Field of the invention The invention relates to regimes of drug administration and drug combinations for use in the treatment of autoimmune diseases.

Background to the Invention Cannabinoids are a class of phenolic compounds abundantly produced in plants of the Cannabis genus. Various cannabinoid compounds have long been known to exhibit psychotropic effects in humans and are widely used recreationally, despite such use being illegal in many jurisdictions. However, recent research has shown that certain cannabinoids are of therapeutic value against a diverse array of pathologies such as inflammatory disorders, neurodegenerative and psychiatric disorders, chronic pain, anxiety and PTSD. While cannabinoids are currently used to combat the wasting, emesis and nausea associated with cancer treatment, evidence exists which suggests that certain cannabinoid compounds may be effective in treating the underlying pathologies of cancers. Presently available evidence suggests that they do this through disruption of cancer cell migration, adhesion, and tumour vascularisation.

An endogenous cannabinoid-mediated signalling system functions in mammals, and on the balance of evidence probably in most other vertebrates too. In humans, two cannabinoid receptors have been identified and named CB1 and CB2, both of which are part of the G protein-coupled receptor superfamily.

Pharmacological evidence to date suggests that activation of CB1 is responsible for the majority of the psychotropic effects of cannabinoid intake, while therapeutic effects are mostly mediated by CB2. As such, ideal therapeutic molecules will activate CB2 in preference to CB1 , alleviating unwanted psychotropic side effects. Cannabidiol (CBD), a component of Cannabis extract, is a cannabinoid with this binding profile which has recently generated considerable interest, but whose full mechanism of action is still being elucidated. In general however, natural cannabinoids do not tend to show high specificity for one or other receptor, but newly developed synthetic cannabinoids are available which bind more specifically.

It is sometimes the case that receptors in one signalling pathway, responsive to a particular ligand, can have their expression levels or downstream effects altered by a change in the signalling output of another seemingly separate signalling pathway. Such cross-modulation has been demonstrated between, for example, the signalling pathways following activation of the Growth Hormone and insulin receptors.

Autoimmune diseases are a broad spectrum of disorders characterised by the abnormal recognition and reaction to self-antigens by the immune system. Pathologically, autoimmune diseases can be caused by genetic factors, environmental factors, infectious agents or a combination of any number of the above. Different autoimmune diseases can be systemic, such as systemic lupus erythematosus or localised to particular regions of the body, such as dermatitis.

Cannabinoids have shown to have a variety effects on body systems. Through the CB1 and CB2 receptors, they exert an effect by modulating neurotransmitter and cytokine release. Current research in the role of cannabinoids in the immune system shows that they possess immunosuppressive properties. They can inhibit proliferation of leucocytes, induce apoptosis of T cells and macrophages and reduce secretion of pro-inflammatory cytokines. In mice models, they are effective in reducing inflammation in arthritis, multiple sclerosis, have a positive effect on neuropathic pain and in type 1 diabetes mellitus (Katchan et al., 2016).

As the underlying causes of many autoimmune diseases remain elusive, the development of preventative measures to combat the onset of many autoimmune diseases is unattainable. There is thus a need to develop curative therapies. Moreover, the link between lifestyle factors and the increasing frequency of diagnosis has created an even greater urgency for developing effective treatments that can reverse or bring under control the symptoms of autoimmune diseases. Summary of the Invention

It has been found by the present inventors that the immunosuppressive properties of cannabinoids is brought about more effectively by combined treatment with low dose naltrexone (LDN) or 6- -naltrexol (6BN), a metabolite of naltrexone. Furthermore, the effectiveness of such treatment is surprisingly dependent on the order in which the two agents are administered, with the most effective regime being a phase of treatment with LDN or6BN followed by a phase of treatment with a cannabinoid.

The inventors have also found that treatment with LDN or 6BN brings about a significant increase in levels of the CB2 receptor in cells, thus allowing the cannabinoid to be more effective.

