WO2009004380A1 - Method and use of circulating levels of endocannabinoid ligands for the determination of patient in need and/or suitable for cb1r antagonist drug treatment as well as method for inducing weight loss/maintenance/gain - Google Patents

Abstract

The present invention relates to patient selection tools and methods (including personalised medicine). In particular, the present invention permits the im proved selection of a patient in need of treatment with a cannabinoid re ceptor (CB1R) antagoni st drug, based on the levels of circulating endogenous cannabinoi d ligands (endocannabi noids). One of the aspects of the invention is a method of select ing a mammal in need of weight loss and/or weight maintenance and/or pr evention of weight gain treatment with a CB1R antagonist drug which comprises determining the leve l of at least one circulating endogenous cannabinoid ligand in the mamma l, whereby to predict an in creased likelihood of response to the CB1R antagonist drug. Some of the benefits of this personalised medicine approach include the ability to identify patients that are more likely to respond favourably to CB1R antagonist drug treatment and to identify pa tients less likely to respond to the drug treatment thus avoiding unnecessary treatment and any side eff ects that may be associated with such ineffective treatment.

Description

METHOD AND USE OF CIRCULATING LEVELS OF ENDOCANNABINOID LIGANDS FOR THE

DETERMINATION OF PATIENT IN NEED AND/OR SUITABLE FOR CB1 R ANTAGONIST

DRUG TREATMENT AS WELL AS METHOD FOR INDUCING

WEIGHT LOSS/MAINTENANCE/GAIN

The present invention relates to patient selection tools and methods (including personalised medicine). In particular, the present invention permits the improved selection of a patient in need of treatment with a cannabinoid (CB) 1 receptor (CB 1 R) antagonist drug, in order to identify patients most likely to benefit from treatment with a CBlR antagonist drug, including for example the possibility to predict an increased likelihood of response to the CBlR antagonist drug and/or to identify patients less likely to respond to the drug thus avoiding unnecessary treatment and any side effects that may be associated with such ineffective treatment. The selection is based on the levels of circulating endogenous cannabinoid ligands (endocannabinoids) previously isolated from the test subject/patient. These endocannabinoids, singly or in combination, can therefore be used as biomarkers of suitability for CBlR antagonist treatment.

CBl antagonist drugs have been proposed for use in a wide variety of indications and the invention can be used for identifying patients suitable for any treatment with a

CBlR antagonist. However, the invention is of particular use in weight treatment, such as weight loss, weight maintenance, prevention of weight gain, obesity settings, and the like; and in treatment of diseases where weight loss can be used as treatment or prevention of other diseases including type 2 diabetes, dyslipidemia, hypertension, sleep apnea, muscle and skeletal diseases, hyperuricemia, and cancer.

The worldwide prevalence of obesity is increasing at an alarming rate and is associated with major adverse consequences for human health and substantial health care costs (Wee et al, Am J Public Health, 1:159-165, 2005). The World Health Organization (WHO) has estimated that worldwide, over one billion adults are overweight and at least 300 million are obese (Bays, Obesity Res., 12:1197-1211, 2004). The obesity epidemic affects all demographic groups including children (Hedley et al., JAMA, 291:2847-2850, 2004). Obesity increases the risk of a variety of associated diseases, e.g. the metabolic syndrome, type 2 diabetes, dyslipidemia, hypertension, coronary artery disease, stroke, gallbladder disease, osteoarthritis, obstructive sleep apnea, asthma, cancer, reproductive and psychological disorders (Park et al., Curr Opin Gastroenterol, 21 :228-233, 2005).

Obesity is also associated with impaired quality of life (QoL) (Fontaine et al., J Fam Pract, 43:265-270, 1996). Due to the epidemic proportions of obesity and the high risk for obesity-associated conditions, it is of utmost importance to find efficient treatment strategies.

Current available drugs offer modest efficacies despite their high cost and sub optimal safety and side effect profiles. Bariatric surgery is the only treatment proven to result in significant long-term weight loss in a majority of patients (Sjostrom et ah, N Engl J Med, 351:2683-2693, 2004). At present, surgery is only recommended for morbidly obese patients or patients at high risk of life-threatening co-morbid conditions. In addition, surgical complications can occur, sometimes life-threatening, and a non-surgical obesity treatment that provides a more pronounced body weight reduction and maintenance would be desirable (Buchwald et ah, JAMA, 292: 1724- 1737, 2004). From studies on anti-obesity drugs it is well known that some patients respond well and obtain good effects (>10% reduction in body weight), some obtain a moderate effect and some will have very limited effect when treated with an anti-obesity drug. The reason for this variation in response to anti-obesity drugs is unknown but it would be highly desirable to be able to identify responders to a certain anti-obesity therapy before initiation of therapy. A successful personalised medicine approach, may help to improve not only efficacy outcome in clinical practice but also strongly enhance the health economic advantages and reduce safety concerns because a smaller patient segment is exposed. However, it has been notoriously difficult to identify markers that predict response, either in terms of efficacy or safety, to anti-obesity treatment.

Locally produced endogenous cannabinoid ligands (endocannabinoids) in the brain have been shown to play a role in the physiological regulation of food intake and body weight (Fride E, Prostaglandins Leukot Essent Fatty Acids, 66(2-3): 221-233, (2002 review)). In addition it has also been shown that pharmacological treatment targeting the CBlR, including smoking or ingestion of exogenous cannabinoid ligands present in extracts derived from the plant Cannabis sativa stimulates appetite (Williams and Kirkham, Psychopharmacology 143:315-317, 2004). It is believed that these effects are mediated by binding of the cannabinoids to the G-protein-coupled CBlR localized in the brain, including regions of the hypothalamus. The CBlRs are also present in the periphery, e.g. in the adipose tissue and in the liver, where they regulate adipogenesis and lipogenesis (Osei-Hyiaman et al., J Clin Invest, 115:1298-1305, 2005). Administration of CBlR antagonist drugs has been shown to decrease food intake and to cause weight loss in both humans and animals. There are several endogenous ligands for the cannabinoid system, including arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) that have been implicated in regulation of feeding in both animals and humans (Cota et al., Int J Obes, 27:289-301, 2003). There have been reports that obese patients possess high levels of circulating endocannabinoids. For example, Bluher et al. (Diabetes, 55:3053-3060, 2006) found that circulating AEA levels were equivalent but that 2-AG levels were elevated in obese subjects compared with lean subjects. Furthermore, they found that CBl receptor expression in adipose tissue was reduced in obese subjects. Cote et al. (Int J. Obesity 31 (4):692-699, 2007) studied circulating levels of 2-AG and AEA and found that 2-AG levels were elevated in obese men with intra-abdominal adiposity, whereas AEA was not elevated. Engeli et al. (Diabetes, 54:2838-2843, 2005) found that concentrations of AEA and 1/2 -AG are increased in obese compared with lean women. However, none of these documents teaches that there are differential (stratified) levels of circulating endocannabinoids within the group of obese or overweight subjects.

There have also been reports of differential amounts of endocannabinoid ligands in other diseases. For example, Hill et al., (Pharmacopsychiatry 41: 48-53; 2008) found that female patients with clinical depression tended to have low circulating levels of 2-AG endocannabinoid. However, none of the documents identified teach that circulating endocannabinoids can be used as a biomarker of susceptibility to treatment with a CB IR antagonist drug.

As the amount of consumed food will impact body weight, the local levels of endogenous ligands in the brain are therefore also linked to body weight control. The local production and destruction of endogenous ligands in the brain is a tightly controlled process (Di Marco and Matias, Nature Neuroscience 8:585-589, 2005). Due to the importance of the tightly controlled, local production of endogenous ligands in the brain for food intake, there has been no reason to believe that circulating (blood) levels of endogenous ligands will be anyhow linked to food intake and body weight control. Surprisingly, the inventors found that circulating levels of endogenous ligands measured before initiation of treatment with a CBlR antagonist drug strongly predicted the body weight loss during treatment. Higher levels of endogenous ligands in the blood before treatment start were associated with larger weight losses during CBlR antagonist treatment. Lower levels of endogenous ligands in the blood before treatment start were associated with smaller weight losses or absence of weight loss during CBlR antagonist drug treatment. However, there is benefit in identifying overweight or obese patients with low circulating levels of endocannabinoids because, for example, such patients can then be deselected for treatment, thus avoiding the patient having to suffer any drug-associated side effects when the likelihood of receiving therapeutic benefit is low.

