METHOD OF PRODUCTION OF PHYTOCANNABINOIDS FOR USE IN MEDICAL TREATMENTS

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. application Ser. No. 16/806,994, filed Mar. 2, 2020, which is a continuation of PCT Application No. PCT/EP2018/077149, filed Oct. 5, 2018, which claims priority from Great Britain Application No. 1717554.8, filed Oct. 25, 2017, the contents of all of which are incorporated herein by reference in their entirety.

DESCRIPTION

Background of the Invention

Phytocannabinoids are a set of compounds present in cannabis plants that, when ingested, interact with the human body to produce a physical or psychological effect. The best known phytocannabinoid is tetrahydrocannabinol (THC). Phytocannabinoids are becoming increasingly commonly used in medical treatments under the umbrella term of medical marijuana. This term covers the use of any phytocannabinoid in order to treat a physical or mental condition. Medical marijuana is an active and growing sector and as such there is need for a reliable and consistent source of phytocannabinoids.

Currently, phytocannabinoids for use as medical marijuana are generally obtained from Cannabis Sativa plants that are grown in a conventional agricultural manner. However, the growth of Cannabis Sativa plants in this manner is less than ideal. The plants will always have variations in phytocannabinoid content, even if cultivated in identical conditions, due to natural genetic variations between plants. Further, variations between weather and/or soil conditions during agricultural growth of plants can lead to variation in phytocannabinoid content of the plants.

Land use is also a problem as it takes land out of use for alternative uses, such as conventional agriculture. Land security can also be a problem, as cannabis plants are valuable to third parties for legal and non-legal uses. Agricultural cannabis cultivation also requires large quantities of water and is a relatively inefficient process.

As an alternative to conventional agricultural growth, cannabis plants are often grown hydroponically indoors. However, such growth is extremely energy intensive and requires large quantities of water. As the plants require artificial lighting in indoor situations, it is also necessary for the ventilation and temperature control to be provided to remove excess heat produced by the lighting. Further, the issue of genetic variation remains present in hydroponically grown plants.

In light of the above, there is a need for a new method of producing phytocannabinoids that is secure, reduces the resource requirements, and can provide consistent amounts of phytocannabinoids.

SUMMARY OF THE INVENTION

The present invention provides a method of producing phytocannabinoids for use in medical treatments by growing cultured Cannabis sativa plant cells through tissue culture, the method comprising the steps of:

• • selecting Cannabis sativa leaf tissue for culture or flower tissue; 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. It will be recognized by individuals in the art that in cell culture, the cells often revert to a pluripotent stem cell which can then be triggered with the correct physical and chemical stimuli to become a different type of cell from the same plant.

The method of the present invention is advantageous as it allows production of a medium of consistent and controllable phytocannabinoids content. In particular, tissue culture allows production of tissue without genetic variation, and control of light exposure allows control of phytocannabinoid production within the tissue. The leaf tissue that is cultured can be from a plant selected to provide the correct phytocannabinoid composition. For example, a leaf tissue that provides maximised or minimised levels of THC and/or cannabis oil may be selected.

After growth of the tissue culture, the resulting cells can either be used directly as a medicament, or the phytocannabinoids can be extracted from the cells for further processing. In contrast to previous methods, the tissue culture produced by the method of the present invention has consistent phytocannabinoid content that can be controlled and, as such, it is possible to use the plant cells of the tissue culture as a medicament without further significant processing. For example, the plant cells of the tissue culture may be freeze-dried and used as a medicament without any further processing or by subsequently dissolving the freeze-dried cells in water.

Phytocannabinoids produced by the method of the present invention may be used in many dosage forms including but not limited to tablets, capsules, lozenges, vapour inhalation products, or other orally taken dosage forms.

In order for the tissue culture of the present invention to grow, it is necessary that the tissue culture is exposed to photosynthetically active radiation (PAR). As will be readily understood, PAR is light that allows the tissue to photosynthesise. Generally, photoreceptors for photosynthesis are most efficient in the blue (400-500 nm) and red (600-700 nm) area of the light spectrum. Far-red (700-800 nm) is critical for flowering of many plants. The (500-600 nm) green area is less understood and even though much of this range is reflected, it is considered beneficial for carotenoids and lycopene (for colour and photo-protection). On that basis, most PAR will comprise blue and red light and may also comprise far-red and/or green light. There are many commercially available lighting systems that are designed and intended for producing PAR for plant growth, and that would be suitable for use in the method of the present invention.

In embodiments of the invention, the light exposure may be controlled such that tissue culture is constantly exposed to PAR during growth of the tissue culture. This can be beneficial as it can maximise the growth of the tissue culture. In particular, as the method of the present invention involves the growth of tissue culture, rather than whole plants, there is no requirement for the PAR to be cycled to replicate natural daylight cycles.

