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

Abstract

The present invention relates to the use of cannabidiol (CBD) 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 with PURA gene mutation. In a further embodiment the types of seizures include tonic, atonic and focal seizures with secondary generalisation. Preferably the dose of CBD is between 5 mg/kg/day to 50 mg/kg/day.

Claims

1. A cannabidiol (CBD) preparation for use in the treatment of seizures associated with PURA mutation.

2. A CBD preparation for use according to claim 1, wherein the seizures associated with PURA mutation are tonic, atonic and focal seizures with secondary generalisation.

3. A CBD preparation for use according to any of the preceding claims, wherein the CBD preparation comprises greater than 95% (w/w) CBD and not more than 0.15% (w/w) tetrahydrocannabinol (THC).

4. A CBD preparation for use according to any of the preceding claims, wherein the CBD preparation comprises greater than or equal to 98% (w/w) CBD and less than or equal to 2% (w/w) other cannabinoids, wherein the less than or equal to 2% (w/w) other cannabinoids comprise the cannabinoids tetrahydrocannabinol (THC); cannabidiol-C1 (CBD-C1); cannabidivarin (CBDV); and cannabidiol-C4 (CBD-C4), and wherein the THC is present as a mixture of trans-THC and cis-THC.

5. A CBD preparation to any of the preceding claims, wherein the CBD preparation is used in combination with one or more concomitant anti-epileptic drugs (AED).

6. A CBD preparation for use according to claim 5, wherein the one or more AED is selected from the group consisting of: levetiracetam, clobazam, and rufinamide.

7. A CBD preparation for use according to any of the preceding claims, wherein the CBD is present is isolated from cannabis plant material.

8. A CBD preparation for use according to any of the preceding claims, wherein at least a portion of at least one of the cannabinoids present in the CBD preparation is isolated from cannabis plant material.

9. A CBD preparation for use according to claims 1 to 6, wherein the CBD is present as a synthetic preparation.

10. A CBD preparation for use according to claim 9, wherein at least a portion of at least one of the cannabinoids present in the CBD preparation is prepared synthetically.

11. A CBD preparation for use according to any of the preceding claims, wherein the dose of CBD is greater than 5 mg/kg/day.

12. A CBD preparation for use according to any of the preceding claims, wherein the dose of CBD is 20 mg/kg/day.

13. A CBD preparation for use according to any of the preceding claims, wherein the dose of CBD is 25 mg/kg/day.

14. A CBD preparation for use according to any of the preceding claims, wherein the dose of CBD is 50 mg/kg/day.

15. A method of treating seizures associated with PURA mutation comprising administering a cannabidiol (CBD) preparation to the subject in need thereof.

Description

DETAILED DESCRIPTION

Preparation of Highly Purified Cbd Extract

[0043] The following describes the production of the highly-purified (>95% w/w) cannabidiol extract which has a known and constant composition.

[0044] In summary the drug substance used is a liquid carbon dioxide extract of high-CBD containing chemotypes of Cannabis sativa L. which had been further purified by a solvent crystallization method to yield CBD. The crystallisation process specifically removes other cannabinoids and plant components to yield greater than 95% CBD. Although the CBD is highly purified because it is produced from a cannabis plant rather than synthetically there is a small number of other cannabinoids which are co-produced and co-extracted with the CBD. Details of these cannabinoids and the quantities in which they are present in the medication are as described in Table A below.

TABLE-US-00001 Composition of highly purified CBD extract Cannabinoid Concentration CBD > 95% w/w CBDA NMT 0.15% w/w CBDV NMT 1.0% w/w Δ.sup.9 THC NMT 0.15% w/w CBD-C4 NMT 0.5% w/w > - greater than NMT - not more than

Preparation of Botanically Derived Purified Cbd

[0045] The following describes the production of the botanically derived purified CBD which comprises greater than or equal to 98% w/w CBD and less than or equal to other cannabinoids was used in the open label, expanded-access program described in Example 1 below.