In addition to its role in the regulation of food intake and body weight, studies have suggested that endocannabinoids may regulate multiple physiological and pathological functions. Examples are given in the following references: Maccarrone M et al., Prostaglandins Leukot Essent Fatty Acids, 66(2-3): 309-317, (2002 review); Cainazzo MM, Eur J Pharmacol, 441(l-2):91-97, (2002); Parolaro D et al, Prostaglandins Leukot Essent Fatty Acids 66(2-3): 319-32, (2002 review).A more detailed list of CBIR-mediated diseases and conditions is given below.

The present invention is based on the discovery of a link between circulating levels of endocannabinoids and susceptibility to treatment with a CBlR antagonist drug. This discovery therefore, provides opportunities, methods and tools for selecting patients for treatment with a CBlR antagonist drug, particularly patients in need of weight loss, weight maintenance, and prevention of weight gain such as overweight and obese patients, while at the same time avoiding treatment of patients less likely to respond therapeutically to the treatment and thus avoiding such patients having to experience or suffer any adverse drug- associated side effects.

CBIR-mediated diseases and conditions

The CB 1 receptor is implicated as a mediator in a wide range of diseases and disorders, and accordingly CBlR antagonist drugs have been proposed for the treatment of obesity or being overweight, (e.g., promotion of weight loss and maintenance of weight loss), prevention of weight gain (e.g., medication-induced or subsequent to cessation of smoking), for modulation of appetite and/or satiety, eating disorders (e.g. binge eating, anorexia, bulimia and compulsive), cravings (for drugs, tobacco, alcohol, any appetizing macronutrients or non-essential food items), for the treatment of psychiatric disorders such as psychotic and/or mood disorders, schizophrenia and schizo-affective disorder, bipolar disorders, anxiety, anxio-depressive disorders, depression, mania, obsessive-compulsive disorders, impulse control disorders (e.g., Gilles de Ia Tourette's syndrome), attention disorders like ADD/ ADHD, stress, and neurological disorders such as dementia and cognitive and/or memory dysfunction (e.g., amnesia, Alzheimer's disease, Pick's dementia, dementia of ageing, vascular dementia, mild cognitive impairment, age-related cognitive decline, and mild dementia of ageing), neurological and/or neurodegenerative disorders (e.g. Multiple Sclerosis, Raynaud's syndrome, Parkinson's disease, Huntington's chorea and Alzheimer's disease), demyelinisation-related disorders, neuroinflammatory disorders (e.g., Guillain-Barre syndrome). They have also been proposed for the prevention or treatment of dependence and addictive disorders and behaviours (e.g., alcohol and/or drug abuse, pathological gambling, kleptomania), drug withdrawal disorders (e.g., alcohol withdrawal with or without perceptual disturbances; alcohol withdrawal delirium; amphetamine withdrawal; cocaine withdrawal; nicotine withdrawal; opioid withdrawal; sedative, hypnotic or anxiolytic withdrawal with or without perceptual disturbances; sedative, hypnotic or anxiolytic withdrawal delirium; and withdrawal symptoms due to other substances), alcohol and/or drug-induced mood, anxiety and/or sleep disorder with onset during withdrawal, and alcohol and/or drug relapse. They have also been proposed for the prevention or treatment of neurological dysfunctions such as dystonias, dyskinesias, akathisia, tremor and spasticity, treatment of spinal cord injury, neuropathy, migraine, vigilance disorders, sleep disorders (e.g., disturbed sleep architecture, sleep apnea, obstructive sleep apnea, sleep apnea syndrome), pain disorders, cranial trauma.

They have also been proposed for the treatment of immune, cardiovascular disorders (e.g. atherosclerosis, arteriosclerosis, angina pectoris, abnormal heart rhythms, and arrhythmias, congestive heart failure, coronary artery disease, heart disease, hypertension, prevention and treatment of left ventricular hypertrophy, myocardial infarction, transient ischaemic attack, peripheral vascular disease, systemic inflammation of the vasculature, septic shock, stroke, cerebral apoplexy, cerebral infarction, cerebral ischaemia, cerebral thrombosis, cerebral embolism, cerebral hemorrhagia, metabolic disorders (e.g. conditions showing reduced metabolic activity or a decrease in resting energy expenditure as a percentage of total fat-free mass, diabetes mellitus, dyslipidemia, fatty liver, gout, hypercholesterolemia, hyperlipidemia, hypertriglyceridemia, hyperuricacidemia, impaired glucose tolerance, impaired fasting glucose, insulin resistance, insulin resistance syndrome, metabolic syndrome, syndrome X, obesity-hypoventilation syndrome (Pickwickian syndrome), type I diabetes, type II diabetes, low HDL- and/or high LDL-cholesterol levels, low adiponectin levels), reproductive and endocrine disorders (e.g. treatment of hypogonadism in males, treatment of infertility or as contraceptive, menstrual abnormalities/emmeniopathy, polycystic ovarian disease, sexual and reproductive dysfunction in women and men (erectile dysfunction), GH-deficient subjects, hirsutism in females, normal variant short stature) and diseases related to the respiratory (e.g. asthma and chronic obstructive pulmonary disease) and gastrointestinal systems (e.g. dysfunction of gastrointestinal motility or intestinal propulsion, diarrhea, emesis, nausea, gallbladder disease, cholelithiasis, obesity-related gastro-esophageal reflux, ulcers). They have also been proposed as agents in treatment of dermatological disorders, cancers (e.g. colon, rectum, prostate, breast, ovary, endometrium, cervix, gallbladder, bile duct), craniopharyngioma, Prader-Willi syndrome, Turner syndrome, Frohlich's syndrome, glaucoma, infectious diseases, urinary tract disorders and inflammatory disorders (e.g. arthritis deformans, inflammation, inflammatory sequelae of viral encephalitis, osteoarthritis) and orthopedic disorders. They have also been proposed for the treatment of (esophageal) achalasia.

The various aspects of the invention can be applied to the identification of suitable patients suffering from any of these diseases, disorders or conditions, and to whom a CBlR antagonist drug is to be administered. In a particular embodiment, it is applied to weight treatment, including the treatment or prophylaxis of obesity or being overweight, (e.g., promotion of weight loss and maintenance of weight loss, prevention of weight gain (e.g., medication-induced or subsequent to cessation of smoking), for modulation of appetite and/or satiety, for eating disorders (e.g. binge eating, anorexia, bulimia and compulsive), and for cravings (for drugs, tobacco, alcohol, any appetizing macronutrients or non- essential food items)),

According to a first aspect of the invention there is provided a method of selecting a mammal in need of weight loss and/or weight maintenance and/or prevention of weight gain treatment with a cannabinoid 1 receptor (CBlR) antagonist drug which comprises determining the level of at least one circulating endogenous cannabinoid ligand in the mammal, whereby to predict an increased likelihood of response to the CBlR antagonist drug. According to a further aspect of the invention there is provided a method of selecting a mammal in need of weight loss and/or weight maintenance and/or prevention of weight gain treatment with a CBlR antagonist drug which comprises determining the level of at least one circulating endogenous cannabinoid ligand in the mammal, whereby to predict an decreased likelihood of response to the CBlR antagonist drug.

The mammal can be any mammal, including without limitation humans, non- human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs and rodents. In a particular embodiment, the mammal is a human.

CBlR

G protein-coupled receptor (GPCR) action generally requires binding of an agonist to the receptor to elicit receptor activation. However, some receptors display spontaneous intrinsic activity without being activated by a ligand, and these are termed "constitutively active". The CBl receptor appears to be constitutively active (Bouaboula et al., J Biol Chem, 272:22330-339, (1997)). CBlR ligands are:

• CBl receptor agonists and partial agonists which activate the receptor; partial agonists activate the receptor, but with lower efficacy than a full agonist. If the efficacy is sufficiently low, partial agonists will approach the profile of a neutral antagonist.

