NEW USE FOR CANNABINOID-CONTAINING PIANT EXTRACTS

FIELD OF INVENTION

The present invention relates to the use of cannabinoid- containing plant extracts in the prevention or treatment of diseases or conditions that are alleviated by blockade of one or more types of TRP channel. Preferably the subset of TRP channel that is blockaded is the TRPA channel. More preferably the TRPA channel is the TRPAl channel. Preferably the diseases or conditions to be prevented or treated include: neuropathic pain, inflammation or vasoconstriction. Alternatively the TRP channel that is blockaded is the TRPM channel. More preferably the TRPM channel is the

TRPM8 channel. Preferably the diseases or conditions to be prevented or treated are cancer. More preferably the cancers to be treated include: cancer of the prostate, cancer of the breast, cancer of the colon, cancer of the lung or cancer of the skin.

Alternatively the TRP channel that is blockaded is the TRPV channel. More preferably the TRPV channel is the TRPVl channel. Preferably the diseases or conditions to be prevented or treated include neuropathic pain, inflammation or vasoconstriction.

BACKGROUND TO THE INVENTION

Transient receptor potential (TRP) channels are known to be at the forefront of mammals sensory systems, and have been found to be involved in the response to temperature, touch, pain, osmolarity, pheromones, taste and other stimuli. It is thought that the role of TRP channels is far broader than simple sensory transduction as they are

able to respond to many stimuli from both inside and outside of the cell.

Mammals are able to detect temperature with specialised neurons in their peripheral nervous system. These neurones are a subset of TRP channels: vanilloid-type channels (TRPV) . Four different TRPV channels (TRPVl-4) have been identified and are implicated in heat sensing. These are temperature sensitive ion channels and are critical contributors to normal pain and temperature sensation. As such they are useful targets for the relief of pain.

A different subset, the TRPM channels (melastatin-type) , in particular the TRPM8 channel, is implicated in sensing cold temperatures of less than 25°C. The combined range of temperatures that these channels are able to detect covers the majority of the relevant 'normal range' temperatures that are sensed by most mammals. Externally applied agents such as menthol, eucalyptol and icilin are able to activate the TRPM8 channels.

Up-regulation of activity of the TRPM8 channel occurs in the presence of certain tumour cells including prostate cancer cell carcinomas and other non-prostatic primary human tumours such as breast, colon, lung and skin cancer.

The subset of TRP channels known as ankyrin-like (TRPA) channels, in particular the TRPAl channels, are cold- activated channels. The TRPAl channels have a lower activation temperature in comparison to the TRPM8 channel. The TRPAl (also known as ANKTMl) channel shares very little amino acid homology with the TRPM8 channel,

and as such is thought to be a distant family member of the TRP channels.

The TRPAl channels have been found to be activated by noxious cold and pungent natural compounds such as those found in cinnamon oil, wintergreen oil, clove oil, mustard oil, raw garlic, camphor and ginger. Bradykinin, which is an inflammatory peptide that acts through the G protein-coupled receptor, is also shown to activate TRPAl.

The topical application of compounds such as mustard oil (allyl isothiocyanate) activates sensory nerve endings; this in turn produces pain, inflammation and a hypersensitivity to both thermal and mechanical stimuli. These effects are caused by activation of TRPAl channels. Cinnamon oil (cinnamaldehyde) has been shown to be the most specific TRPAl activator. It excites the TRPAl channel and is able to elicit nociceptive behaviour in mice. Activation of TRPAl produces a painful sensation and therefore the eiicitation of nociceptive behaviour in mammals provides a model for why noxious cold can be perceived as burning pain, (Bandell at al. Neuron 2004).

The cannabinoid tetrahydrocannabinol (THC) has also been shown to activate the TRPAl channels (Jordt et al. Nature 2004) acting in a similar manner to mustard oil and cinnamaldehyde .

TRPAl is also targeted by environmental irritants, such as acrolein, which account for toxic and inflammatory actions of tear gas, vehicle exhaust, and metabolic byproducts of chemotherapeutic agents.

