Pharmaceutical formulation

Abstract

The invention relates to pharmaceutical formulations, and more particularly to formulations containing cannabinoids for administration via a pump action spray. In particular, the invention relates to pharmaceutical formulations, for use in administration of lipophilic medicaments via mucosal surfaces, comprising: at least one lipophilic medicament, a solvent and a co-solvent, wherein the total amount of solvent and co-solvent present in the formulation is greater than 55% wt/wt of the formulation and the formulation is absent of a self emulsifying agent and/or a fluorinated propellant.

Claims

1. A method of treating spasms associated with multiple sclerosis in a patient in need thereof, wherein the method comprises administering to the patient a liquid pharmaceutical formulation comprising tetrahydrocannabinol (THC) and cannabidiol (CBD), wherein the liquid pharmaceutical formulation comprises in a 1 mL volume: 22.5-27.5 mg/mL of THC based on amount of cannabinoid in a botanical drug substance, and 22.5-27.5 mg/mL of CBD based on amount of cannabinoid in a botanical drug substance, and wherein the liquid pharmaceutical formulation lacks a self-emulsifying agent.

2. The method of claim 1, wherein the liquid pharmaceutical formulation comprises in a 1 mL volume: 22.5-27.5 mg/mL of THC based on amount of cannabinoid in a botanical drug substance, and 25 mg/mL of CBD based on amount of cannabinoid in a botanical drug substance.

3. The method of claim 1, wherein the patient has multiple sclerosis.

4. The method of claim 1, wherein the patient has spasms associated with multiple sclerosis.

5. The method of claim 1, wherein the liquid pharmaceutical formulation is administered to the patient by sub-lingual application.

6. The method of claim 1, wherein the liquid pharmaceutical formulation is administered to the patient by buccal application.

7. The method of claim 1, wherein the liquid pharmaceutical formulation is administered via a pump-action spray.

8. The method of claim 7, wherein the pump-action spray produces a spray comprising particles having a mean aerodynamic particle size of between 15 and 45 microns.

9. The method of claim 8, wherein the pump-action spray produces a spray in which the particles have a mean aerodynamic particle size of between 20 and 40 microns.

10. The method of claim 3, wherein the patient has spasms associated with multiple sclerosis.

11. The method of claim 3, wherein the liquid pharmaceutical formulation is administered to the patient by sub-lingual application.

12. The method of claim 3, wherein the liquid pharmaceutical formulation is administered to the patient by buccal application.

13. The method of claim 3, wherein the liquid pharmaceutical formulation is administered via a pump-action spray.

14. The method of claim 13, wherein the pump-action spray produces a spray comprising particles having a mean aerodynamic particle size of between 15 and 45 microns.

15. The method of claim 14, wherein the pump-action spray produces a spray in which the particles have a mean aerodynamic particle size of between 20 and 40 microns.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1a and 1b illustrate mean plasma concentrations of cannabinoids CBD, THC and 11-hydroxy THC following administration of high CBD (FIG. 1a) and high THC (FIG. 1b) Cannabis extracts to human subjects.

(2) FIG. 2 illustrates mean plasma concentrations of cannabinoids CBD, THC and 11-hydroxy THC following administration of a Cannabis extract containing a 1:1 ratio of THC:CBD to a human subject.

(3) FIG. 3 illustrates cross-sectional area of aerosol plume vs % propylene glycol in propylene glycol/ethanol liquid spray formulations.

(4) FIG. 4 illustrates viscosity as a function of propylene glycol content in propylene glycol/ethanol liquid spray formulations.

(5) FIG. 5 illustrates cross-sectional area of aerosol plume vs viscosity for propylene glycol/ethanol liquid spray formulations.

(6) FIGS. 6 and 6a show results of HPLC analysis of samples drawn from stored, light exposed solutions of THC, before and after charcoal treatment.

(7) FIGS. 7 and 7a show results of HPLC analysis of samples drawn from stored, light exposed solutions of CBD, before and after charcoal treatment.

(8) FIG. 8 shows a summary of steps in production from seed accession to dried Medicinal Cannabis.

(9) FIG. 9 shows a flow chart of the process of manufacturing extract from the High-THC and High-CBD chemovars.