According to a first aspect of the invention, there is provided a pharmaceutical composition comprising naltrexone or a metabolite thereof or an analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorphine, for use in the treatment of an autoimmune disease within a subject, wherein a therapeutically effective amount of the naltrexone or metabolite thereof or analogue is to be administered to the subject in a first treatment phase, wherein after the first treatment phase the subject is to be administered a therapeutically effective amount of a cannabinoid in a second treatment phase.

According to a second aspect of the invention, there is provided a pharmaceutical composition comprising a cannabinoid for use in the treatment of an autoimmune disease within a subject, wherein said subject is characterised in having undergone a first treatment phase during which the subject is administered a therapeutically effective amount of naltrexone or a metabolite thereof or an analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorphine, and wherein following the first treatment phase a therapeutically effective amount of said cannabinoid is to be administered to the subject. According to a third aspect of the invention, there is provided a preparation comprising naltrexone or a metabolite thereof or an analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorphine, and a cannabinoid for use in the treatment of an autoimmune disease within a subject, wherein the naltrexone or metabolite thereof or analogue is provided in a therapeutically effective amount to be administered in a first treatment phase, wherein the cannabinoid is provided in a therapeutically effective amount to be administered in a second treatment phase following the first treatment phase.

According to a fourth aspect of the invention, there is provided a method for determining the suitability of a subject with an autoimmune disease for treatment with a cannabinoid in a second treatment phase, said subject characterised in having undergone a first treatment phase as defined above, the method comprising the steps of: i. contacting a sample obtained from the subject after or during a recovery phase with a probe specific for CB2; ii. determining the concentration of CB2 within the sample; and iii. comparing the concentration of CB2 within the sample with a concentration of CB2 determined from a sample obtained from the subject before the first treatment phase, wherein if the CB2 concentration has increased by at least two-fold after the first treatment phase, the subject is suitable for the second treatment phase.

According to a fifth aspect of the invention, there is provided the use of naltrexone or a metabolite thereof or an analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorphine, in the manufacture of a medicament for the treatment of an autoimmune disease within a subject, wherein the medicament is to be administered to a subject in a first treatment phase of a combined treatment regimen, said treatment regimen comprising the first treatment phase followed by a second treatment phase, wherein the subject is to be administered a cannabinoid in the second treatment phase. According to a sixth aspect of the invention, there is provided the use of a cannabinoid in the manufacture of a medicament for the treatment of an autoimmune disease in a subject, wherein said medicament is to be administered in a second treatment phase of a combined treatment regimen, said treatment regimen comprising a first treatment phase followed by the second treatment phase, wherein the subject is to be administered naltrexone or a metabolite thereof or an analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorphine, in the first treatment phase.

According to a seventh aspect of the invention, there is provided the use of naltrexone or a metabolite thereof or an analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorphine, in the manufacture of a first medicament and the use of a cannabinoid in the manufacture of a second medicament, both medicaments for the treatment of an autoimmune disease, wherein the first and second medicaments are to be administered to a subject with an autoimmune disease in a combined treatment regimen, said treatment regimen comprising a first treatment phase followed by a second treatment phase, wherein the first medicament is to be administered to the subject during the first treatment phase and the second medicament is to be administered to the subject in the second treatment phase.

According to an eighth aspect of the invention there is provided a method for the treatment of an autoimmune disease in a subject comprising administering to the subject in a first treatment phase a therapeutically effective amount of naltrexone or a metabolite thereof or an analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorphine, followed by administering to the subject in a second treatment phase a therapeutically effective amount of a cannabinoid.

Detailed Description of the Invention

The invention provides a specific therapeutic regimen for treating an autoimmune disease in a subject, wherein a cannabinoid is administered after administration of Low Dose Naltrexone (LDN), a metabolite of naltrexone, or an analogue thereof selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorphine.

