Compositions for affecting weight loss comprising an opioid antagonist and bupropion

(56) References cited:

EP-A1- 0 005 636 WO-A-02/24214

WO-A-03/013524 WO-A1-01/52851 WO-A1-99/38504

ΕΡ 2 316 456 Β1

Note: Within nine months ofthe publication ofthe mention ofthe grant ofthe European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall nőt be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).

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Description

Background ofthe Invention

Field of the Invention [0001] The present invention is in the field of pharmaceutical compositions and methods tor the treatment of obesity and tor affecting weight loss in individuals.

Description ofthe Related Art [0002] EP0005636 concerns naltrexone tor inducing anorexia. WO 99/38504 concerns methods of treatment and pharmaceutical compositions employing the compound (-)bupropion. W02002/024214 concerns the use of GLP activators tor the treatment, prevention, diagnosis, and prognosis of bone-related and nutrition-related disorders.

[0003] Obesity is a disorder characterized bythe accumulation of excess tat in the body. Obesity has been recognized as one ofthe leading causes of disease and is emerging as a global problem. Increased instances of complications such as hypertension, non-insulin dependent diabetes mellitus, arteriosclerosis, dyslipidemia, certain forms of cancer, sleep apnea, and osteoarthritis have been related to increased instances of obesity in the generál population.

[0004] Obesity has been defined in terms of body mass index (BMI). BMI is calculated as weight (kg)/[height (m)]². According to the guidelines of the U.S. Centers tor Disease Control and Prevention (CDC), and the World Health Organization (WHO) (World Health Organization. Physical status: The use and interpretation of anthropometry. Geneva, Switzerland: World Health Organization 1995. WHO Technical Deport Serves), tor adults over 20 years old, BMI falls intő one of these categories: below 18.5 is considered underweight, 18.5 - 24.9 is considered normál, 25.0 - 29.9 is considered overweight, and 30.0 and above is considered obese.

[0005] Prior to 1994, obesity was generally considered a psychological problem. The discovery of the adipostatic hormoné leptin in 1994 (Zhang et al., Positional cloning ofthe mouse obese gene and its humán homologue, Natúré 1994; 372:425-432) brought forth the realization that, in certain cases, obesity may have a biochemical basis. Acorollary to this realization was the idea that the treatment of obesity may be achieved by Chemical approaches. Since then, a number of such Chemical treatments have entered the markét. The most famous of these attempts was the introduction of Fen-Phen, a combination offenfluramine and phentermine. Unfortunately, itwasdiscovered thatfenfluramine caused heart-valve complications, which in somé cases resulted in the death ofthe user. Fenfluramine has since been withdrawn from the markét. There has been somé limited success with other combination therapy approaches, particularly in the field of psychological eating disorders. One such example is Devlin, et al., Int. J. Eating Disord. 28:325-332, 2000, in which a combination of phentermine and fluoxetine showed somé efficacy in the treatment of binge eating disorders. Of course, this disorder is an issue tor only a small portion of the population.

[0006] In addition to those individuals who satisfy a strict definition of medical obesity, a significant portion ofthe aduit population is overweight. These overweight individuals would alsó benefit from the availability of an effective weightloss composition. Therefore, there is an unmet need in the art to provide pharmaceutical compositions that can affect weight loss without having other adverse side effects.

Summary ofthe Invention [0007] The invention is defined by the appended claims. The invention comprises a composition tor use in a method of affecting weight loss or treating obesity comprising a first compound and a second compound, wherein the first compound is an opioid antagonist and the second compound is bupropion.

[0008] The invention alsó comprises a composition as defined in the claims tor use in methods of affecting weight loss, increasing energy expenditure, increasing satiety in an individual, or suppressing the appetite of an individual, comprising identifying an individual in need thereof and treating that individual with a composition as defined in the claims to antagonize opioid receptor activity and to enhance α-MSH activity.

Detailed Description ofthe Preferred Embodiments [0009] Arcuate nucleus neurons are known to be responsive to a wide array of hormones and nutrients, including leptin, insulin, gonadal steroids, and glucose. In addition to potential transport mechanisms, peripheral substances may access these neurons via arcuate cell bodies in and projections to the médián eminence, a region considered to be a circumventricular organ, which lacks a blood-brain barrier. Cone et al., The arcuate nucleus as a conduit tor diverse signals relevant to energy homeostasis, Inti Journal of Obesity (2001) 25, Suppl 5, S63-S67.

[0010] Administration of exogenous leptin activates a numberof different neurons in hypothalamic and brainstem cell

ΕΡ 2 316 456 Β1 groups that bear leptin receptor. Leptin-responsive neurons in the arcuate nucleus include both those containing neuropeptide Y (NPY) and agouti-related peptide (AgRP) in the médiai part ofthe nucleus and those containing both proopiomelanocortin (POMC) and its derivatives, including α-melanocyte stimulating hormoné (α-MSH), as well as cocaine and amphetamine-related transcript (CÁRT). Saperetal., The need tofeed: Homeostatic and hedoniccontrol ofeating, Neuron, 36:199-211 (2002).

[0011] The leptin-responsive POMC neurons in the arcuate nucleus are thought to cause anorexia and weigh réduction by means ofthe action of α-MSH on melanocortin 3 and/or 4 receptors (MC3-R, MC4-R). The highest MC3-R expression level is in the hypothalamus and limbic system, whereas MC4-R mRNA is expressed in virtually all major brain regions. Somé ofthe metabolic effects resulting from stimulation of MC4-R are decreased food intake and an increase in energy expenditure through stimulation of thyrotropin-releasing hormoné and activation of the sympathetic nervous system. Targeted deletion ofthe MC4-Rgene produces obesity, hyperphagia, hyperinsulinemia, and reduced energy expenditure. Targeted deletion of MC3-R results in increased adiposity due to decreased energy expenditure. Korner et al., The emerging Science of body weight regulation and its impact on obesity treatment, J. Clin. Invest. 111(5):565-570 (2003). Thus, increased concentrations of α-MSH in the central nervous system (CNS) increase its action on MC3-R and/or MC4-R and result in a suppressed appetite.

[0012] POMC neurons alsó release β-endorphin when they release a-MSH. β-endorphin is an endogenous agonist of the μ-opioid receptors (MOP-R), found on the POMC neurons. Stimulation of MOP-R decreases the release of aMSH. This is a biofeedback mechanism that under normál physiological conditions Controls the concentration of a-MSH in the CNS. Thus, blocking MOP-R by opioid antagonists will break the feedback mechanism, which results in continued secretion of α-MSH and an increase in its concentration in the CNS.

[0013] A second population of neurons in the arcuate nucleus tonically inhibits the POMC neurons. These POMCinhibiting neurons secrete NPY, the neurotransmitter γ-aminobutyric acid (GABA), and AgRP. NPY and GABA inhibit POMC neurons, via NPY Y1 receptors and GABA receptors, respectivley. Thus, within the arcuate nucleus NPY and GABA inhibit the release of α-MSH, and therefore are stimulators of feeding. It is known that leptin inhibits the release of GABA from NPY terminals synapsing onto POMC neurons, whereas ghrelin, an orexigenic peptide, stimulates the ghrelin receptors on NPY neurons and increase the secretion of NPY and GABA onto the POMC cells, which in turn inhibits the release of a-MSH.

[0014] AgRP stimulates food intake in the rat through antagonism ofthe interaction of α-MSH at MC4-R. Expression of the AgRP gene is suppressed by leptin.

[0015] Serotonin, alsó known as 5-hydroxytryptamine or 5-HT, activates the POMC neurons to secrete α-MSH. However, serotonin is taken up and removed from action by specific transporters so that a single serotonin molecule has short term effects. It is known that selective serotonin reuptake inhibitors (SSRIs) prevent the uptake of serotonin and increase its concentrations in the CNS. Thus, SSRIs alsó increase the secretion of α-MSH and its concentrations in the CNS.

[0016] Dopamine alsó increases the activity of POMC neurons to secrete α-MSH. Like serotonin, dopamine is alsó taken up and removed from action so that a single dopamine molecule has short term effect. Dopamine re-uptake inhibitors, which prevent or reduce the uptake of dopamine, can alsó increase the secretion of α-MSH and its concentrations in the CNS.

[0017] Therefore, increased secretion of α-MSH through various mechanisms, such as serotonin re-uptake inhibition, are among the strategies that the methods and pharmaceutical compositions ofthe present invention pursue in order to produee a biochemical anorexigenic effect.

[0018] The present invention provides a multi-faceted combination therapy approach to the problem of weight loss. It addresses nőt just single molecules, messengers, or receptors, bút instead acts on multiple points in the feeding and satiety pathway. Aspects ofthe present invention are directed to increasing the concentrations of α-MSH in the CNS by stimulating the release of α-MSH, suppressing its metabolism, reducing the antagonism of its interaction at MC3/4-R, and suppressing any feedback mechanisms that slow or stop its release. Aspects of the present invention include pharmaceutical compositions whose components aehieve one or more of these functions. The present inventors have discovered that a combination of two or more of the compounds disclosed herein results in a synergistic effect that affects weight loss more quickly and on a more permanent basis.

