Cannabis Research A-Z

Article Date: 11 Nov 2005 - 0:00 PDT

The first study to use a cannabis-based medicine (CBM) for treating rheumatoid arthritis has found that it has a significant effect on easing pain and on suppressing the disease.

Writing in the medical journal Rheumatology [1], the researchers say that although the differences were small and variable in the group of 56 patients they studied, the results are statistically significant and a larger trial is needed to investigate in more detail the effects of CBM on the disease which affects approximately 600,000 people in the UK (1 in 100 of the population).[2]

There is anecdotal evidence that cannabis can provide pain relief for people with rheumatoid arthritis (RA), and in a recent survey 155 (16%) of 947 people who obtained cannabis on the black market for medicinal reasons said they did so to obtain relief from symptoms of RA. However, this study in Rheumatology journal, led by David Blake, Professor of Bone and Joint Medicine at the Royal National Hospital for Rheumatic Diseases (RNHRD), Bath, and the University of Bath, UK, is the first randomised controlled trial to investigate the effect of a CBM on RA. It is published online today (Wednesday 9 November).

In the double-blind trial, the researchers randomised 31 patients to receive the CBM and 27 the placebo. The CBM (brand name: Sativex) was in the form of an easy-to-use mouth spray that patients could administer themselves up to a maximum of six doses a day. The CBM consisted of a blend of whole plant extracts, standardised for content, that delivered approximately equal amounts of two key therapeutic constituents from the cannabis plant: delta-9-Tetrahydrocannabinol (THC) and cannabidiol (CBD). Mouse studies have shown that THC and CBD have anti-inflammatory effects, and that CBD blocked progression of RA and produced improvements in symptoms.

Dr Ronald Jubb, Consultant Rheumatologist, at the University Hospital Birmingham NHS Foundation Trust, UK, said: "Patients had a baseline assessment at the beginning of the trial and then were randomised to receive either the CBM or placebo. Patients only took the doses in the evening in order to minimise possible intoxication-type reactions. The starting dose was one actuation within half an hour of retiring, and this was increased by one actuation every two days to a maximum of six doses according to individual response over a period of two weeks. Stable dosing was then maintained for a further three weeks."

The researchers found that in comparison with the placebo, patients who had taken the CBM had statistically significant improvements in pain on movement, pain at rest, quality of sleep, inflammation (measured by a Disease Activity Score involving 28 joints - DAS 28) and intensity of pain (measured by the Short-Form McGill Pain Questionnaire SF-MPQ).

For instance, on a score of 0-10 where 0 is no pain, CBM patients on average moved from 7 to 4.8 for pain on movement (placebo patients moved from 6.7 to 5.3), 5.3 to 3.1 (placebo 5.3 to 4.1) for pain at rest, and 5.7 to 3.4 (placebo 5.8 to 4.6) for quality of sleep. On the DAS 28 score of 0-10, the CBM patients moved from 5.9 to 5 (placebo 6 to 5.9), and on the SF-MPQ score of 0-100 for intensity of pain at present, the CBM patients moved from 48 to 33, while the placebo patients remained unchanged at 50.

Adverse side effects were mostly mild or moderate (e.g. dizziness, light-headedness, dry mouth, nausea). Of the eight patients who experienced mild dizziness, in four patients this occurred during the initial two-week period when they were gradually increasing the doses, and two occurred two days after this initial period, so these were probably due to patients getting used to the correct dose. No patients taking the CBM had to withdraw from the trial due to adverse side effects, but three did from the placebo group.

Dr Philip Robson, Senior Research Fellow and Consultant Psychiatrist at the Oxford University Department of Psychiatry and Director of the Cannabinoid Research Institute within GW Pharmaceuticals (the manufacturer of Sativex), explained: "Withdrawals from the placebo group were probably due to a psychological effect, a spontaneous occurrence, or a reaction with another medicine."

Dr Jubb said: "The results from the first controlled study of CBM in rheumatoid arthritis are encouraging, with overall improvements in pain on movement and at rest, improvement in the quality of sleep and improvement in the overall condition of the patients' arthritis. Whilst the differences are small and variable across the patient group, they represent benefits of clinical relevance and indicate the need for more detailed investigation through larger trials to see exactly where CBM could be best used with minimum side effects."

If further trials are run, researchers will probably extend the dosing period over the full 24-hour period. Dr Robson said: "The beneficial effects in this study occurred in the context of a dosing regime restricted to evening dosing in order to minimise any possible intoxication-type reactions. However, 24-hour dosing with Sativex, using a self-titration regime, in trials for multiple sclerosis resulted in only minimal intoxication scores."

He continued: "The element that can cause the 'high' in cannabis - THC - also has valuable pharmacological activity. It is thought to be an essential therapeutic component and therefore it can't be removed from the medicine. However, the method of giving the doses, via the mouth spray, and the principle of self-titration, where each patient gradually determined their own optimal dose level up to a maximum of six doses a day, minimised the risk of intoxication."

Dr Robson said that fears that the CBM could be abused by patients hoping to get a "high" were probably unfounded. "It seems that in practice this is a very rare event. More than 1,000-patient years of treatment with Sativex in clinical trials have been accumulated and to date there has not been a single documented case of abuse. The fact is that the motivation of medicinal users of cannabis-based medicine is entirely different from recreational users: the former simply want symptom relief and the ability to go about their normal lives, and for them intoxication would be a distinct disadvantage; for the latter, smoking marijuana is infinitely more intoxicating than Sativex and is still easily available."

[1] Preliminary assessment of the efficacy, tolerability and safety of a cannabis-based medicine (Sativex) in the treatment of pain caused by rheumatoid arthritis. Rheumatology Advance Access published on November 9, 2005.
doi:10.1093/rheumatology/kei183

[2] Rheumatoid arthritis affects three times as many women as men. Prevalence in the UK is approximately 0.5% in men and 1.8% in women, increasing after the age of 64 to 2% in men and 5% in women. There are many more people with less severe forms of RA that do not meet the diagnostic criteria for definite or classical disease.

Emma Mason
wordmason@mac.com
Oxford University Press
http://www3.oup.co.uk/jnls

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Abstract:

Habitual cannabis use has been shown to affect the human immune system, and recent advances in endocannabinoid research provide a basis for understanding these immunomodulatory effects.

Cell-based experiments or in vivo animal testing suggest that regulation of the endocannabinoid circuitry can impact almost every major function associated with the immune system.

These studies were assisted by the development of numerous novel molecules that exert their biological effects through the endocannabinoid system. Several of these compounds were tested for their effects on immune function, and the results suggest therapeutic opportunities for a variety of inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, allergic asthma, and autoimmune diabetes through modulation of the endocannabinoid system.

Publication date: 2006-07-01

1. . R. Blake,
2. P. Robson 
3. M. Ho 
4. R. W. Jubb and
5. C. S. McCabe

Author Affiliations

1. Royal National Hospital for Rheumatic Diseases, Bath, ¹Cannabinoid Research Institute, Oxford Science Park, Oxford, ²Department of Rheumatology, Northampton General Hospital, Northampton and ³Department of Rheumatology, Selly Oak Hospital, Birmingham, UK.

1. Correspondence to: D. R. Blake, Royal National Hospital for Rheumatic Diseases, Upper Borough Walls, Bath BA1 1RL, UK. E-mail: David.Blake@rnhrd-tr.swest.nhs.uk

• Received July 6, 2005.
• Revision received October 5, 2005.

• © The Author 2005. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

J Cell Biochem. 2007 Jan 1;100(1):184-90.

Johnson DR, Stebulis JA, Rossetti RG, Burstein SH, Zurier RB.

Department of Medicine, Division of Rheumatology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.

Eur J Pharmacol 2007 Jun 5.

Cannabinoid CB(2) receptors have been implicated in antinociception in animal models of both acute and chronic pain.

We evaluated the role both cannabinoid CB(1) and CB(2) receptors in mechanonociception in non-arthritic and arthritic rats. The antinociceptive effect of Delta(9)-tetrahydrocannabinol (Delta(9)THC) was determined in rats following administration of the cannabinoid CB(1) receptor-selective antagonist, SR141716A, the cannabinoid CB(2) receptor-selective antagonist, SR144528, or vehicle.