The use of TRPAl-deficient mice has shown that this channel is the sole target through which mustard oil and garlic activate primary afferent nociceptors to produce inflammatory pain. The TRPAl-deficient mice display normal cold sensitivity and unimpaired auditory function, suggesting that this channel is not required for the initial detection of noxious cold or sound. However, these mice exhibit pronounced deficits in bradykinin- evoked nociceptor excitation and pain hypersensitivity. It can therefore be concluded that TRPAl is an important component of the transduction machinery through which environmental irritants and endogenous pro-analgesic agents depolarize nociceptors to elicit inflammatory pain (Bautista et al. Cell 2006) .

Cold hyperalgesia is an enhanced sensitivity to pain and is a well-documented symptom of inflammatory and neuropathic pain; however, the underlying mechanisms of this condition are poorly understood. It has been found that the pharmacological blockade of TRPAl in primary sensory neurons is able to reverse cold hyperalgesia that has been caused by inflammation and nerve injury. Therefore blocking TRPAl in sensory neurons might provide a fruitful strategy for treating cold hyperalgesia caused by inflammation and nerve damage, (Obata et al. J Clin Invest 2005) .

Intracellular Ca ²⁺ activates human TRPAl via an EF-hand domain and cold sensitivity occurs indirectly (and non physiologically) through increased [Ca ²⁺ ] during cooling in heterologous systems. (Zurborg et al. Nature Neurosci 2007) .

The incidence of cold hyperalgesia following L5 spinal nerve ligation (SNL) has been examined, because it is likely that the activation of two distinct populations of TRPAl- and TRPM8-expressing small neurons underlie the sensation of cold. In the nearby uninjured L4 (dorsal route ganglion (DRG) , TRPAl mRNA expression increased in trkA-expressing small-to-medium diameter neurons from the 1st to 14th day after the L5 SNL. This upregulation corresponded well with the development and maintenance of nerve injury-induced cold hyperalgesia of the hind paw.

In contrast, there was no change in the expression of the TRPM8 mRNA/protein in the L4 DRG throughout the 2-week time course of the experiment. In the injured L5 DRG, on the other hand, both TRPAl and TRPM8 expression decreased over 2 weeks after ligation. Furthermore, intrathecal administration of TRPAl, but not TRPM8, antisense oligodeoxynucleotide suppressed the L5 SNL-induced cold hyperalgesia. Increased TRPAl in uninjured primary afferent neurons may contribute to the exaggerated response to cold observed in the neuropathic pain model, (Katsura et al. Exp Neurol 2006) .

Neuropathic pain is a chronic pain that usually is accompanied or caused by tissue injury. With neuropathic pain, the nerve fibres are often damaged, dysfunctional or injured. These damaged nerve fibres send incorrect signals to other pain centres resulting in the chronic pain. The impact of nerve fibre injury includes a change in nerve function both at the site of injury and areas around the injury.

One example of neuropathic pain is called phantom limb syndrome. This occurs when an arm or a leg has been removed because of illness or injury, but the brain still

gets pain messages from the nerves that originally carried impulses from the missing limb. These nerves now misfire and cause pain.

Neuropathic pain often seems to have no obvious cause; but, some common causes of neuropathic pain include: multiple sclerosis, diabetes, back injury, amputation, spinal surgery, HIV infection, shingles, alcoholism and facial nerve problems.

The symptoms of neuropathic pain include shooting and burning pain, tingling and numbness and increased sensitivity to touch or cold.

Current treatments include the use of non-steroidal antiinflammatory drugs and stronger analgesics such as morphine-based drugs. Anti-convulsant and antidepressant drugs are also often used to treat neuropathic pain.

Very often neuropathic pain can be difficult to treat; in this case a pain specialist may use invasive or implantable device therapies to effectively manage the pain. Electrical stimulation of the nerves involved in neuropathic pain generation may significantly control the pain symptoms .

Unfortunately, neuropathic pain often responds poorly to standard pain treatments and occasionally may get worse instead of better over time. For some people, it can lead to serious disability.

Inflammation is the immune systems first response to infection or irritation. Inflammation often causes redness, swelling, pain and dysfunction of the affected area .

Inflammation may also be associated with other symptoms including: fever, chills, fatigue, loss of energy, headaches, loss of appetite and muscle stiffness.