[0019] Thus, in a first embodiment, the present invention is directed to a composition as defined in the claims for use in a method of treating obesity or affecting weight loss comprising a first compound and a second compound, where the first compound is an opioid antagonist and the second compound causes increased agonism ofa melanocortin 3 receptor (MC3-R) or a melanocortin 4 receptor (MC4-R) compared to normál physiological conditions.

[0020] In certain embodiments, the second compound causes increased activity of the POMC neurons, leading to greater agonism at MC3-R and/or MC4-R.

[0021] In certain embodiments the opioid antagonist antagonizes a μ-opioid receptor (MOP-R) in a mammal. The mammal may be selected from the group consisting of mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, primates, such as monkeys, chimpanzees, and apes, and humans.

ΕΡ 2 316 456 Β1 [0022] In somé embodiments the opioid antagonist is selected from the group consisting of alvimopan, norbinaltorphimine, nalmefene, naloxone, naltrexone, methylnaltrexone, and nalorphine, and pharmaceutically acceptable salts or prodrugs thereof.

[0023] In other embodiments, the opioid antagonist is a partial opioid agonist. Compounds of this class have somé agonist activity at opioid receptors. However, because they are weak agonists, they function as de-facto antagonists. Examples of partial opioid agonists include pentacozine, buprenorphine, nalorphine, propiram, and lofexidine.

[0024] The term pharmaceutically acceptable salt refers to a formulation ofa compound that does nőt cause significant irritation to an organism to which it is administered and does nőt abrogate the biological activity and properties ofthe compound. Pharmaceutical salts can be obtained by reacting a compound ofthe invention with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. Pharmaceutical salts can alsó be obtained by reacting a compound of the invention with a base to form a salt such as an ammonium salt, an alkali metál salt, such as a sodium or a potassium salt, an alkaline earth metál salt, such as a calcium óra magnesium salt, a salt of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl) methylamine, and salts thereof with amino acids such as arginine, lysine, and the like.

[0025] A prodrug refers to an agent that is converted intő the parent drug in vivő. Prodrugs are often useful because, in somé situations, they may be easier to administer than the parent drug. They may, fór instance, be bioavailable by órai administration whereas the parent is nőt. The prodrug may alsó have improved solubility in pharmaceutical compositions over the parent drug, or may demonstrate increased palatability or be easier to formulate. An example, without limitation, ofa prodrug would be a compound ofthe present invention which is administered as an ester (the prodrug) to facilitate transmittal across a cell membráné where water solubility is detrimental to mobility bút which then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water-solubility is beneficial. A further example of a prodrug might be a short peptide (polyaminoacid) bonded to an acid group where the peptide is metabolized to provide the active moiety.

[0026] In certain embodiments, the second compound in the pharmaceutical compositions ofthe present invention as defined in the claims triggers the release of α-melanocyte stimulating hormoné (α-MSH). The second compound may increase the extracellular serotonin concentrations in the hypothalamus. In somé aspects disclosed herein, the second compound is selected from the group consisting ofa selective serotonin reuptake inhibitor (SSRI), a serotonin 2C agonist, and a serotonin 1B agonist. In further aspects disclosed herein, the second compound is selected, e.g., from the group consisting of fluoxetine, fluvoxamine, sertraline, paroxetine, citalopram, escitalopram, sibutramine, duloxetine, and venlafaxine, and pharmaceutically acceptable salts or prodrugs thereof.

[0027] The terms serotonin 1B receptor, serotonin 2C receptor, 5-HT1 b receptor, and 5-HT2c receptor refer to receptors found more commonly in rodents. It is understood by those of skill in the art that other mammals have serotonin receptors on various neurons that are analogous in function and form to these receptors. Agonists or antagonists at these non-rodent, preferably humán, serotonin receptors are within the scope ofthe present invention.

[0028] In certain aspects disclosed herein, the second compound suppresses the expression of the AgRP gene or the production or release of agouti-related protein (AgRP). In somé of these aspects, the second compound suppresses the activity of neurons that express AgRP.

[0029] In other aspects disclosed herein, the second compound suppresses the expression of the NPY gene or the production or release of neuropeptide Y (NPY). In somé of these aspects, the second compound suppresses the activity of neurons that express NPY. In further aspects, the second compound is selected from the group consisting of NPY antagonists, ghrelin antagonists, and leptin. In certain other aspects, thesecond compound agonizes NPYY2 receptor. [0030] Other aspects disclosed herein include those in which the second compound is selected from the group consisting of a γ-amino butyric acid (GABA) inhibitor, a GABA receptor antagonist, and a GABA channei antagonist. By GABA inhibitor it is meant a compound that reduces the production of GABA in the cells, reduces the release of GABA from the cells, or reduces the activity of GABA on its receptors, either by preventing the binding of GABA to GABA receptors or by minimizing the effect of such binding. The GABA inhibitor may be a 5-HT 1 b agonist or another agent that inhibits the activity of NPY/AgRP/GABA neurons. In addition, the GABA inhibitor may suppress the expression of the AgRP gene, or the GABA inhibitor may suppress the production or release of AgRP. It is, however, understood that a 5-HTIb agonist may inhibit the NPY/AgRP/GABA neuron (and therefore activate POMC neurons) without acting as an inhibitor of the GABA pathway.

[0031] In certain other aspects disclosed herein the GABA inhibitor increases the expression of the POMC gene. In somé of these aspects, the GABA inhibitor increases the production or release of pro-opiomelanocortin (POMC) protein. In certain other of these aspects, the GABA inhibitor increases the activity on POMC expressing neurons. In somé aspects, the GABA inhibitor is topiramate.

[0032] The second compound as defined in the claims is a dopamine reuptake inhibitor. Phentermine is an example of a dopamine reuptake inhibitor. In certain other embodiments, the second compound is a norepinephrine reuptake inhibitor. Examples of norepinephrine reuptake inhibitors include bupropion, thionisoxetine, and reboxetine. The second

ΕΡ 2 316 456 Β1 compound may be a dopamine agonist. Somé dopamine agonists that are available on the markét include cabergoline, amantadine, lisuride, pergolide, ropinirole, pramipexole, and bromocriptine. The second compound may be a norepinephrine releaser, or a mixed dopamine/norepinephrine reuptake inhibitor.

[0033] In certain other aspects disclosed herein, the second compound is a 5-HT1b agonist, such as sumatriptan, almotriptan, naratriptan, frovatriptan, rizatriptan, zomitriptan, and elitriptan.

[0034] In further aspects disclosed herein, the second compound is an anticonvulsant. The anticonvulsant may be selected from the group consisting ofzonisamide, topiramate, nembutal, lorazepam, clonazepam, clorazepate, tiagabine, gabapentin, fosphenytoin, phenytoin, carbamazepine, valproate, felbamate, levetiracetam, oxcarbazepine, lamotrigine, methsuximide, and ethosuxmide.

[0035] In certain embodiments, the second compound itself may be a combination of two or more compounds. Fór example, the second compound may be a combination ofa dopamine reuptake inhibitor and a norepinephrine reuptake inhibitor, e.g. bupropion and mazindol. Alternatively, the second compound may be a combination of a SSRI and a norepinephrine reuptake inhibitor.

[0036] In certain embodiments, the second compound as defined in the claims is an activator of the POMC neurons. Examples of POMC activators include Ptx1 and interleukin 1 béta, (IL-1 β).

[0037] In another embodiment, the present invention relates to a composition as defined in the claims fór use in a method of affecting weight loss, comprising identifying an individual in need thereof and treating that individual to antagonize opioid receptor activity and to enhance α-MSH activity.

[0038] In certain embodiments, the individual has a body mass index (BMI) greater than 25. In other embodiments, the individual has a BMI greater than 30. In still otherembodiments, the individual has a BMI greater than 40. However, in somé embodiments, the individual may have a BMI less than 25. In these embodiments, it may be beneficial fór health or cosmetic purposes to affect weight loss, thereby reducing the BMI even further.

[0039] In somé embodiments, opioid receptor activity is antagonized by administering an opioid receptor antagonist. The opioid receptor antagonist may be a MOP receptor antagonist. In somé embodiments, the opioid receptor antagonist is selected from alvimopan, norbinaltorphimine, nalmefene, naloxone, naltrexone, methylnaltrexone, and nalorphine, and pharmaceutically acceptable salts or prodrugs thereof.

[0040] In somé ofthe embodiments set forth above, α-MSH activity is enhanced by administering a compound, where the compound triggers reiease of α-MSH or increases the activity of neurons that express α-MSH. In somé aspects disclosed herein, the compound is a selective serotonin reuptake inhibitor (SSRI) or a specific 5-HT receptor agonist. Examples of SSRIs include fluoxetine, fluvoxamine, sertraline, paroxetine, citalopram, escitalopram, sibutramine, duloxetine, and venlafaxine, and pharmaceutically acceptable salts or prodrugs thereof.

[0041] In other aspects disclosed herein, the compound is a γ-amino butyric acid (GABA) inhibitor. The GABA inhibitor may be a 5-HT1b receptor agonist. The GABA inhibitor may suppress the expression of the AgRP gene, or it may suppresses the production or reiease of AgRP. The GABA inhibitor may suppress the expression or reiease of NPY. In certain aspects, the GABA inhibitor suppresses the activity of neurons that express AgRP. Fór example, the GABA inhibitor may be topiramate, 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NNC-711), or vigabatrin.

