment with CBC, and determining a responsive subject as having downregulated expression following CBC treatment.BRIEF DESCRIPTION OF THE FIGURES

[0028] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0029] Figure 1 depicts differential expression of the CALCA gene in the spinal cord of SNL rats pre-surgery vs. post-surgery.

[0030] Figure 2 depicts differential expression of the CALCB gene in the spinal cord of SNL rats pre-surgery vs. post-surgery.

[0031] Figure 3 depicts differential expression of the TRPM8 gene in the spinal cord of SNL rats pre-surgery vs. post-surgery.

[0032] Figure 4 depicts differential expression of the CALCA gene in the spinal cord collected from SNL rats following treatment with Cannabichromene (CBC; 10 mg / Kg; p.o.), morphine (10 mg / Kg; i.p.), or vehicle daily for 20 days.

[0033] Figure 5 depicts differential expression of the CALCB gene in the spinal cord collected from SNL rats following treatment with Cannabichromene (CBC; 10 mg / Kg; p.o.), morphine (10 mg / Kg; i.p.), or vehicle daily for 20 days.

[0034] Figure 6 depicts differential expression of the TRPM8 gene in the spinal cord collected from SNL rats following treatment with vehicle, morphine (10 mg / Kg; i.p.), or Cannabichromene (CBC; 10 mg / Kg; p.o.) daily for 20 days.DETAILED DESCRIPTION

[0035] Generally, the present disclosure provides a cannabichromene formulation for treating migraine, and for preventing, monitoring and managing pain associated with migraine. It has not previously been recognized that cannabichromene can have an effect on migraine treatment and biomarkers thereof when relied upon as the primary cannabinoid in a formulation.

[0036] A formulation is described for treating and preventing migraine comprising cannabichromene as the primary cannabinoid together with an excipient. Methods of use of the formulation, doses and dosage forms are described. Correlations between CALC and TRPM8 gene expressions in migraine pain were found, with downregulated expression resulting from CBC treatment. The use of these biomarkers is described for use in monitoring treatment, prevention or onset of migraine in subjects using CBC.

[0037] The described methods and uses for treatment or prevention of migraine involve modulating the expression of alpha-calcitonin and beta-calcitonin gene-related peptides (a-CGRP and p-CGRP, respectively), neuropeptides that are involved in migraine pathophysiology. Migraine is a common type of headache disorder that stands as the second highest cause of disability with estimated prevalence of about 15% globally. The cannabinoid formulation described herein comprises primarily cannabichromene together with an excipient. The described methods of detecting the correlation between CALCA and CALCB genes with TRPM8 (Transient Receptor Potential Cation Channel Subfamily M Member 8) in spinal cord using RNA sequencing permits monitoring of migraine treatment. This permits an effective non-psychoactive therapy for migraine pain, migraine attacks and migraine prevention, based on the gene expression profile of patients, where patient response to cannabichromene treatment can be predicted by such biomarkers and can benefit personalized therapy.

[0038] A formulation for use in migraine treatment of a subject in need thereof is described. The formulation comprises a primary cannabinoid and an excipient. The primary cannabinoid consists of cannabichromene (CBC). The formulation is essentially free of tetrahydrocannabinol (THC), meaning that small and insignificant amount may bepresent, for example at an amount of 1% or less by weight of the CBC. The management of migraine pain attributable to the singular presence of the CBC as the primary cannabinoid in the formulation has been surprisingly found.

[0039] A formulation is described herein for use in a method of treating migraine by a subject in need thereof, said formulation comprising a primary cannabinoid and an excipient, carrier, or diluent wherein said primary cannabinoid consists of cannabichromene (CBC), and wherein said formulation is essentially free of tetrahydrocannabinol (THC). Essentially free may mean an insignificant amount, for example at 1% by weight or less, 0.5% by weight or less, or 0.1% by weight or less as compared with the weight of the primary cannabinoid.

[0040] The formulation may additionally comprise one or more secondary cannabinoids, preferably cannabidiol (CBD).