Male Sprague-Dawley rats were rendered arthritic using Freund's complete adjuvant and tested for mechanical hyperalgesia in the paw-pressure test.

Arthritic rats had a baseline paw-pressure of 83 +/- 3.6g versus a paw-pressure of 177 +/- 6.42g in non-arthritic rats. SR144528 or SR141716A (various doses mg/kg; i.p.) or 1:1:18 (ethanol:emulphor:saline) vehicle were injected 1 h prior to Delta(9)THC (4mg/kg; i.p) or 1:1:18 vehicle and antinociception determined 30min post Delta(9)THC. AD(50)'s for both antagonists were calculated with 95% confidence limits.

In addition, midbrain and spinal cord were removed for determination of cannabinoid CB(1) and CB(2) receptor protein density in the rats. SR144528 significantly attenuated the antinociceptive effect of Delta(9)THC in the arthritic rats [AD(50) = 3.3 (2.7-4) mg/kg], but not in the non-arthritic rats at a dose of 10/mg/kg. SR141716A significantly attenuated Delta(9)THC-induced antinociception in both the non-arthritic [AD(50) = 1.4 (0.8-2) mg/kg] and arthritic rat [AD(50) = 2.6 (1.8-3.1) mg/kg]. SR141716A or SR144528 alone did not result in a hyperalgesic effect as compared to vehicle.

Our results indicate that the cannabinoid CB(2) receptor plays a critical role in cannabinoid-mediated antinociception, particularly in models of chronic inflammatory pain.

Article Date: 11 Nov 2005

 cannabis-based medicine (CBM) for treating rheumatoid arthritis has found that it has a significant effect on easing pain and on suppressing the disease.

Writing in the medical journal Rheumatology [1], the researchers say that although the differences were small and variable in the group of 56 patients they studied, the results are statistically significant and a larger trial is needed to investigate in more detail the effects of CBM on the disease which affects approximately 600,000 people in the UK (1 in 100 of the population).[2]

There is anecdotal evidence that cannabis can provide pain relief for people with rheumatoid arthritis (RA), and in a recent survey 155 (16%) of 947 people who obtained cannabis on the black market for medicinal reasons said they did so to obtain relief from symptoms of RA. However, this study in Rheumatology journal, led by David Blake, Professor of Bone and Joint Medicine at the Royal National Hospital for Rheumatic Diseases (RNHRD), Bath, and the University of Bath, UK, is the first randomised controlled trial to investigate the effect of a CBM on RA. It is published online today (Wednesday 9 November).

In the double-blind trial, the researchers randomised 31 patients to receive the CBM and 27 the placebo. The CBM (brand name: Sativex) was in the form of an easy-to-use mouth spray that patients could administer themselves up to a maximum of six doses a day. The CBM consisted of a blend of whole plant extracts, standardised for content, that delivered approximately equal amounts of two key therapeutic constituents from the cannabis plant: delta-9-Tetrahydrocannabinol (THC) and cannabidiol (CBD). Mouse studies have shown that THC and CBD have anti-inflammatory effects, and that CBD blocked progression of RA and produced improvements in symptoms.

Dr Ronald Jubb, Consultant Rheumatologist, at the University Hospital Birmingham NHS Foundation Trust, UK, said: "Patients had a baseline assessment at the beginning of the trial and then were randomised to receive either the CBM or placebo. Patients only took the doses in the evening in order to minimise possible intoxication-type reactions. The starting dose was one actuation within half an hour of retiring, and this was increased by one actuation every two days to a maximum of six doses according to individual response over a period of two weeks. Stable dosing was then maintained for a further three weeks."

The researchers found that in comparison with the placebo, patients who had taken the CBM had statistically significant improvements in pain on movement, pain at rest, quality of sleep, inflammation (measured by a Disease Activity Score involving 28 joints - DAS 28) and intensity of pain (measured by the Short-Form McGill Pain Questionnaire SF-MPQ).

For instance, on a score of 0-10 where 0 is no pain, CBM patients on average moved from 7 to 4.8 for pain on movement (placebo patients moved from 6.7 to 5.3), 5.3 to 3.1 (placebo 5.3 to 4.1) for pain at rest, and 5.7 to 3.4 (placebo 5.8 to 4.6) for quality of sleep. On the DAS 28 score of 0-10, the CBM patients moved from 5.9 to 5 (placebo 6 to 5.9), and on the SF-MPQ score of 0-100 for intensity of pain at present, the CBM patients moved from 48 to 33, while the placebo patients remained unchanged at 50.

Adverse side effects were mostly mild or moderate (e.g. dizziness, light-headedness, dry mouth, nausea). Of the eight patients who experienced mild dizziness, in four patients this occurred during the initial two-week period when they were gradually increasing the doses, and two occurred two days after this initial period, so these were probably due to patients getting used to the correct dose. No patients taking the CBM had to withdraw from the trial due to adverse side effects, but three did from the placebo group.

Dr Philip Robson, Senior Research Fellow and Consultant Psychiatrist at the Oxford University Department of Psychiatry and Director of the Cannabinoid Research Institute within GW Pharmaceuticals (the manufacturer of Sativex), explained: "Withdrawals from the placebo group were probably due to a psychological effect, a spontaneous occurrence, or a reaction with another medicine."

Dr Jubb said: "The results from the first controlled study of CBM in rheumatoid arthritis are encouraging, with overall improvements in pain on movement and at rest, improvement in the quality of sleep and improvement in the overall condition of the patients' arthritis. Whilst the differences are small and variable across the patient group, they represent benefits of clinical relevance and indicate the need for more detailed investigation through larger trials to see exactly where CBM could be best used with minimum side effects."

If further trials are run, researchers will probably extend the dosing period over the full 24-hour period. Dr Robson said: "The beneficial effects in this study occurred in the context of a dosing regime restricted to evening dosing in order to minimise any possible intoxication-type reactions. However, 24-hour dosing with Sativex, using a self-titration regime, in trials for multiple sclerosis resulted in only minimal intoxication scores."

He continued: "The element that can cause the 'high' in cannabis - THC - also has valuable pharmacological activity. It is thought to be an essential therapeutic component and therefore it can't be removed from the medicine. However, the method of giving the doses, via the mouth spray, and the principle of self-titration, where each patient gradually determined their own optimal dose level up to a maximum of six doses a day, minimised the risk of intoxication."

Dr Robson said that fears that the CBM could be abused by patients hoping to get a "high" were probably unfounded. "It seems that in practice this is a very rare event. More than 1,000-patient years of treatment with Sativex in clinical trials have been accumulated and to date there has not been a single documented case of abuse. The fact is that the motivation of medicinal users of cannabis-based medicine is entirely different from recreational users: the former simply want symptom relief and the ability to go about their normal lives, and for them intoxication would be a distinct disadvantage; for the latter, smoking marijuana is infinitely more intoxicating than Sativex and is still easily available."

[1] Preliminary assessment of the efficacy, tolerability and safety of a cannabis-based medicine (Sativex) in the treatment of pain caused by rheumatoid arthritis. Rheumatology Advance Access published on November 9, 2005.
doi:10.1093/rheumatology/kei183

[2] Rheumatoid arthritis affects three times as many women as men. Prevalence in the UK is approximately 0.5% in men and 1.8% in women, increasing after the age of 64 to 2% in men and 5% in women. There are many more people with less severe forms of RA that do not meet the diagnostic criteria for definite or classical disease.

Emma Mason
wordmason@mac.com
Oxford University Press
http://www3.oup.co.uk/jnls

Nov. 8, 2005 - A spray containing two chemicals extracted from marijuana improved pain and sleep in rheumatoid arthritis (RA) patients, British researchers report.

The study, which appears in Rheumatology, was small, brief, and likely the first of its kind, note the researchers. They write that the "encouraging" results warrant larger, longer studies.

The spray, called Sativex, is made by GW Pharmaceuticals, the British drug company that funded the study. It is sprayed into the mouth and the medication is absorbed under the tongue or the inside part of the cheek.

One of the researchers is GW Pharmaceuticals' medical director. Two others disclose having received honoraria from GW Pharmaceuticals.

Spray Study

The study included 58 RA patients. They had no history of psychiatric disorders, substance misuse, epilepsy, or severe heart, kidney, or liver problems.

First, patients rated their pain at rest, during movement, and first thing in the morning. They also rated their quality of sleep.