[0042] In certain embodiments, the invention set forth above is practiced with the proviso that the individual is nőt suffering from Prader-Willi syndrome or binge eating disorder. Thus, somé embodiments of the invention are to be distinguished from combination therapy involving SSRI anti-depressants (e.g., fluoxetine) used to treat physiological eating disorders such as binge eating disorder or Prader-Willi syndrome. In these embodiments, the target population is the population of individuals needing or desiring weight loss, apart from needing treatment fór Prader-Willi syndrome or binge eating disorder.

[0043] Individuals suffering from depression may gain weight as a result of their depression. In addition, certain depressed individuals gain weight as a side effect ofthe depression therapy. In certain embodiments, the invention set forth above is practiced with the proviso that the individual is nőt suffering from depression. In somé embodiments, the individual’s overweight state was nőt caused by treatment fór depression.

[0044] In other embodiments, the invention set forth above is practiced with the proviso that if the opioid receptor is antagonized using naltrexone, then reiease of α-MSH is nőt stimulated with fluoxetine. However, the combination of naltrexone with fluoxetine may be used to affect weight loss in individuals who wish to lose weight, whether or nőt they are clinically categorized as obese. These individuals may include those with BMI of greater than 25, or those individuals with BMI of less than 25 who still wish to lose additional weight. This particular combination may alsó be used fór the treatment of generál obesity. In certain aspects, the individual who wishes to lose additional weight does nőt suffer from binge eating disorder.

[0045] In somé embodiments, the use ofthe claimed composition in the above method comprises administering to the individual a first compound and a second compound, where the first compound is an opioid antagonist and the second compound enhances α-MSH activity.

[0046] In somé embodiments the first compound and the second compound are administered more or less simulta5

ΕΡ 2 316 456 Β1 neously. In other embodiments the first compound is administered prior to the second compound. In yet other embodiments, the first compound is administered subsequent to the second compound.

[0047] In certain embodiments, the first compound and the second compound are administered individually. In other embodiments, the first compound and the second compound are covalently linked to each other such that they form a single Chemical entity. The single Chemical entity is then digested and is metabolized intő two separate physiologically active Chemical entities, one ofwhich is the first compound and the other one is the second compound.

[0048] Compositions may be a combination ofthe following compounds:

a SSRI in combination with a dopamine reuptake inhibitor, a dopamine/norepinephrine reuptake inhibitor, a norepinephrine reuptake inhibitor, an opioid antagonist, a partial opioid agonist, GABA inhibitor, a peripherally acting weight loss agent such as metformin, or a peptide, such as PYY, PYY3-361 or leptin;

Serotonin in combination with a dopamine reuptake inhibitor, a dopamine/norepinephrine reuptake inhibitor, an opioid antagonist, a partial opioid agonist, or a GABA inhibitor;

a dopamine reuptake inhibitor in combination with a norepinephrine reuptake inhibitor, a norepinephrine releaser, a norepinephrine agonist, an opioid antagonist, a partial opioid agonist, a GABA inhibitor, an adenosine compound, a cholinergic receptor antagonist, or a peptide, such as PYY, PYY3-36, or leptin;

a dopamine/norepinephrine reuptake inhibitor in combination with an opioid antagonist, a partial opioid agonist, a GABA inhibitor, or a peripherally acting weight loss agent such as metformin;

a dopamine agonist in combination with an opioid antagonist, a partial opioid agonist, a GABA inhibitor, or a peptide, such as PYY, PYY3-36, or leptin.

[0049] Examples of norepinephrine agonists include phendimetrazine and benzphetamine. Examples of adenosine compounds include all xanthine derivatives, such as adenosine, caffeine, theophylline, theobromine, and aminophylline. An example of a cholinergic receptor antagonist is nicotine.

[0050] In another embodiment, the present invention relates to a composition as defined in the claims fór use in a method of increasing satiety in an individual comprising identifying an individual in need thereof and treating that individual to antagonize opioid receptor activity and to enhance α-MSH activity.

[0051] In somé embodiments, the use ofthe claimed composition in the above method comprises administering to the individual a first compound and a second compound, where the first compound is an opioid antagonist and the second compound enhances α-MSH activity.

[0052] In somé embodiments the first compound and the second compound are administered nearly simultaneously. In other embodiments the first compound is administered prior to the second compound. In yet other embodiments, the first compound is administered subsequent to the second compound.

[0053] In yet another embodiment, the present invention relates to a composition as defined in the claims fór use in a method of suppressing the appetite ofan individual comprising identifying an individual in need thereof and treating that individual to antagonize opioid receptor activity and to enhance α-MSH activity.

[0054] In somé embodiments, the use ofthe claimed composition in the above method comprises administering to the individual a first compound and a second compound, where the first compound is an opioid antagonist and the second compound enhances α-MSH activity.

[0055] In somé embodiments the first compound and the second compound are administered nearly simultaneously. In other embodiments the first compound is administered prior to the second compound. In yet other embodiments, the first compound is administered subsequent to the second compound.

[0056] In another embodiment, the present invention relates to a composition as defined in the claims fór use in a method of increasing energy expenditure in an individual comprising identifying an individual in need thereof and treating that individual to antagonize opioid receptor activity and to enhance α-MSH activity.

[0057] In somé embodiments, the use ofthe claimed composition in the above method comprises administering to the individual a first compound and a second compound, where the first compound is an opioid antagonist and the second compound enhances α-MSH activity.

[0058] In somé embodiments the first compound and the second compound are administered nearly simultaneously. In other embodiments the first compound is administered prior to the second compound. In yet other embodiments, the first compound is administered subsequent to the second compound.

[0059] In certain embodiments disclosed herein, an individual is given a pharmaceutical composition as defined in the claims comprising a combination of two or more compounds to affect weight loss. In somé of these embodiments, each compound is a separate Chemical entity. However, in other embodiments, the two compounds are joined together by a Chemical linkage, such as a covalent bond, so that the two different compounds form separate parts ofthe same molecule. The Chemical linkage is selected such that after entry intő the body, the linkage is broken, such as by enzymatic action, acid hydrolysis, base hydrolysis, or the like, and the two separate compounds are then formed.

[0060] In another aspect, the present disclosure relates to synthetic routes to növel molecules in which an opioid

ΕΡ 2 316 456 Β1 antagonist is linked by a flexible linker to a selective serotonin reuptake inhibitor (SSRI).

[0061] Data from previous structure-activity relationship (SÁR) studies within the family of μ opioid antagonists may be used as a guide to determine which antagonists to use and the optimál position or positions on the antagonist molecules to attach the tether such that potency and selectivity of the antagonist will remain high. Similarly, SÁR data within the family of SSRIs may be used as a guide to determine which inhibitors to use and the optimál position or positions on the inhibitors to attach the tether such that potency and selectivity remain high. The tether or linker moiety is chosen from among those of demonstrated utility tor linking bioactive molecules together. Disclosed herein are representative opioid antagonists, linkers and SSRI molecules that can be attached together in different combinations to form heterobivalent therapeutic molecules.

[0062] Structure-activity relationships ofthe opioid agonists and antagonists have been reviewed. See tor example, Zimmerman, D. M.; Leander, J. D. J. Med. Chem. 1990, 33, 895; Portoghese, P. S. J. Med. Chem. 1992, 35, 1927; Carroll, F. I. J. Med. Chem. 2003, 46, 1. The opioid antagonists, nalmefene (1), naltrexone (2), naloxone (3) and naltrexamine (4) are thebaine-derived structures that share a common opiate-type template. μ-Subtype selective opioid antagonists are of considerable current interest as agents tor the treatment of obesity (Glass, M. J.; Billington, C. J.; Levine, A. S. Neuropeptides 1999, 33, 350) and CNS disorders (Reneric, J. P.; Bouvard, M.P. CNS Drugs 1998,10, 365).

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β-naltrexamine [0063] /V-Methyl and /\/-2-phenylethyl substituted opioids tend to show opioid agonist activity whereas /V-allyl and /V-cycIopropylmethyl substituted analogs tend to show opioid antagonist activity. Any /V-attached linker moiety will be larger than methyl. Provided that the linker moiety does nőt mimic 2-phenylethyl, such linked opioids are expected to behave as opioid antagonists. Therefore, the nitrogén atom of nalmefene and naltrexone (and naloxone) is a suitable site tor attachment of a linker moiety. Less SÁR information is available with regard to substitution at other sites on these opioids, however, attachment ofthe linker unit to one or the other ofthe carbon atoms bearing one or more hydrogen atoms remains an option.