[0041] The formulation may comprise the one or more secondary cannabinoids in an amount of up to 15% by weight of the primary cannabinoid.

[0042] The formulation may be prepared in a dosage form selected from the group consisting of a pill, tablet, gel capsule, syrup, oil-based spray, topical or nasal formulation, and liquid oil form.

[0043] The formulation may provide a total amount of from about 10 mg to about 200 mg of primary cannabinoid per dose, preferably from about 10 mg to about 100 mg, or about 20 mg to about 50 mg. On a daily basis, amounts used can vary widely depending on the individual’s tolerance, body weight, and the extent of the pain endured from migraine.

[0044] A method for migraine treatment is described herein for use by a subject in need thereof, comprising administering to said subject an effective amount of a formulation comprising a primary cannabinoid and an excipient, wherein said primary cannabinoid consists of cannabichromene (CBC), and wherein said formulation is essentially free of tetrahydrocannabinol (THC).

[0045] In the method described, the formulation may additionally comprise one or more secondary cannabinoids, preferably cannabidiol (CBD).

[0046] The formulation used in the method may involve the one or more secondary cannabinoids being present in the formulation in an amount of up to 15% by weight of the primary cannabinoid. The method may involve administration in a dosage form selected from the group consisting of a pill, tablet, gel capsule, syrup, oil-based spray, topical or nasal formulation, and liquid oil form.

[0047] The method may comprise administration to the subject an amount of the formulation that provides to the subject a total amount of from about 10 mg to about 200mg of primary cannabinoid per dose, preferably from about 10 mg to about 100 mg or from about 20 mg to about 50 mg per dose.

[0048] Primary Cannabinoid. The term “primary” is meant to indicate the cannabinoid that is primarily responsible for the intended effect of migraine treatment, as described herein. CBC is the primary cannabinoid, and it has been found to be effective when used without significant amounts of other cannabinoids in the context of migraine treatment and / or pain management attributable to migraine. If another cannabinoid is present in the formulation in a lower amount, the quantity present would not render it a “primary” cannabinoid. But other cannabinoids can be present in the formulation as secondary cannabinoids.

[0049] Cannabinoid Sources. The primary cannabinoid, CBC, may be present in the formulation from natural sources, such as from one or more cannabis plants, an in particular extracts thereof. Or the primary cannabinoid may be obtained from one or more isolated sources, or from a synthetic source where one or more of the desired cannabinoids is synthesized. A blend of natural and synthetic cannabinoids may be used so that a natural source with a variable content (due to growing conditions or other reasons), may be standardized to pre-determined amounts using adjustment with synthetic or isolated sources.

[0050] An extract may be obtained from a plant that is specially modified or grown under conditions conducive to production of a cannabinoid ratio particularly suited to the desired primary cannabinoid ratio, without needing to dramatically alter or supplement the amount of any of the primary cannabinoids present.

[0051] If purification of cannabinoids is desired extraction methods such as an ethanolic extraction, or a CO2based extraction may be used.

[0052] Cannabinoids may be incidentally present in the formulation, and if present, the quantities of such additional cannabinoid ingredients would not reduce or significantly influence the migraine treatment feature of the formulation.

[0053] Subjects and Populations. The formulation may be used by humans or by pets (companion animals such as dogs or cats), as well as for working animals such as horses, where migraine has been detected.

[0054] Subjects in need of a therapeutic effect for migraine treatment may use the formulation prior to, during, or after the migraine event or as need arises. Migraine pain can be debilitating for a number of reasons. Management with the formulation described herein can avoid problems or side effects inherent with other migraine medications.

[0055] Further, the formulation may be used prophylactically to lessen the pain that is anticipated when an individual feels a migraine coming on.

[0056] Biomarkers of migraine shown herein to be responsive to CBC treatment may be monitored in samples from individuals susceptible to migraine to compare with a self-reference to as to determine efficacy of treatment and monitor for migraine onset or improvement while a subject is undergoing treatment or prophylactic use.