The phased administration of LDN and then a cannabinoid has been found by the inventors to inhibit an autoimmune disease more effectively than separate or simultaneous administration, or phased administration of a cannabinoid and then LDN.

It was also found that levels of the CB2 receptor increased significantly after treatment with LDN. Without wishing to be bound by theory, this increase in CB2, which is the therapeutic target of cannabinoids such as CBD, is feasibly a contributing factor to the increase in the effectiveness of the treatment with CBD.

The skilled person will be able to carry out the phased administration of the therapeutic agents as described. The invention can be further understood with reference to the following definitions:

As used herein, “naltrexone” refers to the compound 17-cyclopropylmethyl-4.5a- epoxy-3,14-dihydroxymorphinan-6-one (with lUPAC name (4R,4aS,7aR,12bS)-3- (cyclopropylmethyl)-4a,9-dihydroxy-2,4,5,6,7a, 13-hexahydro-1 H-4, 12- methanobenzofuro[3,2-e]isoquinoline-7-one) and pharmaceutically acceptable salts, solvates, hydrates, racemates, stereoisomers, clathrates, polymorphs and prodrugs thereof. Analogues thereof are also envisaged for use as per the invention. Suitable analogues include methylnaltrexone, naloxone, nalmefene and nalorphine.

The naltrexone is typically in its hydrochloride salt form.

“Low Dose Naltrexone” (LDN) refers to naltrexone administered in “low” doses of less than 0.5 mg/kg, preferably less than 0.2 mg/kg, more preferably between 0.01 mg/kg and 0.08 mg/kg, even more preferably between 0.03 mg/kg and 0.06 mg/kg, most preferably between 0.04 mg/kg and 0.05 mg/kg. Typically, a low dose is up to 3mg per day in total, per patient. Metabolites of naltrexone include 6-p-naltrexol, 2-hydroxy-3-methoxy-6p-naltrexol and 2-hydroxy-3 methoxy-naltrexone.

The preferred metabolite of naltrexone is 6-p-naltrexol (6BN), which as used herein refers to the compound N-cyclopropylmethyl-7,8-dihydro-14- hydroxynorisomorphine (with lUPAC name (4R,4aS,7R,7aR,12bS)-3- (cyclopropylmethyl)-l ,2,4,5,6,7,7a, 13-octahydro-4, 12-methanobenzofuro[3,2- e]isoquinoline-4a,7,9-triol), and pharmaceutically acceptable salts, solvates, hydrates racemates, stereoisomers, clathrates, polymorphs, and prodrugs thereof.

The 6BN or analogues etc, may also be administered at “low dose”. In this context, a “low dose” can be the same as that outlined above for naltrexone.

Cannabinoids are a class of compounds understood by the skilled person which comprise those abundantly made by plants of the Cannabis genus, as well as endocannabinoids which are synthesised in animals. Synthetic compounds active against CB receptors are also envisaged. When used herein the term can refer to any cannabinoid, but is preferably selected from the list containing cannabidiol, cannabidiolic acid, cannabinol, cannabigerol, cannabivarin, tetrahydrocannabivarin, cannabidivarin, cannabichromene, arachidonoylethanolamine, 2-arachidonoylglycerol, 2-arachidonoyl glyceryl ether, A/-arachidonoyl dopamine, virodhamine, dronabinol, nabilone, rimonabant, R-(+)- Met-anandamide, WIN-55,212-2, HU-210, JWH-133, SR141716, SR144528 or combinations thereof, or pharmaceutically acceptable salts, solvates, hydrates, racemates, stereoisomers, clathrates, polymorphs, prodrugs, and analogues thereof which bring about equivalent effects.