• CBl receptor inverse agonists, which interact with the receptor and exerts the opposite effect of an agonist, thus both blocking the action of an agonist (or partial agonist) and attenuating constitutive receptor activity. Partial inverse agonists deactivate the receptor, but with lower efficacy than a full inverse agonist. As is well known and frequently used in the art, CBl receptor inverse agonists are also referred to as CBl receptor antagonists.

• CBl receptor neutral antagonists, which block the action of an agonist or inverse agonist, but do not change the constitutive receptor activity; they may also be low efficacy partial agonists that behave as neutral antagonists. As used herein, to "block" the activity of a CBlR means either to deactivate its basal constitutively active state, i.e., decrease its cellular function to less than the measured basal level in the absence of CBl receptor agonism in the particular environment in which it is found and/or to make it unable to perform its cellular function even in the presence of an exogenous CBlR agonist or a natural binding ligand. A natural binding ligand is an endogenous molecule acting as an agonist for the receptor, thus in the present case an "endocannabinoid" . Compounds useful to the present invention block the CBlR by suppressing or attenuating its natural signal function in the presence or absence of CBlR agonists including endocannabinoids. The term "cannabinoid CBl receptor antagonists" as used herein, therefore encompasses CBl receptor partial agonists, CBl receptor inverse agonists, CBl receptor inverse partial agonists and CBl receptor neutral antagonists. The inventors have discovered that mammals with high circulating levels of one or more of the endogenous cannabinoid ligand(s) have an increased likelihood of responding to a CBlR antagonist drug. The circulating levels of one or more of the endogenous cannabinoid ligand(s) may therefore be used to identify subjects suitable to be treated with an CBlR antagonist drug. The inventors have discovered that mammals with low circulating levels of one or more of the endogenous cannabinoid ligand(s) have a decreased likelihood of responding to a CBlR antagonist drug with regard to weight reduction. They may however have an increased likelihood of suffering from adverse events associated with said treatment and so can be de-selected from treatment

The link between circulating levels of an endocannabinoid and likelihood of responding to drug treatment realises the possibility of pre-selecting patients for treatment with a CBlR antagonist drug. An example of when such pre-selection would be useful is in patient selection for inclusion in a clinical trial involving a putative CBlR antagonist therapeutic drug.

According to another aspect of the invention there is provided a method of identifying one or more patients most likely to benefit from treatment with a CB IR antagonist drug comprising measuring the levels of one or more endogenous cannabinoid ligands in a blood sample previously isolated from a patient and identifying whether or not the patient is likely to benefit from CBlR antagonist treatment according to the levels present. With weight treatments, patients with high levels being more likely to benefit from weight reduction and low level patients will benefit from not having to be subjected to treatment and thus unnecessary suffering and risk from adverse effects. According to a further aspect of the invention there is provided a method of identifying whether or not an obese or overweight patient would benefit from or respond to treatment with a CBlR antagonist comprising determining the level of at least one circulating endogenous cannabinoid ligand in the patient and based on the level present determining whether or not the patient is likely to benefit from or respond to treatment with a CBlR antagonist drug.

The various aspects of the invention can be used in conjunction with any suitable CBlR antagonist. The following is a representative, non-limiting list of publications that disclose CBlR antagonist compounds and their uses. It is known that certain CBlR antagonists are useful in the treatment of obesity, psychiatric and neurological disorders (WO01/70700, EP 658,546 and EP 656,354). Pyrazoles having anti-inflammatory activity are disclosed in WO 95/15316, WO96/38418, WO97/11704, WO99/64415, EP 418 845 and WO2004050632. l,5-Diarylpyrazole-3-carboxamide derivatives are disclosed in US 5,624,941, WO01/29007, WO2004/052864, WO03/020217, US 2004/0119972, Journal of Medicinal Chemistry, 46(4):642-645 2003, Bioorganic & Medicinal Chemistry Letters, 14(10):2393-2396 2004, Biochemical Pharmacology, 60(9): 1315-1323 2000, Journal of Medicinal Chemistry, 42(4):769-776 1999 and U.S. Pat. Appl. Publ. US 2003199536.

WO 03/007887 and WO03/075660 disclose certain 4,5-diarylimidazole-2- carboxamide CBl receptor antagonists. WO03/27076 and WO 03/63781 disclose certain l,2-diarylimidazole-4-carboxamide CBl receptor antagonists. WO03/40107 and WO2006/067443 disclose certain 1 ,2-diarylimidazole-4-carboxamides as being useful in the treatment of obesity and obesity-related disorders. l,5-Diarylpyrazole-3-carboxamide derivatives having a fluorinatedalkylsulphonyloxy phenyl substituent are disclosed in WO2005/080343 and WO2006/067428. WO2005/ 095354 and WO2007/031720 disclose certain 1 ,2-diarylimidazole-4-carboxamides having a fluorinatedalkylsulphonyloxy phenyl substituent as being useful in the treatment of obesity and obesity-related disorders.

WO2007039740 (PCT/GB2006/003695) discloses 4, 5, 6, 7 -tetrahydropyrrole [3,2- c]pyridin-4-one and 4, 5 -dihydropyrrolo[3,2-c]pyridin-4-one compounds and processes for preparing such compounds, their use as CBl antagonists in the treatment of obesity, psychiatric and neurological disorders, to methods for their therapeutic use and to pharmaceutical compositions containing them. In one embodiment, the CBlR antagonist is a CBlR antagonist as described in the patent references listed earlier. A particular CBlR antagonist is 'rimonabant' (aka SRl 41716 /Acomplia^® ; (5-(4-chlorophenyl)- 1 -(2,4-dichlorophenyl)-4-methyl-N-( 1 - piperidinyl)-lH-pyrazole-3-carboxamide, CAS NO: 158681-13-1) which is described in EP 656,354 Further particular CB IR antagonists include 'taranabant' ((MK-0364) described in J. Med. Chem. 2006, 49 7584 and references therein); CP945598 (l-[9-(4- chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethylaminopiperidine-4-carboxylic acid amide HCl; disclosed in WO2005 049615), SR147778 (SR147778 [5-(4- bromophenyl)- 1 -(2,4-dichlorophenyl)-4-ethyl-N-( 1 -piperidiny I)- 1 H-pyrazole-3 - carboxamide], 'surinabant'; disclosed in J Pharmacol Exp Ther. 2004 Sep;310(3):905- 914), AVE-1625 ((N- { l-[Bis-(4-chlorophenyl)-methyl]-azetidin-3-yl}-N-(3,5- difluorophenyl)-methylsulfonamide; disclosed in WO2006 040464) and SLV319 (disclosed in J. Med. Chem 2004, 47, 627-643).

Other CBlR antagonists are disclosed in Hertzog (Expert Opinion Therapeutic Patents 14(10): 1435-1452, 2004).

In one embodiment the CBlR antagonist is a compound of formula (I)

I in which

R¹ represents a C3-6alkyl group optionally substituted by one or more fluoro; R² represents H and R³ represents cyclohexyl optionally substituted by hydroxy or R¹ and R together with the nitrogen atom to which they are attached represent a piperidine ring which is optionally substituted by hydroxy;

represents a group of formula a, b or c

a b _or c in which the bond marked * is attached to the phenyl ring carrying the sulphonyloxy group and the other bond marked # is attached to NR²R³; is an optional additional bond between positions 6 and 7; R⁴ and R⁵ independently represent H, bromo, chloro or fluoro; and

R⁶ represents methyl or hydroxymethyl; n and m independently represent 0 orl; or a pharmaceutically acceptable salt thereof.