Inflammation is caused by chemicals from white blood cells being released into the blood or affected tissues in an attempt to rid the body of foreign substances. This release of chemicals increases the blood flow to the area and may result in redness and warmth. Some of the chemicals cause leakage of fluid into the tissues, resulting in swelling. The inflammatory process may stimulate nerves and cause pain.

Inflammation of the joints can also occur, this is caused by an increased number of cells and inflammatory substances within the joint causing irritation, wearing down of cartilage (cushions at the end of bones) and swelling of the joint lining.

Inflammation can also affect organs as part of an autoimmune disorder. For example: Inflammation of the heart (myocarditis) , which may cause shortness of breath or leg swelling; Inflammation of the small tubes that transport air to the lungs, which may cause an asthma attack; Inflammation of the kidneys (nephritis) , which may cause high blood pressure or kidney failure; and Inflammation of the large intestine (colitis) may cause cramps and diarrhoea.

Pain may not be a primary symptom of the inflammatory disease, since many organs do not have many pain- sensitive nerves. Treatment of organ inflammation is directed at the cause of inflammation whenever possible.

There are a number of treatment options for inflammatory diseases including medications, rest and exercise, and surgery to correct joint damage. The type of treatment prescribed will depend on several factors including the type of disease, the person's age, type of medications he or she is taking, overall health, and medical history and severity of symptoms.

There are many medications available to decrease joint pain, swelling and inflammation and prevent or minimize the progression of the inflammatory disease. The medications include: Non-steroidal anti-inflammatory drugs, corticosteroids and anti-malarial medications.

Blockade of the TRPAl channel has been shown to relieve cold hyperalgesia. This is an enhanced sensitivity to pain, and is a well-documented symptom of inflammatory and neuropathic pain and as such it is thought that agents that are able to blockade the TRPAl channels could be useful treatments for neuropathic pain and inflammation.

Blockade of the TRPAl channels also results in vasodilation, therefore agents that are able to produce such an effect might also be useful as vasodilators. Vasodilators are often used to treat conditions such as hypotension or blood clots when there is a requirement to dilate the blood vessels.

Cannabinoids are a group of chemicals known to activate cannabinoid receptors in cells. These chemicals, which are found in cannabis plants, are also produced endogenously in humans and other animals, these are

termed endocannabinoids . Synthetic cannabinoids are chemicals with similar structures to plant cannabinoids or endocannabinoids.

Plant cannabinoids can also be isolated such that they are "essentially pure" compounds. These isolated cannabinoids are essentially free of the other naturally occurring compounds, such as, other minor cannabinoids and molecules such as terpenes. Essentially pure compounds have a degree of purity up to at least 95% by total weight.

The cannabinoid tetrahydrocannabinol (THC) has been shown to activate the TRPAl channels (Jordt et al. Nature 2004) by acting in a similar manner to mustard oil and cinnamaldehyde . The type of THC used in this study was synthetic THC. Synthetic THC such as dronabinol can cause many side effects in users. Such side effects include: palpitations, tachycardia, facial flush, abdominal pain, nausea, vomiting, amnesia, anxiety/nervousness, ataxia, confusion, depersonalization, dizziness, euphoria, hallucination, paranoia, somnolence, hypotension, diarrhoea, depression, nightmares and vision difficulties .

Surprisingly the applicants have found that the administration of cannabinoid-containing plant extracts, are efficacious in the blockade of TRPVl, TRPM3 and TRPAl channels. In particular cannabinoid-containing plant extracts comprising as a predominant cannabinoid either tetrahydrocannabinol (THC) , tetrahydrocannabinolic acid (THCA) , cannabidiol (CBD) , cannabidiolic acid (CBDA) , cannabigerol (CBG) or cannabichromene (CBC) were particularly efficacious.

The term "cannabinoid-containing plant extract" is taken herein to refer to one or more plant extracts from the cannabis plant. A cannabinoid-containing plant extract contains in addition to one or more other cannabinoids, one or more non-cannabinoid components which are co- extracted with the cannabinoids from the plant material. The degree of purity obtained and the respective ranges of additional cannabinoids in the cannabinoid-containing plant extract will vary according to the starting plant material and the extraction methodology used.