DETAILED DESCRIPTION OF THE INVENTION

(10) Development of Pump-Action Spray Formulations

(11) Initially the applicant looked at cannabinoid uptake in patients by applying drops sublingually (BDS dissolved in a mixture of a glycerol/propylene glycol and ethanol) THC 5 mg/ml, CBD 5 mg/ml and THC/CBD 5 mg/ml plus 5 mg/ml.

(12) The results are noted in Table 3 below:

(13) TABLE-US-00004 TABLE 3 Initial absorption: 20 min T max: approx 2 hours C max: 6 ng/ml THC, 2 ng/ml CBD AUC 0-12: approx 16 ng .Math. h/ml THC, 8 ng .Math. h/ml CBD following a dose of approx 20 mg of each cannabinoids Plasma levels after 6 hours were about 1 ng/ml THC and 0.5 ng/ml CBD

(14) The proportion of 11 hydroxy tetrahydro cannabinol to THC (AUC 0-12) was about 1.9 indicating a significant amount of oral ingestion may have occurred.

(15) On moving to a pump action sublingual spray (following problems solubilising cannabinoids with hydrofluorocarbon propellant systems) the applicant obtained the results noted in Table 4. The solvent system comprised 50:50 ethanol to propylene glycol (v/v ratio) with THC 25 mg/ml; CBD 50 mg/ml and THC/CBD 25 mg/ml plus 50 mg/ml respectively.

(16) TABLE-US-00005 TABLE 4 Initial absorption: 60 min T max: approx 3 hours C max: 6 ng/ml THC, 8 ng/ml CBD AUC 0-12: approx 16 ng .Math. h/ml THC, 22 ng .Math. h/ml CBD following a dose of approx 21 mg of THC and 35 mg CBD Plasma levels after 6 hours were about 1 ng/ml THC and 1 ng/ml CBD

(17) The proportion of 11 hydroxy tetrahydro cannabinol to THC (AUC 0-12) was about 1.6. The profile for each cannabinoid was similar irrespective of the formulation (THC, CBD, THC plus CBD).

(18) After accounting for the different dosages, whilst the extent of absorption was comparable to the drops, the rate of absorption was slower and the proportion metabolised reduced.

(19) Despite the slower rate of absorption the pump spray mechanism and the ethanol/propylene glycol carrier system provided the opportunity to administer sufficient cannabinoids, in a flexible dose form with accuracy and advantageously with reduced metabolism.

(20) The data obtained is illustrated in FIGS. 1a, 1b and 2, which show the mean plasma concentrations for the formulations identified with reference to Tables 3 and 4.

(21) That effective delivery of the cannabinoids can be achieved in a vehicle consisting of ethanol and propylene glycol is illustrated by the plasma levels shown in FIGS. 1a, 1b and 2. These show, respectively, formulations containing the high THC and high CBD formulations in FIGS. 1a and 1b. Similarly, the effectiveness of a defined ratio formulation THC:CBD 1:1 is illustrated in FIG. 2.

(22) Significantly the ethanol/propylene glycol system was found to only work with a pump action spray within quite narrow limits.

(23) The findings giving rise to the development of pump spray formulations, as exemplified in formulations 1-4 below, are set out below:

Example 1-Significance of Particle Size

(24) Applicant observed that the propellant aerosols that were developed suffered from “bounce back” and this appeared to be a function of delivery speed and particle size.

(25) Applicant determined that, in contrast to the propellant driven system, a pump spray could deliver an aerosol plume in which the particle size could be controlled to generate a particle size of between 20 and 40 microns (thus maximising the amount of material hitting the sublingual/buccal mucosa and thus the amount of cannabinoids that can be absorbed). To produce particles of the appropriate size the viscosity of the formulation needed to be carefully controlled. If the formulation was too viscous droplet formation was hindered, a jet formed and the valve blocked; If the formulation was not viscous enough they got excessive nebulisation, a plume of broad cross sectional area formed, and the spray was no longer directed solely onto the sublingual/buccal mucosa. This could result in the formulation pooling and some of the formulation being swallowed. In both cases the result is unsatisfactory.