In certain embodiments, where the agent is 6- -naltrexol, 6- -naltrexol is to be administered in an amount effective to increase the blood plasma concentration of 6- -naltrexol to at least 0.34 ng/ml, preferably at least 3.4 ng/ml, more preferably at least 34 ng/ml, or most preferably at least 340 ng/ml. In certain embodiments, 6- -naltrexol is to be administered in an amount effective to increase the blood plasma concentration of d-b-naltrexol to within the range of 0.3 ng/ml to 3,400 ng/ml, preferably to within the range of from 34 ng/ml to 3,400 ng/ml, more preferably within the range of 340 ng/ml to 3,400 ng/ml. The amount effective to achieve such an amount can be determined using any number of conventional techniques known to the person skilled in the art. For example, the skilled person could perform mass spectrometry on a blood plasma sample obtained from the subject in order to determine the increase in the concentration of 6- -naltrexol within the sample after administration of an amount of 6-b- naltrexol. The effective amount is the amount determined to bring about the desired increase in blood plasma concentration. Typically, the naltrexol will be administered in an amount up to 3mg per day per patient.

The cannabinoid can be administered at conventional amounts based on the particular cannabinoid and the details of the patient. In certain embodiments, the cannabinoid is to be administered each day in doses of between 10 mg and 1000 mg, preferably between 200 and 800 mg, more preferably between 300 and 500 mg.

In another embodiment, the therapy may also include administering Vitamin D to the subject. The Vitamin D may be administered to the subject before, during or following the treatment regimen. During the treatment regimen the Vitamin D may be administered to the subject at any time, such as during the first treatment phase or during the second treatment phase. Preferably, the administration of Vitamin D in the patient should occur before the disclosed treatment regimen begins.

During the first treatment phase, the Vitamin D may be administered to the patient before and/or after administration of the naltrexone or a metabolite thereof or an analogue thereof. The Vitamin D and the naltrexone or a metabolite thereof or an analogue thereof may be administered simultaneously, sequentially or separately, preferably simultaneously. The Vitamin D may also be continued to be administered to the patient after treatment with the naltrexone ceases, so the Vitamin D is administered in the first and second treatment phases. During the second treatment phase, the Vitamin D may be administered to the patient before and/or after administration of the cannabinoid. The Vitamin D and the cannabinoid may be administered simultaneously, sequentially or separately, preferably simultaneously. The Vitamin D may also be continued to be administered to the patient after treatment with the cannabinoid ceases, so the Vitamin D is also administered following the treatment regimen.

Preferably during the treatment regimen the Vitamin D should be administered to the subject on a daily basis to maintain the correct Vitamin D levels throughout all phases of treatment.

“Simultaneous” administration, as defined herein, includes the administration of substances within about 2 hours or about 1 hour or less of each other, even more preferably at the same time.

“Separate” administration, as defined herein, includes the administration of substances, more than about 12 hours, or about 8 hours, or about 6 hours or about 4 hours or about 2 hours apart.

“Sequential” administration, as defined herein, includes the administration of substances each in multiple aliquots and/or doses and/or on separate occasions.

As used herein, “vitamin D” refers to vitamin D and any intermediate or product of a metabolic pathway of vitamin D that result in a metabolite that is capable of boosting the cytostatic effect of the active. Metabolite may refer to a vitamin D precursor, which can be incorporated into a vitamin D synthetic pathway occurring naturally within the subject to undergo the therapy of the invention. Alternatively, metabolite may refer to a molecule derived from an anabolic or catabolic process that utilizes vitamin D. Non-limiting examples of vitamin D metabolites include ergocalciferol, cholecalciferol, calcidiol, and calcitriol, 1a-hydroxycholecalciferol, 25-hydroxycholecalciferol, 1 a,25-hydroxycholecalciferol,

24,25-hydroxycholecalciferol. An “active” metabolite is a metabolite that can be used in the context of the present invention. Dosage regimes of vitamin D or active metabolites thereof will be well known to the person skilled in the art. The term vitamin D also encompasses pharmaceutically acceptable salts of any of the above. A particularly suitable metabolite of vitamin D for use in the present invention is calcitriol.