In another embodiment, the CBlR antagonist is a compound selected from: 1-propanesulfonic acid, 3,3,3-trifluoro-, 4-[l-(2,4-dichlorophenyl)-3-[[(2- hydroxycyclohexyl)amino]carbonyl]-4-(hydroxymethyl)-lH-pyrazol-5-yl]phenyl ester;

1-propanesulfonic acid, 3,3,3-trifluoro-, 4-[3-[(cyclohexylamino)carbonyl]-l-(2,4- dichlorophenyl)-4-(hydroxymethyl)-lH-pyrazol-5-yl]phenyl ester;

1-propanesulfonic acid, 3,3,3-trifluoro-, 4-[l-(2,4-dichlorophenyl)-4,5,6,7-tetrahydro-3- methyl-4-oxo-5-(l-piperidinyl)-lH-pyrrolo[3,2-c]pyridin-2-yl]phenyl ester;

1-propanesulfonic acid, 3,3,3-trifluoro-, 4-[l-(2,4-dichlorophenyl)-4-methyl-3-[(l- piperidinylamino)carbonyl]-lH-pyrazol-5-yl]phenyl ester;

1 -propanesulfonic acid, 4-[ 1 -(2,4-dichlorophenyl)-4-methyl-3-[(l - piperidinylamino)carbonyl]-lH-pyrazol-5-yl]phenyl ester; 3,3,3-trifluoropropane-l-sulfonic acid , 4-[2-(2,4-dichlorophenyl)-5-methyl-4-(piperidin-l- ylcarbamoyl)imidazol-l-yl]phenyl ester; or

3, 3,3-trifluoropropane-l -sulfonic acid 4-[l-(2-chloro-4-fluorophenyl)-3-methyl-4-oxo-5- piperidin-l-yl-4,5,6,7-tetrahydro-lH-pyrrolo[3,2-c]pyridin-2-yl]phenyl ester or a pharmaceutically acceptable salt thereof.

It is contemplated that any reasonably potent CBlR antagonist (with a Ki value of <10μM in a GTPγS assay using CP-55940 as agonist ligand (Ryberg et al. FEBS Letters, 579:259-264 (2005)), would be eligible for the proposed personalised medicine approaches of the invention.

Endocannabinoids

Endogenous cannabinoids (endocannabinoids), are unsaturated fatty acid derivatives that act as ligands for the cannabinoid receptors (DiMarzo Biochim Biophys Acta 1392:153-157, 1998). They include arachidonic acid metabolites such as: anandamide (aka arachidonoyl ethanolamide; AEA), 2-arachidonoylglycerol (2-AG) and noladin ether (for review see Cannabinoid physiology and pharmacology: 30 years of progress (Howie tt et al., Neuropharmacology 47:345-358, 2004). Virodhamine, palmitoylethanolamide and oleoylethanolamide are recent additions to an increasing number of characterised endocannabinoids. Furthermore, there are also ethanolamides of long chain fatty acids

(C 16 - C22) identified as endocannabinoids which binds to the CBl receptor. Examples of such compounds are, but not limited to, Dihomo-γ-linolenoyl ethanolamide, Arachidonoyl Ethanolamide, Dihomo-γ-Linolenic Acid methyl ester, Docosatetraenoyl Ethanolamide, Leukotriene B4 Ethanolamide, Prostaglandin D2 Ethanolamide, Prostaglandin El Ethanolamide, Prostaglandin E2 Ethanolamide, R-I Methanandamide, R-2

Methanandamide, R-Palmitoyl-(2 -methyl) Ethanolamide, S-I Methanandamide, Stearoyl Ethanolamide, Docosahexaenoyl Ethanolamide, Linoleoyl ethanolamide, 1-Linoleoyl Glycerol, α-Linolenoyl Ethanolamide, Mead acid ethanolamide, . The levels of any of these endocannabinoids may serve to predict susceptibility to response to a CBlR antagonist according to the invention.

In a particular embodiment, the endocannabinoid for use in the invention is selected from the group consisting of: anandamide (arachidonoyl ethanolamide (AEA)), 2- arachidonoylglycerol (2-AG), noladin ether, palmitoylethanolamine, virodhamine and oleoyl ethanolamide. Arachidonoyl ethanolamide (AEA) and 2-arachidonoylglycerol (2- AG) are particularly useful biomarkers of susceptibility to CBlR antagonists. Data interpretation

The various aspects of the invention rely on the determination of the levels of at least one endocannabinoid. In one embodiment, at least one of the endogenous cannabinoid ligands being measured/detected is AEA or 2-AG. In another embodiment both AEA and 2-AG are measured.

The inventors have found that there is a predictive potential in the amount of endogenous cannabinoid ligands circulating in the blood for determining the effect of CBlR antagonist treatment. They have found patient selection methods of the invention work based on the levels of a single endocannabinoid (e.g. AEA or 2-AG). See Figures 1 and 2 herein. However, superior results, in terms of more accurate prediction or patient stratification, may be obtained by mathematically combining the data from two or more of the endocannabinoids. See, for example Figures 3 and 4 herein. This type of mathematical construct is defined herein as a biomarker composite score or composite score. The composite score may include adjustments for other relevant covariates, such as age or gender or normal weight gain.

The biomarker composite score can be constructed in many ways. A linear combination of endocannabinoids can be written as:

∑K₀ + K, * L, (1) ι=l

where L₁ is the endocannabinoid ligand i, K₁ is the corresponding coefficient and Ko is the intercept. The class of models described by this equation even includes those where certain of the L₁ refer to endocannabinoid ligands, while other L₁ denote covariates such as age or gender.

More complex biomarker composite scores can be constructed by adding cross and squared terms to (1). Below is the equation for first and second degree (squared) terms, but cubic or higher terms could easily be added.

±(K₀ +K₁ ^♦!,) + £ ∑X * L₁ *L_J + ±K_u ⁺L] (2)

I=I i=l j=ι+l i=l Two examples of different biomarker composite scores are shown in Figures 3 and 4. In Figure 3, a linear combination of AEA and 2-AG was used. In Figure 4, the cross (multiplier) term between AEA and 2-AG was added. These additions enhanced the performance of the prediction. The choice of which particular mathematical model to use with the endocannabinoid data can be selected or identified by the person of skill in the art.

The detected amount of the endocannabinoid can be compared to control subjects. This could be carried out on control subjects at the same time as the test subjects, or it could be historical control data. The historical control values (predetermined reference values) can be derived from measurements of the endocannabinoid(s) in comparable biological samples taken from the general population or a selected population of mammalian subjects, such as an overweight or obese population. Such pre-determined reference values may be the culmination of data from large samples. A difference in the amount of the biomarker between the subject's sample, either the direct value or composite score, and the predetermined value or composite score can, for example, be used to stratify the patient into a likely responder or non-responder groups. Alternatively, the value or level or amount of each endocannabinoid, or the composite score generated therefrom, in the circulation of a particular patient from a test group of patients (e.g. a panel of potential patients for inclusion in a clinical trial) can be compared to those of the other group members.

In a particular embodiment, said group of patients are overweight or obese subjects. In a particular embodiment, if the patient has high circulating levels of endogenous cannabinoid ligand(s) they are more likely to benefit from CBlR antagonist treatment than a patient with low levels. In other embodiments, if the endocannabinoid level/levels (or composite score) places the patient/individual in, increasing order of preference, in the upper 50^th, 40^th, 30^th, 20^th, 15^th, 10^th or 5^th percentile, the patient/individual is characterised or identified as one more likely to benefit from CBlR antagonist treatment and/or is treated with a CBlR antagonist drug. In one embodiment, if the levels of circulating endogenous cannabinoid ligand(s) are in the upper 30^th percentile (top 30%), or the composite score places the patient in the upper 30^th percentile range, the patient is treated with an CBlR antagonist. The methods are particularly suitable for assessing the likelihood of response to a CBlR antagonist for treating weight disorders.

In other embodiments, if the endocannabinoid level/levels (or composite score) places the patient/individual in, increasing order of preference, in the lower 40^th, 30^th, 20^th, 15^th, 10^th or 5 ^th percentile, the patient/individual is characterised or identified as one less likely to benefit from CBlR antagonist treatment.

As used herein, overweight and obese subject are identified based on body mass index (BMI) values set by the World Health Organisation (WHO). At present, an overweight person is one who has a BMI > 25 kg/m² and an obese person is one with a BMI > 30 kg/m². However, it is known that the overweight and obese BMI cut-off values may be changed in the future. In addition, persons at risk for cardiovascular disease that would benefit from a body weight reduction could in addition to BMI also or preferably be defined based on the amount of visceral adipose tissue and/or waist circumference and/or waist-to-hip ratio. The cut-off values differ for gender and for ethnical groups.