[0057] Mode and Forms of Delivery. The formulation is amenable to oral delivery, such as in a pill, tablet, gel capsules, syrup, oil-based spray, topical or nasal formulation, or liquid oil form. The oral form may be provided in a food or as a food supplement, which may be added to a food to be more palatable or readily consumed by a subject. Topical or nasal absorption is possible, such as in the form of a cream, gel or spray. A fat-soluble carrier, or nano- or micro-particles or emulsions may be used so that the highly fat-soluble cannabinoids can be more readily absorbed. The formulation may be prepared as an injectable, for intravenous, intramuscular, or intraocular delivery. The formulation may be delivered in a vapor, such as by vaping, in a vaporizer or puffer, or may be heated to cause volatilization and inhalation which could be considered as “smoking”.

[0058] Dosages. CBC is the primary cannabinoid in the dosage form of the formula. Other cannabinoids may be present in the formulation. On a per dosage basis, the total amount of primary cannabinoid may range from 10 mg - 200 mg, for example 10 mg - 100 mg, or 20 mg - 50 mg per dose. If delivered in a liquid such as an oil, amounts may be expressed on a mg / mL basis, such as from 1 mg / mL - 200 mg / mL, for example 1 mg / mL - 100 mg / mL, or 5 mg / mL - 50 mg / mL, such that requisite doses may be calculated on a volume basis. Dosages may be used as needed depending on the severity of the migraine experienced, but an individual may wish to use the formulation on an as-needed basis, ranging from once per day (or less, if not needed) to more frequently such as taking 6 doses per day, with a frequency of every 4 hours.

[0059] An exemplary formulation may be a solid dosage form such as a pill, tablet, or granule-containing capsule. Alternatively, the formulation may be liquid-based, and may contain isolated or synthetic primary cannabinoid, or may be an oil-based extract of cannabis with significant quantities of CBC. The formulation may be in liquid forms such as oil, and oil-based spray, or a liquid-containing gel capsule (soft-gel capsule). A topical or nasal formulation may be prepared, such as a cream, gel or spray. If liquid-containing or gel-containing capsules are used, these may be limited in volume, for example an approximate volume of 200 pL. The milligram quantity stated above as a dosage range may be included in each such capsule, or the capsules may be formulated so as to be less concentrated in units of mg / mL. When less concentrated capsules areused, then the appropriate dosage is delivered by increasing the number of capsules consumed per dose.

[0060] An individual with migraine may consume orally, on an as-needed or regular basis such as every 6-hours, a dose of the following oil-based cannabinoid formulation. For example, the formulation may comprise 20 mg / mL CBC, and 1 mg / mL CBD, in an oil-based liquid. At the appropriate interval, the individual may take 1 mL orally. As an exemplary regime: initially, the individual may begin by consuming 1 mL of the formulation as-needed or at a frequency of twice per day. The dose may be titrated to a higher amount over time as the individual becomes accustomed to the formulation, until a dose of 1 to 2 mL, taken from 4 to 6 times per day is reached.

[0061] Carriers, Excipients, Diluents and Other Formulation Ingredients.The formulation may incorporate any acceptable carriers, excipients, diluents, or other ingredients (such as colouring, for example), as may be known in formulating drugs or cannabinoid formulations. Such ingredients may include starch, cellulose, alginates, colloidal silicon, lubricants such as stearates, salts, aqueous and non-aqueous (fat soluble) ingredients. The usual formulation considerations would be brought to bear, as one of skill in the art would understand.

[0062] Biomarker Monitoring. Evaluation of biomarkers indicative of migraine status, such as CALCA, CALCB, or TRPM8 gene expression in a body fluid, the downregulation of which is indicative of improvement in migraine status. Persons readily responding to CBC treatments can be identified, and also the need for or efficacy of treatment or prevention strategies involving CBC can be evaluated.