In certain embodiments, the vitamin D product is to be administered to the patient in an amount sufficient to bring the subject’s blood vitamin D concentration to at least 40 ng/ml, more preferable at least 50 ng/ml. Preferably, the blood vitamin D concentration is raised to within a range of from 40 to 220 ng/ml, more preferably the blood vitamin D concentration is raised to within a range of from 40 to 90 ng/ml.

A sufficient amount can be determined by the skilled person by making a routine assessment of certain parameters of the patient to undergo the administration, such as, but not limited to, age, weight, gender, history of illness and/or other lifestyle factors including smoking, alcohol consumption and the level of exercise. Furthermore the skilled person can ascertain whether a dose has been sufficient to raise the vitamin D blood concentration to a sufficient amount by performing routine biochemical and analytical assays on a biological sample obtained from the subject. Preferably, the sample upon which said analysis is to be performed is blood. Examples of such well known assays include but are not limited to mass spectrometry, where the level of vitamin D or active metabolites thereof can be quantitatively measured. A sufficient amount is therefore an amount that achieves the desired blood vitamin D concentration. The desired concentration can be achieved after single administration or after repeated administrations of a dose of vitamin D or an active metabolite thereof. Where the vitamin D product and the naltrexone product are to be administered simultaneously, it is immaterial whether the vitamin D blood concentration is within the desired range prior to administration of the naltrexone product, provided that the vitamin D product is administered in an amount sufficient to raise the blood vitamin D concentration to within the desired concentration range. Other methods for determining the concentration of vitamin D or active metabolites thereof within a biological sample obtained from the patient will be well known to the skilled person. In certain embodiments, the amount of the vitamin D sufficient to raise the blood vitamin D concentration to beyond a certain level is referred to as the “therapeutically effective amount” of the vitamin D product. In a preferred embodiment, the Vitamin D administered to the subject is calcitriol. As used herein, “calcitriol” refers to the active metabolite of vitamin D, specifically vitamin D3. Calcitriol is also called 1 ,25-dihydroxyvitamin-D3 or 1 ,25- dihydroxycholecalciferol. Its empirical formula is C27H44O3.

Preferably, the calcitriol is administered and absorbed in the oral cavity of the patient. The term oral cavity has its normal meaning in the art and is intended to cover the mouth, the lips, the hard palate, the soft palate, the retromolar trigone, the tongue, gingiva (gums), buccal mucosa (the inner lining of the lips and cheeks), and floor of the mouth under the tongue.

Absorption in the oral cavity can be achieved according to any known methods. Absorption of calcitriol in the oral cavity may be achieved by the sublingual, sublabial, gingival or buccal route. For example, calcitriol can be formulated in an orally disintegrating form that disintegrates and dissolves in the mouth without water before swallowing. It may dissolve in this way within 60 seconds or less, preferably less than 10 seconds.

Preferably, the calcitriol is an orally disintegrating tablet or coating, an orodisperse tablet or coating, a mouth-dissolving tablet or coating, a quick-dissolve tablet or coating, a fast-melt tablet or coating, a rapid-disintegrating tablet or coating; or a freeze-dried wafer.

An orally disintegrating tablet/coating is a solid dosage form that contains medicinal substances and disintegrates rapidly (within seconds) without water when placed on the tongue. The drug is released, dissolved, or dispersed in the saliva.

A quick-dissolving tablet/coating (also known as a fast-dissolving, fast-dissolving multiparticulate, rapid-dissolving, mouth-dissolving, fastmelting, or orodispersing tablets) is a solid dosage form that does not require water for swallowing. The tablet dissolves within 60 seconds when placed in the mouth. The active ingredients are absorbed through mucous membranes in the mouth. A freeze-dried wafer is a quick-dissolving, thin matrix that contains a medicinal agent (in this case calcitriol) that does not need water for swallowing. The wafer disintegrates instantaneously in the oral cavity and releases calcitriol, which dissolves or disperses in the saliva.