[0064] Both nalmefene and naltrexone are potent μ-opioid antagonists. The only structural difference is that nalmefene has a methylene group in piacé ofthe ketone oxygen atom in naltrexone. It is thus postulated that significant changes in structure at the ketone oxygen site in naltrexone do nőt significantly affect antagonist potency. Therefore, a linker may be attached to the methylene group in nalmefene without significant reduction in antagonist potency. Carbonyl derivatives of naloxone are well known and include symmetrical azine (=N-N=), mixed azine (Schmidhammer, H.; Kaspar, F.; Marki, A.; Borsodi, A. Helv. Chim. Acta 1994, 77, 999), hydazone (Hahn, E. F.; Itzhak, Y.; Nishimura, S.; Johnson, N.; Pasternak, G. W. J. Pharm. Exper. Therapeutics 1985, 235, 846-50), semicarbazone and thiosemicarbazone derivatives (Kolb, V. M.; Komán, A.; Neil, A. Pharmaceutical Rés. 1985, 6,266-71). Naloxazone, the hydrazone of naloxone, is an irreversible, selective and long acting antagonist ofthe μ-1 subclass ofthe opioid receptors (Pasternak, G. W.; Hahn, E. F. J. of Med. Chem. 1980,23, 674-6). Certain ofthe derivatives are potent μ. opioid antagonists while others are potent agonists. [0065] Naltrexamine (4) has been linked by attachment of its primary amino group to a wide variety of other molecules producing, tor example, a fluorogenic opioid receptor affinity label (Le Bourdonnec, B.; El Kouhen, R.; Lunzer, Μ. M.; Law, P. Y.; Loh, Η. H.; Portoghese, P. S.; J. Med. Chem.; 2000; 43; 2489-2492), an extensive series of nonequilibrium opioid agonists and antagonists (Sayre, L. M.; Larson, D. L.; Takemori, A. E.; Portoghese, P. S. J. Med. Chem. 1984, 27,1325), and a series of potent bivalent opioid antagonists (Erez, M.; Takemori, A. E.; Portoghese, P. S. J.Med. Chem. 1982, 25, 847-849). Consequently, the primary amino group of naltrexamine constitutes a suitable site tor attachment of a linker moiety.

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L &gt;

fluoxetine

N-methylfluoxetine paroxetine [0066] A limited SÁR fór fluoxetine (5) has been published in USP 4,214,081. /V-Methylfluoxetine (6) shows comparable potency and selectivity to that of fluoxetine toward inhibition of serotonin reuptake. Therefore, attachment of a linker to the nitrogén atom of fluoxetine can result in retention ofthe potency and selectivity of fluoxetine itself. However, the present disclosure is nőt limited to the fluoxetine series of SSRIs. It is envisaged that a variety of SSRI molecules such as paroxetine (Dechant, K. L.; Clissold, S. P. Drugs, 1991, 41, 225-253) or one or the other of the bivalent SSRIs described by Kozikowski et al. (Tamiz, A. P.; Zhang, J.; Zhang, M.; Wang, C. Z.; Johnson, K. M.; Kozikowski, A. P. J. Am. Chem. Soc. 2000, 122, 5393-5394; Tamiz, A. P.; Bandyopadhyay, B. C.; Zhang, J.; Flippen-Anderson, J. L.; Zhang, M.; Wang, C. Z.; Johnson, K. M.; Telia, S.; Kozikowski, A. P. J. Med. Chem. 2001,44, 1615-1622) may alsó be utilized to construct the heterobivalent therapeutic molecules of this invention.

[0067] Exam ples oflinkers reported in the scientific literature include methylene(CH₂)_n linkers(Hussey, S. L.; Muddana, S. S.; Peterson, B. R.; J. Am. Chem. Soc. 2003; 125; 3692-3693; Tamiz, A. P.; Bandyopadhyay, B. C.; Zhang, J.; FlippenAnderson, J. L.; Zhang, M.; Wang, C. Z; Johnson, K. M.; Tellar, S.; Kozikowski, A. P. J. Med. Chem. 2001,44,1615-1622), oligo ethyleneoxy O(-CH₂CH₂O-)_n units used to link naltrexamine to other opioids, glycine oligomers of the formula NH-(COCH₂NH)_nCOCH₂CH₂CO-(NHCH₂CO)_nNH- used to link opioid antagonists and agonists together ((a) Portoghese, P. S.; Ronsisvalle, G.; Larson, D. L.; Yim, C. B.;Sayre, L. M.; Takemori, A. E. Life Sci. 1982, 31, 1283-1286. (b) Portoghese, P. S.; Larson, D. L.; Sayre, L. M.; Yim, C. B.; Ronsisvalle, G.; Tam, S. W.; Takemori, A. E. J. Med. Chem. 1986, 29,1855-1861), hydrophilic diamines used to link opioid peptides together (Stepinski, J.; Zajaczkowski, I.; KazemBek, D.; Temeriusz, A.; Lipkowski, A. W.; Tam, S. W. Internat. J. of Peptide &amp; Protein Rés. 1991, 38, 588-92), rigid double stranded DNA spacers (Paar, J. M.; Harris, Ν. T.; Holowka, D.; Baird, B. J. Immunoi. 2002, 169, 856-864) and the biodegradable linker poly (L-lactic acid) (Kiok, H.-A.; Hwang, J. J.;lyer, S. N.; Stupp, S. I. Macromolecules 2002, 35, 746-759). The attachment of the tether to the antagonist can result in the antagonist achieving a favorable binding orientation. The linker itself may or may nőt be biodegradable. The linker may take the form of a prodrug and be tunable fór optimál release kinetics of the linked drugs. The linker may be either conformationally flexible throughout its entire length or else a segment ofthe tether may be designed to be conformationally restricted (Portoghese, P. S.; Ronsisvalle, G.; Larson, D. L.; Takemori, A. E. J. Med. Chem. 1986, 29, 1650-1653).

[0068] In Scheme 1 below, naltrexone (2) is used in the linking reaction. As a consequence ofthe Wittig reaction, a double bond replaces the carbonyl group in naltrexone. The net result is fluoxetine linked with a flexible methylene linker to a nalmefene molecule by way of the nalmefene double bond.

ΕΡ 2 316 456 Β1

Scheme 1

<img file="HUE034290T2_D0004.tif" />

[0069] Reductive amination offluoxetine with an ω-bromoaldehyde such as 11-bromoundecanal 6 (n = 9) gives bromoamine 7 (n = 9), best stored as the hydrobromide salt to prevent an unwanted slow macrocyclization side reaction by way of attack ofthe free amino group on the carbon bearing the bromine atom. Reaction of 7 with triphenylphosphine gives the intermediate phosphonium salt, which upon rection with butyllithium generates the eorresponding ylid 8 (n = 9). A Wittig reaction between 8 and the ketone group of naltrexone (2) gives the linked molecule 9 containing a fluoxetine unit coupled to what is now a nalmefene unit. The expected mixture of cis, trans isomers about the newiy introduced double bond is separable by standard chromatographic techniques. If racemic fluoxetine is used, then a mixture of two optically active diastereomers of 9 will be produced owing to the fact that a single enantiomer 2 of naltrexone was used. Chemists skilled in the art will recognize that the (CH₂)g linker may be varied in length and/or contain substituents by beginning with a different bromoaldehyde. Thus, pharmacological properties may be optimized. Molecule 9 is stable under physiological conditions. Opioid antagonist activity will be due to the covalently linked nalmefene unit and nőt due to free nalmefene released as a result of somé cleavage reaction. Similarly, SSRI activity will be due to the covalently linked fluoxetine unit and nőt due to free fluoxetine released as a result of somé cleavage reaction.

[0070] An analogous reaction sequence may be used in which the bromoaldehyde is derived from an oligo ethylene glycol as shown in Scheme 2 below. For example, tetraethylene glycol (10 n = 2) is converted intő bromide 11 (n = 2), which is then oxidized under Swern conditions to aldehyde 12 (n = 2). Substitution of aldehyde 12 for aldehyde 6 in Scheme 1 will give a series of irreversibly linked molecules in which the linker is more hydrophilic than that in molecules 9. Generation of the ylid in the oligo ethylene glycol series and the subsequent Wittig reaction is performed at reduced temperature to avoid β-elimination ofthe alkoxy group. If racemic fluoxetine is used, then a mixture of two optically active diastereomers of 13 will be produced owing to the fact that a single enantiomer 2 of naltrexone was used. Chemists skilled in the art will recognize that the (OCH₂CH₂)_n linker may be varied in length by beginning with a different bromoaldehyde 12. Thus, pharmacological properties may be optimized. Molecule 13 is stable under physiological conditions.

ΕΡ 2 316 456 Β1

Scheme 2

<img file="HUE034290T2_D0005.tif" />

[0071] In Scheme 3, another linking method beginning with tetraethylene glyeol is illustrated as an example ofa variety of oligo ethylene glyeols that may be used. Adapting the chemistry of Sashiwa et al. (Sashiwa, H.; Shigemasa, Y.; Roy, R. Macromolecules 2000, 33, 6913), tetraethylene glyeol may be converted intő acetal 14 (n = 2) and subsequently intő aldehyde 15. Reductive amination of fluoxetine with aldehyde 15 gives the fluoxetine derivative 16. Reduction of azide 16 to amine 17 and then reductive amination with naltrexone gives molecule 18 in which a fluoxetine unit is linked irreversibly by a flexible oligo ethyleneoxy unit to β-naltrexamine (after séparation of the α and β isomers). If racemic fluoxetine is used, then a mixture of two optically active diastereomers of 18 will be produced owing to the fact that a single enantiomer 2 of naltrexone was used. Chemists skilled in the art will recognize that the (OCH₂CH₂)_n linker may be varied in length by beginning with a different oligo ethylene glyeol 10. Thus, pharmacological properties may be optimized. Molecule 18 should be stable under physiological conditions.