[0046] In summary the drug substance used in the trials is a liquid carbon dioxide extract of high-CBD containing chemotypes of Cannabis sativa L. which had been further purified by a solvent crystallization method to yield CBD. The crystallisation process specifically removes other cannabinoids and plant components to yield greater than 95% CBD w/w, typically greater than 98% w/w.

[0047] The Cannabis sativa L. plants are grown, harvested, and processed to produce a botanical extract (intermediate) and then purified by crystallization to yield the CBD (botanically derived purified CBD).

[0048] The plant starting material is referred to as Botanical Raw Material (BRM); the botanical extract is the intermediate; and the active pharmaceutical ingredient (API) is CBD, the drug substance.

[0049] All parts of the process are controlled by specifications. The botanical raw material specification is described in Table B and the CBD API is described in Table C.

TABLE-US-00002 CBD botanical raw material specification Test Method Specification Identification: -A Visual Complies -B TLC Corresponds to standard (for CBD & CBDA) -C HPLC/UV Positive for CBDA Assay: CBDA + CBD In-house (HPLC/UV) NLT 90% of assayed cannabinoids by peak area Loss on Drying Ph.Eur. NMT 15% Aflatoxin UKAS method NMT 4 ppb Microbial: - TVC - Fungi - E.coli Ph.Eur. NMT10.sup.7cfu/g NMT10.sup.5cfu/g NMT10.sup.2cfu/g Foreign Matter: Ph.Eur. NMT 2% Residual Herbicides and Pesticides Ph.Eur. Complies

TABLE-US-00003 Specification of an exemplary botanically derived purified CBD preparation Test Test Method Limits Appearance Visual Off-white / pale yellow crystals Identification A HPLC-UV Retention time of major peak corresponds to certified CBD Reference Standard Identification B GC-FID/MS Retention time and mass spectrum of major peak corresponds to certified CBD Reference Standard Identification C FT-IR Conforms to reference spectrum for certified CBD Reference Standard Identification D Melting Point 65 - 67° C. Identification E Specific Optical Rotation Conforms with certified CBD Reference Standard; -110° to -140° (in 95% ethanol) Total Purity Calculation ≥ 98.0% Chromatographic Purity 1 HPLC-UV ≥ 98.0% Chromatographic Purity 2 GC-FID/MS ≥ 98.0 % CBDA CBDV THC CBD-C4 HPLC-UV NMT 0.15% w/w 0.2-1.0% w/w 0.01-0.1% w/w 0.3-0.5% w/w Residual Solvents: Alkane Ethanol GC NMT 0.5% w/w NMT 0.5% w/w Residual Water Karl Fischer NMT 1.0% w/w

[0050] The purity of the botanically derived purified CBD preparation was greater than or equal to 98%. The botanically derived purified CBD includes THC and other cannabinoids, e.g., CBDA, CBDV, CBD-C1, and CBD-C4.

[0051] In some embodiments, the CBD preparation comprises not more than 0.15% THC based on total amount of cannabinoid in the preparation. In some embodiments, the CBD preparation comprises about 0.01% to about 0.1% THC based on total amount of cannabinoid in the preparation. In some embodiments, the CBD preparation comprises about 0.02% to about 0.05% THC based on total amount of cannabinoid in the preparation.

[0052] In some embodiments, the CBD preparation comprises about 0.2% to about 1.0% CBDV based on total amount of cannabinoid in the preparation. In some embodiments, the CBD preparation comprises about 0.2% to about 0.8% CBDV based on total amount of cannabinoid in the preparation.

[0053] In some embodiments, the CBD preparation comprises about 0.3% to about 0.5% CBD-C4 based on total amount of cannabinoid in the preparation. In some embodiments, the CBD preparation comprises about 0.3% to about 0.4% CBD-C4 based on total amount of cannabinoid in the preparation.

[0054] In some embodiments, the CBD preparation comprises about 0.1% to about 0.15% CBD-C1 based on total amount of cannabinoid in the preparation.

[0055] 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 CBD. Only the (-)-trans isomer of CBD is believed to occur naturally. During purification, the stereochemistry of CBD is not affected.