[0063] Migraine Types. Migraine types may be classified according to accompanying symptoms. Migraines may be classified as migraine with or without aura (the two most common classifications) or as migraine with typical aura, migraine with brainstem aura, hemiplegic migraine, retinal migraine, or chronic migraine, with the term “aura” indicating neurological symptoms accompanying the migraine, such as vision blurring, or tingling or numbness in limbs.

[0064] Example 1

[0065] Formulation for Treating Migraine

[0066] Migraine is a complex and heterogeneous neurological disorder that leads to significant debilitation in millions of people worldwide.

[0067] CALCA and CALCB are the encoding genes for alpha-calcitonin and betacalcitonin gene-related peptide (a-CGRP and p-CGRP, respectively), neuropeptides that are involved in migraine pathophysiology. CALCA and CALCB are associated with eachother in migraine because they both influence the levels of CGRP and its receptors in the trigeminovascular system, which mediates the pain and inflammation associated with migraine attacks. Human CALCA and CALCB differ only by three amino acids, and, in consequence, similar biological activities are shared. CALCA is considered the principal form of CGRP found in the central and peripheral nervous systems, while CALCB is found mainly in the enteric nervous system (Ocheretyaner ER., et al, 2022).The CALCB region has been identified as a migraine risk locus in a genome-wide association study (GWAS) with over a hundred thousand migraine cases (Hautakangas et al. 2022). p-CGRP is a splice variant of alpha-calcitonin gene-related peptide (a- CGRP), which is encoded by the CALCA gene. Both a-CGRP and p-CGRP can bind to the same receptors and have similar biological effects, such as vasodilation, inflammation, and pain modulation (Messoud A, et al., 2021).

[0068] The regulation of CALCA (CGRP-1) expression occurs mainly at the transcriptional level. CGRP exerts biological action through the interaction with its complex heterotrimeric G-protein coupled receptor, and other proteins (Ocheretyaner et al., 2022). CALCB (CGRP-2) is located on the 11th chromosome but in a distinct site from CALCA (Alevizaki M, et al, 1986). CGRP may also play a role in the transition of acute migraine condition to chronic, as chronic migraine patients have higher CGRP levels than episodic migraine patients (Ocheretyaner et al., 2022). Further, CALCA and CALCB may also have distinct roles in migraine subtypes (Ocheretyaner et al., 2022). In a research study that mapped the cell types and gene-expression profiles across the trigeminal nerve in mice and humans using single-cell RNA sequencing, CALCA and CALCB were shown to be expressed in different subsets of neurons, with distinct molecular signatures and functional properties (Fila et al., 2022).

[0069] The formulation described herein exerts down regulation of CALCA and CALCB genes at the central nervous system level. Inhibition or blockage of CGRP activity, as the key neurotransmitters involved in the pathogenesis of migraines, makes Cannabichromene a promising potential novel drug for the reduction of migraine attacks and migraine prevention. Furthermore, due to the abundant expression of the members of Transient receptor potential (TRP) channels in the primary sensory neurons and their primary role in several models of pain diseases, including inflammatory, neuropathic cancer pain, and migraine pain (lannone et al., 2022), DEG (differentially expressed genes) of TRP encoding genes was also screened. TRP channels play a key role in how pain signals are generated and transmitted and can offer potential targets for pain management (Spekker et al., 2023). Studies support that blocking CGRP receptors can reduce the activity of TRP channels. Similarly, TRP channels can also trigger the releaseof CGRP (Weyer et al., 2017). TRPM8 antagonists have shown therapeutic effects for chronic pain, migraine or inflammation by suppressing cold pressor response to cold- induced allodynia, or to chemotherapy-induced cold allodynia (Spekker et al., 2023). It has been demonstrated that cutaneous TRPM8 is involved in the regulation of autonomic and behavioral thermos-effectors associated with body temperature maintenance and TRPM8 antagonists have shown body temperature decrease in rodents (Almedia et al., 2012). TRP channels and their involvement in migraine therapy is supported by the clinical success of CGRP receptor antagonism and induction of CGRP release by activation of TRP channels (Dussor et al., 2014)

[0070] Objectives. In this study, the analgesic effect of CBC on migraine pain was investigated by differential gene expression profiling in a gold standard rat model of pain treated with CBC formulation. Gene expression profiling of dorsal root ganglia and spinal cord tissue samples was conducted on samples collected from the CBC-treated or control animals to evaluate the differential gene expression in robust biomarkers of migraine pain, CALCA and CALCB (Calcitonin related polypeptide Alpha and Beta) genes.