Scheme 3

<img file="HUE034290T2_D0006.tif" />

H

<img file="HUE034290T2_D0007.tif" />

fluoxetine reductive amination

<img file="HUE034290T2_D0008.tif" />

<img file="HUE034290T2_D0009.tif" />

fluoxetine naltrexamine [0072] Scheme 4 illustrates a synthetic route to fluoxetine linked to nalmefene by way of the /V-cyciopropyl group of nalmefene. The readily available f-butyldimethylsilyl protected noroxymorphone (19) is synthesized from morphine (Ninan, A.; Sainsbury, M. Tetrahedron 1992, 48, 6709-16), and then subjected to a reductive amination reaction with the commercially available cyclopropanecarboxaldehyde 20 (Aldrich, largely trans) giving ester21. Wittig methyleneation gives ester 22, which is hydrolyzed to give acid 23. Activation of acid 23 with an appropriate carbodiimide and then

ΕΡ 2 316 456 Β1 /V-acylation of fluoxetine derivative 17 (Scheme 3) gives 25, deprotection of which with Bu₄NF gives the növel molecule 26. Chemists skilled in the art will recognize that the (OCH₂CH₂)_n linker may be varied in length by beginning with a different aldehyde azide 15 in the synthesis of 17. Thus, pharmacological properties may be optimized. Molecule 26 should be stable under physiological conditions.

[0073] Alternatively, ester 22 may be reduced to aldehyde 24 using DIBAL at-78 °C. Reductive amination of aldehyde 24 with amine 17 gives molecule 27 after removal ofthe TBDMS protecting group. Chemists skilled in the art will recognize that the (OCH₂CH₂)_n linker may be varied in length by beginning with a different aldehyde azide 15 in the synthesis of 17. Thus, pharmacological properties may be optimized. Molecule 27 should be stable under physiological conditions.

Scheme 4

EtO₂C,

TBDMSO

TBDMSO

OH

H₂C=PPh₃

NH reductive amination

Wittig methylenation

CO₂Et protected noroxymorphone

RO,.

H?N oh] ΐΛ

<img file="HUE034290T2_D0010.tif" />

Q OH

TBDMSO

H,C'

HoC ,?^HL

F.C' &gt; COR

R = OEt

R = OH

R = H carboxyl activation of 23 by a suitable carbodiimide or reductive amination with aldehyde 24

F.C

<img file="HUE034290T2_D0011.tif" />

R = TBDMS, X = O

R=H,X = O

R=H,X = H,H / \ f o\ / o

F.C [0074] Ifthe Wittig methyleneation step is omitted in the above sequence, then an analóg of 26, namely ketone 28, is formed in which the methylene group of 26 is replaced by a carbonyl group. The result is a naltrexone unit linked to a fluoxetine unit by way of a flexible, hydrophilic (CH₂CH₂O)_n linker in the form of compound 28. Chemists skilled in the art will recognize that the (OCH₂CH₂)_n linker may be varied in length by beginning with a different aldehyde azide 15 in

ΕΡ 2 316 456 Β1 the synthesis of 17. Thus, pharmacological properties may be optimized. Molecule 28 is stable under physiological conditions.

[0075] Scheme 5 illustrates how fluoxetine may be linked to β-naltrexamine using a combination of linkers, namely the flexible glycine-based linkers 29 exploited by Portoghese et al. and the oligo ethylene glyeol linkers used in the schemes above. Thus carboxyl activation of 29 with a suitable carbodiimide followed by monocondensation with βnaltrexamine gives amidé 30. Reactivation of 30 followed by condensation with amine 17 (Scheme 3) gives molecule 31. Portoghese reports thatsymmetrical amides derived from linker 29 and β-naltrexamine are effective μ-opioid receptor antagonists. Chemists skilled in the art will recognize that the -NH-(COCH₂NH)_n_₁COCH2CH2CO-(NHCH₂CO)_nNHlinker may be varied in length by beginning with a different glycine-based linking unit 29 in the synthesis of 30. Thus, pharmacological properties may be optimized. Molecule 31 is stable under physiological conditions.

<img file="HUE034290T2_D0012.tif" />

CO(NHCH₂CO)_nOH

CO(NHCH₂CO)_nOH

<img file="HUE034290T2_D0013.tif" />

carboxyl activation of Srbo^Vd“ _rCO(NHCH₂CO)„,NHCH₂CO CO(NHCH₂CO)_nOH

<img file="HUE034290T2_D0014.tif" />

[0076] Reaction of bromide 7 (Scheme 1) with Mg in dry THF will give Grignard reagent 32, reaction ofwhich with the carbonyl group of naltrexone gives adduct 33 after separation ofthe two diastereomers produced at the newly created chiral center. Adduct 33 contains a fluoxetine segment linked to a /V-cyclopropylmethyl-normorphine unit by way of a flexible methylene linker. Chemists skilled in the art will recognize that the (CH₂)g linker may be varied in length by beginning with a different bromoaldehyde fór the synthesis of bromide 7. Thus, pharmacological properties may be optimized. Molecule 33 is stable under physiological conditions.

EP 2 316 456 Β1

Scheme 6

<img file="HUE034290T2_D0015.tif" />

MgBr

Naltrexone [0077] Throughout the above schemes, one should be able to employ /V-desmethylfluoxetine (34), or any other derivative of fluoxetine, in piacé of fluoxetine. The resulting linked fluoxetine unit is identical to that of fluoxetine itself except that the methyl group of fluoxetine is replaced by a longer chain that is part of the linker. When necessary due to the use of strongly basic reagents or when chemoselectivity toward a primary amino group elsewhere in the molecule is required, one may protect the intermediate fluoxetine secondary amino group by use ofthe /V-[2-(trimethylsilyl)ethoxy] methyl (SEM) group (Zeng, Z.; Zimmerman, S. C. Tetrahedron Lett. 1988, 29, 5123) as illustrated in Scheme 7.

ΕΡ 2 316 456 Β1

CH₂OCH2CH₂Si(CH3)3

Ο. Ν _Η

Scheme Ί

Q&gt;

F,C^X (CH₃)₃SiCH₂CH₂OCH₂CI (SEM chloride) /\/-desmethylfluoxetine (34) f,ct

Reductive amination with 6

F3C

<img file="HUE034290T2_D0016.tif" />

<img file="HUE034290T2_D0017.tif" />

SEM

N_ Br n

1. Mg, dry THF

2. Naltrexone

<img file="HUE034290T2_D0018.tif" />

N

HCI, EtOH fluoxetine segment

F,C

<img file="HUE034290T2_D0019.tif" />

flexible methylene linker N-cyclopropylmethylnormorphine segment [0078] In another aspect, the invention relates to a pharmaceutical composition as defined in the claims comprising a combination of an opioid antagonist and a compound that causes increased agonism of a melanocortin 3 receptor (MC3-R) or a melanocortin 4 receptor (MC4-R) compared to normál physiological conditions, as described above, or comprising a linked molecule, as described herein, and a physiologically acceptable carrier, diluent, or excipient, or a combination thereof.

[0079] The term pharmaceutical composition refers to a mixture of a compound of the invention with other Chemical components, such as diluents or carriers. The pharmaceutical composition facilitates administration ofthe compound to an organism. Multiple techniques of administering a compound exist in the art including, bút nőt limited to, órai, injection, aerosol, parenteral, and topical administration. Pharmaceutical compositions can alsó be obtained by reacting compounds with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.

[0080] The term carrier defines a Chemical compound that facilitates the incorporation of a compound intő cells or tissues. For example dimethyl sulfoxide (DMSO) is a commonly utilized carrier as itfacilitates the uptake of many organic compounds intő the cells or tissues of an organism.

[0081] The term diluent defines Chemical compounds diluted in water that will dissolve the compound of interest as well as stabilize the biologically active form ofthe compound. Salts dissolved in buffered Solutions are utilized as diluents in the art. One commonly used buffered solution is phosphate buffered saline because it mimics the salt conditions of humán blood. Since buffer salts can control the pH ofa solution at low concentrations, a buffered diluent rarely modifies the biological activity ofa compound.

[0082] The term physiologically acceptable defines a carrier or diluent that does nőt abrogate the biological activity and properties ofthe compound.

[0083] The pharmaceutical compositions described herein can be administered to a humán patient per se, or in pharmaceutical compositions where they are mixed with other active ingredients, as in combination therapy, or suitable

ΕΡ 2 316 456 Β1 carriers or excipient(s). Techniques fór formulation and administration of the compounds of the instant application may be found in Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th edition, 1990.

[0084] Suitable routes of administration may, tor example, include órai, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.

[0085] Alternately, one may administerthe compound in a local rather than systemic manner, tor example, via injection of the compound directly in the renal or cardiac area, often in a depót or sustained release formulation. Furthermore, one may administerthe drug in a targeted drug delivery system, tor example, in a liposome coated with a tissue-specific antibody. The liposomes will be targeted to and taken up selectively by the organ.

[0086] The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping ortabletting processes.

[0087] Pharmaceutical compositions tor use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate Processing ofthe active compounds intő preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art; e.g., in Remington’s Pharmaceutical Sciences, above.

