USE OF CANNABIDIVARIN IN THE TREATMENT OF SEIZURES ASSOCIATED WITH RARE EPILEPSY SYNDROMES RELATED TO GENETIC ABNORMALITIES

Abstract

The present invention relates to the use of cannabidivarin (CBDV) for the treatment of seizures associated with rare epilepsy syndromes. In particular the seizures associated with rare epilepsy syndromes that are treated are those which are experienced in patients diagnosed with Rett syndrome. In a further embodiment the types of seizures include focal motor seizures with impairment, focal non-motor seizures with impairment, generalised motor seizures, generalised non-motor seizures, unknown onset motor seizures, and non-motor seizures. Preferably the dose of CBDV is between 2.5 mg/kg/day to 10 mg/kg/day.

Claims

1. A method for treating seizures associated with Rett syndrome comprising administering a cannabidivarin (CBDV) preparation.

2. The method of claim 1, wherein the seizures associated with Rett syndrome are focal motor seizures with impairment, focal non-motor seizures with impairment, generalised motor seizures, generalised non-motor seizures, unknown onset motor seizures, and non-motor seizures.

3. The method of claim 1, wherein the CBDV preparation comprises greater than 95% (w/w) CBDV and not more than 1.5% (w/w) tetrahydrocannabinol (THC).

4. The method of claim 1, wherein the CBDV preparation comprises greater than or equal to 95% (w/w) CBDV and less than or equal to 5% (w/w) other cannabinoids, wherein the less than or equal to 5% (w/w) other cannabinoids comprise the cannabinoids tetrahydrocannabinol (THC); tetrahydrocannabivarin (THCV); cannabidiol-C1 (CBD-C1); cannabidiol (CBD); cannabidivarin acid (CBDVA) and cannabidiol-C4 (CBD-C4).

5. The method of claim 1, wherein the CBDV preparation is used in combination with one or more concomitant anti-epileptic drugs (AED).

6. The method of claim 5, wherein the one or more AED is selected from the group consisting of: primidone, carbamazepine, zonegran, phenobarbitone, lamotrigine, levetiracetam, sodium valproate and clobazam.

7. The method of claim 1, wherein the CBDV is present is isolated from cannabis plant material.

8. The method of claim 1, wherein at least a portion of at least one of the cannabinoids present in the CBDV preparation is isolated from cannabis plant material.

9. The method of claim 1, wherein the CBDV is present as a synthetic preparation.

10. The method of claim 9, wherein at least a portion of at least one of the cannabinoids present in the CBDV preparation is prepared synthetically.

11. The method of claim 1, wherein the dose of CBDV is greater than 2.5 mg/kg/day.

12. The method of claim 1, wherein the dose of CBDV is 10 mg/kg/day.

13. (canceled)

Description

DETAILED DESCRIPTION

Overview of the Process

[0064] The following describes the production of the botanically derived purified CBDV (>95% w/w) which has a known and constant composition which was used in the Example below.

[0065] Plant material harvested from the Cannabis sativa L. plant was subjected to liquid carbon dioxide extraction, to produce a botanical extract containing CBDV in addition to other cannabinoids and non-cannabinoid components. The extract was then further purified by a solvent crystallization method to yield botanically derived purified CBDV. The crystallization process specifically removed other cannabinoids and plant components to yield greater than 95% (w/w) CBDV.

[0066] Both the botanical starting material and the botanical extract may be controlled by specifications.

[0067] An exemplary CBDV preparation of botanically derived purified CBDV is described in Table 1.1 below. In some embodiments, the isomeric content for each cannabinoid may also be specified.