Detection methods

The endocannabinoids are unsaturated fatty acid derivatives and any method or technique capable of detecting the amount of endocannabinoid, either directly or indirectly, in a blood sample or fraction thereof (e.g. plasma or serum) can be employed in aspects of the invention.

Some conventional techniques for use in the methods of the invention include mass spectrometry, chromatographic separations, displacement methods, binding assays (e.g., immunoassays), competitive inhibition assays, and so on. Any effective method in the art for measuring the level of a lipid or low molecular weight marker is included in the invention. It is within the ability of one of ordinary skill in the art to determine which method would be most appropriate for measuring a specific marker. Thus, for example, a robust ELISA assay may be best suited for use in a physician's office while a measurement requiring more sophisticated instrumentation may be best suited for use in an analytical laboratory. Regardless of the method selected, it is important that the measurements are reproducible. The endocannabinoid markers of the invention can be measured by mass spectrometry, which allows direct measurements of analytes with high sensitivity and reproducibility. A number of mass spectrometric methods are available and could be used to accomplish the measurement. Electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photo ionization (APPI) are different ionization techniques applicable for endocannabinoids. These techniques can be used on single- (MS) or triple-quadrupole (MS/MS) instruments that allow quantification of differences in relative concentration of various species in one sample against another and, by using appropriate internal standards, also absolute quantification of the endocannabinoids present. Matrix-assisted laser desorption ionization (MALDI) or the related SELDI technology also could be used to make a determination of whether a endocannabinoid was present, and the relative or absolute level of the endocannabinoid. Moreover, time-of-flight (TOF) mass spectrometers and Fourier transform mass spectrometers (FTMS) are capable of extremely high resolving power which allow measurements of high accuracy and resolution of low abundant species.

The endocannabinoid markers can be measured using mass spectrometry in connection with a separation technology, such as liquid chromatography-mass spectrometry (LC/MS, LC/MS/MS, LC/MSⁿ) or gas chromatography-mass spectrometry (GC/MS, GC/MS/MS, GC/MSⁿ), and the analytes in a sample can be specified both by a specific retention time and mass-to-charge ratio m/z.

As will be appreciated by one of skill in the art, many other separation technologies may be used in connection with mass spectrometry. For example, a vast array of separation columns is commercially available. In addition, separations may be performed using custom chromatographic surfaces (e.g., a bead on which a marker specific reagent has been immobilized). Molecules retained on the media subsequently may be eluted for analysis by mass spectrometry.

Analysis by liquid chromatography-mass spectrometry produces a mass intensity spectrum, the peaks of which represent various components of the sample, each component having a characteristic mass-to-charge ratio (m/z) and retention time (r.t). The presence of a peak with the m/z and retention time of a biomarker indicates that the marker is present. The peak representing a marker may be compared to a corresponding peak from another spectrum (e.g., from a control sample) to obtain a relative measurement. Any normalization technique in the art (e. g., an internal standard) may be used when a quantitative measurement is desired. The retention time depends to some degree on the conditions employed in performing the liquid chromatography separation.

The markers of the invention may also be detected or measured using a number of chemical derivatization or reaction techniques known in the art. Detection may then be performed using detectors other than mass spectrometry, such as fluorescence detection of tagged molecules, nuclear magnetic resonance (NMR), capillary UV, evaporative light scattering or electrochemical detection.

Other suitable methods that may be employed include radioimmunoassay, enzyme - linked immuno-sorbent assays (ELISA/EIA), and sandwich assays, see U.S. Patent Nos. 4,376,110 and 4,486,530.

In other embodiments, the level of the endocannabinoid markers may be determined using a standard immunoassay, such as sandwiched enzyme-linked immunosorbent assay (ELISA) using matched antibody pairs and, for example, chemiluminescent detection. See also "A Practical Guide to ELISA" by D. M. Kemeny, Pergamon Press, Oxford, England. Commercially available or custom monoclonal or polyclonal antibodies are typically used. However, the assay can be adapted for use with other reagents that specifically bind to the marker. Standard protocols and data analysis are used to determine the marker concentrations from the assay data. A number of the assays discussed above employ a reagent that specifically binds to the endocannabinoids marker ("marker specific reagent"). Any molecule that is capable of specifically binding to a marker is included within the invention. In some embodiments, the marker specific reagents are antibodies or antibody fragments. In other embodiments, the marker specific reagents are non-antibody species. The term "antibodies" is meant to include polyclonal antibodies, monoclonal antibodies, and the various types of antibody constructs such as for example F(ab')₂, Fab and single chain Fv. Affinity of binding can be determined using conventional techniques, for example those described by Scatchard et al, Ann. N.Y. Acad. ScL, (1949) 51:660. Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice or rats, using procedures that are well-known in the art. In general, antigen is administered to the host animal typically through parenteral injection. The immunogenicity of antigen may be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant. Following booster immunizations, small samples of serum are collected and tested for reactivity to antigen.

Monoclonal antibodies may be readily prepared using well-known procedures, see for example, the procedures described in U.S. Patent Nos. RE 32,011; 4,902,614; 4,543,439 and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.), (1980).

A marker specific reagent may be identified and produced by any method accepted in the art. Methods for identifying and producing antibodies and antibody fragments specific for an analyte are well known. Examples of other methods used to identify marker specific reagents include binding assays and design methods based on an analysis of the structure of the marker.

According to one embodiment, the amount of circulating endocannabinoid ligand is detected by immunoassay. Particular immunoassay techniques that can be employed include enzyme linked immunosorbant assay (ELISA/EIA) and radio immuno assay (RIA).

In certain embodiments of the invention, the endocannabinoid levels may be detected indirectly. For example, the enzyme fatty acid amide hydrolase (FAAH) degrades AEA, and it is believed that reduced levels of FAAH correlate with elevated levels of AEA. Detection of levels of FAAH may therefore be considered indicative of levels of AEA.

Similarly, detection of monoacylglycerol lipase, the degrading enzyme of 2-AG, may be considered indicative of levels of 2-AG.

As noted above, the various aspects of the invention can be applied to select patients for inclusion in a clinical trial, in particular a clinical trial of a CBlR antagonist drug.

According to another aspect of the invention there is provided a method of stratifying a group of patients to identify one or more patients suitable for treatment with a CBlR antagonist drug, comprising determining the amount or level of one or more circulating endogenous cannabinoid ligands present in blood previously isolated from the patients.

In one particular embodiment, the group of patients are overweight or obese subjects. According to another aspect of the invention there is provided a method of inducing weight loss and/or weight maintenance and/or prevention of weight gain in a patient in need thereof, comprising determining the level of a one or more circulating endogenous cannabinoid ligands in said patient and selecting an appropriate therapy based on the level of endogenous cannabinoid ligand(s) detected.

According to one particular embodiment, if the patient has high levels of circulating endogenous cannabinoid ligand(s) they are treated with a CBlR antagonist drug. Aspects of the invention also provide the use of an endocannabinoid in a method for assessing the likelihood of effective treatment with a CBlR antagonist.

According to another aspect of the invention there is provided the use of circulating levels of an endogenous cannabinoid ligand as a biomarker of susceptibility to effective weight loss or appetite suppression treatment with a CBlR antagonist drug. According to another aspect of the invention there is provided the use of circulating levels of one or more endogenous cannabinoid ligand(s) to select patients for inclusion in a CBlR antagonist drug clinical trial or select patient for treatment with a CBlR antagonist drug.

In one embodiment, the patients for inclusion in the trial or for treatment with the CBlR antagonist drug have circulating levels of the endocannabinoid ligand(s) that positions the patient in, increasing order of preference, the upper 50^th, 40^th, 30^th, 20^th, 15^th, 10^th or 5^th percentile of the initial group.

In a particular embodiment, the initial group are overweight or obese patients.

The various aspects of the invention rely on the measurement of the levels of one or more endocannabinoids in the circulatory (blood) system. It will be apparent to the person skilled in the art that such measurements can be made on whole blood, or a blood fraction, such as a serum or plasma fraction.