(26) In fact, it turned out that for the solvent of preferred choice, ethanol, and the co-solvent of preferred choice, propylene glycol, the working range was fairly narrow as demonstrated below:

(27) The viscosity of different combinations of ethanol/propylene glycol were studied and their spray performance with a vp7/100 valve (Valois) compared. The results are tabulated in Table 5 below:

(28) TABLE-US-00006 TABLE 5 Propylene Relative viscosity glycol/ethanol (run time in sec) Spray performance 100/0  442 Jet formed 80/20 160 Jet formed 60/40 80 Some jetting 50/50 62 Good aerosol plume 40/60 44 Good aerosol plume 20/80 26 Good aerosol plume  0/100 16 Good aerosol plume

(29) From this data it appeared that addition of propylene glycol at greater than 60/40 would not be acceptable. These result, when read alongside U.S. Pat. No. 3,560,625, could have suggested that the said solvent/co-solvent combination would be no good. However, applicant found that patients could tolerate ethanol levels of this order when presented in the given formulations.

(30) The effect of viscosity on aerosol plume was quantified by spraying the various formulations at a standard distance of 0.5 cm onto disclosing paper. The distance represents the typical distance between the nozzle of the pump action spray unit and the sub lingual cavity in normal use. The paper was photocopied and the image of the plume excised and weighed to give a relative cross sectional area. The relative value was then converted into a real cross sectional area by dividing this value by the weight per cm.sup.2 of the photocopier paper (determined by weighing a known area of paper). The results are given in Table 6 below:

(31) TABLE-US-00007 TABLE 6 Propylene Area of cross section glycol/ethanol of spray plume 100/0   3.5 cm.sup.2 80/20 14.2 cm.sup.2 60/40 17.9 cm.sup.2 50/50 20.7 cm.sup.2 40/60 29.4 cm.sup.2 20/80 54.4 cm.sup.2  0/100 93.8 cm.sup.2
This data is illustrated in FIG. 3.

(32) Additionally plots of viscosity of mixtures of ethanol and propylene glycol content FIG. 4 and plume cross section as a function of viscosity FIG. 5 are given.

(33) The figures emphasise the dramatic and undesirable changes in properties which occur outside the narrow range of ethanol/propylene glycol wt/wt of 60/40 and 40/60, and more particularly still 55/45 to 45/55, most preferably about 50/50.

(34) Other factors are also significant in ensuring the combination is used in a narrow range. Increasing the ethanol levels beyond 60 vol % gives rise to irritation and at propylene glycol levels approaching 60% and as low as 55%, in the case of BDS, non polar derivatives present in the BDS begin to precipitate out on prolonged ambient storage.

(35) Other co-solvents which might be used would be expected to have similar limitations. The more viscous the co-solvent the greater the problem of producing a plume forming spray, and the more polar, the greater the risk that precipitation will be exacerbated.

(36) However, because the combination of ethanol/propylene glycol is able to dissolve up to 50 mg/ml (i.e. therapeutically desirable levels of cannabinoids), is non irritating, pharmaceutically acceptable, and the propylene glycol also acts as a penetration enhancer maximising bioavailability of the cannabinoids it is particularly advantageous.

(37) The mean particle size of the preferred compositions have been shown to be 33 μm when tested using a Malvern Marsteriser. The droplets, which are considerably greater than 5 μm, therefore minimise the risk of inhalation of aerosol.

Example 2-Effect of Water when the Cannabinoids are Present in a BDS

(38) The presence of greater than 5% water in the formulation was shown to cause precipitation of the BDS as illustrated by the investigation described in Table 7 below:

(39) TABLE-US-00008 TABLE 7 Sequential addition of water was made to 5 ml 25 mg/ml THC and 5 ml 25 mg/ml CBD in an ethanol/propylene glycol formulate (50/50). Final Approx final solvent Vol of water vol ratio % vol Water/ added ml ml propylene glycol/ethanol observation 0 5 0/50/50 Solution 0.05 5.05 1/49.5/49.5 Ppt forms but re- dissolves on mixing 0.21 5.26 5/47.5/47.5 Ppt forms. Solution remains cloudy after mixing
Indeed because of this observation the use of anhydrous ethanol is preferred.
Example formulations (non-limiting) according to the invention are as follows:

(40) TABLE-US-00009 COMPOSITION 1 (General) AMOUNT PER UNIT COMPONENT (1 ml) FUNCTION Active THC (BDS) 25-50 mg/ml Active CBD (BDS) 25-50 mg/ml Excipient Propylene Glycol 0.5 ml/ml Co solvent Peppermint oil 0.0005 ml/ml Flavour Ethanol (anhydrous) qs to 1 ml Solvent