Cannabinoid-containing plant extracts may be obtained by various means of extraction of cannabis plant material. Such means include but are not limited to: supercritical or subcritical extraction with CO ₂ , extraction with hot gas and extraction with solvents.

SUMMARY OF INVENTION

According to the first aspect of the present invention there is provided the use of one or more cannabinoid- containing plant extracts in the manufacture of a pharmaceutical formulation for use in the prevention or treatment of diseases or conditions that are alleviated by blockade of one or more types of TRP channel.

Preferably the subset of TRP channel that is blockaded is the TRPA channel.

More preferably the TRPA channel is the TRPAl channel.

Preferably the diseases or conditions to be prevented or treated include: neuropathic pain, inflammation or vasoconstriction.

Alternatively the subset of TRP channel that is blockaded is the TRPM channel.

Preferably the TRPM channel is the TRPM8 channel.

Preferably the diseases or conditions to be prevented or treated are cancer.

More preferably the cancer is taken from the group: cancer of the prostate, cancer of the breast, cancer of the colon, cancer of the lung or cancer of the skin.

Alternatively the subset of TRP channel that is blockaded is the TRPV channel.

More preferably the TRPV channel is the TRPVl channel.

Preferably the diseases or conditions to be prevented or treated include: neuropathic pain, inflammation or vasoconstriction.

Preferably the cannabinoid-containing plant extract comprises one or more of: tetrahydrocannabinol (THC); cannabidiol (CBD) , cannabigerol (CBG) ; cannabichromene

(CBC); tetrahydrocannabidivarin (THCV); tetrahydrocannabinolic acid (THCA) ; cannabidivarin (CBDV) and cannabidiolic acid (CBDA) .

The cannabinoid-containing plant extract may be extracted from a cannabis plant using the subcritical CO ₂ extraction

technique as described in the applicants granted United Kingdom patent GB2391865.

Another cannabis plant extraction technique is extraction with hot gas as described in the applicants granted United Kingdom patent GB2376464.

Preferably the one or more cannabinoid-containing plant extract comprises tetrahydrocannabinol (THC) as a predominant cannabinoid.

Preferably the one or more cannabinoid-containing plant extract comprises cannabidiol (CBD) as a predominant cannabinoid.

Preferably the one or more cannabinoid-containing plant extract comprises cannabichromene (CBC) as a predominant cannabinoid.

Preferably the one or more cannabinoid-containing plant extract comprises tetrahydrocannabinolic acid (THCA) as a predominant cannabinoid.

Preferably the one or more cannabinoid-containing plant extract comprises cannabidiolic acid (CBDA) as a predominant cannabinoid.

Alternatively the one or more cannabinoid-containing plant extract may comprise a combination of a CBD- containing plant extract and a THC-containing plant extract.

Preferably the cannabinoids are present as a cannabis based medicine extract (CBME) .

A CBME is a plant extract from the cannabis plant and as such depending on the extraction technique used will comprise all of the "naturally extracted" cannabis plant components.

Alternatively the cannabinoid-containing plant extract is isolated or substantially pure.

Isolated or substantially pure cannabinoids will be substantially free of other non-target cannabinoids and other non-cannabinoid components such as terpenes. The isolated or substantially pure cannabinoids may be of natural i.e. plant origin or they may be synthetically produced compounds.

The process disclosed in the applicants granted United Kingdom patent GB2393721 describes a process for preparing substantially pure cannabinoids.

"Substantially pure" is defined herein as preparations of cannabinoid compounds or derivatives thereof having a chromatographic purity of greater than 95%, preferably greater than 96%, more preferably greater than 97%, more preferably greater than 98%, more preferably greater than 99% and most preferably greater than 99.5%, as determined by area normalisation of an HPLC profile.

In one embodiment the cannabinoid-containing plant extract is packaged for delivery in a titratable dosage form.

The term "titrate" is defined as meaning that the patient is provided with a medication that is in such a form that smaller doses than the unit dose can be taken.

A "unit dose" is herein defined as a maximum dose of medication that can be taken at any one time or within a specified dosage period such as 3 hours.