In order to grow the tissue culture sufficiently, it is generally preferred that the PAR is controlled to provide at least 0.2 moles of photons per day, and preferably 0.5 moles of photons per day. The precise amount of PAR provided in any embodiment of the method of the present invention will be dependent on the amount of tissue culture that is exposed to the PAR. A larger amount of tissue culture will generally require a larger amount of PAR in order to grow sufficiently. The amount of PAR can be governed by the cell density. For instance, the cell density can be sufficiently low for light to pass through the media to all cells and is not reflected back by having too high a cell density at the walls of the culture vessel.

Whilst PAR is required to grow the tissue culture, the control of phytocannabinoids in the tissue culture can be controlled by controlling the exposure of the tissue culture to UV light. In particular. it is believed that phytocannabinoids, including THC, are formed in cannabis to protect the plant from UV light, as each THC molecule has several ring structures that act to protect the plant from UV light. Therefore, exposure to UV light is believed to increase the production of THC in cannabis plants, and the same mechanism would occur in cannabis tissue culture.

In order to increase the production of THC and other phytocannabinoids in the tissue culture in embodiments of the present invention, the light exposure is controlled such that the tissue culture is exposed to UV light during growth of the tissue culture. In alternative embodiments, in order to minimise the production of THC and other phytocannabinoids in the tissue culture, the light exposure may be controlled such that exposure of the tissue culture to UV light is minimised. However, UV radiation is important in inducing production of phenolics, anthocyanins (coloration), antioxidants and vitamins that inhibit mold growth in the tissue culture. Therefore, it may be necessary that the tissue culture is exposed to a relatively small amount of UV radiation in order to allow the tissue to generate such chemicals. For example, if UV radiation is minimized, it may be controlled such that the tissue culture is exposed to less than 0.05 moles of photons per day.

Maximised THC content may be preferred if the tissue culture is being grown in order to produce THC for use in the treatment of chronic pain or other similar conditions. Minimised THC content may be preferred if the tissue culture is being grown in order to produce cannabis oils for use in the treatment of Parkinson's disease.

UVA light, consisting of light of wavelength 315-400 nm, may result in increased levels of THC, other phytocannabinoids, and other chemicals crucial to tissue growth. Therefore, it may be preferable that the light exposure is controlled such that the tissue culture is exposed to UVA light during growth of the tissue culture.

Exposure to UVB light, consisting of light of wavelength 280-315 nm, results in increased production of THC, other phytocannabinoids, and other chemicals crucial to tissue growth within the cannabis plant. Therefore, it may be preferable that the light exposure is controlled such that tissue culture is exposed to UVB light during growth of the tissue culture. It is generally believed that UVB light is more effective than UVA light in increasing levels of THC within cannabis. Therefore, it may be preferable that the levels of UVB light are maximised in methods according to the present invention where high THC levels are desired, and that levels of UVB light are minimised in methods according to the present invention where low THC levels are desired.

In order to maximise THC levels in the tissue culture, it can be generally beneficial to maximise the intensity of UV light to which the tissue culture is exposed. However, intensities of UV light that are too high can damage the tissue culture. In particular, constant exposure of UV light at intensities of 1200 lumens and above can damage a tissue culture, whilst even periodic exposure to UV light at intensities above 2000 lumens can damage the tissue culture. Therefore, in embodiments of the present invention it may be preferable that 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 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. Each period of exposure to UV light may be equal to or less than one hour and each period of darkness may be equal to or less than one hour. Advantageously, each period of exposure to UV light will be followed by a period of darkness that is equal in duration to the previous period of exposure to UV light. It may also be advantageous that each period of exposure to UV light is of equal duration.

In relation to the period of exposure of the tissue culture to UV light, it is to be understood that a period of darkness is a period in which the tissue culture is exposed to substantially no UV light, but it may still be exposed to normal PAR or other light. That, in this context “darkness”, is to be understood to mean the substantial absence of UV light.

In alternative embodiments of the method of the present invention, 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, or equal to or less than 600 lumens.

During growth of the tissue culture, it is preferable that the tissue culture is maintained at an optimum temperature to promote tissue growth. For example, the tissue culture may be maintained at a temperature between 25° C. and 30° C., for example 27° C.

In the method of the present invention, the tissue culture may be grown for any suitable period of time in order to allow for the desired amount of tissue culture to be grown. For example, the tissue culture may be grown for between 10 and 28 days. In embodiments of the invention the tissue culture may be grown for 14 days.