(41) TABLE-US-00010 COMPOSITION 2 (High THC) AMOUNT PER UNIT COMPONENT (1 ml) FUNCTION Active THC (BDS) 25 mg/ml Active Excipient Propylene Glycol 0.5 ml/ml Co solvent Peppermint oil 0.0005 ml/ml Flavour Ethanol (anhydrous) qs to 1 ml Solvent

(42) TABLE-US-00011 COMPOSITION 3 (High CBD) AMOUNT PER UNIT COMPONENT (1 ml) FUNCTION Active CBD (BDS) 25 mg/ml Active Excipient Propylene Glycol 0.5 ml/ml Co solvent Peppermint oil 0.0005 ml/ml Flavour Ethanol (anhydrous) qs to 1 ml Solvent

(43) TABLE-US-00012 COMPOSITION 4 (THC/CBD substantially 1:1) AMOUNT PER UNIT COMPONENT (1 ml) FUNCTION Active THC (BDS) 25 mg/ml Active CBD (BDS) 25 mg/ml Active Excipient Propylene Glycol 0.5 ml/ml Co solvent Peppermint oil 0.0005 ml/ml Flavour Ethanol (anhydrous) qs to 1 ml Solvent

Example 3

(44) The following example illustrates the application of liquid spray formulations to the buccal mucosae and the blood levels produced by buccal absorption in comparison with sublingual administration.

(45) The following liquid formulations suitable for buccal administration contain self-emulsifying agents, and hence do not fall within the scope of the present invention. Nevertheless, the general principles illustrated by use of these compositions applies equally to the delivery formulations according to the invention. Solutions were produced by dissolving (at a temperature not exceeding 50° C.) the following ingredients (quantitative details are expressed as parts by weight):—

(46) TABLE-US-00013 A B C D E Glyceryl monostearate 2 — 2 — 2 (self-emulsifying) Glyceryl monooleate — 2 — 2 — (self-emulsifying) Cremophor RH40 20 30 30 20 30 CBME-G1 to give THC 5 10 — — — CBME-G5 to give CBD — — 5 10 — CBME-G1 and G5 to — — — — 10 each give THC & CBD α-Tocopherol 0.1 0.1 0.1 0.1 0.1 Ascorbyl palmitate 0.1 0.1 0.1 0.1 0.1 Ethanol BP to produce 100 100 100 100 100

(47) Cannabis Based Medicine Extract (CBME) is an extract of Cannabis which may be prepared by, for example, percolation with liquid carbon dioxide, with the removal of ballast by cooling a concentrated ethanolic solution to a temperature of −20° C. and removing precipitated inert plant constituents by filtration or centrifugation.

(48) The product formed by mixing these ingredients is dispensed in 6 ml quantities into a glass vial and closed with a pump action spray. In use, the dose is discharged through a break-up button or conventional design. Proprietary devices that are suitable for this purpose are Type VP7 produced by Valois, but similar designs are available from other manufacturers. The vial may be enclosed in secondary packaging to allow the spray to be directed to a particular area of buccal mucosa. Alternatively, a proprietary button with an extension may be used to direct the spray to a preferred area of buccal mucosa.

(49) Each 1 ml of product contains 50-100 mg of Δ.sup.9-tetrahydrocannabinol (THC) and/or cannabidiol (CBD). Each actuation of the pump delivers a spray which can be directed to the buccal mucosae. In the above formulations CBMEs of known cannabinoid strength are used. CBME-G1 is an extract from a high THC-yielding strain of Cannabis, and CBME-G5 is from a high CBD-yielding variety. It will be clear to a person skilled in the art that purified cannabinoids, and extracts containing the cannabinoids, can be made formulated as described above by quantitative adjustment.

(50) Although solutions of CBME in ethanol alone can be used as a spray, the quantity of cannabinoid that can be delivered is limited by the aggressive nature of pure ethanol in high concentration as a solvent. This limits the amount that can be applied to the mucosae without producing discomfort to the patient. When a group of patients received THC or CBD in a solution of the type described above, directing the spray either sublingually or against the buccal mucosa, the patients uniformly reported a stinging sensation with the sublingual application, but mild or no discomfort when the same solution was sprayed onto the buccal mucosa. Spraying small quantities of this type of formulation onto the buccal mucosa does not appreciably stimulate the swallowing reflex. This provides greater dwell time for the formulation to be in contact with the buccal surface.