[0071] Study Procedure. Adult CD® Sprague Dawley rats (male and female) were obtained from Charles River, acclimatized, and then underwent sciatic nerve ligation (SNL) or Sham. Surgical procedures were performed as per Spinal Nerve Ligation (SNL) Model of Charles River, Canada (www.criver.com). After a 7-day post-op care, the success of SNL surgery was validated by behavioral assessments, such as electronic von Frey and the SNL animals were then randomly distributed to undergo treatments. The animals received Vehicle, Morphine (10 mg / Kg; i.p.), or Cannabichromene (CBC) (10 mg / Kg; p.o.) daily for 20 days.

[0072] Following 20 days of daily treatment, tissue samples from the dorsal root ganglion (DRG) and spinal cord (SC) tissue samples were collected from 50 rats, collected in sterile Hibernate A (BrainBits, www.Transnetyx.com) containing B27 (GIBCO™) and GLUTAMAX™ supplement (GIBCO™). The experiments were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, Canada, 2011) and in accordance with European Union Directive 2010 / 63 and the Dutch law. The experiments were approved by the Animal Welfare Body (Instantie voor Dierenwelzijn, the Netherlands). Five samples were taken from each tissue type for the following groups:

[0073] Pre-surgery baseline (day -5):

[0074] - Pre-treatment group (nDRG = 5 and nSc = 5)

[0075] Post-surgery pain baseline (day 7):

[0076] - Post-treatment group (nDRG = 5 and nSc = 5)

[0077] 20-day chronic daily treatment (day 28):

[0078] - Vehicle group (nDRG = 5 and nSc = 5)

[0079] - Morphine group (nDRG = 5 and nSc = 5)

[0080] - CBC group (nDRG = 5 and nsc = 5)

[0081] Therefore, a total of 30 DRG samples and 30 SC samples (a total of 60 samples) were harvested and processed for RNA sequencing.

[0082] RNA was then extracted from tissue samples within two batches, i.e. dorsal root ganglion (DRG) and spinal cord (SC) using a combined Trizol / RNEASY® method and the RNEASY® Mini kit (Qiagen). RNA QC was performed by microcapillary electrophoresis using an Agilent 2100 Bioanalyzer instrument. The RNA libraries were then prepared using the TruSeq Stranded Total RNA Kit (www.lllumina.com) with RiboZero Gold (www.lllumina.com). Briefly, Ribosomal RNA (rRNA) were removed from RNA samples using biotinylated, target-specific oligonucleotides covalently bound to Ribo-Zero rRNA removal beads. Following purification, RNA samples were fragmented into small pieces using divalent cations under elevated temperature. RNA fragments were then retro-transcribed into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The cDNA fragments were then subjected to the addition of a single "A" nucleotide and subsequent ligation of adapter. The products were purified and enriched by PCR to create the final cDNA library. Libraries underwent paired-end sequencing on a NOVASEQ™ 6000 instrument (www.lllumina.com), with about 26.7 million paired end reads and 2 x 150 bases. After an internal quality control of the libraries, the samples were pooled (i.e. multiplexed) per batch and each batch will be sequenced on one NOVASEQ™ 6000 sequencing lane (i.e. one batch = one lane). After sequencing, data is demultiplexed using the unique indexes.

[0083] Quality Control and Exploratory Data Analysis: Upstream quality control. 100 paired FASTQ files, corresponding to 50 dorsal root ganglion (DRG) samples and 50 spinal cord (SC) samples, were received for quality control (QC) evaluation. A preliminary QC of the raw reads was performed prior to further processing.