The blood sample could be taken just before the above measurement, but it could also be a blood sample that has been taken from the subject in question a long time before and have been stored in an appropriate way prior to the above measurement. It may also be necessary to use appropriately standardised protocols for measurement of endocannabinoid ligand values. For example, Vogeser et al. (Clin Chem and Lab Medicine 44(4):488-491, 2006) investigated the impact of different sample handling conditions on measured plasma anandamide concentrations and found that anandamide is released from blood cells ex vivo at a very high rate. Storage of blood isolated from a test patient for differing periods of time, and/or under different conditions relative to those employed on other samples may results in skewed data on the actual levels of the circulating endocannabinoid.

For the avoidance of doubt, the methods of the invention may not involve diagnosis practised on the human body. The methods of the invention are preferably conducted on a sample that has previously been removed from the individual. The kits of the invention, however, may include means for extracting the sample from the individual. The invention also provides a kit for use in identifying the likely response that a mammal, in particular a human, will have upon administration of a CBlR antagonist, the kit comprising reagents for detection of levels of an endocannabinoid in a sample, particularly a blood sample. The kit may further comprise instructions for use of the reagents. The reagents may comprise antibodies or antibody fragments against the endocannabinoid, or any other binding member capable of binding to the endocannabinoid. Further the antibody or antibody fragment or binding member may be suitably labelled, for example with a fluorescent or radioactive label. Further the kit may comprise means for taking the blood sample from a subject mammal; for example a syringe, swabs, ethanol wipes, sterile tubes and the like.

According to a further aspect of the invention there is provided a method of treating a patient in need of treatment with a CBlR antagonist comprising measuring the amount of one or more endocannabinoids in the circulatory system of the patient, comparing said amount with a predetermined reference value and if the measured amount is higher than the predetermined reference value administering to said patient of a suitable amount of a CBlR antagonist. According to a further aspect of the invention there is provided the use of a CBlR antagonist in the treatment of obese or overweight patients identified as having a high level of at least one circulating endogenous cannabinoid ligand relative to the obese or overweight population as a whole.

According to a further aspect of the invention there is provided the use of a CBlR antagonist in the manufacture of a medicament for the treatment of obese or overweight patients identified as having a high level of at least one circulating endogenous cannabinoid ligand relative to the obese or overweight populations as a whole. The CBlR antagonist compound for use in treating a patient according to the invention will normally be administered via the oral, parenteral, intravenous, intramuscular, subcutaneous or in other injectable ways, buccal, rectal, vaginal, transdermal and/or nasal route and/or via inhalation, in the form of pharmaceutical preparations comprising the active ingredient or a pharmaceutically acceptable addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated and the route of administration, the compositions may be administered at varying doses. Suitable daily doses of the CBlR antagonist compound, for use in the therapeutic treatment of humans according to the methods of the invention, are about 0.001-10 mg/kg body weight, preferably 0.01-3 mg/kg body weight.

Oral formulations are preferred particularly tablets or capsules which may be formulated by methods known to those skilled in the art to provide doses of the active compound in the range of 0.5mg to 500mg for example 1 mg, 2 mg, 4 mg, 6 mg, 10 mg, 15mg, 20mg, 25mg, 50mg, lOOmg and 300mg.

The invention will be further described with reference to the following non-limiting Example and Figures in which:

Figure 1 shows loss of body weight upon CBl receptor antagonist (rimonabant) treatment as a function of the pre -treatment level of AEA. The dashed line represents the least-squares linear regressionfit to the data (p=0.031; r =0.15).

Figure 2 shows loss of body weight upon CBl receptor antagonist (rimonabant) treatment as a function of the pre -treatment level of 2-AG. The dashed line represents the least-squares linear regressionfit to the data (p=0.0013; r²=0.31). Figure 3 is a scatter plot showing loss of body weight upon CBl receptor antagonist

(rimonabant) treatment on the vertical axis and biomarker composite score, as assessed by the equation combining the AEA and 2-AG pre-treatment levels, on the horizontal axis. The dashed line represents the least-squares linear regression fit to the data (p=8.2e-05; r²=0.50). Note that here the x-values are equivalent to fitted values of a regression fit to the y-axis. Figure 4 is a scatter plot showing loss of body weight upon CBl receptor antagonist (rimonabant) treatment on the vertical axis and biomarker composite score, as assessed by the equation combining the AEA and 2-AG pre-treatment levels, on the horizontal axis. The dashed line represents the least-squares linear regression fit to the data (p=1.5e-06; r²=0.68). Note that here the x-values are equivalent to fitted values of a regression fit to the y-axis.

Examples: Example 1 The study was carried out in male rats of the Sprague-Dawley strain (Rheoscience,

Ledøje, Denmark). The rats were housed individually (1 rat/cage) under a normal light cycle (lights on 6 AM-6 PM) at controlled temperature conditions with ad lib. access to water and a high energy diet (4.41 kcal/g - energy content %: carbohydrate 51.4 kcal %, fat 31.8 kcal %, protein 16.8 kcal %; diet #12266B; Research Diets, New Jersey, USA). For the experiment, 15+15 subjects were used. These were selected from a cohort of rats selectively bred to display either an enhanced likelihood of developing diet- induced obesity (DIO), or an enhanced likelihood of being resistant to diet- induced obesity (DR). Animals for the study were selected so that body weights of each group displayed the largest possible variation (weight ranges at study start: DIO 392-626g and DR 395-459g). The rats were 19 and 20 weeks old, respectively, at initiation of the study, a time point at which they had received access to the high-energy diet (cf. above) for 15 and 16 weeks, respectively.

The animals received one gavage administration of the CBl receptor antagonist (/inverse agonist) rimonabant (5-(4-chlorophenyl)-l-(2,4-dichlorophenyl)-4-methyl-N-(l- piperidinyl)-lH-pyrazole-3-carboxamide, CAS NO: 158681-13-1), at lOμmoles/kg daily at 2 pm (9 hours into the light phase). The dose chosen was in the range known from the literature to decrease appetite and body weight in obese rodents (e.g., Colombo et al., Life Sci.;63:PLl 13-7, 1998). ). Dosing volume was kept at 5 ml/kg throughout the study. The experiment was preceded by a 3 -day run-in period with mock gavages. From day -1 and onwards, 24-hour food intake and body weight was measured twice weekly. Two days (day -2) prior to the beginning of the dosing period and again 14 days after dosing, food was withdrawn at 6 am and eight hours later (three hours before the end of the light phase) fasting blood samples were collected. Blood samples were collected by tail cut in one 1.2 ml EDTA tube, one 500 μl EDTA tube and one 300 μl Heparin tube per animal. All tubes were pre-chilled on ice and returned to ice immediately after sampling. Blood was centrifuged, starting maximum 5 minutes after sampling, at a speed corresponding to 2000 RCF in the bottom of the tubes for 10 minutes, in order to minimize release of endocannabinoids from blood cells (Vogeser et al, Clin Chem Lab Med. 44(4):488-491 (2006)). Plasma from the top ³A was then pipetted off into new pre-chilled 0.5 ml polypropylene microtubes (0.5 ml, 30x8 mm 0, SARSTEDT, Germany) and immediately frozen at -80 degrees Celsius (250 μl + 100 μl from the 1.2 ml EDTA tube, 100 μl + 100 μl from the 500 μl EDTA tube and 100 μl from the 300 μl Heparin tube).

To 100 μl plasma was added 10 ng AEA-D8 and 100 ng 2-AG-D5 (Cayman Chemical, Ann Arbor, Mi, USA) as internal standards and 600 μl methyl tert-butyl ether / isohexane (50:50, by vol). After 10 min of extraction, the supernatant was transferred to new tubes, evaporated to dryness under nitrogen, dissolved in 50 μl 75% acetonitril in water and placed in the refrigerated autosampler (10 ⁰C). 20 μl was then injected on a HyPurity C18 column (50*2.1 mm, 3 μm) (Thermo Electron Corp.). Gradient mobile phases consisted of A (63 % water, 5 mM ammonium acetate, 0.02 % formic acid and 37% acetonitril) and B (1 % water, 5 mM ammonium acetate, 0.02% formic acid, 50 % acetonitril, 49 % 2-propanol) delivered at 0.3 ml/min. A linear gradient from 40% B to 90% B in 5 min. was used. The tandem MS was operated in electrospray positive multiple reaction monitoring mode at a source temperature of 120⁰C, desolvation temperature of 450⁰C and a cone voltage of 27 V. Cone and desolvation gas flows were set to 100 and 900 L/hr. The transitions monitored were m/z 348 to 62 for AEA, m/z 356 to 63 for AEA-D8, m/z 379 to 287 for 2-AG and m/z 384 to 287 for 2-AG-D5. The collision energy used was 16 eV for all analytes.