(51) Formulations were administered to a group of 13 human subjects so that they received 4 mg THC, 4 mg of CBD or placebo (vehicle alone) via a sublingual tablet, sublingual pump-action spray or buccal route.

(52) Absorption [area under the absorption curve (AUC)] of cannabinoid and primary metabolite were determined in samples of blood taken after dosing. The following Table 8 gives these as normalised mean values.

(53) TABLE-US-00014 TABLE 8 Route of Administration PAS sublingual Sublingual tablet Oropharyngeal Analyte in Plasma AUC AUC AUC THC 2158.1 1648.4 1575 11-OH THC 3097.6 3560.5 2601.1 CBD 912 886.1 858

(54) These results show that the total amounts of cannabinoid absorbed by sublingual and buccal (oropharyngeal) routes are similar but that there is a substantial (approximately 25%) reduction in the amount of 11-hydroxy (11-OH) metabolite detected after oropharyngeal (buccal) administration. This finding is not inconsistent with reduced swallowing (and subsequent reduced hepatic) metabolism of the buccal formulation.

(55) It is known that the 11-hydroxy metabolite of THC (11-OH THC) is possibly more psychoactive than the parent compound. It is therefore desirable to minimise the amount of this metabolite during administration, and this is likely to be achieved by using a formulation and method of application which reduces the amount of a buccal or sublingual dose that is swallowed. The pump action spray appears to offer a simple means of reducing the amount of material that is swallowed and metabolised by absorption from the intestinal tract below the level of the oropharynx.

Example 4-Growing of Medicinal Cannabis

(56) Plants are grown as clones from germinated seed, under glass at a temperature of 25° C.±1.5° C. for 3 weeks in 24 hour daylight; this keeps the plants in a vegetative state. Flowering is induced by exposure to 12 hour day length for 8-9 weeks.

(57) No artificial pesticides, herbicides, insecticides or fumigants are used. Plants are grown organically, with biological control of insect pests.

(58) The essential steps in production from seed accession to dried Medicinal Cannabis are summarised in FIG. 8.

Example 5-Determination of Cannabinoid Content in Plants and Extracts

(59) Identity by TLC

(60) a) Materials and methods

(61) Equipment Application device capable of delivering an accurately controlled volume of solution i.e., 1 μl capillary pipette or micro litre syringe. TLC development tank with lid Hot air blower Silica gel G TLC plates (SIL N-HR/UV254), 200 μm layer with fluorescent indicator on polyester support. Dipping tank for visualisation reagent. Mobile phase 80% petroleum ether 60:80/20% Diethyl ether. Visualisation reagent 0.1% w/v aqueous Fast Blue B (100 mg in 100 ml de-ionised water). An optional method is to scan at UV 254 and 365 nm.
b) Sample preparation

(62) i) Herbal Raw Material

(63) Approximately 200 mg of finely ground, dried Cannabis is weighed into a 10 ml volumetric flask. Make up to volume using methanol:chloroform (9:1) extraction solvent.

(64) Extract by ultrasound for 15 minutes. Decant supernatant and use directly for chromatography.

(65) ii) Herbal Drug Extract

(66) Approximately 50 mg of extract is weighed into a 25 ml volumetric flask. Make up to volume using methanol solvent. Shake vigorously to dissolve and then use directly for chromatography.

(67) c) Standards

(68) 0.1 mg/ml delta-9-THC in methanol.

(69) 0.1 mg/ml CBD in methanol.

(70) The standard solutions are stored frozen at −20° C. between uses and are used for up to 12 months after initial preparation.

(71) d) Test Solutions and Method

(72) Apply to points separated by a minimum of 10 mm.

(73) i) either 5 μl of herb extract or 1 μl of herbal extract solution as appropriate,

(74) ii) 10 μl of 0.1 mg/ml delta-9-THC in methanol standard solution,

(75) iii) 10 μl of 0.1 mg/ml CBD in methanol standard solution.

(76) Elute the TLC plate through a distance of 8 cm, then remove the plate. Allow solvent to evaporate from the plate and then repeat the elution for a second time (double development).

(77) The plate is briefly immersed in the Fast Blue B reagent until the characteristic re/orange colour of cannabinoids begins to develop. The plate is removed and allowed to dry under ambient conditions in the dark.

(78) A permanent record of the result is made either by reproduction of the image by digital scanner (preferred option) or by noting spot positions and colours on a tracing paper.