Production of CBD Botanical Drug Substance

[0056] An overview of the steps to produce a botanical extract, the intermediate, are as follows: [0057] a) Growing [0058] b) Direct drying [0059] c) Decarboxylation [0060] d) Extraction - using liquid CO.sub.2 [0061] e) Winterization using ethanol [0062] f) Filtration [0063] g) Evaporation

[0064] High CBD 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.

[0065] Decarboxylation of CBDA to CBD was carried out using heat. BRM was decarboxylated at 115° C. for 60 minutes.

[0066] 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 CBD 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 CBD Preparation

[0067] The manufacturing steps to produce the botanically derived purified CBD preparation from BDS were as follows: [0068] a) Crystallization using C.sub.5-C.sub.12 straight chain or branched alkane [0069] b) Filtration [0070] c) Vacuum drying

[0071] 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 <10mb at a temperature of 60° C. until dry. The botanically derived purified CBD 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.

Physicochemical Properties of the Botanically Derived Purified CBD

[0072] The botanically derived purified CBD used in the clinical trial described in the invention comprises greater than or equal to 98% (w/w) CBD and less than or equal to 2% (w/w) of other cannabinoids. The other cannabinoids present are THC at a concentration of less than or equal to 0.1% (w/w); CBD-C1 at a concentration of less than or equal to 0.15% (w/w); CBDV at a concentration of less than or equal to 0.8% (w/w); and CBD-C4 at a concentration of less than or equal to 0.4% (w/w).

[0073] The botanically derived purified CBD used additionally comprises a mixture of both trans-THC and cis-THC. It was found that the ratio of the trans-THC to cis-THC is altered and can be controlled by the processing and purification process, ranging from 3.3:1 (trans-THC:cis-THC) in its unrefined decarboxylated state to 0.8:1 (trans-THC:cis-THC) when highly purified.

[0074] Furthermore, the cis-THC found in botanically derived purified CBD is present as a mixture of both the (+)-cis-THC and the (-)-cis-THC isoforms.

[0075] Clearly a CBD preparation could be produced synthetically by producing a composition with duplicate components.

[0076] Example 1 below describes the use of a botanically derived purified CBD in an open label, expanded-access program to investigate the clinical efficacy and safety of purified pharmaceutical cannabidiol formulation (CBD) in the treatment of seizures associated with PURA mutation.

EXAMPLE 1: CLINICAL EFFICACY AND SAFETY OF PURIFIED PHARMACEUTICAL CANNABIDIOL (CBD) IN THE TREATMENT OF PATIENTS DIAGNOSED WITH PURA MUTATION

Study Design

[0077] The subject was required to be on one or more AEDs at stable doses for a minimum of two weeks prior to baseline and to have stable vagus nerve stimulation (VNS) settings and ketogenic diet ratios for a minimum of four weeks prior to baseline.

[0078] The patient was administered botanically derived purified CBD in a 100 mg/mL sesame oil-based solution at an initial dose of 10 milligrams per kilogram per day (mg/kg/day) in two divided doses. Dose was then increased weekly by 5 mg/kg/day to a goal of 25 mg/kg/day.

[0079] A maximum dose of 50 mg/kg/day could be utilised for the patient if they were tolerating the medication but had not achieved seizure control; the patient had further weekly titration by 5 mg/kg/day.

[0080] There was one patient in this study, and they received CBD for 24 weeks. Modifications were made to concomitant AEDs as per clinical indication.