TABLE-US-00001 TABLE 1.1 Specification of an exemplary botanically derived purified CBDV preparation Test Test Method Limits Appearance Visual Off-white/pale yellow crystals Identification A HPLC-UV Retention time of major peak corresponds to certified CBDV Reference Standard Identification B GC-FID/MS Retention time and mass spectrum of major peak corresponds to certified CBDV Reference Standard Identification C FT-IR Conforms to reference spectrum for certified CBDV Reference Standard Identification D Melting Point 115-118° C. Identification E Specific Optical Conforms with certified CBDV Rotation Reference Standard; −110° to −140° (in 95% ethanol) Total Purity Calculation ≥95.0% Chromatographic HPLC-UV ≥95.0% Purity 1 Chromatographic GC-FID/MS ≥95.0% Purity 2 CBD HPLC-UV NMT 4.0% w/w CBD-C4 NMT 0.2% w/w CBD-C1 NMT 0.15% w/w CBDVA NMT 0.15% w/w Δ.sup.9 THC NMT 0.05% w/w THCV NMT 0.01% w/w Residual Solvents: Alkane GC NMT 0.5% w/w Ethanol NMT 0.5% w/w Residual Water Karl Fischer NMT 1.0% w/w i.NMT—Not more than

[0068] The purity of the botanically derived purified CBDV preparation was greater than or equal to 95%. The botanically derived purified CBDV includes THC and other cannabinoids, e.g., CBD, CBDVA, THCV, CBD-C1, and CBD-C4.

[0069] Distinct chemotypes of the Cannabis sativa L. plant have been produced to maximize the output of the specific chemical constituents, the cannabinoids. Certain chemovars produce predominantly CBDV. Only the (−)-trans isomer of CBDV is believed to occur naturally. During purification, the stereochemistry of CBDV is not affected.

Production of CBDV Botanical Drug Substance

[0070] An overview of the steps to produce a botanical extract, the intermediate, are as follows: [0071] a. Growing [0072] b. Direct drying [0073] c. Decarboxylation [0074] d. Extraction—using liquid CO.sub.2 [0075] e. Winterization using ethanol [0076] f. Filtration [0077] g. Evaporation

[0078] High CBDV chemovars were grown, harvested, dried, baled and stored in a dry room until required. The botanical raw material (BRM) was finely chopped using an Apex mill fitted with a 1 mm screen. The milled BRM was stored in a freezer prior to extraction.

[0079] Decarboxylation of CBDVA to CBDV was carried out using heat. BRM was decarboxylated at 115° C. for 60 minutes.

[0080] Extraction was performed using liquid CO.sub.2 to produce botanical drug substance (BDS), which was then crystalized to produce the test material. The crude CBDV BDS was winterized to refine the extract under standard conditions (2 volumes of ethanol at −20° C. for approximately 50 hours). The precipitated waxes were removed by filtration and the solvent was removed to yield the BDS.

Production of Botanically Derived Purified CBDV Preparation

[0081] The manufacturing steps to produce the botanically derived purified CBDV preparation from BDS were as follows: [0082] a. Crystallization using C.sub.5-C.sub.12 straight chain or branched alkane [0083] b. Filtration [0084] c. Vacuum drying

[0085] The BDS produced using the methodology above was dispersed in C.sub.5-C.sub.12 straight chain or branched alkane. The mixture was manually agitated to break up any lumps and the sealed container then placed in a freezer for approximately 48 hours. The crystals were isolated via vacuum filtration, washed with aliquots of cold C.sub.5-C.sub.12 straight chain or branched alkane, and dried under a vacuum of <10 mb at a temperature of 60° C. until dry. The botanically derived purified CBDV preparation was stored in a freezer at −20° C. in a pharmaceutical grade stainless steel container, with FDA food grade approved silicone seal and clamps.

[0086] Clearly a CBDV preparation could be produced synthetically by producing a composition with duplicate components.

[0087] Example 1 below describes the use of a botanically derived purified CBDV in an open label, phase I trial to investigate the clinical efficacy and safety of purified pharmaceutical cannabidivarin formulation (CBDV) in the treatment of patients diagnosed with Rett syndrome.

Study Design

[0088] Subjects were required to be on one or more AEDs at stable doses for a minimum of 4 weeks prior to baseline. Subjects had to have been diagnosed with intractable epilepsy: failed adequate trial of two, or more, standard anticonvulsants, and four or more quantifiable seizures less than 8 weeks prior to screening, and two or more quantifiable seizures in prospective baseline phase.

[0089] Patients were administered botanically derived purified CBDV in a 50 mg/mL sesame oil-based solution which was titrated to an initial dose of 2.5 milligrams per kilogram per day (mg/kg/day). Doses were then increased to a goal of 10 mg/kg/day.