(79) Assay THC, THCA, CBD, CBDA and CBN by HPLC

(80) a) Materials and Methods

(81) Equipment: HP 1100 HPLC with diode array detector and autosampler. The equipment is set up and operated in accordance with in-house standard operating procedures (SOPlab037) HPLC column Discovery C8 5 μm, 15×0.46 cm plus Kingsorb ODS2 precolumn 5 μm 3×0.46 cm. Mobile Phase Acetonotrile:methanol:0.25% aqueous acetic acid (16:7:6 by volume) Column Operating 25° C. Temperature Flow Rate 1.0 ml/min Injection Volume 10 μl Run time 25 mins Detection Neutral and acid cannabinoids 220 nm (band width 16 nm) Reference wavelength 400 nm/bandwidth 16 nm Slit 4 nm Acid cannabinoids are routinely monitored at 310 nm (band width 16 nm) for qualitative confirmatory and identification purposes only. Data capture HP Chemistation with Version Δ7.01 software
b) Sample Preparation Approximately 40 mg of Cannabis Based Medicinal Extract is dissolved in 25 ml methanol and this solution is diluted to 1 to 10 in methanol. This dilution is used for chromatography. 0.5 ml of the fill solution, contained within the Pump Action Sublingual Spray unit, is sampled by glass pipette. The solution is diluted into a 25 ml flask and made to the mark with methanol. 200 μl of this solution is diluted with 800 μl of methanol. Herb or resin samples are prepared by taking a 100 mg sample and treating this with 5 or 10 ml of Methanol/Chloroform (9/1 w/v). The dispersion is sonicated in a sealed tube for 10 minutes, allowed to cool and an aliquot is centrifuged and suitably diluted with methanol prior to chromatography.
c) Standards
External standardisation is used for this method. Dilution of stock standards of THC, CBD and CBN in methanol or ethanol are made to give final working standards of approximately accurately 0.1 mg/ml. The working standards are stored at −20° C. and are used for up to 12 months after initial preparation.
Injection of each standard is made in triplicate prior to the injection of any test solution. At suitable intervals during the processing of test solutions, repeat injections of standards are made. In the absence of reliable CBDA and THCA standards, these compounds are analysed using respectively the CBD and THC standard response factors.
The elution order has been determined as CBD, CBDA, CBN, THC and THCA. Other cannabinoids are detected using this method and may be identified and determined as necessary.
d) Test Solutions
Diluted test solutions are made up in methanol and should contain analytes in the linear working range of 0.02-0.2 mg/ml.
e) Chromatography Acceptance Criteria:
The following acceptance criteria are applied to the results of each sequence as they have been found to result in adequate resolution of all analytes (including the two most closely eluting analytes CBD and CBDA)

(82) i) Retention Time Windows for Each Analyte: CBD 5.4-5.9 minutes CBN 7.9-8.7 minutes THC 9.6-10.6 minutes

(83) ii) Peak Shape (Symmetry Factor According to BP Method) CBD<1.30 CBN<1.25 THC<1.35

(84) iii) A number of modifications to the standard method have been developed to deal with those samples which contain late eluting impurity peaks e.g., method CBD2A extends the run time to 50 minutes. All solutions should be clarified by centrifugation before being transferred into autosampler vials sealed with teflon faced septum seal and cap.

(85) iv) The precolumn is critical to the quality of the chromatography and should be changed when the back pressure rises above 71 bar and/or acceptance criteria regarding retention time and resolution, fall outside their specified limits.

(86) f) Data Processing

(87) Cannabinoids can be subdivided into neutral and acidic—the qualitative identification can be performed using the DAD dual wavelength mode. Acidic cannabinoids absorb strongly in the region of 220 nm-310 nm. Neutral cannabinoids only absorb strongly in the region of 220 nm.
Routinely, only the data recorded at 220 nm is used for quantitative analysis.
The DAD can also be set up to take UV spectral scans of each peak, which can then be stored in a spectral library and used for identification purposes.
Data processing for quantitation utilises batch processing software on the Hewlett Packard Chemstation.
a) Sample Chromatograms
HPLC sample chromatograms for THC and CBD Herbal Drug extracts are provided in the accompanying Figures.

Example 6-Preparation of the Herbal Drug Extract

(88) A flow chart showing the process of manufacture of extract from the High-THC and High-CBD chemovars is given in FIG. 9.