[0088] Fór injection, the agents ofthe invention may be formulated in aqueous Solutions, preferably in physiologically compatible buffers such as Hanks’s solution, Ringer’s solution, or physiological saline buffer. Fór transmucosal administration, penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art.

[0089] Fór órai administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pilis, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, tor órai ingestion by a patient to be treated. Pharmaceutical preparations tor órai use can be obtained by mixing one ormore solid excipient with pharmaceutical combination ofthe invention, optionally grinding the resulting mixture, and Processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, tor example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such asthecross-linked polyvinyl pyrrolidone, agar, or alginic acid óra salt thereof such as sodium alginate. [0090] Dragee cores are provided with suitable coatings. Fór this purpose, concentrated sugár Solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gél, polyethylene glycol, and/or titanium dioxide, lacquer Solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings tor identification or to characterize different combinations of active compound doses. [0091] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with fillér such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations tor órai administration should be in dosages suitable tor such administration.

[0092] Fór buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0093] Fór administration by inhalation, the compounds tor use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orothersuitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin tor use in an inhaleror insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0094] The compounds may be formulated tor parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations tor injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, Solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agentssuch assuspending, stabilizing and/ordispersing agents. [0095] Pharmaceutical formulations tor parenteral administration include aqueous Solutions ofthe active compounds in water-soluble form. Additionally, suspensions ofthe active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity ofthe suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension

ΕΡ 2 316 456 Β1 may alsó contain suitable stabilizers or agents which increase the solubility ofthe compounds to allow forthe preparation of highly concentrated Solutions.

[0096] Alternatively, the active ingredient may be in powder form fór constitution with a suitable vehicle, e.g., sterilé pyrogen-free water, before use.

[0097] The compounds may alsó be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

[0098] In addition to the formulations described previously, the compounds may alsó be formulated as a depót preparation. Such long acting formulations may be administered by implantation (fór example subcutaneously or intramuscularly) or by intramuscular injection. Thus, fór example, the compounds may be formulated with suitable polymeric or hydrophobic materials (fór example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, fór example, as a sparingly soluble salt.

[0099] A pharmaceutical carrier fór the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. A common cosolvent system used is the VPD co-solvent system, which is a solution of 3% w/v benzyl alcohol, 8% w/v ofthe nonpolar surfactant Polysorbate 80™, and 65% w/v polyethylene glycol 300, made upto volume in absolute ethanol. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity ofthe co-solvent components may be varied: fór example, other low-toxicity nonpolar surfactants may be used instead of POLYSORBATE 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and othersugarsor polysaccharides may substitute fór dextrose.

[0100] Alternatively, other delivery systems fór hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers fór hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide alsó may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their Chemical natúré, release the compounds fór a few weeks up to over 100 days. Depending on the Chemical natúré and the biological stability of the therapeutic reagent, additional strategies fór protein stabilization may be employed.

[0101] Many ofthe compounds used in the pharmaceutical combinations ofthe invention may be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts may be formed with many acids, including bút nőt limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free acid or base forms.

[0102] Pharmaceutical compositions suitable fór use in the present invention include compositions where the active ingredients are contained in an amount effective to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount of compound effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light ofthe detailed disclosure provided herein.

[0103] The exact formulation, route of administration and dosage fór the pharmaceutical compositions ofthe present invention can be chosen by the individual physician in view ofthe patient’s condition. (See e.g., Fingl et al. 1975, in The Pharmacological Basis of Therapeutics, Ch. 1 p. 1). Typically, the dose rangé ofthe composition administered to the patient can be from about 0.5 to 1000 mg/kg of the patient’s body weight. The dosage may be a single one or a series of two or more given in the course ofone or more days, as is needed by the patient. Note that fór almost all ofthe specific compounds mentioned in the present disclosure, humán dosages fór treatment of at least somé condition have been established. Thus, in most instances, the present invention will use those same dosages, or dosages that are between about 0.1% and 500%, more preferably between about 25% and 250% ofthe established humán dosage. Where no humán dosage is established, as will be the case fór newly-discovered pharmaceutical compounds, a suitable humán dosage can be inferred from ED₅₀ or ID₅₀ values, or other appropriate values derived from in vitro or in vivő studies, as qualified by toxicity studies and efficacy studies in animals.

[0104] Although the exact dosage will be determined on a drug-by-drug basis, in most cases, somé generalizations regarding the dosage can be made. The daily dosage régimén fór an aduit humán patient may be, fór example, an órai dose of between 0.1 mg and 500 mg of each ingredient, preferably between 1 mg and 250 mg, e.g. 5 to 200 mg or an intravenous, subcutaneous, or intramuscular dose of each ingredient between 0.01 mg and 100 mg, preferably between 0.1 mg and 60 mg, e.g. 1 to 40 mg of each ingredient ofthe pharmaceutical compositions of the present invention or a pharmaceutically acceptable salt thereof calculated as the free base, the composition being administered 1 to 4 times per day. Alternatively the compositions ofthe invention may be administered by continuous intravenous infusion, preferably at a dose of each ingredient up to 400 mg per day. Thus, the totál daily dosage by órai administration of each ingredient will typically be in the rangé 1 to 2000 mg and the totál daily dosage by parenteral administration will typically be in the rangé 0.1 to 400 mg. Suitably the compounds will be administered fór a period of continuous therapy, fór

ΕΡ 2 316 456 Β1 example fór a week or more, or fór months or years.

[0105] Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the modulating effects, or minimál effective concentration (MEC). The MEC will vary fór each compound bút can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

[0106] Dosage intervals can alsó be determined using MEC value. Compositions should be administered using a régimén which maintains plasma levels above the MEC fór 10-90% ofthe time, preferably between 30-90% and most preferably between 50-90%.

[0107] In cases of local administration or selective uptake, the effective local concentration of the drug may nőt be related to plasma concentration.

[0108] The amount of composition administered will, of course, be dependent on the subject being treated, on the subject’s weight, the severity ofthe affliction, the manner of administration and the judgment ofthe prescribing physician. [0109] The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may fór example comprise metál or plastic foil, such as a blister pack. The páckor dispenser device may be accompanied by instructions fór administration. The páckor dispenser may alsó be accompanied with a notice associated with the Container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug fór humán or veterinary administration. Such notice, fór example, may be the labeling approved by the U.S. Food and Drug Administration fór prescription drugs, orthe approved product insert. Compositions comprising a compound ofthe invention formulated in a compatible pharmaceutical carrier may alsó be prepared, placed in an appropriate Container, and labeled fór treatment of an indicated condition.

[0110] It will be understood by those of skill in the art that numerous and various modifications can be made without departing from the spirit ofthe present invention. Therefore, it should be clearly understood that the forms ofthe present invention are illustrative only and are nőt intended to limit the scope ofthe present invention.

Somé Embodiments ofthe Invention [0111] Somé ofthe embodiments ofthe present invention are as follows:

In the first embodiment, the invention relates to a composition as defined in the claims fór use in a method of affecting weight loss or treating obesity comprising a first compound and a second compound, wherein said first compound is an opioid antagonist and said second compound causes increased agonism of a melanocortin 3 receptor (MC3R) or a melanocortin 4 receptor (MC4-R) compared to normál physiological conditions.

[0112] In the second embodiment, the invention relates to the composition ofthe first embodiment, wherein said opioid antagonist antagonizes an opioid receptor in a mammal.

[0113] In the third embodiment, the invention relates to the composition ofthe second embodiment, wherein said opioid receptor is selected from a μ-opioid receptor (MOP-R), a κ-opioid receptor, and a δ-opioid receptor.

[0114] In the fourth embodiment, the invention relates to the composition ofthe second embodiment, wherein said opioid antagonist antagonizes a μ-opioid receptor (MOP-R) in a mammal.

[0115] In the fifth embodiment, the invention relates to the composition ofthe first embodiment, wherein said opioid antagonist is selected from the group consisting of alvimopan, norbinaltorphimine, nalmefene, naloxone, naltrexone, methylnaltrexone, and nalorphine, and pharmaceutically acceptable salts or prodrugs thereof.

[0116] In the sixth embodiment, the invention relates to the composition ofthe first embodiment, wherein said opioid antagonist is a partial opioid agonist.

[0117] In the seventh embodiment, the invention relates to the composition of the sixth embodiment, wherein said partial opioid agonist is selected from the group consisting of pentacozine, buprenorphine, nalorphine, propiram, and lofexidine.

[0118] In the eighth embodiment, the invention relates to the composition ofthe first embodiment, wherein said second compound triggers the release of α-melanocyte stimulating hormoné (a-MSH).

[0119] In the ninth embodiment, the disclosure relates to the composition ofthe eighth embodiment, wherein said second compound increases the extracellular serotonin concentrations in the hypothalamus.

[0120] In the tenth embodiment, the disclosure relates to the composition of the ninth embodiment, wherein said second compound is selected from the group consisting of a selective serotonin reuptake inhibitor (SSRI), a serotonin 2C agonist, and a serotonin 1B agonist.