Arachidonoyl ethanolamide (AEA) and 2-arachidonoyl glycerol (2-AG) were analyzed in plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS). A Quattro Premier XE mass spectrometer (Waters) was used in conjunction with an HPl 100 LC pump (Agilent Technologies) and a Waters 2111 C Sample Manager. The same methodology was also used for analysis of other endocannabinoids like oleoyl ethanolamide (OEA) and palmitoyl ethanolamide (PEA).

At the time of blood sampling (days -2 and 14), the rats were weighed. In order to isolate the treatment effect, body weight data was pre-processed by subtracting the natural weight gain for the rats being about 19 weeks old. A normal daily weight gain of 3.1 g and 1.8 g was used for the DIO and DR rats, respectively.

Baseline levels for two of the endocannabinoids were highly correlated to treatment response measured as body weight reduction during the treatment period. Both levels of AEA (Figure 1) and 2-AG (Figure 2) predicted the body weight reduction. Combining the levels of AEA and 2-AG into a biomarker composite score further improved the prediction of treatment outcome (Figure 3). To decide systematically which terms to include in the biomarker composite score or, in other words, how to rank all possible linear regression models with various subset combinations of AEA, 2-AG, OEA and PEA, we used a branch-and-bound algorithm (Furnival and Wilson, Technometrics, 42 (l):69-79, 2000). The model, or biomarker composite score, was then selected using the model with the lowest Bayesian Information Criterion (BIC, Schwartz, Ann Stat, 6:461-464, 1978) value. The BIC value is a statistical criterion for model selection. The BIC is a decreasing function of the residual sum of squares (the goodness of fit), and an increasing function of the number of variables included. The resulting model is shown in Figure 4. In addition to the previous model, the final composite score includes a cross term between AEA and 2- AG. In other settings, we might choose an alternative method of model selection that did not employ either BIC or all subsets regression.

Linear regression modelling and the branch-and-bound algorithm (in the leaps package) were performed using the software R, version 2.4.1 (www.R-project.org). As an example of responders and non-responders, rats with the highest quartile of the final biomarker composite score had a mean body weight decrease of 61.8 ± 4.2 g and rats with the lowest quartile of the biomarker composite score had a mean body weight decrease of 8.3 ± 3.1 g (mean±SEM). Embodiments

1. A method of selecting a mammal in need of weight loss and/or weight maintenance and/or prevention of weight gain treatment with a cannabinoid 1 receptor (CBlR) antagonist drug which comprises determining the level of at least one circulating endogenous cannabinoid ligand in the mammal, whereby to predict an increased likelihood of response to the CBlR antagonist drug.

2. The method according to embodiment 1, wherein the mammal is a human.

3. The method according to embodiment 1, wherein the CBlR antagonist is an inverse agonist.

4. The method according to embodiment 1, wherein the CBlR antagonist is a neutral antagonist.

5. The method according to any of embodiments 1 to 4, wherein if the mammal has high circulating levels of the endogenous cannabinoid ligand(s) they have an increased likelihood of responding to a CBlR antagonist drug.

6. A method of identifying patients most likely to benefit from treatment with a CBlR antagonist drug comprising measuring the levels of one or more endogenous cannabinoid ligands in a blood sample previously isolated from a patient and identifying whether or not the patient is likely to benefit from CBlR antagonist treatment according to the levels present.

7. The method according to any of embodiments 1 - 6, wherein at least one of the endogenous cannabinoid ligands is AEA or 2-AG.

8. The method according to any of embodiments 1 - 7,wherein the levels of at least two endogenous cannabinoid ligands are determined. 9. The method according to any of embodiments 1 - 8, wherein the CBlR antagonist drug is selected from: rimonabant, taranabant, surinabant, CP945598 and SLV319.

10. The method according to embodiment 9, wherein the drug is rimonabant.

11. The method according to embodiment 6, wherein if the patient has high circulating levels of endogenous cannabinoid ligand(s) they are likely to benefit from CBlR antagonist treatment.

12. The method according to embodiment 6, wherein if the patient has low circulating levels of endogenous cannabinoid ligand(s) they are less likely to benefit from CBlR antagonist treatment.

13. The method according to embodiment 6, wherein if the levels of circulating endogenous cannabinoid ligand(s) are in the upper 30^th percentile, or the composite score places the patient in the highest 30^th percentile range, the patient is treated with a CBlR antagonist.

14. The method according to embodiment 6, wherein if the levels of circulating endogenous cannabinoid ligand(s) are in the lower 30^th percentile, or the composite score places the patient in the lowest 30^th percentile range, the patient will not benefit and/or benefit less from treatment with a CBlR antagonist.

15. The method according to embodiment 6, wherein the group of patients are overweight or obese subjects.

16. A method according to any of embodiments 1 - 15, wherein the amount of circulating endocannabinoid ligand is detected using mass spectrometry.

17. The method according to embodiment 16, which uses liquid chromatography mass spectrometry (LC/MS, LC/MS/MS, LC/MSⁿ) or gas chromatography mass spectrometry (GC/MS, GC/MS/MS, GC/MSⁿ). 18. A method according to any of embodiments 1 - 17, wherein the amount of circulating endocannabinoid ligand is detected following derivatization with fiuorogenic reagents for chromatographic systems.

19. A method according to any of embodiments 1 - 18, wherein the amount of circulating endocannabinoid ligand is detected by a fluorescence displacement assay.

20. A method according to any of embodiments 1 - 19, wherein the amount of circulating endocannabinoid ligand is detected by immunoassay.

21. The method according to embodiment 20, wherein the immunoassay is enzyme linked immunosorbent assay (ELISA/EIA), radio immunoassay (RIA), fluorescence immunoassay, luminescence immunoassay, electrochemical luminescence immunossay or SELDI-based immunoassay in combination with mass spectrometry.

22. The method according to any of embodiments 1 - 21, wherein the amount of circulating endocannabinoid ligand is detected by using the AEA degrading enzyme, fatty acid amide hydrolase (FAAH) and/or the 2-AG degrading enzyme, monoacylglycerol lipase.

23. The method according to any of embodiments 1 - 22,wherein the method is used to select patients for inclusion in a clinical trial.

24. A method of stratifying a group of patients to identify one or more patients suitable for treatment with a CBlR antagonist drug, comprising determining the amount or level of one or more circulating endogenous cannabinoid ligands present in blood previously isolated from the patients. 25. The method according to embodiment 24, wherein the group of patients are overweight or obese subjects.

26. A method of inducing weight loss and/or weight maintenance and/or weight gain in a patient in need thereof, comprising determining the level of a one or more circulating endogenous cannabinoid ligands in said patient and selecting an appropriate therapy based on the level of endogenous cannabinoid ligand(s) detected.

27. The method according to embodiment 26, wherein if the patient has high levels of circulating endogenous cannabinoid ligand(s) they are treated with a CBlR antagonist drug.

28. Use of circulating levels of an endogenous cannabinoid ligand as a biomarker of susceptibility to effective weight loss or appetite suppression treatment with a

CBlR antagonist drug.

29. Use of circulating levels of one or more endogenous cannabinoid ligand(s) to select patients for inclusion in a CBlR antagonist drug clinical trial or select patient for treatment with a CBlR antagonist drug.

30. Use according to embodiment 29, wherein the patients for inclusion in the trial or for treatment with the CBlR antagonist drug have circulating levels of the endocannabinoid ligand(s) that positions the patient in the upper 50^th percentile of the initial group.