Next, the patients were given one of two sprays to use every evening for about a month. Sativex was one of those sprays. The other was an empty spray (placebo).

Sativex was given to 31 patients. The other 27 patients got the placebo. No one knew which patients had gotten Sativex.

Sativex contains THC (delta-9-tetrahydrocannabinol) and CBD (cannabidiol). Those are key therapeutic compounds in cannabis that have been shown by other studies to produce effects on pain and inflammation, write the researchers.

They included rheumatologist David R. Blake of the Royal National Hospital for Rheumatic Diseases in Bath, England.

Study's Results

Compared with the placebo group, patients taking Sativex had notable improvements in pain (including pain during movement and pain at rest), sleep quality, and RA disease activity, the researchers report.

Morning pain didn't change much but was "surprisingly low" to begin with, write Blake and colleagues.

The sprays were only used in the evening to minimize any intoxication. The most common side effects with Sativex were dizziness (eight patients, or 26% of the Sativex group), dry mouth (four patients, or 13%), and lightheadedness (three patients, or 10% of those taking Sativex).

A compound found in cannabis as well as in herbs such as basil and oregano could help to treat inflammatory bowel diseases and arthritis, Swiss scientists believe. 

(E)-beta-caryophyllene (BCP) is an aromatic sesquiterpene that has used for many years as a food additive because of its peppery flavour. The researchers now say that it interacts selectively with one of two cannabinoid receptors, CB₂, blocking the chemical signals that lead to inflammation without triggering cannabis's mood-altering effects.

Many cannabinoids bind to the CB₂ receptor, but few target it selectively. Most also interact with CB₁, which is responsible for cannabis' psychoactive properties. CB₁ is found in brain tissue, whereas CB₂ is found only in cells elsewhere in the body.

The compound is the only product identified in nature that activates CB₂ selectively. 'There are many compounds that have been designed synthetically that are CB₂ selective, but they are made in the lab,' says Jürg Gertsch, who led the study at the Swiss Federal Institute of Technology.

According to Gretsch, BCP is potent enough to have an impact at normal dietary levels. Herbs such as basil and oregano contain large amounts of the compound, he says, suggesting that the Mediterranean diet may protect against Crohn's disease and other inflammatory bowel diseases. 'If somebody eats a lot of herbs containing essential oils then it's possible they could get enough to reach a therapeutic dose,' Gertsch says.

The team screened a whole library of natural products for cannabinoid activity, which led finally to fractionation of Cannibis sativa  essential oil and identification of BCP as the active component. They then tested the compound by using it to treat mice with swollen paws and found that in around 70 per cent of cases, small doses of BCP were enough to make the inflammation subside. BCP may also account for the for the anti-inflammatory properties of copaiba oil, an essential oil prescribed by doctors and healers in Brazil, which contains large quantities of the compound. 

Birgit Kraft, who studies the therapeutic effects of marijuana at the Medical University of Vienna, says selective CB₂ agonists are keenly sought after by clinicians, as they may be candidates for treatment of rheumatoid arthritis, as well as bowel disease.

Hayley Birch

INFLAMMATION, Vol 12, No. 4, 1988 

E.A. Formukong, A.T. Evans, and F.J. Evans

Department of Pharmacognosy, The School of Pharmacy University of London,

29-39 Brunswick Square London, WC11N 1AX, England 

Abstract---Two extracts of Cannabis sativa herb, one being cannabinoid--free (ethanol) and the other containing the cannabinoids (petroleum), were shown to inhibit PBQ- induced writhing in mouse when given orally and also to antagonize tetradecanoylphorbol acetate (TPA) -induced erythema of mouse skin when applied topically. With the exception of cannabinol (CBN) and delta-1-tetrahydrocannabinol (delta-1-THC), the cannabinoids and olivetol (their biosynthetic precursor) demonstrated activity in the PBQ test exhibiting their maximal effect at doses of about 100 mcg/kg. Delta-1-THC only became maximally effective in doses of 10 mg/kg.

This higher dose corresponded to that which induced catalepsy and is indicative of a central action. CBN produce a 40% inhibition of PBQ-induced writhing. Cannabidiol (CBD) was the most effective of the cannabinoids at doses of 100 mcg/kg. Doses of cannabinoids that were effective in the analgesic test orally were used topically to antagonize TPA-induced erythema of skin. The fact that delta-1-THC and CBN were the least effective in this test suggests a structural relationship between analgesic activity and antiinflammatory activity among the cannabinoids related to their peripheral actions and separate from the central effects of delta-1-THC.  

INTRODUCTION  

Various preparations of Cannabis sativa have been employed for their medicinal effects, including antipyretic, antirheumatic, antiallergic, and analgesic purposes. Extracts of Cannabis have been shown to possess analgesic activity (2, 3), and delta-1-tetrahydrocannabinol (delta-1-THC), the psychoactive component of Cannabis has also been shown to possess this activity in various models. In addition, cannabinol (CBN) but not cannabidiol (CBD) was shown to exhibit analgesic activity in vivo.

It is possible that the antiinflammatory and antiasthmatic properties of this herb are mediated through effects on arachidonate metabolism. However, constituents of Cannabis are known to stimulate  and inhibit prostaglandin (PG) release by influencing enzymes of this pathway.

A cannabinoid or an extract of Cannabis with little or no central effects could be of use therapeutically. In this paper, we have examined the antiinflammatory potential of two extracts of Cannabis, pure cannabinoids and olivetol (a cannabinoid biosynthetic precursor) in two models of inflammation, in an attempt to separate on a structural basis the peripheral from the central action of these phenolic drugs.  

MATERIALS AND METHODS  

The folowing were used: aspirin (Sigma Chemical Co., Poole, Dorset.), tripotassium citrate (analytical grade), all cannabinoids except CBG (Sigma), and CBG (Makor Chemicals, Jerusalem, Israel).

Preparation of Drugs: PBQ Test. Cannabinoids and cannabis extracts were suspended in a 1% ethanolic solution containing 2.5% w/v Tween. Aspirin was dissolved in a 40 mg/ml solution of tripotassium citrate.

Phenyl Benzoquinone Writhing (PBQ) and Preparation of PBQ Solution. A 0.04% solution of PBQ was prepared immediately before use by dissolving PBQ in warm ethanol and diluting with water at 40 degrees C ( 15) bringing the ethanolic concentration to 5% (16). The bottle was stoppered, foil paper wrapped around it, and the solution maintained at 34 degrees C. Deterioration of the solution occurs if left exposed to light and air.

Administration of Drugs. Male CDI male (Charles River) weighing 18-20 g were starved overnight for the experiment. Animals were placed in a thermostatically controlled environment maintained at 34 degrees C. Mice were orally administered test drug 20 min before the intraperitoneal injection of PBQ (4 mg/kg). Five minutes after injection, a hand tally counter was used to record the number of stretching movements for each mouse in a 5-min period. Control animals were only administered the vehicle. Note less than five animals were used per dose.

Statistical Analysis. Results are expressed as mean percentage inhibition of control (+SEM) in the case of PBQ test. IC-50s were obtained from graphs relating probit percentage inhibition (ordinate) against log dose (abscissa). The IC-50 is that dose of drug which would inhibit PBQ-induced writhing by 50%.

Tetradecanoyl phorbol-acetate-induced (TPA) Erythema of Mouse Ear. In order to exclude the possibility of a central mechanism of action (see Discussion), compounds also were tested for their ability to inhibit TPA-induced erythema on mouse ears in 100% of the animals was chosen as the challenging dose for inhibition studies, measured 4 h after application.

Test drugs were dissolved in ethanol and 5 ul applied to the inner ear of the mouse 15 min before the application of 1 mcg TPA in 5 ul acetone. Only one dose of test dug was used for this experiment, 100 mcg/mcl ethanols, except trifluoperazine at 1 mg/5 ul. The other ear acted as a control.

The results were expressed as percentage inhibition, taken to mean the complete suppression of erythema in the test animals, as described in reference 19. 

RESULTS 

PBQ-Induced Writhing. CBD, CBG, olivetol, ethanolic extract, and petroleum spirit extract produced significant inhibition at doses up to 10 mg/kg (Figures 1-3). CBN was only marginally active (Table 1.)

Delta-1-THC was fully effective only at concentrations above 10 mg/kg Figure 2).