This calcitriol may be administered sublingually, sublabially or buccally to the subject by rapidly dissolving in the oral cavity when it comes in contact with saliva. Preferably, it is absorbed sublingually.

The calcitriol may be formulated according to known methods such as lyophilisation (freeze-drying), cotton candy process, moulding, sublimation and direct compression. In a preferred embodiment, the calcitriol is formulated as a lyophilised dosage form. Preferably it is a lyophilised liquid solution, suspension or emulsion. This may be achieved according to any known methods for producing dosage forms that dissolve in the oral cavity such as using the following technology: Zydis® , QuickSolv® , Lyoc®, Flashdose® , OraSolve® , Ziplet technology, Frosta® , DuraSolve® , Wowtab®, Durasolv®, Flashtab®, Oraquick®, RapiTab® and Nanomelt® (by Elan).

It has been discovered that the patient absorbing calcitriol in the oral cavity overcomes the problems of variable bioavailability that occurs when calcitriol is swallowed in a tablet. When swallowed, calcitriol has variable bioavailability which means the desired dosage may not be achieved. One way to mitigate this problem is to increase the dose per tablet to give the top-end of the dose. However, by doing this, there would be a danger of causing renal damage from toxicity. It has been discovered that administering calcitriol in the oral cavity allows the desired dosage to be achieved without causing any adverse symptoms associated with renal damage, such as fatigue, loss of appetite or leg swelling. Absorption in the oral cavity also avoids drug metabolism via the stomach and intestines, which means a more efficient delivery of calcitriol. As used herein, a “preparation” can refer to a substance or a collection of substances, in the form of one or more compositions intended for use either simultaneously or non-simultaneously.

The method of administration is not particularly limited for either therapeutic agent, but in various embodiments of the invention, LDN and cannabinoids are administered via the oral, buccal, sublingual, nasal, pulmonary, intravenous, rectal, topical, and transdermal routes. For LDN administration is preferably oral, and for cannabinoids it is preferably sublingual.

The treatment regimen envisaged comprises a “first treatment phase” and a “second treatment phase”. In the first treatment phase, a therapeutically effective amount of LDN, a metabolite thereof, or an analogue of either is administered. In the second treatment phase, an effective amount of one or more cannabinoids is administered.

It is preferred that the second treatment phase begins only after 1 to 7 days have elapsed from the start of the first treatment phase, more preferably 1 to 4 days, most preferably 1 to 2 days, a “day” being a continuous period of 24 hours.

In a further preferred embodiment of the invention, there is a “recovery phase" in between the end of the first treatment phase and the start of the second treatment phase. During the recovery phase no LDN or cannabinoid is administered. In one embodiment the recovery phase has a duration of at least two days, preferably no more than 1 week apart, most preferably the duration is two days.

As used herein, the phrase “pathological condition” refers to the autoimmune disease to be treated by any aspect of the invention.

The phrases “autoimmune disease” or ““autoimmune disorder” are well known in the art, and refer to conditions arising from an abnormal immune response to a normal body part. There are over 80 recognised autoimmune conditions, and the underlying causes of many remain unknown. The development of particular autoimmune disorders is linked to the presence of genetic risk factors, whereas others are associated with exposure to specific environmental or chemical factors. Certain lifestyle factors, such as smoking, exercise, sleep and diet have also been linked to an increased risk of developing certain autoimmune disorders. It is envisaged that the treatment of any autoimmune disorder would benefit from the therapy disclosed herein. In a particular feature of the invention, the autoimmune disease to be treated is selected from the list consisting of hepatitis, ankylosing spondylitis, Lyme disease, systemic lupus erythematosus, fibromyalgia, myasthenia gravis, Guillain-Barre syndrome, multiple sclerosis, Behget's disease, chronic fatigue syndrome or psoriasis.