[0121] In the eleventh embodiment, the disclosure relates to the composition ofthe tenth embodiment, wherein said second compound is selected from the group consisting of fluoxetine, fluvoxamine, sertraline, paroxetine, citalopram,

EP 2 316 456 Β1 escitalopram, sibutramine, duloxetine, and venlafaxine, and pharmaceutically acceptable salts or prodrugs thereof. [0122] In the twelfth embodiment, the disclosure relates to the composition of the first embodiment, wherein said second compound suppresses the expression of the AgRP gene or the production or release of agouti-related protein (AgRP).

[0123] In the thirteenth embodiment, the disclosure relates to the composition of the first embodiment, wherein said second compound suppresses the activity of neurons that express AgRP.

[0124] In the fourteenth embodiment, the disclosure relates to the composition of the first embodiment, wherein said second compound suppresses the expression of the NPY gene or the production or release of neuropeptide Y (NPY). [0125] In the fifteenth embodiment, the disclosure relates to the composition of the first embodiment, wherein said second compound suppresses the activity of neurons that express NPY.

[0126] In the sixteenth embodiment, the disclosure relates to the composition ofthe first embodiment, wherein said second compound is selected from the group consisting of NPY Y1 receptor antagonists, ghrelin antagonists, and leptin. [0127] In the seventeenth embodiment, the disclosure relates to the composition ofthe first embodiment, wherein said second compound agonizes NPY Y2 receptor.

[0128] In the eighteenth embodiment, the disclosure relates to the composition of the first embodiment, wherein said second compound is selected from the group consisting of a γ-amino butyric acid (GABA) inhibitor, a GABA receptor antagonist, and a GABA channel antagonist.

[0129] In the nineteenth embodiment, the disclosure relates to the composition ofthe eighteenth embodiment, wherein said GABA inhibitor is a 5-HT1 b agonist, which may be selected from sumatriptan, almotriptan, naratriptan, frovatriptan, rizatriptan, zomitriptan, and elitriptan.

[0130] In the twentieth embodiment, the disclosure relates to the composition ofthe eighteenth embodiment, wherein said GABA inhibitor suppresses the expression of the AgRP gene.

[0131] In the twenty first embodiment, the disclosure relates to the composition ofthe eighteenth embodiment, wherein said GABA inhibitor suppresses the production or release of AgRP.

[0132] In the twenty second embodiment, the disclosure relates to the composition ofthe eighteenth embodiment, wherein said GABA inhibitor increases the expression ofthe POMC gene.

[0133] In the twenty third embodiment, the disclosure relates to the composition ofthe eighteenth embodiment, wherein said GABA inhibitor increases the production or release of α-MSH from pro-opiomelanocortin (POMC) neurons.

[0134] In the twenty fourth embodiment, the disclosure relates to the composition of the eighteenth embodiment, wherein said GABA inhibitor increases the activity of POMC expressing neurons.

[0135] In the twenty fifth embodiment, the disclosure relates to the composition ofthe eighteenth embodiment, wherein the GABA inhibitor is topiramate.

[0136] In the twenty sixth embodiment, the invention relates to the composition ofthe first embodiment, wherein said second compound is a dopamine reuptake inhibitor.

[0137] In the twenty seventh embodiment, the disclosure relates to the composition ofthe twenty sixth embodiment, wherein said dopamine reuptake inhibitor is phentermine.

[0138] In the twenty eighth embodiment, the invention relates to the composition ofthe first embodiment, wherein said second compound is a norepinephrine reuptake inhibitor.

[0139] In the twenty ninth embodiment, the invention relates to the composition of the twenty eighth embodiment, wherein said norepinephrine reuptake inhibitor is bupropion.

[0140] In the thirtieth embodiment, the invention relates to the composition of the first embodiment, wherein said second compound may be a dopamine agonist.

[0141] In the thirty first embodiment, the disclosure relates to the composition of the thirtieth embodiment, wherein said dopamine agonist is selected from the group consisting of cabergoline, amantadine, lisuride, pergolide, ropinirole, pramipexole, and bromocriptine.

[0142] In the thirty second embodiment, the invention relates to the composition ofthe first embodiment, wherein said second compound may be a norepinephrine releaser.

[0143] In the thirty third embodiment, the disclosure relates to the composition ofthe thirty second embodiment, wherein said norepinephrine releaser is diethylpropion.

[0144] In the thirty fourth embodiment, the invention relates to the composition of the first embodiment, wherein said second compound is a combination of a dopamine reuptake inhibitor and a norepinephrine reuptake inhibitor.

[0145] In the thirty fifth embodiment, the invention relates to the composition ofthe thirty fourth embodiment, wherein said second compound is bupropion.

[0146] In the thirty sixth embodiment, the disclosure relates to the composition of the first embodiment, wherein said second compound is a combination ofa SSRI and a norepinephrine reuptake inhibitor.

[0147] In the thirty seventh embodiment, the disclosure relates to the composition of the thirty sixth embodiment, wherein said second compound is selected from sibutramine, venlafaxine, and duloxetine.

[0148] In the thirty eighth embodiment, the disclosure relates to the composition ofthe first embodiment, wherein said

ΕΡ 2 316 456 Β1 first compound is naltrexone and said second compound is fluoxetine.

[0149] In the thirty ninth embodiment, the disclosure relates tothe composition ofthe thirty eighth embodiment, wherein the naltrexone is in a time-release formulation whereas the fluoxetine is in an immediate release formulation.

[0150] In the fortieth embodiment, the invention relates to a composition as defined in the claims fór use in a method of affecting weight loss, comprising identifying an individual in need thereof and treating that individual to antagonize opioid receptor activity and to enhance α-MSH activity.

[0151] In the forty first embodiment, the invention relates to the composition fór use according to the method of the fortieth embodiment, wherein said individual has a body mass index greater than 25.

[0152] In the forty second embodiment, the invention relates to the composition fór use according to the method of the fortieth embodiment, wherein opioid receptor activity is antagonized by administering an opioid receptor antagonist. [0153] In the forty third embodiment, the invention relates to the composition fór use according to the method of the forty second embodiment, wherein the opioid receptor antagonist is a MOP receptor antagonist.

[0154] In the forty fourth embodiment, the invention relates to the composition fór use according to the method ofthe fortieth embodiment, wherein the opioid receptor antagonist is selected from alvimopan, norbinaltorphimine, nalmefene, naloxone, naltrexone, methylnaltrexone, and nalorphine, and pharmaceutically acceptable salts or prodrugs thereof. [0155] In the forty fifth embodiment, the invention relates to the composition fór use according to the method of the forty second embodiment, wherein said opioid receptor antagonist is a partial opioid agonist.

[0156] In the forty sixth embodiment, the invention relates to the composition fór use according to the method ofthe forty fifth embodiment, wherein said partial opioid agonist is selected from the group consisting of pentacozine, buprenorphine, nalorphine, propiram, and lofexidine.

[0157] In the forty seventh embodiment, the invention relates to the composition fór use according to the method of the fortieth embodiment through the forty fifth embodiment, wherein α-MSH activity is enhanced by administering a compound, wherein said compound triggers release of α-MSH or increases the activity of neurons that express a-MSH. [0158] In the forty eighth embodiment, the disclosure relates to the method ofthe forty seventh embodiment, wherein said compound is a selective serotonin reuptake inhibitor (SSRI) or a specific 5-HT receptor agonist.

[0159] In the forty ninth embodiment, the disclosure relates to the method of the forty eighth embodiment, wherein said 5-HT receptor is selected from 5-HT1b receptor and 5-HT2c receptor.

[0160] In the fiftieth embodiment, the disclosure relates to the method ofthe forty eighth embodiment, wherein said SSRI is selected from fluoxetine, fluvoxamine, sertraline, paroxetine, citalopram, escitalopram, sibutramine, duloxetine, and venlafaxine, and pharmaceutically acceptable salts or prodrugs thereof.

[0161] In the fifty first embodiment, the disclosure relates to the method of the forty seventh embodiment, wherein said compound is a γ-amino butyric acid (GABA) inhibitor.

[0162] In the fifty second embodiment, the disclosure relates to the method ofthe fifty first embodiment, wherein said GABA inhibitor is a 5-HT 1 b receptor agonist.

[0163] In the fifty third embodiment, the disclosure relates to the method of the fifty first embodiment, wherein said GABA inhibitor suppresses the expression of the AgRP gene.

[0164] In the fifty fourth embodiment, the disclosure relates to the method of the fifty first embodiment, wherein said GABA inhibitor suppresses the production or release of AgRP.

[0165] In the fifty fifth embodiment, the disclosure relates to the method ofthe forty eighth embodiment, wherein said 5-HT agonists inhibits the NPY/AgRP/GABA neurons.

[0166] In the fifty sixth embodiment, the disclosure relates to the method of the fifty first embodiment, wherein said GABA inhibitor suppresses the activity of neurons that express AgRP.

[0167] In the fifty seventh embodiment, the disclosure relates to the method ofthe fifty first embodiment, wherein said GABA inhibitor is topiramate.

[0168] In the fifty eighth embodiment, the invention relates to the composition fór use according to the method ofthe forty seventh embodiment, wherein said compound may be a dopamine reuptake inhibitor, a norepinephrine reuptake inhibitor, a dopamine agonist, a norepinephrine releaser, a combination of a dopamine reuptake inhibitor and a norepinephrine reuptake inhibitor, and a combination ofa SSRI and a norepinephrine reuptake inhibitor.