The ethanolic and petroleum extract, CBD, olivetol, CBG, and cannflavon were more potent than aspirin. The petroleum spirit extract was about four times more potent than the ethanolic extract, which was virtually equipotent with CBD. Cannflavon, isolated from the ethanolic extract was 14 times less potent than the ethanolic extract of the dried herb (Table 2).

There was a decline in response following the administration of doses greater than 0.1 mg/kg of some substances. This is most evident in the bell shaped dose-response curve of the petroleum spirit extract (Figure 1). The activity of the ethanolic extract and CBD was also found to decrease slightly at higher dose levels. (Figures 1 and 2)

TPA-Induced Erythema. In general, the ability of compounds to inhibit TPA-induced erythema correlated well with their potency in the PBQ-writhing test. Thus, CBN and delta-1-THC were the least active followed by CBG, CBD, and cannflavon. Again, the extracts were the most active (Table 3). Twenty-four hours after application, the ethanolic extract still produced 16% inhibition of TPA-induced erythema of the animals. All other substances were without activity after 24 h.

All substances were more active than trifluoperazine, 1 mg/5ul, a known phorbol ester antagonist both in vivo (19) and in vitro (20).  

DISCUSSION

The PBQ-induced writhing response is believed to be produced by the liberation of endogenous substance(s), notably metabolites of the arachidonic cascade (21, 22). However, the PBQ test is not specific for weak analgesics such as the nonsteroidal antiinflammatory drugs, as it also detects centrally active analgesics (16, 17). Therefore, in the elucidation of the action of the cannabinoids as inflammatory drugs, it was necessary to perform more than one test. In this case, peripheral rather than central action was confirmed in the mouse ear erythema assay.

TPA-induced erythema was inhibited by the extracts cannflavon, cannabinoids, and olivetol. The activity of TPA has been shown to be dependent upon PG release in mouse epidermis (23) and mouse peritoneal macrophages (24) possibly via the initial stimulation of protein kinase C (for a review see reference 25). It has also been shown that compounds that show moderate to very potent antiinflammatory potential in standard in vivo inflammation models will also inhibit TPA-induced edema of the mouse ear (26), and phorbol-ester-induced erythema (19).

It is possible that the cannabinoids and their extracts are inhibiting both PBQ-induced writhing and TPA-induced erythema by effects on arachidonate release and metabolism. Cannabinoids and olivetol have been shown to inhibit PG mobilization (11, 12) and synthesis (14). The noncannabinoid constituents of Cannabis, for example, cannflavon, have been shown to be mainly cyclooxygenase inhibitors (14). Cannabinoids, however, stimulate and inhibit phospholipase A2 (PLA2) activity (13), as well as inducing an inhibition of cyclooxygenase and lipoxygenase (14). The activity of Cannabis herb or resin is complex, in that activities can be demonstrated on at least three major enzymes of the arachidonate cascade.

The mechanism by which delta-1-THC inhibits PBQ-induced writhing may differ from that of the other substances. At concentrations greater than 10 mg/kg, delta-1-THC may be inhibiting PBQ-induced writhing by acting on central rather than peripheral functions. It is possible that prostaglandins modulate certain inhibitory pathways in the brain, bringing about an increase in the pain threshold. This dose of delta-1-THC is capable of bringing about the cataleptic effect (27), which is a standard test for central involvement. Central analgesics have higher efficacies than peripheral ones, and this may explain the effectiveness of delta-1-THC (Figure 2). The central involvement of delta-1-THC is perhaps the primary reason why delta-1-THC was recognized as an analgesic before other cannabinoids.

Our results suggest that the response of the ethanolic extract cannot be solely due to cannflavon. Other structurally related phenolic substances, known to be present in this complex extract, may account for the higher activity seen either due to cumulative or synergistic effects upon cyclooxygenase. The activity of the petroleum ether extract is likely to be largely due to the presence of CBD and CBN. GLC analysis of the extract has shown that this extract contained 14.13% CBD, 9.08% CBN, and 6.68% delta-1-THC (27). On the basis of our results, it is possible to separate the centrally active cannabinoid delta-1-THC from peripherally active compounds of the herbal extracts. An attempt has been made to differentiate them structurally (Table 3). It can be seen that the olivetolic nucleus together with a free C-5 hydroxyl group are structural requirements for peripheral effects, involving both cyclooxygenase and lipoxygenase inhibition (14). Substances possessing this structure possess antiinflammatory and analgesic activities without central hallucinogenic effects. Delta-1-THC and CBN, which are cyclized derivatives exhibiting no C-5 hydroxyl moiety, have little if any peripheral action.

The traditional use of Cannabis as an analgesic, antiasthmatic, and antirheumatic drug is well established. Our results would suggest that cultivation of Cannabis plants rich in CBD and other phenolic substances would be useful not only as fiber-producing plants but also for medicinal purposes in the treatment of certain inflammatory disorders. 

Acknowledgments----We are grateful to the Medicinal Research Council and the Government of Cameroon for financial support.  

REFERENCES 

1. Pars, H.G., R.J. Razdan, and J.F. Howes. 1977. Potential therapeutic agents derived from the cannabinoid nucleus. Adv. Drug. Res. 11.  

2. O.L. Davies, J. Raventos, and A.L. Walpole, 1946. A method for evaluation of analgesic activity using rats. Br. J. Pharmacol. 1: 255-264. 

3. Gill, E.W., W.D.M. Paton, and R.G. Pertwee, 1970. Preliminary experiments on the chemistry and pharmacology of Cannabis. Nature 228: 134-136. 

4. Dewey, W.L., L.S. Harris, and J.S. Kennedy, 1972. Some pharmacological and toxicological effects of 1-trans-delta-8- and 1-trans-delta-9-THC in laboratory rodents. Arch. Int. Pharmacodyn. 196: 133-145.  

5. Chesher, G.B., C.J. Dahl, M. Everingham, D.M. Jackson, H. Marchant-Williams, and G.A. Starmer, 1973. The effect of cannabinoids on intestinal mobility and their antinociceptive effect in mice. Br. J. Pharmacol. 49: 588-594. 

6. Buxbaum, D., E. Sanders-Bush, and D.H. Efron. 1969. Analgesic activity of tetrahydrocannabinol in the rat and mouse. Fed. Proc. 28: 735.  

7. Sanders, J., D.M. Jackson, and G.A. Starmer. 1979. Interactions among the cannabinoids in the antagonism of abdominal constriction response in the mouse. Psychopharmacology 61: 281-285.  

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9. Burstein, S., and S.A. Hunter. 1978. Prostaglandins and Cannabis VI. Release of arachidonic acid from HeLa cells by delta-1-THC and other cannabinoids. Biochem. Pharmacol. 27: 1275-1280.  

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 12. Barrett, M.L., D.Gordon, and F.J. Evans. 1985. Isolation from Cannabis sativa L of cannflavin: A novel inhibitor of prostaglandin production. Biochem. Pharmacol. 34: 2019-2024.  

13. Evans, A.T., E.A. Formukong, and F.J. Evans. 1987. Activation of phospholipase A2 by cannabinoids. Lack of correlation with CNS effects. FEBS Lett. 211: 119-122.

 14. Evans, A.T., E.A. Formukong, and F.J. Evans. 1987. Actions of Cannabis constituents on enzymes of prostaglandin synthesis: Antiinflammatory potential. Biochm. Pharmacol. 36: 2035-2037.  

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17. Hendershot, L.C., and J. Forsaith. 1959. Antagonism of the frequency of phenylbenzoquinone induced writhing in the mouse by weak analgesics and nonanalgesics. J. Pharmacol. Exp. Ther. 125: 237-240.  

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 19. Williamson, E.M., and F.J. Evans. 1981. Inhibition of erythema induced by proinflammatory esters of 12-deoxyphorbol. Acta Pharmacol. Toxicol. 481: 47-52.

 20. Williamson, E.M., J. Westwick, V.V. Kakkar, and F.J. Evans. 1981. Studies on the mechanism of action of 12-DOPP, a potent platelet aggregating phorbol ester. Biochem. Pharmacol. 30: 2691-2696.  