In order to promote growth of the tissue culture, it may be preferable that the tissue culture is agitated during its growth, for example by positioning the tissue culture on a shaker during the growth.

It may also be preferable that the CO₂ content of the environment in which the tissue culture is grown is controlled to increase tissue growth. This can be done in any manner apparent to the person skilled in the art.

The tissue culture produced by the method of the present invention may be used in any suitable way. For example, the method may further comprise the step of collecting and freeze-drying the tissue culture after growing. The freeze-dried tissue culture may then be used as a medicament or as part of a medicament. Alternatively, the desired active ingredient, for example THC or any other phytocannabinoid, may be extracted from the tissue culture after growth.

In order to provide consistency in the method of the present invention, it may be advantageous that some or all of the leaf tissue culture selected for culture was previously grown according to the method of the present invention. That is, genetic consistency can be assured by continuing to use the same tissue culture for subsequent tissue culture growth after an initial run of the method of the present invention.

During growth of the tissue culture, it can be preferable that air is added to the liquid-based medium in order to keep the tissue cells oxygenated. For example, it may be preferable to keep the saturated oxygen level above 20%, or more preferably 25%, in the liquid based medium.

Any suitable liquid based medium may be used to grow the tissue culture. In embodiments of the invention, the liquid based medium may be formed of a Murashige and Skoog solution, with potential further additions to adjust the pH level and/or with the potential addition of sucrose or an equivalent substance.

An example of an embodiment of the method of the present invention is described below. It is to be understood that this is provided as an example only and is not intended to be limiting on the scope of the application. Unless specifically indicated, any specific step of the method may be used in any method of the present invention.

EXAMPLE

i) Liquid Media

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%

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

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

Reagents

Liquid media (as prepared in the manner set out above)

Cannabis sativa leaf tissue

Equipment

Sterile glass beakers

Sterile distilled water

Sterile scalpel Sterile tweezers

10% bleach solution

70% ethanol solution

1M NaOH solution

0.1M NaOH solution

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) The sterile washed leaf tissue was asceptically cut into disc shapes in a sterile laminar flow cabinet;
• 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

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%

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

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

Reagents

Friable callus

70% ethanol

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

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, or until the density of cells in the culture ranges from about 10,000 cells/mL to about 100,000 cells/mL, from about 20,000 cells/mL to about 90,000 cells/mL, from about 30,000 cells/mL to about 80,000 cells/mL, from about 40,000 cells/mL to about 70,000 cells/mL, from about 50,000 cells/mL to about 60,000 cells/mL, from about 10,000 cells/mL to about 60,000 cells/mL, from about 20,000 cells/mL to about 70,000 cells/mL, from about 30,000 cells/mL to about 80,000 cells/mL, from about 40,000 cells/mL to about 90,000 cells/mL, or from about 50,000 cells/mL to about 100,000 cells/mL;
• 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, or until the density of cells in the culture ranges from about 10,000 cells/mL to about 100,000 cells/mL, from about 20,000 cells/mL to about 90,000 cells/mL, from about 30,000 cells/mL to about 80,000 cells/mL, from about 40,000 cells/mL to about 70,000 cells/mL, from about 50,000 cells/mL to about 60,000 cells/mL, from about 10,000 cells/mL to about 60,000 cells/mL, from about 20,000 cells/mL to about 70,000 cells/mL, from about 30,000 cells/mL to about 80,000 cells/mL, from about 40,000 cells/mL to about 90,000 cells/mL, or from about 50,000 cells/mL to about 100,000 cells/mL, use the cell suspension culture for further subcultures or harvest cells.

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. In some aspects, the concentration of cannabinoids using this lighting regime ranges from about 0.01% (g/mL) to about 5% (g/mL), from about 0.05% (g/mL) to about 4% (g/mL), from about 0.1% to about 3% (g/mL), from about 0.5% (g/mL) to about 4% (g/mL), from about 0.1% (g/mL) to about 2% (g/mL), from about 0.5% (g/mL) to about 1%. (g/mL), from about 0.05% (g/mL) to about 1%. (g/mL), from about 0.1% (g/mL) to about 1.5%. (g/mL), or from about 1% (g/mL) to about 2%. (g/mL).

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. In some aspects, the concentration of cannabinoids using this lighting regime ranges from about 1% (g/mL) to about 60% (g/mL), from about 5% (g/mL) to about 50% (g/mL), from about 10% (g/mL) to about 40% (g/mL), from about 20% (g/mL) to about 40% (g/mL), from about 40% (g/mL) to about 60% (g/mL), from about 1% (g/mL) to about 50% (g/mL), or from about 20% (g/mL) to about 50% (g/mL).