[0084] Overall, read quality was consistently high; however, all samples showed a high proportion of adapter content, identified by FastQC as "Illumina Universal Adapter". Therefore, the parameters of STAR were adjusted to allow soft clipping of up to 50% of the read. Approximately 60-70% of read-pairs were uniquely mapped to a region of the genome in each sample.

[0085] Quality Control and Exploratory Data Analysis: Downstream quality control. Quality control assessment and exploratory data analysis were performed by manually inspecting density, MA and PCA plots, the correlation heatmap and PCA association plots as well as using automated outlier tests, namely distance, Kolomogorov- Smirnov, correlation and Hoeffding's D. Samples were classed as outliers if they failed two or more tests. All samples derived from DRG tissue passed the QC at both the raw and normalised data stages. Two samples derived from SC tissue failed the QC at the raw stage (R02_30 and R02_38). However, after normalisation both samples passed all four outlier tests and were retained for analysis. Therefore, all 100 samples were included in the downstream statistical contrasts.

[0086] In the principal component analysis of the normalised dorsal root ganglion (DRG) data, clustering by timepoint was observed. Timepoint was significantly associated with principal component 1 (FDR-adjusted P < 0.05), which explained approximately 33% of the variance within the data.

[0087] Association Tests. Differential expression analyses were performed to identify genes that were significantly differentially expressed between groups in a number of comparisons:- comparisons to identify gene expression changes associated with the SNL surgery; and- comparisons to identify gene expression changes associated with treatment.

[0088] As few differentially expressed genes (DEGs) were identified using an FDR-adjusted R value threshold, an unadjusted statistical significance threshold of P < 0.05 was used to define significantly differentially expressed genes for the functional enrichment analyses. At this statistical threshold, there is an opportunity to observe similar patterns under a null model given the number of tests performed.

[0089] Functional Enrichment. Reactome pathway and Gene Ontology (GO) analyses was performed using differentially expressed genes identified at the unadjusted P < 0.05 threshold. A threshold of unadjusted P < 0.05 was also used to define significant enrichment of DEGs within a pathway.

[0090] Congruence Analysis. Congruence analysis was performed to evaluate the level of overlap in gene expression changes between i) DRG tissue and spinal cord tissue, and ii) the different treatments vs vehicle controls, within DRG tissue and SC tissue separately.

[0091] Results. RNA sequencing is utilized herein, providing a powerful tool for identifying the effect of cannabichromene on differentially expressed genes involved in migraine.

[0092] A great number of gene expression changes in the study were observed in dorsal root ganglion (DRG) tissue samples from before and after SNL surgery. A high number of differentially expressed genes (DEGs) were detected in DRG tissue suggestive of the nature of SNL and the nerve injury due to surgery (Ti-Yen et al., 2020) with strong inflammatory profile (Wang et al., 2021 ; Matson et al., 2020). Therefore, spinal cord tissue was used for further data analyses and the investigation of the differentially expressed genes associated with the treatments. Very few DEGs were observed at an adjusted statistical threshold of less than 0.05. Therefore, an unadjusted statistical threshold of 0.05 was used to define significant features for the enrichment and congruence analysis. Given the size of the dataset, the association method was able to be used to detect trends in the expression of calcitonin gene-related peptide calcitonin gene-related peptide (CGRP) encoding genes. In the Association method for data analysis, normalized data provided the input for statistical hypothesis testing, in which genes that were significantly different between sample groups were identified. Statistical comparisons were performed using linear modelling, as implemented in the Bioconductor package limma at www.bioconductor.org (Ritchie et al., 2015). Significance values Upvalues) were adjusted for multiple testing, by controlling the false discovery rate. For each comparison (e.g., group A vs. group B), a positive log2(fold change) indicates up- / down- regulation in group A relative to group B and vice versa. For the purpose of this patent, unadjusted p-values are reported where trends are observed.