21. Collier, H.O.J., L.C. Dineen, C.A. Johnson, and C. Schneider. 1968. Abdominal constriction response and its suppression by analgesic drugs in the mouse. Br. J. Pharmacol. Chemother. 32: 295-310.   (22)

 23. Marks, F., G. Furstenberger, and E. Kownatzki, 1981. Prostaglandin E-mediated mitogenic stimulatin of mouse epidermis in vivo by divalent cation ionophore A23187 and by tumor promoter 12-O-tetradecanoyl phorbol-13-acetate. Cancer Res. 41: 696-702.  

24. Humes, J.L., S. Sadowski, M. Galavage, M. Goldenberg, E. Bubers, R.J. Bonney, and F.A. Kuehl, 1982. Evidence for two sources of arachidonic acid for oxidative metabolism by mouse peritoneal macrophages. J. Biol. Chem. 257: 1291-1594.

 25. Edwards, M.C., and F.J. Evans. 1987. Activity correlations in the phorbol ester series. Bot. J. Linn. Soc. 94: 231-246.  

26. Calson, R.P., L. O'Neill-David, J. Chary, and A.J. Lewis. 1985. Modulation of mouse ear edema by cyclooxygenase and lipoxygenase inhibitors and other pharmacological agents. Agents Actions 17: 197-204.  

27. Formukong, E.A., A.T. Evans, F.J. Evans. 1987. Inhibition of the cataleptic effect of delta-1-tetrahydrocannabinol by noncataleptic constituents of Cannabis sativa L. J. Pharm. Pharmacol. (in press).

Patent 6132762 Issued on October 17, 2000. Estimated Expiration Date:  May 5, 2018.

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More ...

Inventor

• Cristobal, Walter

Application

US Classes:

Examiners

Attorney, Agent or Firm

• Pangrle; Brian J., Ownbey; Nancy E., Peacock; Deborah A.

Foreign Patent References

• 2 274 588 GB. 08/09/1994

International Class

A61K 009/70

Abstract

Other References

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April 15, 2003 -- Cannabis may offer hope to people with autoimmune disorders such as lupus and rheumatoid arthritis. Cannabis seems to decrease inflammation in the body by suppressing certain parts of the immune system. Researchers are hoping this finding will lead to new treatments.

Previous studies have hinted at immune system abnormalities among cannabis users -- specifically, in the function of immune system cells called T lymphocytes and natural killer cells. While these cells help the body fight infections, no direct link with lowered immunity has yet been shown.

In this study, researchers tested the blood of 29 cannabis smokers -- 13 occasional users and 16 regular users (weekly or daily use). They compared the results with a group of 32 nonsmokers.

Again, researchers found that cannabis smokers had fewer immune-enhancing natural killer cells and lymphocytes, and higher levels of a protein that may promote tumor growth, called interleukin-10.

These changes can dampen the immune system's response to infection, increasing susceptibility to infections and promoting growth of tumors, states lead researcher Roberta Pacifici, PhD, with the Instituto Superiore di Sanita in Rome, Italy.

But researchers also say this finding could lead to new treatments for people with autoimmune disorders. Current treatments suppress the immune system -- thereby calming the abnormal immune response that plagues people with the conditions.

Cannabis lowers levels of the inflammation-promoting protein interleukin-2 and raises levels of the anti-inflammatory protein interleukin-10. Both of these findings could be of potential benefit for treating autoimmune disorders one day.

SOURCE: The Journal of the American Medical Association, April 16, 2003.

This article has been cited by other articles in PMC.

Introduction

Cannabis sativa is the source of at least 60 distinct alkaloids, which make up a group of compounds called cannabinoids. Cannabinoids can be categorized depending on whether they are synthetic, plant-derived (phytocannabinoids) or naturally produced in the body (endocannabinoids). The endocannabinoid system is believed to play an important role in health and disease where it can ameliorate the severity of disorders, such as pain, multiple sclerosis, schizophrenia and emesis (Di Marzo and Petrocellis, 2006; Pertwee, 2006a). Overproduction of endocannabinoids, however, could have a detrimental effect on the body, leading to problems such as obesity, infertility and inflammatory disorders. Thus, strategic control of the endocannabinoid system with selective agonists or antagonists may prove to have therapeutic value in the treatment of various diseases.

Two cannabinoid receptors have been cloned, viz. CB₁ and CB₂ (Matsuda et al., 1990; Munro et al., 1993). These G_i/o protein-coupled receptors are distributed throughout the body and are involved in the control of miscellaneous physiological processes, such as pain perception, appetite and vasoregulation. CB₁ receptors are predominantly found on nerve terminals in the central and peripheral nervous systems (Tsou et al., 1998; Pertwee, 1999a; Farquhar-Smith et al., 2000), although they have also been localized in non-neuronal tissues, such as the pituitary gland, spleen and immunocytes (Bouaboula et al., 1993). The primary location of CB₂ receptors is on immunocytes (Pertwee, 1999b, 2006a), but they have also been identified on peripheral nerves (Griffin et al., 1997) and in the central nervous system (Van Sickle et al., 2005). A plethora of cannabinoid ligands have been developed with fairly high selectivity for CB₁ and CB₂ receptors (Pertwee, 1999b, 2006b). Two selective CB₂ receptor agonists are JWH015 ((2-methyl-1-propyl-1H-indol-3-yl)-1-napthalenylmethanone) and JWH133 ((6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran). The K_i values for JWH015 are 383 and 13.8nM for the CB₁ and CB₂ receptors, respectively (Chin et al., 1999), while JWH133 has K_i of 677 and 3.4nM for the CB₁ and CB₂ receptors, respectively (Huffman et al., 1999). For antagonists, the aminoalkylindole AM630 (6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl) methanone) has been shown to be an effective blocker of CB₂ receptors (Hosohata et al., 1997).

Cannabinoids are known to exert potent cardiovascular effects in vivo. For example, systemic administration of the endocannabinoid anandamide, which acts on CB₁ and CB₂ receptors, causes hypotension and bradycardia (Varga et al., 1996; Högestatt and Zygmunt, 2002). Moreover, anandamide has been shown to produce vasodilatation in the brain (Ellis et al., 1995), liver (Garcia et al., 2001), kidney (Deutsch et al., 1997), heart (Wagner et al., 2001; Ford et al., 2002) and mesentery (Randall et al., 1996; White and Hiley, 1998; Jarai et al., 1999). A number of studies using more selective cannabinoid analogues have confirmed that CB₁ receptor activation leads to relaxation of vascular smooth muscle and systemic hypotension (for review, see Randall et al., 2004). Indeed in the rat knee joint, local administration of the selective CB₁ receptor agonist, arachidonyl-2-chloroethylamide causes an increase in synovial blood flow (Baker and McDougall, 2004). Less is known about the vasomotor effects of CB₂ receptor agonists. Wagner et al. (2005) reported that in vitro perfusion of rat hearts with JWH133 caused an increase in coronary blood flow that was insensitive to AM630 antagonism. In vivo assessment of CB₂ receptor agonists on tissue blood flow have so far not been reported.

Evidence has been emerging in recent years that suggests that cannabinoids are able to bind to and activate the transient receptor potential vanilloid-1 (TRPV1) channel. TRPV1 channels are non-selective cation channels expressed on the axon terminals of a subgroup of primary afferent neurons (Caterina et al., 1997; Szallasi, 2002). In joints, activation of TRPV1 channels by local capsaicin treatment leads to inflammatory changes, including increased synovial blood flow (Karimian et al., 1995; Varga et al., 2005). Interestingly, the CB₁ receptor agonist arachidonyl-2-chloroethylamide has also been shown to act on TRPV1 channels to cause synovial hyperaemia in the rat knee (Baker and McDougall, 2004). Whether CB₂ receptors are similarly coupled to TRPV1 channels has never been explored.

The current study was undertaken to assess the vasomotor effects of the two CB₂ receptor agonists, JWH015 and JWH133, on knee joint blood flow in vivo. The role of TRPV1 channels in these vasoregulatory processes was also investigated. Further studies were carried out to examine the effect of joint inflammation on CB₂ receptor-mediated responses using animal models of acute and chronic arthritis.

Methods

All experimental procedures received prior approval from the University of Calgary Animal Care Committee, which is in accordance with the Canadian Council for Animal Care. Adult male Wistar rats (242–495g) were obtained from Charles River Laboratories (Montreal, Quebec, Canada) and housed in pairs at room temperature under a 12h light–dark cycle, with free access to water and standard rat chow. Animals were randomly assigned to one of three experimental groups: naïve control, acutely inflamed and chronically inflamed.

