Method of production of phytocannabinoids for use in medical treatments

Abstract

A method of producing cannabinoids for use in medical treatments by growing cultured Cannabis sativa plant cells through tissue culture, the method of comprising the steps of: selecting Cannabis sativa leaf tissue for culture; and growing a tissue culture from the selected leaf tissue in a liquid based medium whilst controlling the light exposure of the tissue culture to control the cannabinoid content of the tissue culture. Control of the light exposure can enable the phytocannabinoid content of the grown tissue culture to be tailored to the use intended for the tissue culture. For example, the THC content of the tissue culture can be controlled to be maximised or minimized depending on the intended use. Use of tissue culture is beneficial as compared to prior art methods as it allows for genetic consistency and reduces the resources necessary to produce plant cells containing phytocannabinoids.

Claims

1. A method of producing a cannabinoid from Cannabis plant cells, the method comprising the steps of: selecting a Cannabis tissue for culture; growing a cell suspension culture from the selected Cannabis tissue in a liquid based medium whilst controlling the light exposure of the cell suspension culture to control the content of the cannabinoid produced by the cell suspension culture, the light exposure comprising UV light; and collecting and drying cells from the cell suspension culture after growing the cell suspension culture.

2. A method of claim 1 further comprising dissolving the dried cells in water.

3. A method of claim 1 further comprising using the dried cells as a medicament or as part of a medicament.

4. A method according to claim 1, wherein the light exposure is controlled such that tissue culture is constantly exposed to PAR during growth of the tissue culture.

5. A method according to claim 4, wherein the PAR is controlled to provide at least 0.2 moles of photons per day.

6. A method according to claim 4, wherein the PAR is controlled to provide 0.5 moles of photons per day.

7. A method according to claim 1, wherein the light exposure is controlled such that the tissue culture is exposed to UV light during growth of the tissue culture.

8. A method according to claim 7, wherein the light exposure is controlled such that the tissue culture is exposed to UVA light during growth of the tissue culture.

9. A method according to claim 7, wherein the light exposure is controlled such that tissue culture is exposed to UVB light during growth of the tissue culture.

10. A method according to claim 7, wherein the UV light is controlled during growth of the tissue culture such that the tissue culture is exposed to UV light of an intensity greater or equal to 1200 lumens but equal to or less than 2000 lumens and the UV light exposure is cycled through alternating periods of exposure and darkness; wherein each period of exposure is at least 30 minutes and each period of darkness is at least 30 minutes.

11. A method according to claim 10, wherein each period of exposure is equal to or less than one hour and each period of darkness is equal to or less than one hour.

12. A method according to claim 7, wherein the intensity of the UV light is less than or equal to 2000 lumens.

13. A method according to claim 7, wherein the UV light has an intensity equal to or less than 600 lumens.

14. A method according to claim 7, wherein the UV light is controlled during growth of the tissue culture such that the tissue culture is constantly exposed to UV light of an intensity equal to or less than 1200 lumens.

15. A method according to claim 1, wherein during growing the tissue culture is maintained at a temperature between 25° C. and 30° C.

16. A method according to claim 15, wherein during growing the tissue culture is maintained at a temperature of 27° C.

17. A method according to claim 1, wherein the tissue culture is grown for between 10 and 28 days.

18. A method according to claim 1, wherein the tissue culture is grown for 14 days.

19. A method according to claim 1, wherein the tissue culture is agitated during growth of the tissue culture.

20. A method according to claim 1, wherein the CO2 content of the environment in which the cell suspension culture is grown is controlled to increase tissue growth.

21. A method of claim 1 wherein the selected the Cannabis leaf tissue was previously grown in a liquid based medium whilst controlling the light exposure of the tissue culture to control the cannabinoid content of the tissue culture.

22. A method of claim 1, wherein the cannabinoid is THC.

23. A method of claim 1, wherein the Cannabis plant cells are Cannabis sativa plant cells and the Cannabis tissue is Cannabis sativa tissue.