(89) The resulting extract is referred to as a Cannabis Based Medicine Extract and is also classified as a Botanic Drug Substance, according to the US Food and Drug Administration Guidance for Industry Botanical Drug Products.

Example 7

(90) High THC Cannabis was grown under glass at a mean temperature of 21+2° C., RH 50-60%. Herb was harvested and dried at ambient room temperature at a RH of 40-45% in the dark. When dry, the leaf and flower head were stripped from stem and this dried biomass is referred to as “medicinal Cannabis”.

(91) Medicinal Cannabis was reduced to a coarse powder (particles passing through a 3 mm mesh) and packed into the chamber of a Supercritical Fluid Extractor. Packing density was 0.3 and liquid carbon dioxide at a pressure of 600 bar was passed through the mass at a temperature of 35° C. Supercritical extraction is carried out for 4 hours and the extract was recovered by stepwise decompression into a collection vessel. The resulting green-brown oily resinous extract is further purified. When dissolved in ethanol BP (2 parts) and subjected to a temperature of −20° C. for 24 hours a deposit (consisting of fat-soluble, waxy material) was thrown out of solution and was removed by filtration. Solvent was removed at low pressure in a rotary evaporator. The resulting extract is a soft extract which contains approximately 60% THC and approximately 6% of other cannabinoids of which 1-2% is cannabidiol and the remainder is minor cannabinoids including cannabinol. Quantitative yield was 9% w/w based on weight of dry medicinal Cannabis.

(92) A high CBD chemovar was similarly treated and yielded an extract containing approximately 60% CBD with up to 4% tetrahydrocannabinol, within a total of other cannabinoids of 6%. Extracts were made using THCV and CBDV chemovars using the general method described above.

(93) A person skilled in the art will appreciate that other combinations of temperature and pressure (e.g. in the range +10° C. to 35° C. and 60-600 bar) can be used to prepare extracts under supercritical and subcritical conditions.

Example 8-The Effects of Light on the Stability of the Alcoholic Solutions of THC, CBD or THCV

(94) The following example includes data to support the packaging of liquid dosage forms in amber glass, to provide some protection from the degradative effects of light on cannabinoids.

(95) Further credence is also given to the selection of the lowest possible storage temperature for the solutions containing cannabinoid active ingredients.

(96) Background and Overview:

(97) Light is known to be an initiator of degradation reactions in many substances, including cannabinoids. This knowledge has been used in the selection of the packaging for liquid formulations, amber glass being widely used in pharmaceutical presentations as a light exclusive barrier.

(98) Experiments were set up to follow the effects of white light on the stability of methanolic solutions of THC, CBD or THCV. Following preliminary knowledge that light of different wavelengths may have differing effects on compound stability (viz. tretinoin is stable only in red light or darkness), samples were wrapped in coloured acetate films or in light exclusive foil. A concurrent experiment used charcoal treated CBME to study the effects of the removal of plant pigments on the degradation process.

(99) Materials and Methods:

(100) Cannabinoids: 1 mg/ml solutions of CBME were made up in AR methanol. Methanolic solutions of CBME (100 mg/ml) were passed through charcoal columns (Biotage Flash 12AC 7.5 cm cartridges, b/no. 273012S) and were then diluted to 1 mg/ml. Solutions were stored in soda-glass vials, which were tightly screw capped and oversealed with stretch film. Tubes were wrapped in coloured acetate films as follows:

(101) Red, Yellow, Green, and Cyan

(102) Solutions were also filled into the amber glass U-save vials; these were sealed with a septum and oversealed. One tube of each series of samples was tightly wrapped in aluminium foil in order to completely exclude light. This served as a “dark” control to monitor the contribution of ambient temperature to the degradation behaviour. All of the above tubes were placed in a box fitted with 2×40 watt white Osram fluorescent tubes. The walls of the box were lined with reflective foil and the internal temperature was monitored at frequent intervals.

(103) A further tube of each series was stored at −20° C. to act as a pseudo to the reference sample; in addition, one tube was exposed directly to light without protection. Samples were withdrawn for chromatographic analysis at intervals up to 112 days following the start of the study. The study was designated AS01201/AX282.

(104) Samples of the test solutions were withdrawn and diluted as appropriate for HPLC and TLC analysis. HPLC was carried out in accordance with TM GE.004.V1 (SOPam058). TLC was performed on layers on Silica gel (MN SilG/UV) in accordance with TM GE.002.V1 (SOPam056).