Induction of joint inflammation

Two distinct models of arthritis were used in the present investigation based on the different phases of inflammation. Thus, the kaolin/carrageenan arthritis model produces an acute inflammatory reaction within 24h after induction, while adjuvant monoarthritis is more suitable to assess the chronic phase of joint inflammation. Prior to arthritis induction, knee joint diameters were measured using electronic digital callipers (Mitutoyo Instruments, Tokyo, Japan) oriented medio laterally across the joint line between the femoral condyle and the tibial plateau. Diameters were compared before and upon completion of inflammation development, which is at 24h for the kaolin/carrageenan model and at 1 week for the adjuvant monoarthritis model.

In the acutely inflamed group of rats, inflammation was induced in the right knee joint by kaolin and carrageenan. Under isoflurane anaesthesia (2–5% isoflurane; 100% O₂ at 1lmin⁻¹), 0.2ml of 2% kaolin was injected through a 27-gauge needle into the posterior and anterior synovial cavity, followed by repeated limb extensions and flexions for 10min to ensure adequate dispersion of the suspension within the joint and to cause articular abrasion. Subsequently, 0.2ml of 2% carrageenan was injected into the joint by the same procedure. These animals were allowed to recover for 24h prior to blood flow assessment.

For chronic inflammation, rats were anaesthetized with 2–5% isoflurane (100% O₂ at 1lmin⁻¹), and a localized monoarthritis was induced as previously described (Donaldson et al., 1993; McDougall et al., 1995). The right knee joint was shaved and 0.2ml of Freund's complete adjuvant (heat-killed Mycobacterium tuberculosis, 1mgml⁻¹) was injected through a 27-gauge needle into the knee joint with 0.1ml being introduced into the posterior compartment of the joint and 0.1ml being injected anteriorly. Animals were allowed to recover for 1 week prior to the blood flow experiments. Sham-injected animals were not tested as an intra-articular injection of sterile saline has been shown to have no effect on synovial blood flow or vasoreactivity (Karimian et al., 1995).

Surgical preparation of animals

On the day of experimentation, animals were deeply anaesthetized with urethane (25% stock solution, 2gkg⁻¹, i.p.). Depth of anaesthesia was confirmed by a lack of a pinch withdrawal reflex applied to the hindpaw as well as an absence of a corneal blink reflex. A longitudinal incision was made in the neck and the trachea was isolated and cannulated to permit unobstructed breathing. The right carotid artery was then isolated and cannulated with heparinized saline (100Uml⁻¹)-filled polyethylene tubing (Portex Fine Bore Tubing, 0.5mm ID, 1.00mm OD; SIMS Portex Ltd., Kent, UK). The carotid cannula was attached to a pressure transducer (1050 Pressure Transducer; Stoelting Co., Wood Dale, IL, USA) and mean arterial pressure (MAP) was recorded continuously throughout the experiment via a blood pressure monitor (Pressure Monitor BP-1; World Precision Instruments, Sarasota, FL, USA). The animal was then placed in dorsal recumbency on an electric-heating blanket (TR-100; Fine Science Tools Inc., Vancouver, British Columbia, Canada) and the internal body temperature was maintained at 37°C as measured by a rectally inserted electronic thermometer. An ellipse of skin covering the anteromedial region of the right knee joint was removed and the underlying tissue was kept moist by regular superfusion of 37°C physiological saline (0.9% NaCl).

Measurement of knee joint blood flow

Knee joint blood flow was measured by laser Doppler perfusion imaging (LDI) as previously described (Karimian et al., 1995; McDougall et al., 1995). The technique involved placing a laser Doppler perfusion imager (MoorLDI V.2; Moor Instruments Ltd., Devon, UK) 30cm above the exposed rat knee and angling the scanner head to avoid reflectance artefact. A low-power (2mW) red He–Ne laser (λ=633nm) was directed onto the surface of the knee joint and by means of a motor-controlled mirror the laser beam scanned in a raster pattern across the joint capsule. Careful placement of opaque black cloth at the margins of the knee ensured that only the joint capsule was scanned. The image resolution was set at 42 pixels × 55 pixels with a scan speed of 4mspixel⁻¹. At each pixel point, photons in the incident laser beam underwent Doppler shift by virtue of erythrocytes flowing in the tissue. These Doppler-shifted photons were captured by a photodetector in the scanner head and a perfusion value was calculated based on the velocity and concentration of erythrocytes circulating in the microvasculature. A two-dimensional colour-coded image of tissue perfusion was then constructed and stored for later offline analysis.

To achieve a more accurate appreciation of the time course for cannabinoid activity, the imager was set to the single-point measurement mode to maximize the temporal resolution of the instrument. In these experiments, the laser beam remained stationary at a discrete locus on the surface of the joint capsule, and blood flow measurements were continuously acquired. The precise positioning of the laser beam was randomly chosen, but was usually placed in the vicinity of an observable microvascular network. A steady blood flow level was measured for about 1min before a cannabinoid agonist was topically applied to the knee joint. Blood flow was then measured continually over the succeeding 5min. In subsequent scanning experiments, the timeline for image acquisition was designed so as to coincide with the maximal vasomotor effect of the test agents.

Experimental protocol

All test reagents were placed in a water bath and gently warmed to 37°C. A control LDI scan of the knee was taken and then the test drug was topically applied to the surface of the joint capsule as a 0.1ml bolus. The CB₂ receptor agonists tested in this study were JWH015 (10⁻¹⁴–10⁻¹¹mol) and JWH133 (10⁻¹⁴–10⁻¹²mol). Blood flow scans of the joint were then taken at 0, 1.5, 2, 3 and 5min following drug application. A cumulative concentration–response curve to each agonist was generated in separate groups of animals and sequential doses of agonist were applied to the joint following each 5-min scan. In other experiments, each dose of the CB₂ receptor agonist was co-administered with the selective CB₂ receptor antagonist AM630 (10⁻⁸mol; 0.1ml bolus topical). The concentration of AM630 was based on previous successful antagonism in the rat cardiovascular system (Ford et al., 2002). To determine whether the transient potential vanilloid receptor-1 (TRPV1) is involved in CB₂ receptor-mediated vasoregulation, additional experiments were undertaken in which animals were pretreated with the TRPV1 receptor antagonist SB366791. At 20min following an intraperitoneal injection of SB366791 (500μgkg⁻¹), concentration–response effects of JWH133 were assessed. This treatment regimen with SB366791 has previously been shown to successfully block TRPV1 receptors in the rat knee joint (Varga et al., 2005). It should be pointed out that JWH015 proved to be a weak, non-selective agonist for the CB₂ receptor and as such was neither used in the SB366791 experiments nor tested in the inflammatory models.

At the conclusion of the experiment, the animal was killed by anaesthetic overdose (pentobarbital sodium, 240mg intracardiac) and a final scan of the joint was performed. This dead scan constituted a ‘biological zero' measurement, which corresponded to tissue optical noise and residual Brownian motion. This ‘biological zero', which was typically about 5–10% of the basal perfusion level, was subtracted from all image perfusion values prior to data processing.

Image analysis and statistics

Knee joint LDI scans were analysed by proprietary perfusion image-processing software (Moor Instruments Ltd.). For each image, an average blood flow value for the entire anteromedial knee joint capsule was calculated and expressed in arbitrary perfusion units (PUs). Blood flow changes in response to drug administration were expressed as percentage change in perfusion between control and test scans. The maximal vasomotor effect (E_max) and the EC₅₀ were calculated from a sigmoidal nonlinear regression curve fit. All data sets conformed to a Gaussian distribution and as such were tested with parametric statistical analyses using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). ANOVA was used to assess significant differences in blood flows within (one-way ANOVA) and between (two-way ANOVA) different animal groups. A Bonferroni post hoc test was used to determine whether groups of data were significantly different at a specific concentration of cannabinoid. A significance level of P<0.05 was used for each test and all reported values represent means±s.e.m. for ‘n' observations.