[0090] There were five patients in this study, and each received CBDV for various durations of time. Modifications were made to concomitant AEDs as per clinical indication.

[0091] Seizure frequency, intensity, and duration were recorded by caregivers in a diary during a baseline period of at least 28 days. Changes in seizure frequency relative to baseline were calculated after at least 2 weeks and at defined timepoints of treatment.

Statistical Methods:

[0092] Patients may be defined as responders if they had more than 50% reduction in seizure frequency compared to baseline. The percent change of seizure frequency for the end of the treatment period was calculated as follows:

[00001]$\%\mathrm{reduction}\mathrm{seizure}\mathrm{frequency}=\frac{\begin{array}{c}(\left(\mathrm{monthly}\mathrm{seizure}\mathrm{frequence}\mathrm{Baseline}\right)-\\ \left(\mathrm{monthly}\mathrm{seizure}\mathrm{frequency}\mathrm{End}\right))\end{array}}{\left(\mathrm{monthly}\mathrm{seizure}\mathrm{frequency}\mathrm{Baseline}\right)}\times 100$

Results

Patient Description

[0093] The five patients enrolled in the open label, phase I trial were diagnosed with Rett syndrome. These patients experienced a range of different seizure types including focal seizures with impairment, generalised seizures, seizures of unknown onset, non-motor seizures and other seizure types.

[0094] The age of patients ranged from 6-13 years, all five were female as detailed in Table 1 below.

TABLE-US-00002 TABLE 1 Patient demographics, seizure type and concomitant medication Patient Age Concomitant Number (years) Sex Seizure types AEDs 1 11 F Focal seizures with PRI, CAR, impairment (motor) ZON, PHE 2 13 F Focal seizures with PHE, LAM, impairment (non-motor); LEV, VAL Other 3 12 F Focal seizures with LEV, CLO impairment (motor); Generalised tonic-clonic seizures 4 F 13 Focal seizures with VAL, CLO impairment (motor); Other generalized motor 5 F 6 Generalised tonic-clonic VAL, CLO, seizures; LEV Other generalized (motor); Unknown onset (motor) PRI = primidone, CAR = carbamazepine, ZON = zonegran, PHE = phenobarbitone, LAM = lamotrigine, LEV = levetiracetam, VAL = sodium valproate, CLO = clobazam

Study Medication and Concomitant Medications

[0095] Patients on the study were titrated up to various doses of CBDV.

[0096] Patients were taking an average of three AEDs.

Clinical Changes

[0097] Tables 2A-2E illustrate the seizure frequency for each patient.

TABLE-US-00003 TABLE 2A Seizure frequency data for Patient 1 Onset Movement Baseline /14 months Focal Motor 32 192 seizures with Non-motor 0 2 impairment Generalised Motor tonic-clonic 0 0 seizures Motor other 0 8 Unknown Motor tonic-clonic 0 7 onset Motor other 0 3 Non-motor 0 0 Other 0 1 Total 32.0/month 15.2/month Mean seizure frequency reduction 52.5%

[0098] Patient 1 was treated for 14 months and experienced a 52.5% reduction in seizures over the treatment period.

TABLE-US-00004 TABLE 2B Seizure frequency data for Patient 2 Onset Movement Baseline /11 months Focal Motor 0 0 seizures with Non-motor 32 61 impairment Generalised Motor tonic-clonic 0 0 seizures Motor other 0 0 Unknown Motor tonic-clonic 0 0 onset Motor other 0 0 Non-motor 0 0 Other 3 18 Total 35.0/month 7.2/month Mean seizure frequency reduction 79.4%

[0099] Patient 2 was treated for 11 months and experienced a 79.4% reduction in seizures over the treatment period.

TABLE-US-00005 TABLE 2C Seizure frequency data for Patient 3 Onset Movement Baseline /12 months Focal Motor 1 3 seizures with Non-motor 0 0 impairment Generalised Motor tonic-clonic 5 4 seizures Motor other 0 0 Unknown Motor tonic-clonic 0 0 onset Motor other 0 0 Non-motor 0 0 Other 0 0 Total 6.0/month 0.6/month Mean seizure frequency reduction 90.0%

[0100] Patient 3 was treated for 12 months and experienced a 90.0% reduction in seizures over the treatment period.