[0093] Figure 1 depicts differential expression of the CALCA gene in the spinal cord of SNL rats pre-surgery vs. post-surgery.

[0094] Figure 2 depicts differential expression of the CALCB gene in the spinal cord of SNL rats pre-surgery vs. post-surgery.

[0095] Figure 3 depicts differential expression of the TRPM8 gene in the spinal cord of SNL rats pre-surgery vs. post-surgery.

[0096] RNAseq data analysis indicated a trend for upregulation of CALCA (p = 0.277) and CALCB (p = 0.268) gene expression in the spinal cord of the animals that underwent Sciatic Nerve Ligation (SNL) surgery with 3.8- and 3.5- fold increase in CALCA and CALCB expression respectively, when compared to pre-surgery (Figure 1, Figure 2). The same trend was observed in upregulation of TRPM8, a TRP channel involved in cold allodynia and cold hyperalgesia, where there was a 3-fold increase in the expression of TRPM8 encoding gene post-surgery (p = 0.26; Figure 3).

[0097] Figure 4 depicts differential expression of the CALCA gene in the spinal cord collected from SNL rats following treatment with Cannabichromene (CBC; 10 mg / Kg; p.o.), morphine (10 mg / Kg; i.p.), or vehicle daily for 20 days.

[0098] Figure 5 depicts differential expression of the CALCB gene in the spinal cord collected from SNL rats following treatment with Cannabichromene (CBC; 10 mg / Kg; p.o.), morphine (10 mg / Kg; i.p.), or vehicle daily for 20 days.

[0099] Figure 6 depicts differential expression of the TRPM8 gene in the spinal cord collected from SNL rats following treatment with vehicle, morphine (10 mg / Kg; i.p.), or Cannabichromene (CBC; 10 mg / Kg; p.o.) daily for 20 days.

[0100] Data obtained in this example illustrate downregulation of CALCA gene (- 1.37-fold change; p = 0.79) and CALCB gene (-2.6-fold change; p = 0.36) in the spinal cord of animals treated with CBC daily for 20 days when compared with vehicle (Figure 4, Figure 5). Additionally, the same trend was detected in the expression of TRPM8 encoding genes (-1.9-fold change; p = 0.49), confirming a trend in downregulation of TRPM8 (Figure 6). This finding indicates a likelihood of correlation between CALC and TRPM8 gene expressions in migraine pain, and offers a useful biomarker for CBC activity for the prevention and management of migraine pain.

[0101] In contrast, the DEGs following daily treatment with morphine was associated with a trend upregulation of CALCA (2.9-fold change; p = 0.31) and CALCB gene (1.9-fold change; p = 0.50) in the spinal cord compared to vehicle, although the differences were not statistically significant (Figure 4, Figure 5). Similarly, a trend was observed in TRPM8 upregulation following repeated 20-day morphine treatment (2.48- fold change; p = 0.29 Figure 6).

[0102] Conclusion. The DEG findings from RNAseq of the spinal cord of SNL rat pain model treated with cannabichromene (CBC; 10 mg / Kg; p.o. daily for 20 days) indicate a trend in downregulation of CALCA, CALCB, and TRPM8 encoding gene. On the contrary, daily treatment of SNL rat pain model with morphine (10 mg / Kg; i.p. daily for 20 days) showed a trend toward upregulation of CALCA, CALCB, and TRPM8.

[0103] These results support the finding that downregulation of CALCA and CALCB, which in turn, can lead to the inhibition of calcitonin gene-related peptide (CGRP) release. CGRP is one of the most promising biomarkers for migraine and is involved in the activation of the trigeminovascular system and regulation of pain and inflammation associated with migraine attacks. Downregulation of CALCA and CALCB can lead to reduction or inhibition of CGRP release during the onset of migraine.

[0104] These findings reveal novel biomarkers for CBC activity in the management of migraine pain. CALCA, CALCB, and TRPM8 can be used as biomarkers to confirm the underlying mechanism of CBC activity for the prevention or treatment of migraine pain.