Drugs and reagents

JWH015, JWH133 and AM630 were all purchased from Tocris Bioscience, Ellisville, MO, USA; SB366791, Freund's complete adjuvant, λ-carrageenan, kaolin, urethane and heparin were all obtained from Sigma-Aldrich Ltd., Oakville, Ontario, Canada. SB366791 and all cannabinoid agonists and antagonists were dissolved in dimethyl sulphoxide and cremophor, diluted to their working concentrations, aliquoted and stored at either 4°C or −20°C as required. The final concentration of vehicle was maintained at 2% dimethyl sulphoxide and 1% cremophor for each concentration. This vehicle formulation has previously been found to have no significant effect on joint blood flow (Baker and McDougall, 2004).

Results

CB₂ receptor effects in normal knee joints

LDI-determined basal blood flow in normal rat knees was 421±26.8 PU (n=52). Application of the CB₂ receptor agonists JWH015 and JWH133 onto the surface of normal rat knees caused a marked but transient increase in synovial blood flow. Continuous measurement of joint blood flow at a single discrete location indicated that vasodilatation was rapid in onset in response to CB₂ receptor activation, with maximal blood flow being reached approximately 1.5min after drug application (Figure 1). Synovial blood flow subsequently returned to control levels at around the 4-min time point. Concentration–response curves were constructed for JWH015 and JWH133 and both agonists showed a concentration-dependent effect (Figure 2). The calculated E_max for JWH015 was 18.5±3.2% with an EC₅₀ of 1.9 × 10⁻¹⁴mol. JWH133 on the other hand had an E_max of 36.4±5.3% and an EC₅₀ of 7.9 × 10⁻¹⁴mol. The selective CB₂ receptor antagonist AM630 had no effect on JWH015-mediated vasodilatation (P=0.81, two-way ANOVA; n=9–10; Figure 2a). Conversely, co-administration of AM630 with JWH133 significantly inhibited the vasodilatatory effect of the cannabinoid (P<0.0001, two-way ANOVA; n=11–38; Figure 2b).

Figure 1 Single-point measurements of knee joint blood flow at a discrete locus on the joint capsule showing typical responses to topical application of the CB2 receptor agonists JWH015 (a) and JWH133 (b). Blood flow gradually rose, with maximal vasodilatation (more ...)

Figure 2 Concentration–response effects of JWH015 (a) and JWH133 (b) on synovial blood flow. The selective CB2 receptor antagonist AM630 (10−8mol) was unable to block the vasodilator effect of JWH015, but significantly attenuated the hyperaemic (more ...)

Administration of 10⁻¹²mol JWH133 without previous application of the lower concentrations of the cannabinoid still elicited around a 40% increase in joint blood flow (data not shown), indicating that tachyphylaxis was not occurring in these experiments.

Since JWH015 only produced a weak, non-selective effect on synovial blood flow, the subsequent experiments were only carried out with the more efficacious JWH133.

Role of TRPV1 in CB₂ receptor-mediated vasodilatation

To determine if the TRPV1 ion channel is involved in the activity shown by JWH133, animals were treated with the TRPV1 antagonist SB366791. As shown in Figure 3, the vasodilator effect of JWH133 was blocked in SB366791-treated animals. A two-way ANOVA confirmed that the JWH133 concentration–response curves in SB366791-treated vs non-treated animals were statistically different from each other (P<0.0001; n=12–38).

Figure 3 Vasodilator effect of JWH133 is attenuated in animals pretreated with the TRPV1 antagonist SB366791 (500μgkg−1). ***P<0.001, **P<0.01, *P<0.05 two-way (more ...)

Joint oedema following inflammation

Comparison of joint diameters before and after inflammation induction confirmed oedema formation in the animal models of arthritis. In kaolin/carrageenan acutely inflamed knees, joint diameter increased from 9.0±0.07 to 15.1±0.44mm, while chronically inflamed adjuvant monoarthritic knees showed an increase in knee diameter from 9.0±0.13 to 12.2±0.42mm. Both increases were found to be statistically significant (P<0.0001, paired Student's t-test; n=10–13 knees).

Assessment of JWH133 in inflamed knee joints

Basal blood flow to acutely inflamed knees was 566±65 PU (n=10). Administration of JWH133 to acutely inflamed rat knee joints failed to elicit any significant change in synovial blood flow (Figure 4). A two-factor ANOVA confirmed that the vasodilator effect of JWH133 seen in normal joints was abrogated in the acute inflammatory model (P<0.0001, two-way ANOVA; n=8–12).

Figure 4 Effect of joint inflammation on JWH133-mediated vasodilatation. The hyperaemic effect of JWH133 was significantly reduced in both acute and chronically inflamed knees (***P<0.001, *P<0.05 two-way ANOVA followed (more ...)

In chronically inflamed joints, the hyperaemic effect of JWH133 was also significantly attenuated compared to normal control knees (P<0.0001, two-way ANOVA; Figure 4). Basal blood flow to adjuvant monoarthritic knees was 513±47.5 PU (n=10).

Effect of AM630 on normal, acutely and chronically inflamed knees

Topical administration of the selective CB₂ receptor antagonist AM630 (10⁻⁸mol) had no effect on synovial blood flow in normal (P=0.29), acutely inflamed knees (P=0.81) or chronically inflamed (P=0.05) rat knee joints (Figure 5). Thus, AM630 does not appear to be acting as an inverse or partial agonist to increase blood flow in this model system.

Figure 5 Effect of topical application of the selective CB2 antagonist AM630 (10−8mol) on knee joint blood flow. AM630 had no effect on joint blood flow in normal, acutely inflamed or chronically inflamed knees. Data are mean % of control (more ...)

Blood pressure effects

As shown in Table 1, local administration of all of the test compounds had no significant effect on MAP. Thus, all reported vasomotor changes are a direct effect of the drugs on articular blood vessels and are not a secondary consequence of systemic changes in blood pressure.

Table 1 MAP taken before (control) and at 1.5min following topical application of the CB2 receptor agonists JWH015 and JWH133, and the CB2 receptor antagonist AM630

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Cannabidiol (CBD) is one of the major components of Cannabis sativa, marijuana. Marijuana contains approximately 80 constituents, termed cannabinoids. CBD is not psychoactive, unlike the other major component of cannabis, Δ⁹-tetrahydrocannabinol (Δ⁹THC). A vast literature documents the immune modulating effects of cannabinoids, in vivo and in vitro, mainly of Δ⁹THC and synthetic analogues such as CP55,940 (reviewed in ref. 6). A nonexhaustive list of in vitro effects includes inhibition of the proliferative responses of T lymphocytes (7), inhibition of cytotoxic T cell activity (8), suppression of macrophage function and antigen presentation (9, 10), and inhibition of NO production by macrophages (11). Reports on the in vitro effects of CBD on immune cells are scarce and include the modulation of tumor necrosis factor (TNF), IL-1, and IFN-γ by human peripheral blood mononuclear cells (12, 13) and the suppression of chemokine production by a human B cell line (14). These potentially anti-inflammatory properties of CBD, together with the lack of psychotropic effect and low toxicity (15), prompted us to test the potential of CBD as a therapeutic agent in collagen-induced arthritis (CIA).

CIA, a murine model for rheumatoid arthritis (RA), is elicited by immunizing DBA/1 mice with type II collagen (CII) in complete Freund's adjuvant (16). The immune response to CII involves both humoral and cellular mechanisms (17, 18), and the cellular response is T helper 1-mediated. CIA is characterized by rapid onset of clinical joint inflammation, resulting in destruction of joint tissues and cartilage/bone erosions. Suppression of the inflammatory process by blocking TNF with mAbs has proven an effective treatment of CIA (20, 21), and these findings led to the successful use of TNF blockade in multiple phase I, II, and III clinical trials with RA patients (reviewed in ref. 22), thus validating the predictive value of CIA as a model for RA. In the present study, we report that CBD has a beneficial therapeutic action on established CIA, and we explore its mode of action.

Results

Systemic Administration of CBD Has a Dose-Dependent Therapeutic Effect on CIA.