Description

EXAMPLE

(1) i) Liquid Media

(2) Starting Media 0.44% Murashige and Skoog basal powdered medium 1.0% NAA (naphthalene acetic acid) 0.004% stock solution 3.0% sucrose Distilled water to 100%

(3) Equipment Glass bottle with cap Magnetic stirrer Sterile plastic plant culture dishes Glass pipettes pH meter Autoclave Laminar flow cabinet Balance Nescofilm Phytagel 1M NaOH solution 0.1M NaOH solution

(4) The liquid media was prepared in the following manner: a) The starting media was Murashige and Skoog (MS) media with 3% sucrose and 1% naphthalene acetic acid (from concentrated stock solution of 0.004% w/v); b) The media was then pH adjusted to pH 5.75 and solidified with 0.2% phytagel; c) The media was then autoclaved for 20 minutes at 121° C. and then poured into sterile plastic plant tissue culture dishes.
ii) Culture Initiation

(5) Reagents Liquid media (as prepared in the manner set out above) Cannabis sativa leaf tissue

(6) Equipment Sterile glass beakers Sterile distilled water Sterile scalpel Sterile tweezers 10% bleach solution 70% ethanol solution 1M NaOH solution 0.1M NaOH solution

(7) The culture was initiated in the following manner: a) The leaf tissue of Cannabis sativa was sterilised by immersion in 70% ethanol for 2 minutes, followed by immersion in 10% bleach solution for 10 minutes; b) The leaf tissue was then washed three times with sterile (autoclaved) distilled water; c) laminar flow cabinets; d) The leaf tissue slices were placed onto the prepared plates containing callus induction media, and plates were sealed with Nescofilm®. e) The plates were placed in the dark at 27° C. and callus formation began to appear after about 1 month.
iii) Media Preparation for Cultures

(8) Reagents 3% sucrose 0.44% Murashige and Skoog basal powdered medium 1% naphthalene acetic acid (NAA) 0.004% stock solution 0.01% vitamin solution (0.05% pyridoalhydrochlorid, 0.1% thiamine dichloride, and 0.05% g nicotinic acid) 1M NaOH solution 0.1M NaOH solution Distilled water to 100%

(9) Equipment 1 L glass bottle Magnetic stirrer 20×250 m conical 20 sheets of foil approximately 20 cm×20 cm Glass pipettes pH meters Autoclave Laminar flow cabinet Balance

(10) The media was prepared in the following manner: a) Mix 3% sucrose, 0.44% Murashige and Skoog basal powder, 1% NAA stock, and 0.01% vitamin solution and prepare to 100% with distilled water; b) Mix using a magnetic stirrer until all dry components dissolved, then pH adjust with 1M and 0.1M NaOH to a pH of 5.75; c) Take 20×250 ml conical flasks, to each add 50 ml media and seal neck of flask with foil; sterilize in autoclave at 121° C., 103 kPa for 25 minutes; d) Immediately following sterilization place flasks in laminar flow cabinet and allow to cool to ambient temperature. iv) Inoculation and subculture of established cultures

(11) Reagents Friable callus 70% ethanol

(12) Equipment Laminar flow cabinet Bunsen burner Prepared media 20 sterile sheets of foil approximately 20 cm×20 cm Several pairs of tweezers or small forceps Wide spatulas with holes Broad spectrum PAR lighting UVA and UVB lighting

(13) The inoculation and subculture of established cultures was carried out in the following manner: a) Sterilize inside of laminar flow cabinet with 70% ethanol; b) Sterilize all tweezers and spatulas by dipping in 70% ethanol, then flaming till red hot. Allow to cool inside laminar flow cabinet; c) Remove foil from prepared media flask; d) Take sterilized tweezers and remove thumbnail sized pieces of friable callus from the plant tissue. Break up into finely dispersed cells and add to flask. Aim to add approximately 5 g of tissue to 50 ml media (10% w/v); e) Flame the neck of the flask and cover with a sterile sheet of foil; f) Place the flask on a shaker at 120 rpm, in a dark room heated to 27° C. Leave until a thick dispersed cell suspension culture can be observed, approximately 2 weeks; g) Remove foil from prepared media flask; h) Remove foil from flask containing dispersed cell suspension cultures (produced by inoculation at point f); i) Take wide spatula with holes, sterilize, allow to cool, and scoop out the cells. Add these cells to the fresh media. Aim to add approximately 5 g tissue to 50 ml of media; j) Flame the neck of the flask and cover with a sterile sheet of foil; k) Place the flask on a shaker at 120 rpm in, subject to one of the two lighting regimes set out below, and heated to 27° C. for 14 days; and l) After 14 days use the cell suspension culture for further subcultures or harvest cells.

(14) Lighting Regime 1 Constant exposure to PAR at a rate of 0.5 moles of photons per day; and Constant exposure to UVB and UVA radiation at an intensity of approximately 500 lumens.

(15) Lighting Regime 2 Constant exposure to PAR at a rate of 0.5 moles of photons per day; and Periodic exposure to UVB and UVA radiation at an intensity of approximately 1500 lumens, the periodic exposure consisting of alternating 1 hour periods of exposure and 1 periods in which there is no UVB and UVA exposure.