31. Use according to embodiment 29, wherein the patients for inclusion in the trial or for treatment with the CBlR antagonist drug have circulating levels of the endocannabinoid ligand(s) that positions the patient in the upper 40^th percentile of the initial group. 32. Use according to embodiment 29, wherein the patients for inclusion in the trial or for treatment with the CBlR antagonist drug have circulating levels of the endocannabinoid ligand(s) that positions the patient in the upper 30^th percentile of the initial group.

33. The use according to any of embodiments 29 to 32, wherein the initial group are overweight or obese patients.

Claims

1. Use of an endogenous cannabinoid ligand as a biomarker of suitability for treatment with a cannabinoid 1 receptor (CBlR) antagonist drug.

2. The use as claimed in claim 1 , wherein the endogenous cannabinoid ligand is used as a biomarker of susceptibility to effective weight loss or appetite suppression treatment with a CBlR antagonist drug.

3. The use as claimed in claim 1 or 2, wherein the levels of the endogenous cannabinoid ligand is measured in a blood sample previously isolated from a patient and the levels present indicate whether or not the patient is likely to benefit from CBlR antagonist treatment.

4. The use as claimed in claim 3, wherein the patient is selected from a group of patients that are overweight or obese subjects.

5. The use as claimed in claim 2, wherein if the patient has high circulating levels of endogenous cannabinoid ligand(s) they are likely to benefit from CBlR antagonist treatment.

6. The use as claimed in any of the preceding claims wherein the endogenous cannabinoid ligand is AEA or 2-AG.

7. The use as claimed in any of the preceding claims wherein the levels of at least two endogenous cannabinoid ligands are determined.

8. The use as claimed in claim 7, wherein a biomarker composite score of the levels of the at least two endogenous cannabinoid ligands is generated.

9. The use as claimed in any of the preceding claims, for selecting patients for inclusion in a CBlR antagonist drug clinical trial or selecting patient for treatment with a CBlR antagonist drug.

10. The use as claimed in any of the preceding claims, wherein the CBlR antagonist is selected from: rimonabant, taranabant, surinabant, CP945598 and SLV319.

11. The use as claimed in any of claims 1 - 9, wherein the CBlR antagonist is a compound selected from: 1-propanesulfonic acid, 3,3,3-trifluoro-, 4-[l-(2,4-dichlorophenyl)-3-[[(2- hydroxycyclohexyl)amino]carbonyl]-4-(hydroxymethyl)-lH-pyrazol-5-yl]phenyl ester; 1- propanesulfonic acid, 3,3,3-trifluoro-, 4-[3-[(cyclohexylamino)carbonyl]-l-(2,4- dichlorophenyl)-4-(hydroxymethyl)-lH-pyrazol-5-yl]phenyl ester; 1-propanesulfonic acid, 3,3,3-trifluoro-, 4-[l-(2,4-dichlorophenyl)-4,5,6,7-tetrahydro-3-methyl-4-oxo-5-(l- piperidinyl)-lH-pyrrolo[3,2-c]pyridin-2-yl]phenyl ester; 1-propanesulfonic acid, 3,3,3- trifluoro-, 4-[l-(2,4-dichlorophenyl)-4-methyl-3-[(l-piperidinylamino)carbonyl]-lH- pyrazol-5-yl]phenyl ester; 1-propanesulfonic acid, 4-[l-(2,4-dichlorophenyl)-4-methyl-3- [(l-piperidinylamino)carbonyl]-lH-pyrazol-5-yl]phenyl ester; 3,3,3-trifluoropropane-l- sulfonic acid , 4-[2-(2,4-dichlorophenyl)-5-methyl-4-(piperidin-l-ylcarbamoyl)imidazol-l- yl]phenyl ester; and 3,3,3-trifluoropropane-l-sulfonic acid 4-[l-(2-chloro-4-fluorophenyl)- 3 -methyl-4-oxo-5-piperidin- 1 -yl-4,5 ,6,7-tetrahydro- 1 H-pyrrolo[3 ,2-c]pyridin-2-yl]phenyl ester, or a pharmaceutically acceptable salt thereof.

12. A method of selecting a mammal in need of treatment with a cannabinoid 1 receptor (CBlR) antagonist drug which comprises determining the level of at least one circulating endogenous cannabinoid ligand in the mammal, whereby to predict an increased likelihood of response to the CBlR antagonist drug.

13. The method as claimed in claim 12, wherein the mammal is a human.

14. The method as claimed in claim 12, wherein the mammal is in need of weight loss and/or weight maintenance and/or prevention of weight gain treatment.

15. The method as claimed in claim 12, 13 or 14, wherein if the mammal has high circulating levels of the endogenous cannabinoid ligand(s) they have an increased likelihood of responding to a CBlR antagonist drug.

16. The method as claimed in any of claims 12 to 15, wherein the levels of endocannabinoid ligand(s) is detected by immunoassay.

17. The method as claimed in claim 16, wherein the immunoassay is selected from: enzyme linked immunosorbent assay (ELISA/EIA), radio immunoassay (RIA), fluorescence immunoassay, luminescence immunoassay, electrochemical luminescence immunossay and SELDI-based immunoassay in combination with mass spectrometry.

18. The method as claimed in any of claims 12 to 17, wherein the amount of circulating endocannabinoid ligand is detected by using the AEA degrading enzyme, fatty acid amide hydrolase (FAAH) and/or the 2-AG degrading enzyme, monoacylglycerol lipase.

19. A method of stratifying a group of patients to identify one or more patients suitable for treatment with a CBlR antagonist drug, comprising determining the amount or level of one or more circulating endogenous cannabinoid ligands present in blood previously isolated from the patients.

20. The method as claimed in claim 19, wherein the group of patients are overweight or obese subjects.

21. A method of inducing weight loss and/or weight maintenance and/or weight gain in a patient in need thereof, comprising determining the level of a one or more circulating endogenous cannabinoid ligands in said patient and selecting an appropriate therapy based on the level of endogenous cannabinoid ligand(s) detected.

22. The method as claimed in claim 21, wherein if the patient has high levels of circulating endogenous cannabinoid ligand(s) they are treated with a CBlR antagonist drug.

23. The method as claimed in any of claims 12 to 22, wherein the CBlR antagonist is selected from: rimonabant, taranabant, surinabant, CP945598 and SLV319.

24. The method as claimed in any of claims 12 to 22, wherein the CBlR antagonist is a compound selected from: 1-propanesulfonic acid, 3,3,3-trifluoro-, 4-[l-(2,4-dichlorophenyl)-3-[[(2- hydroxycyclohexyl)amino]carbonyl]-4-(hydroxymethyl)-lH-pyrazol-5-yl]phenyl ester; 1- propanesulfonic acid, 3,3,3-trifluoro-, 4-[3-[(cyclohexylamino)carbonyl]-l-(2,4- dichlorophenyl)-4-(hydroxymethyl)-lH-pyrazol-5-yl]phenyl ester; 1-propanesulfonic acid, 3,3,3-trifluoro-, 4-[l-(2,4-dichlorophenyl)-4,5,6,7-tetrahydro-3-methyl-4-oxo-5-(l- piperidinyl)-lH-pyrrolo[3,2-c]pyridin-2-yl]phenyl ester; 1-propanesulfonic acid, 3,3,3- trifluoro-, 4-[l-(2,4-dichlorophenyl)-4-methyl-3-[(l-piperidinylamino)carbonyl]-lH- pyrazol-5-yl]phenyl ester; 1-propanesulfonic acid, 4-[l-(2,4-dichlorophenyl)-4-methyl-3- [(l-piperidinylamino)carbonyl]-lH-pyrazol-5-yl]phenyl ester; 3,3,3-trifiuoropropane-l- sulfonic acid , 4-[2-(2,4-dichlorophenyl)-5-methyl-4-(piperidin-l-ylcarbamoyl)imidazol-l- yl]phenyl ester; and 3,3,3-trifluoropropane-l-sulfonic acid 4-[l-(2-chloro-4-fluorophenyl)- 3 -methyl-4-oxo-5-piperidin- 1 -yl-4,5 ,6,7-tetrahydro- 1 H-pyrrolo[3 ,2-c]pyridin-2-yl]phenyl ester, or a pharmaceutically acceptable salt thereof.