CBD at the doses of both 20 mg/kg per day and 10 mg/kg per day had a slight therapeutic effect on CIA, whereas the lower dose of 5 mg/kg caused an optimal suppression of disease (Fig. 1A). The therapeutic action of CBD was lost when the dose was further lowered to 2.5 mg/kg per day. The dose-dependent effects of CBD were confirmed in the homologous CIA model, a chronic relapsing arthritis with a clinical pattern that more closely resembles human disease. Thus, the clinical score in these mice typically goes up and down for several weeks. Overall, the arthritis is chronic relapsing and progressive (30, 31). It was found that 5 mg/kg i.p. CBD was optimal in suppressing the arthritis (Fig. 1B). The area under the curve, which reflects overall disease severity over 28 days, was 38.4 in the controls and 37.3 in the 10 mg/kg group and was reduced to 28.9 in the 5 mg/kg-treated group. CBD treatment caused no obvious side effects in these mice.

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Figure 1

From the first clinical signs of arthritis, mice were treated daily with CBD, i.p. at the following doses: 20 mg/kg (■), 10 mg/kg (▵), 5 mg/kg (▴), or 2.5 mg/kg (○). Mice treated with the vehicle alone served as controls (□). Each point is the mean of n mice ± SEM. (A) Shown is the clinical score over 10 days of classical CIA in three pooled experiments (controls, n = 23; CBD 20 mg/kg, n = 17; CBD 10 mg/kg, n = 15; CBD 5 mg/kg, n = 15; CBD 2.5 mg/kg, n = 9). * denotes a P value < 0.05 (Mann–Whitney U test). (B) An experiment in chronic relapsing homologous CIA, where control, n = 6; CBD 10 mg/kg, n = 6, and CBD 5 mg/kg, n = 6. The area under the curve was 38.4 for the control group, 37.3 for the 10 mg/kg group, and 28.9 for the 5 mg/kg group (not significant).

Oral Administration of CBD Has an Equally Potent Therapeutic Effect on Established Arthritis.

Daily oral gavage of CBD immediately after onset of arthritis resulted in suppression of acute CIA (Fig. 2A). The optimal dose was 25 mg/kg, although the higher dose of 50 mg/kg worked almost as well. The 25 mg/kg dose was used in a chronic experiment in homologous CIA and was shown to effectively suppress progression of disease over a study period of 4 weeks (Fig. 2B). The area under the curve was reduced from 72.3 in the controls to 49.7 in the treated animals (P < 0.05).

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Figure 2

From the first clinical signs of arthritis, mice were given CBD by oral gavage, at the following doses: 50 mg/kg (▵), 25 mg/kg (▴), or 10 mg/kg (○). Mice treated with olive oil served as controls (□). Each point is the mean of n mice ± SEM. (A) Shown is the clinical score over 10 days of classical CIA (controls, n = 6; CBD 50 mg/kg, n = 6; CBD 25 mg/kg, n = 6; CBD 10 mg/kg, n = 6). * denotes a P value < 0.05 (Mann–Whitney U test). (B) An experiment in chronic relapsing homologous CIA, where control, n = 6 and CBD 25 mg/kg, n = 6. The area under the curve was 72.3 for the control group and 49.7 for the CBD-treated group (P < 0.05, Mann–Whitney U).

Effect of CBD on Joint Damage.

Joints in the hind paws of control mice and mice treated with CBD were assessed for hyperplasia and destruction. Table 1 shows the results in acute CIA. First, in the i.p. experiments none of the control mice had normal feet, whereas 34% of the feet in mice treated with 5 mg/kg CBD were completely protected. Sixty nine percent of all of the feet in the control mice were moderately or severely affected (31% and 38%, respectively), whereas in mice treated with 5 mg/kg CBD this was lowered to 40% (20% in each category). For the oral treatment protocol, it was found that 25 mg/kg was the optimal dose. Although no normal feet were found in this treatment group, only 25% were severely affected, as compared with 75% in the controls and 50% in the 50 mg/kg group (Table 1). Thus, the histological findings confirm that CBD at an i.p. dose of 5 mg/kg or an oral dose of 25 mg/kg has an optimal therapeutic effect on acute CIA. Those optimal doses, when tested in the chronic model, gave a good protection against histological damage as well (Table 2): 5 mg/kg CBD i.p. and 25 mg/kg CBD orally increased the number of normal hind paws to 30% and 36%, respectively, as compared with 0% in both control groups. It should be noted that the lesions in this model are generally less severe.

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Table 1

Histology of the hind feet in acute bovine CIA

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Table 2

Histology of the hind feet in chronic homologous CIA

Spontaneous TNF Release by Synovial Cells from Arthritic Animals Is Suppressed After CBD Treatment.

The synovium is the most critical site of cytokine production in arthritis, and synovial cells from arthritic mice at day 10 are known to spontaneously produce large amounts of TNF when cultured in vitro (26). Synovial cells were taken from control arthritic mice and mice that had been treated with 5 mg/kg i.p. CBD at day 10 of arthritis. It was found that synovial cells from CBD-treated mice released significantly less TNF when cultured in vitro (Fig. 3). The results in Fig. 3 are from cultures with standardized cell numbers, thus the in vivo local TNF levels must be a lot lower in the treated animals, which had fewer cells in their synovium (Fig. 3). As a comparison, TNF release by synovial cells taken from healthy age-matched nonimmunized mice was included in Fig. 3.

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Figure 3

Synovial cells were isolated from arthritic mice at day 10 of arthritis, either from control mice or mice treated with CBD, 5 mg/kg i.p. A control group of nonimmunized age-matched mice was included. Total cell number per synovial membrane was on average 50% reduced in the CBD-treated mice compared with the arthritic controls. Cultures were standardized for cell numbers. Supernatants were assessed at 24 h for bioactive TNF by the Walter and Eliza Hall Institute assay. Each dot is the mean of triplicate cultures from one individual mouse, the dots marked with * are a pool of three mice. Thus in total 10 mice per treatment group were tested. Five healthy mice were tested. The horizontal bar is the median.

CBD Treatment Suppresses ex Vivo LNC Proliferation and IFN-γ Production.

Draining LNCs from mice on day 3 of arthritis, both control mice and CBD-treated mice (5 mg/kg per day i.p.) were stimulated in vitro with bovine CII. Table 3 shows the antigen-specific cell proliferation and IFN-γ production. It was found that CBD treatment attenuated cell proliferation (unstimulated proliferation, and to a greater extent CII-specific proliferation) and CII-specific IFN-γ release.

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Table 3

In vivo treatment with CBD suppresses CII-specific T helper 1 responses

CBD Blocks Mitogen-Induced Lymphocyte Proliferation As Well As Antigen-Specific LNC Proliferation.

Purified lymphocytes from spleens from 10-week-old DBA/1 mice were stimulated with Con A, in the presence of increasing concentrations of CBD (0–10 μg/ml). Fig. 4A shows a dose-dependent suppression of Con A-induced proliferation. This finding was reproduced with lymphocytes from C57BL mice, indicating that the effect is not strain-dependent. Likewise, draining LNCs from control arthritic DBA/1 mice taken at day 3 of arthritis were stimulated in vitro with 50 μg/ml bovine CII in the presence of increasing CBD concentrations (Fig. 4B), and again, a dose-dependent suppression of cell proliferation by CBD was seen.

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Figure 4

(A) The effect of CBD on Con A-induced proliferation of splenic lymphocytes. (B) The effect of CBD on CII-specific proliferation of LNCs from arthritic mice. CBD was added in vitro at the concentrations shown for 72 h (□). For the control cultures, the vehicle (ethanol) was used at corresponding dilutions (○). Each point represents the mean ± SD of triplicate cultures. A is a representative experiment of five experiments and B of three experiments.

CBD Suppresses the Production of Reactive Oxygen Intermediates by Granulocytes.

Treatment of mouse granulocytes with 6 μg/ml CBD suppressed the production of Zymosan-induced reactive oxygen intermediates, as assessed by chemiluminescence (Table 4). The inhibitory effect was optimal when the granulocytes were pretreated with CBD before Zymosan stimulation.

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Table 4

CBD inhibits Zymosan-induced release of reactive oxygen intermediates by mouse peritoneal granulocytes

Systemic Administration of CBD Blocks LPS-Induced Serum TNF.

High levels of serum TNF in C57BL/6 mice were measured by bioassay 90 min after i.p. injection of LPS. Simultaneous injection of CBD, either i.p. or s.c., effectively abrogated the rise in serum TNF (Table 5).

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Table 5

LPS-induced rise in serum TNF is blocked by simultaneous administration of CBD

• Received March 10, 2000.

• Copyright © The National Academy of Sciences