Titration of doses is beneficial to the patient as they are able to increase the dose incrementally until the drug is efficacious. It is understandable that not all patients will require exactly the same dose of medication, for example patients of a larger build or faster metabolism may require a higher dose than that required by a patient that is of a smaller build. Different patients may also present with different degrees of complaints and as such may require larger or smaller doses in order to treat the complaint effectively. The benefits of a titratable dosage form over a standard dosage form, which would have to be split into a partial dose, are therefore evident.

Unit dose ranges for the cannabinoid-containing plant extract may be determined by reference to the cannabinoid content which is preferably in the range of between 5 and lOOmg of the total cannabinoids .

Preferably the pharmaceutical formulations are packaged for delivery such that delivery is targeted to an area selected from one or more of the following: sublingual; buccal; oral; rectal; nasal; parenteral and via the pulmonary system.

More preferably the pharmaceutical formulations are in the form selected from one or more of the following: gel; gel spray; tablet; liquid; capsule, by injection and for vaporisation.

Additionally the pharmaceutical formulation further comprises one or more carrier solvents. Preferably the carrier solvents are ethanol and/or propylene glycol. More preferably the ratio of ethanol to propylene glycol is between 4:1 and 1:4. More preferably still the ratio is substantially 1:1.

The cannabinoid-containing plant extracts are used in the manufacture of a pharmaceutical formulation for use in the prevention or treatment of diseases or conditions that are alleviated by blockade of the TRP channels.

Preferably the diseases or conditions that are alleviated by blockade of the TRP channels are taken from the group: neuropathic pain; inflammation and vasoconstriction.

Preferably the neuropathic pain alleviated by blockade of the TRP channel is taken from the group: multiple sclerosis; diabetes; back injury; amputation; spinal surgery; HIV infection; shingles; alcoholism and facial nerve problems .

Preferably the inflammation alleviated by blockade of the TRP channel is taken from the group: inflammatory disease; rheumatoid arthritis; autoimmune disorders such as myocarditis; inflammation of the small tubes that transport air to the lungs; nephritis and colitis.

Preferably the vasoconstriction alleviated by blockade of the TRP channel is taken from the group: high blood pressure and blood clots.

In a further aspect of the present invention, there is provided a method of preventing or treating diseases or conditions that are alleviated by blockade of one or more types of TRP channel, comprising administering a pharmaceutically effective amount of a cannabinoid- containing plant extract to a subject in need thereof.

The discussion of the first aspect of the invention applies mutatis mutandis to this aspect of the invention. As discussed in further detail herein, by "treatment" is meant at least improvement, preferably cure of the condition in question.

Certain aspects of this invention are further described, by way of example only.

SPECIFIC DESCRIPTION

The applicants have conducted experiments using cannabinoid-containing plant extracts on rat recombinant TRPVl, TRPM8 channels and TRPAl (also known as ANKTMl) channels, both were stably expressed in HEK293 cells. The TRPAl channel is the receptor for mustard oil isothiocyanates and other plant natural products such as cinnamaldehyde. The TRPM8 channel is the receptor fro menthol and icilin. The intracellular Ca ²⁺ concentration was determined before and after the addition of various concentrations of test compounds.

Surprisingly it was discovered that the cannabinoid- containing plant extracts were able to blockade the channels tested and produced estimated EC ₅₀ values in the 100 nM range, and below.

Example 1:

The effects of cannabinoid-containing plant extracts on intracellular Ca ²⁺ concentration

Materials and methods

Compounds

For the experiments with the TRPAl channels allyl isothiocyanate (mustard oil) and cinnamaldehyde were used as positive controls to which the values obtained from cannabinoid-containing plant extracts were compared to. For the experiments with the TRPM8 channels menthol and iciϊin were used to activate the channels. For the experiments with the TRPVl channels ionomycin was used to activate the channels. The cannabinoid-containing plant extracts were produced from cannabis plants using the subcritical CO ₂ extraction technique as described in the applicants granted United Kingdom patent GB2391865. The cannabinoids were then purified further using the method disclosed in the applicants granted United Kingdom patent GB2393721 to produce substantially pure cannabinoids. Un- purified plant extracts of THC and CBD were also tested in the experiments with the TRPAl channels.