(105) Two further TLC systems were utilised in order to separate degradation products:

(106) a) SilG/UV, stationary phase, hexane/acetone 8/2 v/v mobile phase

(107) b) RPC18 stationary phase, acetonitrile/methanol/0.25% aqueous acetic acid 16/7/6 by volume

(108) Visualisation of cannabinoids was by Fast Blue B salt.

(109) Results and Discussion:

(110) HPLC Quantitative Analysis:

(111) The results from the HPLC analysis of samples drawn from the stored, light exposed solutions, are plotted and presented as FIGS. 6 and 6a (THC before and after charcoal treatment), and FIGS. 7 and 7a (CBD before and after charcoal treatment).

(112) It can be seen from FIGS. 6 and 6a that there are significant improvements to the stability of THC in all solutions, except those stored in the dark (at ambient temperature) and at −20° C. (and hence which are not under photochemical stress). Even storage in amber glass shows an improvement when un-treated extract is compared with charcoal treated extract. This, however, may reflect in an improvement of the thermal stability of the charcoal treated extract.

(113) FIGS. 7 and 7a present similar data for CBD containing extracts, from which it can be seen that this cannabinoid is significantly more sensitive to the effects of light than is THC. In the absence of charcoal, all exposures, except in amber glass, light excluded (foil) and −20° storage, had degraded to non-detectable levels of CBD before 40 days. This improved to figures of between 42 and 62 days following charcoal treatment. Amber glass protected CBD showed an improvement from ˜38% residual compound at 112 days without charcoal clean up, to approximately 64% at the same time after charcoal treatment. There was also an improvement in the stability of CBD in light excluded solution after charcoal treatment. This can only reflect a reduction in either thermo-oxidative degradation, or a residual photochemical degradation initiated by light (and/or air) during CBME and solution preparation.

(114) Thin Layer Chromatography Qualitative Analysis:

(115) The evaluation of the light degraded solutions using thin layer chromatography, used both the existing normal phase system (i.e. Silica stationary phase and hexane/diethyl ether as mobile phase) and two additional systems, capable of resolving more polar or polymeric products formed during the degradation processes.

(116) Thus, chromatography using the hexane/diethyl ether system, showed that for THC by day 112, there was a reduction in the intensity of the THC and secondary CBD spots with all of the colour filtered lights (data not shown). At the same time, there was an increase in the intensity of Fast Blue B staining material running at, or close to, the origin. Foil protected solution exhibited none of these effects.

CONCLUSIONS AND RECOMMENDATIONS

(117) Cannabinoids are known to be degraded by a number of natural challenges, viz. light, heat, oxygen, enzymes etc. It is most likely that in an extract of herbal plant material, which has not been subjected to extensive clean-up procedures, that some of these processes may still be able to continue. Paradoxically, it is also likely that the removal of cannabinoids from the presence of any protection agents within the plant tissue, may render the extract more likely to suffer from particular degradation pathways.

(118) Packaging into amber glass vials, conducting formulation manufacture in amber filtered light, and the storage of plant extracts and pharmaceutical formulations at temperatures as low as possible compatible with manufacturing and distribution requirements and patient compliance eliminates, or at least reduces, the effect of light on degradation of cannabinoids. These actions dramatically improved the storage stability of both plant extracts and finished products.

(119) It was interesting to note that CBD appeared to be markedly less stable than THC, when subjected to photochemical stress. This is the opposite of the finding for the relative thermo-oxidative stabilities, in which THC is the less stable. This seems to indicate that, although polymeric degradation products may be the common result of both photochemical and thermo-oxidative degradation, the exact details of the mechanism are not identical for the two processes.

(120) Among the conclusions that can be drawn are the following:

(121) 1] The choice of amber glass for the packaging of the dose solutions provides improved stability, but minor improvements can be made by additional light exclusion measures.

(122) 2] The drying process and subsequent extraction and formulation of Cannabis extracts should indeed be carried out in low intensity, amber filtered light.

(123) 3] Consideration should be given to the blanketing of extracts under an inert atmosphere (e.g. Nitrogen).

(124) 4] Clean-up of Cannabis extracts by simple charcoal filtration after winterisation, may yield substantial improvements to product shelf-life.

(125) Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

(126) All references disclosed herein are incorporated by reference in their entirety.