As used herein, the terms “therapy” and "treating" and "treatment" and "to treat" refer to both 1) therapeutic measures that cure, slow down, and/or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In some instances, a subject is successfully "treated" for an autoimmune disease according to the novel applications of the present invention if the patient shows one or more of the following: for example, a reduction in swelling, pruritus, fatigue, general malaise, fever, skin lesions, hair loss or weight loss. In some instances treatment may result in halting the further progression of symptoms of the disorder or the emergence of symptoms of the disorder.

As used herein, the term "subject" refers to any animal, including but not limited to humans, non-human primates, horses, canines, felines, rodents, and other vertebrates which are to be the recipient of a treatment for cancer. The terms "subject" and "patient" are used interchangeably herein.

In another embodiment, the autoimmune disease for treatment is selected from the list consisting of hepatitis, ankylosing spondylitis, Lyme disease, systemic lupus erythematosus, fibromyalgia, myasthenia gravis, Guillain-Barre syndrome, multiple sclerosis, Behget's disease, chronic fatigue syndrome or psoriasis. Preferably, the autoimmune disease is psoriasis. In one aspect of the invention is provided a method for determining the suitability of a subject, who has an autoimmune disease and has already been treated with LDN, for treatment with a cannabinoid. In this aspect, a sample is to be obtained from the subject and contacted with a CB2-specific probe. The concentration of CB2 in the sample is thereby determined and compared with a reference measurement made before the first treatment phase.

As used herein, “suitability” refers to the property of having a high probability of treatment, as defined above, being successful, compared to those subjects deemed unsuitable by the test. A subset of subjects undergoing testing by the method envisaged in one aspect of this invention will be deemed to be unsuitable for the second phase of treatment. In no way is any part of the invention disallowed for use on such subjects. The method of determining suitability is intended to be considered by the skilled person alongside other tests known to them and exercising their intuition and judgement.

In certain embodiments, the biological “sample” obtained from the subject for use in the method is blood, plasma, serum, lymph fluid, a tissue, or cells derived from a tissue sample.

As used herein, the “probe” is any moiety which, on contacting the sample obtained from the subject, allows in some way the measurement of the concentration of CB2 in the sample. In one embodiment, this probe is an antibody or other CB2-binding molecule used in the provision of a purer solution of CB2 which can be analysed spectrophotometrically and through calibration using methods known to the skilled person, the concentration of CB2 in the original sample can be determined.

In another aspect is provided the use of naltrexone, metabolites thereof, or analogue selected from the group consisting of methylnaltrexone, naloxone, nalmefene and nalorphine, and cannabinoids in the manufacture of a medicament to be administered as part of a “combined treatment regimen”, which as used herein refers to the regimen consisting of the first treatment phase, preferable recovery phase, and second treatment phase, as defined above for the treatment of an autoimmune disease.

Example

One illustrative and non-limiting experiment was carried out on cultures of MCF7 cells. In brief, either LDN at 10nM or 6BN at 1 mM or 10 pM was administered in vitro to a culture for two days, after which the expression levels of CB1 , CB2, and GAPDH (as a loading control) were analysed via Western blot and subsequent quantification via density measurement of the bands.

In full, MCF7 cells were seeded into 6-well plates at a density of 1 x 105 /well and left to adhere over-night. Naltrexone (10 nM) or d-b-naltrexol (1 pM or 10 pM) were added to the cells before harvesting cells for western blot analysis. Primary probing was performed with specific antibodies generated against CBR-1 and CBR-2. Anti-GAPDH was used as a loading control. All antibodies were used at a dilution of 1 :1000, followed by the appropriate HRP conjugated secondary antibodies also at a dilution of 1 : 1000. Bands were visualised by the SuperSignal chemiluminescent detection system, and densitometry of band intensity was determined using Adobe Photoshop CS3, v10.0, and normalised to the loading control.

In response to all three treatments, levels of CB2 rose relative to the controls in which no drug was administered. For CB2, the rise was highest in response to administration of 10 pM 6BN. This can be seen in Figure 1.

References

Katchan V et al. , Autoimmun. Rev., 2016, 15(6):513-28