TABLE-US-00006 TABLE 2D Seizure frequency data for Patient 4 Onset Movement Baseline /12.5 months Focal Motor 3 73 seizures with Non-motor 0 0 impairment Generalised Motor tonic-clonic 0 0 seizures Motor other 4 4 Unknown Motor tonic-clonic 0 0 onset Motor other 0 0 Non-motor 0 0 Other 0 6 Total 7.0/month 6.6/month Mean seizure frequency reduction 5.7%

[0101] Patient 4 was treated for 12.5 months and experienced a 5.7% reduction in seizures over the treatment period.

TABLE-US-00007 TABLE 2E Seizure frequency data for Patient 5 Onset Movement Baseline /11 months Focal Motor 0 0 seizures with Non-motor 0 0 impairment Generalised Motor tonic-clonic 25 38 seizures Motor other 18 18 Unknown Motor tonic-clonic 0 0 onset Motor other 328 17 Non-motor 0 6 Other 0 0 Total 371.0/month 7.2/month Mean seizure frequency reduction 98.1%

[0102] Patient 5 was treated for 11 months and experienced a 98.1% reduction in seizures over the treatment period.

[0103] Overall, patients reported reductions of 5.7-98.1% in seizures over the period of treatment with CBDV.

[0104] CBDV was effective in reducing the frequency of all seizure types: focal motor seizures with impairment, focal non-motor seizures with impairment, generalised motor seizures, generalised non-motor seizures, unknown onset motor seizures, non-motor seizures and other types.

[0105] Significantly, three patients (patients 3-5) suffered from generalised seizures and all three experienced a reduction in these seizures. Four patients (patient 1-4) suffered from focal seizures with impairment and three (patients 1-3) of these patients experienced a reduction in their seizures.

CONCLUSIONS

[0106] These data indicate that CBDV was able to significantly reduce the number of seizures associated with Rett syndrome. Clearly the treatment is of significant benefit in this difficult to treat epilepsy syndrome given the high responder rate experienced in four of the five patients.

[0107] Significantly, it was found that four of the five patients had more than 50% reduction in seizure frequency compared to baseline after treatment with CBDV whilst three experienced more than 75% reduction.

[0108] In conclusion, this study signifies the use of CBDV for treatment of seizures associated with Rett syndrome. Seizure types include focal motor seizures with impairment, focal non-motor seizures with impairment, generalised motor seizures, generalised non-motor seizures, unknown onset motor seizures, and non-motor seizures for which seizure frequency rates decreased by significant rates, by up to 98%.

REFERENCES

[0109] 1. https://clinicaltrials.gov/ct2/show/NCT03848832?term=cannabidiol&cond=Rett+Syndrome Accessed: 10 Jul. 2020. [0110] 2. Vigli et al. (2018) “Chronic Treatment With the Phytocannabinoid Cannabidivarin (CBDV) Rescues Behavioural Alterations and Brain Atrophy in a Mouse Model of Rett Syndrome” Neuropharmacology [0111] 3. Zamberletti et al. (2019) “Cannabidivarin completely rescues cognitive deficits and delays neurological and motor defects in male Mecp2 mutant mice” Journal of Psychopharmacology [0112] 4. Amada et al. (2013) “Cannabidivarin (CBDV) suppresses pentylenetetrazole (PTZ)-induced increases in epilepsy-related gene expression”, pages 1-18, PeerJ, vol. 1. [0113] 5. Hill et al. (2013) “Cannabidivarin-rich cannabis extracts are anticonvulsant in mouse and rat via a CB1 receptor-independent mechanism”, pages 679-692. British Journal of Pharmacology, vol. 170 [0114] 6. Hill et al. (2012) “Cannabidivarin is anticonvulsant in mouse and rat”, pages 1629-1642, British Journal of Pharmacology, vol. 167 [0115] 7. Huizenga et al. (2019) “Preclinical safety and efficacy of cannabidivarin for early life seizures”, pages 189-198, Neuropharmacology, vol. 148. [0116] 8. European Medicines Agency (2018) “Public summary of opinion on orphan designation: Cannabidivarin for the treatment of Rett syndrome.”