[0081] 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

[0082] Patients may be defined as responders if they had more than 50% reduction in seizure frequency compared to baseline. The percent change in seizure frequency was calculated as follows:

[00001]$\begin{array}{l}\%\text{change seizure frequency}=\\ \frac{\left(\begin{array}{c}\left(\text{weekly seizure frequency}timeinterval\right)\text{-}\\ \left(\text{weekly seizure frequency}Baseline\right)\end{array}\right)}{\left(\text{weekly seizure frequency}Baseline\right)}\times 100\end{array}$

[0083] The percent change of seizure frequency may be calculated for any time interval where seizure number has been recorded. For the purpose of this example the percent change of seizure frequency for the end of the treatment period was calculated as follows:

[00002]$\begin{array}{l}\%\text{reduction seizure frequency}=\\ \frac{\left(\begin{array}{l}\left(\text{weekly seizure frequency}Baseline\right)\text{-}\\ \left(\text{weekly seizure frequency}End\right)\end{array}\right)}{\left(\text{weekly seizure frequency}Baseline\right)}\times 100\end{array}$

RESULTS

Patient Description

[0084] One patient enrolled in the open label, expanded-access program had a PURA mutation (autosomal dominant mutation of PURA on chromosome 5q31.3). The patient experienced several different seizure types including tonic, atonic and focal seizures with secondary generalisation and was taking several concomitant AEDs.

[0085] The patient was 8 years old and she was female as detailed in Table 1 below.

TABLE-US-00004 Patient demographics, seizure type and concomitant medication Patient Number Age (years) Sex Seizure types Concomitant AEDs 1 8.07 F Tonic, atonic, focal with secondary generalisation CLB, LEV, RFN LEV = levetiracetam, CLB = clobazam, RFN = rufinamide

Study Medication and Concomitant Medications

[0086] The patient on the study was titrated up to 25 mg/kg/day of CBD.

[0087] The patient was on three concomitant AEDs at the time of starting CBD.

Clinical Changes

[0088] Table 2 illustrates the seizure frequency for the patient as well as the dose of CBD given.

TABLE-US-00005 Seizure frequency data for Patient 1 Patient 1 Time Seizure Type Dose CBD (mg/kg/day) Tonic Atonic Focal with secondary generalisation Baseline 68.0 68.0 168.0 - 2 weeks 7.6 28.0 72.8 15.0 4 weeks 11.2 18.8 20.4 25.0 8 weeks 5.0 16.0 16.0 25.0 12 weeks 10.8 4.8 1.9 25.0 16 weeks 15.2 6.4 0.0 25.0 24 weeks 5.4 2.9 0.0 25.0

[0089] Patient 1 was treated for 24 weeks and experienced a 92.1% reduction in tonic seizures, a 95.7% reduction in atonic seizures and a 100% reduction in focal seizures with secondary generalisation over the treatment period.

[0090] Overall, the patient reported reductions of 92-100% in seizures over period of treatment with CBD. Significantly, the patient became seizures free in their focal seizures with secondary generalisation after 16 weeks of treatment with CBD.

[0091] CBD was effective in reducing the frequency of the following seizure types: tonic, atonic and focal seizures with secondary generalisation.

CONCLUSIONS

[0092] These data indicate that CBD was able to significantly reduce the number of seizures associated with PURA mutation. Clearly the treatment is of significant benefit in this difficult to treat epilepsy syndrome given the high response rate experienced in the patient.

[0093] In conclusion, this study signifies the use of CBD for treatment of seizures associated with PURA mutation. Seizure types include tonic, atonic and focal seizures with secondary generalisation for which seizure frequency rates decreased by significant rates, by 92-100%.

REFERNCES

[0094] 1. Shimojima et al. (2018) “Infantile spasms related to a 5q3 I.2 - q3 I.3 microdeletion including PURA.” Human Genome Variation, 2018, 5; 18007 [0095] 2. Lalani et. al. (2014) “Mutations in PURA cause profound neonatal hypotonia, seizures, and encephalopathy in 5q3 I .3 microdeletion syndrome.” Am J Hum Genet, 2014, 95(5); 579 - 83 [0096] 3. Reijnders et al. (2018) “PURA syndrome: clinical delineation and genotype-phenotype study in 32 individuals with review of published literature.” J Med Genet, 2018, 55; 104-113 [0097] 4. Bonaglia et al. (2015) “Long-term follow-up of a patient with 5q3 I .3 microdeletion syndrome and the smallest de novo 5q3 I .2q3 I .3 deletion involving PURA.” Mol Cytogenet, 2015, 8;89