Permanent transfection of HEK-293 cells with rat TRPAl, TRPVl or TRPM8 cDNA

HEK293 (human embryonic kidney) cells were plated on 100 mm diameter Petri dishes and transfected at about 80%

confluence with Lipofectamine 2000 (Invitrogen) using a plasmid containing the rat TRPAl, TRPVl or TRPM8 cDNA, according to the manufacturer's protocol. A stably transfected clone was selected by Geneticin G-418 (Invitrogen) 600 μg/ml. Stable transfection was checked by quantitative real time-PCR (RT-PCR) . PCR analysis on the DNA from TRPA1/TRPV1/TRPM8-HEK293 cells demonstrated full integration of gene into HEK293 cell genome (not shown) .

Experiments in HEK-293 cells over-expressing the rat TRPAl, TRPVl or TRPM8 channel

TRPA/TRPV1/TRPM8-HEK-293 cells were plated on 100 mm diameter Petri dishes and after 3 days loaded for 1 hour at room temperature with the cytoplasmic calcium indicator Fluo4-AM (4 μM, Molecular Probes) dissolved in Tyrode' buffer (NaCl 145 mM; KCl 2.5 mM; CaCl ₂ 1.5 mM; MgCl ₂ 1.2 mM; D-Glucose 10 mM; HEPES 10 mM pH 7.4) containing Pluronic (0.02%, Molecular Probes). The cells were washed twice in Tyrode buffer, resuspended and transferred to the quartz cuvette of the spectrofluorimeter (Perkin -Elmer LS 50B) (λ excitation = 488 nm; λ emission = 516 nm) . Intracellular Ca ²⁺ concentration was determined before and after the addition of various concentrations of test compounds. EC ₅₀ values were determined as the concentration of test substances required to produce half-maximal increases in intracellular Ca ²⁺ concentration. Curve fitting and parameter estimation was performed with Graph Pad Prism ^® . The same compounds were tested also on non-transfected HEK 293 cells.

Results

As can be observed by looking at the values in Tables 1 and 2 derived from the experiments, the log EC50% values for the cannabinoids tested demonstrate that the cannabinoids tested were able to produce a blockade of the TRP channels.

Table 1: TRPAl channel

The values obtained for the cannabinoids were highly comparable to that of mustard oil and cinnamaldehyde, and their potency can be ranked as follows: THC extract > CBC > CBD > THC > THCA > CBD extract > CBDA > mustard oil > cinnamaldehyde. In particular, THC extract, CBC and CBD exhibited EC ₅₀ values in the 60-100 nM range of concentrations. These data suggest that TRPAl might be one of the molecular targets underlying some of the pharmacological actions of phytocannabinoids .

These data are significant, as at the present time there are few useful treatment options for patients suffering form neuropathic pain, inflammation or vasoconstriction and the use of cannabinoids in the production of a pharmaceutical formulation that could be used to treat such conditions would prove useful.

Table 2: TRPM8 channel blockade

The table above details the potency of the cannabinoids at blockade of the TRPM8 channel. The values obtained demonstrate that the cannabinoids were all effective at blockade and as such TRPM8 antagonists might provide new therapeutic tools for the treatment of cancers where TRPM8 activity is essential the cancer cells survival.

It has previously been shown that cannabinoid-containing plant extracts can be used either alone or in combination to usefully treat various diseases and conditions. These data presented herein provide evidence for the use of cannabinoid-containing plant extracts for the treatment

of diseases and conditions that are alleviated by blockade of the TRP channels. Herein it has been demonstrated that all of the cannabinoids tested produced an activation of the TRPAl channels and as such could be useful in the prevention or treatment of diseases or conditions that are alleviated by activation of the TRPAl channels. It has also been demonstrated that the cannabinoids tested are able to antagonize the TRPM8 channels and as such are potentially of use in the prevention or treatment of diseases or conditions that are alleviated by antagonism of the TRPM8 channels.

Table 3: TRPVl channel blockade

The table above details the potency of the cannabinoids at blockade of the TRPVl channel. The values obtained demonstrate that the cannabinoids were all effective at blockade and as such TRPVl antagonists might provide new

therapeutic tools for the treatment of patients suffering from neuropathic pain, inflammation or vasoconstriction.