[0105] In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the embodiments. However, it will be apparent to one skilled in the art that these specific details are not required. References cited herein are incorporated by reference.

[0106] The above-described embodiments are intended to be examples only. Alterations, modifications and variations can be made to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

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Claims

WHAT IS CLAIMED IS:1 . A formulation for use in a method of treatment or prophylaxis of migraine in a subject in need thereof, said formulation comprising a primary cannabinoid and one or more excipient, diluent or carrier, wherein said primary cannabinoid is cannabichromene (CBC).

2. The formulation for use according to claim 1 , wherein the formulation is essentially free of tetrahydrocannabinol (THC).

3. The formulation for use according to claim 1 , additionally comprising one or more secondary cannabinoids, preferably cannabidiol (CBD).

4. The formulation for use of claim 3, wherein the one or more secondary cannabinoids is present in an amount of up to 15% by weight of the primary cannabinoid.

5. The formulation for use according to claim 1 , wherein: the formulation is prepared in a dosage form selected from the group consisting of a pill, tablet, gel capsule, syrup, oil-based spray, topical or nasal formulation, and liquid oil form; and / or the formulation provides a total amount of from about 10 mg to about 200 mg of primary cannabinoid per dose, preferably from about 10 mg to about 100 mg per dose, or from about 20 mg to about 50 mg per dose.

6. A method for treatment or prophylaxis of migraine in a subject in need thereof, comprising administering to said subject an effective amount of a formulation comprising a primary cannabinoid and an excipient, diluent or carrier, wherein said primary cannabinoid is cannabichromene (CBC).

7. The method for treatment or prophylaxis of migraine according to claim 6, wherein said formulation is essentially free of tetrahydrocannabinol (THC).

8. The method for treatment or prophylaxis of migraine according to claim 6, wherein the formulation additionally comprises one or more secondary cannabinoids, preferably cannabidiol (CBD).

9. The method according to claim 8, wherein the one or more secondary cannabinoids is present in the formulation in an amount of up to 15% by weight of the primary cannabinoid.

10. The method according to claim 6, wherein: the formulation is administered in a dosage form selected from the group consisting of a pill, tablet, gel capsule, syrup, oil-based spray, topical or nasal formulation, and liquid oil form; and / or the formulation provides to the subject a total amount of from about 10 mg to about 200 mg of primary cannabinoid per dose, preferably from about 10 mg to about 100 mg per dose, or from about 20 mg to about 50 mg per dose.11 . The method for treatment or prophylaxis of migraine according to claim 6, further comprising the step of evaluating CALC or TRPM8 gene expression following administration to determine migraine status.

12. Use of a formulation for treating migraine in a subject in need thereof, or for preparation of a medicament for treating migraine in a subject in need thereof, said formulation comprising an effective amount of a primary cannabinoid and an excipient, diluent or carrier, wherein said primary cannabinoid consists of cannabichromene (CBC).

13. The use of claim 12, wherein said formulation is essentially free of tetrahydrocannabinol (THC).

14. The use of claim 12, wherein said formulation additionally comprising one or more secondary cannabinoids, preferably cannabidiol (CBD).

15. The use of claim 14, wherein the one or more secondary cannabinoids is present in an amount of up to 15% by weight of the primary cannabinoid.

16. The use according to claim 12, wherein: the formulation is prepared in a dosage form selected from the group consisting of a pill, tablet, gel capsule, syrup, oil-based spray, topical or nasal formulation, and liquid oil form; and / orthe formulation provides a total amount of from about 10 mg to about 200 mg of primary cannabinoid per dose, preferably from about 10 mg to about 100 mg per dose, or from about 20 mg to about 50 mg per dose.

17. A method of identifying a subject responsive to CBC for treatment or prophylaxis of migraine comprising evaluation of CALC and TRPM8 gene expression, upon treatment with CBC, and determining a responsive subject as having downregulated expression following CBC treatment.

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