[0169] In the fifty ninth embodiment, the invention relates to the composition fór use according to the method ofthe fifty eighth embodiment, wherein said compound is nőt phentermine.

[0170] In the sixtieth embodiment, the invention relates to the composition fór use according to the method of the fortieth embodiment, with the proviso that the individual is nőt suffering from Prader-Willi syndrome.

[0171] In the sixty first embodiment, the invention relates to the the composition fór use according to method of the fortieth embodiment, with the proviso that if the opioid receptor is antagonized using naltrexone, then release of a-MSH is nőt stimulated with fluoxetine.

[0172] In the sixty second embodiment, the invention relates to the composition fór use according to the method of the fortieth embodiment, wherein said treating step comprises administering to said individual a first compound and a second compound, wherein said first compound is an opioid antagonist and said second compound enhances a-MSH

ΕΡ 2 316 456 Β1 activity.

[0173] In the sixty third embodiment, the invention relates to the composition fór use according to the method ofthe sixty second embodiment, wherein said first compound and said second compound are administered nearly simultaneously.

[0174] In the sixty fourth embodiment, the invention relates to the composition fór use according to the method ofthe sixty third embodiment, wherein said first compound is administered prior to said second compound.

[0175] In the sixty fifth embodiment, the invention relates to the composition fór use according to the method of the sixty fourth embodiment, wherein said first compound is administered subsequent to said second compound.

[0176] In the sixty sixth embodiment, the invention relates to a composition as defined in the claims fór use in a method of increasing satiety in an individual comprising identifying an individual in need thereof and treating that individual to antagonize opioid receptor activity and to enhance α-MSH activity.

[0177] In the sixty seventh embodiment, the invention relates to the composition fór use according to the method of the sixty sixth embodiment, wherein said treating step comprises administering to said individual a first compound and a second compound, wherein said first compound is an opioid antagonist and said second compound enhances a-MSH activity.

[0178] In the sixty eighth embodiment, the invention relates to the composition fór use according to the method ofthe sixty seventh embodiment, wherein said first compound and said second compound are administered nearly simultaneously.

[0179] In the sixty ninth embodiment, the invention relates to the composition fór use according to the method ofthe sixty seventh embodiment, wherein said first compound is administered prior to said second compound.

[0180] In the seventieth embodiment, the invention relates to the composition fór use according to the method ofthe sixty seventh embodiment, wherein said first compound is administered subsequent to said second compound.

[0181] In the seventy first embodiment, the invention relates to a composition as defined in the claims fór use in a method of increasing energy expenditure in an individual comprising identifying an individual in need thereof and treating that individual to antagonize opioid receptor activity and to enhance α-MSH activity.

[0182] In the seventy second embodiment, the invention relates to the composition fór use according to the method ofthe seventy first embodiment, wherein said treating step comprises administering to said individual a first compound and a second compound, wherein said first compound is an opioid antagonist and said second compound enhances aMSH activity.

[0183] In the seventy third embodiment, the invention relates to the composition fór use according to the method of the seventy second embodiment, wherein said first compound and said second compound are administered nearly simultaneously.

[0184] In the seventy fourth embodiment, the invention relates to the composition fór use according to the method of the seventy second embodiment, wherein said first compound is administered prior to said second compound.

[0185] In the seventy fifth embodiment, the invention relates to the composition fór use according to the method of the seventy second embodiment, wherein said first compound is administered subsequent to said second compound. [0186] In the seventy sixth embodiment, the invention relates to a composition as defined in the claims fór use in a method of suppressing the appetite ofan individual comprising identifying an individual in need thereof and treating that individual to antagonize opioid receptor activity and to enhance α-MSH activity.

[0187] In the seventy seventh embodiment, the invention relates to the composition fór use according to the method ofthe seventy sixth embodiment, wherein said treating step comprises administering to said individual a first compound and a second compound, wherein said first compound is an opioid antagonist and said second compound enhances aMSH activity.

[0188] In the seventy eighth embodiment, the invention relates to the composition fór use according to the method of the seventy seventh embodiment, wherein said first compound and said second compound are administered nearly simultaneously.

[0189] In the seventy ninth embodiment, the invention relates to the composition fór use according to the method of the seventy seventh embodiment, wherein said first compound is administered prior to said second compound.

[0190] In the eightieth embodiment, the invention relates to the composition fór use according to the method ofthe seventy seventh embodiment, wherein said first compound is administered subsequent to said second compound. [0191] In the eighty first embodiment, the disclosure relates to a method of affecting weight loss in an individual comprising identifying an individual in need thereof and treating that individual with a combination of naltrexone and fluoxetine, provided that the individual does nőt sufferfrom Prader-Willi syndrome or binge eating disorder.

[0192] In the eighty second embodiment, the disclosure relates to the method ofthe eighty first embodiment, wherein the individual has a BMI greater than 30.

[0193] In the eighty third embodiment, the disclosure relates to the method of the eighty first embodiment, wherein the individual has a BMI greater than 25.

ΕΡ 2 316 456 Β1 [0194] In the eighty fourth embodiment, the disclosure relates to the method of the eighty first embodiment, wherein the naltrexone is in a time-release formulation whereas the fluoxetine is in an immediate release formulation.

[0195] In the eighty fifth embodiment, the disclosure relates to the method of the eighty fourth embodiment, wherein the plasma concentration level of both naltrexone and fluoxetine foiiow a similar concentration profile.

[0196] In the eighty sixth embodiment, the disclosure relates to the method ofthe eighty fourth embodiment, wherein the naltrexone and the fluoxetine are administered substantially simultaneously.

[0197] In the eighty seventh embodiment, the disclosure relates to the method ofthe eighty fourth embodiment, wherein the naltrexone is administered prior to the fluoxetine.

[0198] In the eighty eighth embodiment, the disclosure relates to the method ofthe eighty fourth embodiment, wherein the naltrexone is administered subsequent to the fluoxetine.

Examples [0199] The invention is defined in the claims. The examples below are non-limiting and are merely representative of various aspects ofthe invention and associated disclosure.

Example 1: Combination of fluoxetine and naltrexone:

[0200] Individuals having a BMI of greater than 25 are identified. Each individual is instructed to take one 20 mg tablet of fluoxetine (PROZAC®) on a daily basis, in addition to one 50 mg tablet of naltrexone on a daily basis.

[0201] The individuals are monitored for a period of months. It is recommended that the dosage be adjusted so that each individual loses weight at a rate of 10% of initial weight every 6 months. However, the rate of weigh loss for each individual may be adjusted by the treating physician based on the individual’s particular needs.

[0202] lf the initial dosage is nőt effective, then the fluoxetine dosage can be increased by 20 mg per day, though never exceeding 80 mg totál per day. lf the initial dosage results in a more rapid weight loss than the above rate, the dosage of each of fluoxetine or naltrexone can be reduced.

[0203] Fluoxetine has a physiological haif life of about 9 hours, whereas that of naltrexone is about 1.5 hours. Thus, in somé cases, it is beneficial to administer one dose of fluoxetine per day in conjunction with two or three or more doses of naltrexone throughout the day. Naltrexone may alsó be in a time-release formulation where the dose is administered once a day, bút naltrexone gradually enters the blood stream throughout the day, or in the course of a 12 hour period.

Example 2: Combination of fluoxetine and nalmefene:

[0204] Individuals having a BMI of greater than 25 are identified. Each individual is instructed to take one 20 mg tablet of fluoxetine (PROZAC®) on a daily basis. In addition, each individual is injected with 1 mL of a solution of 100 μg of nalmefene in 1 mL of saline, intravenously, intramuscularly, or subcutaneously.

[0205] The individuals are monitored for a period of months. It is recommended that the dosage be adjusted so that each individual loses weight at a rate of 10% of initial weight every 6 months. However, the rate of weigh loss for each individual may be adjusted by the treating physician based on the individual’s particular needs.

[0206] lf the initial dosage is nőt effective, then the fluoxetine dosage can be increased by 20 mg per day, though never exceeding 80 mg totál per day. In addition, the dosage of nalmefene may be increased up to 2 mL of a solution of 1 mg of nalmefene in 1 mL of saline. lf the initial dosage results in a more rapid weight loss than the above rate, the dosage of each of fluoxetine or nalmefene can be reduced.

Example 3: Combination of fluoxetine and naloxone:

[0207] Individuals having a BMI of greater than 25 are identified. Each individual is instructed to take one 20 mg tablet of fluoxetine (PROZAC®) on a daily basis. In addition, each individual is injected with 1 mL of a solution of 400 μg of naloxone in 1 mL of saline, intravenously, intramuscularly, or subcutaneously.

[0208] The individuals are monitored for a period of months. It is recommended that the dosage be adjusted so that each individual loses weight at a rate of 10% of initial weight every 6 months. However, the rate of weigh loss for each individual may be adjusted by the treating physician based on the individual’s particular needs.

[0209] lf the initial dosage is nőt effective, then the fluoxetine dosage can be increased by 20 mg per day, though never exceeding 80 mg totál per day. lf the initial dosage results in a more rapid weight loss than the above rate, the dosage of each of fluoxetine or nalmefene can be reduced.

ΕΡ 2 316 456 Β1

Example 4: Combination of opioid antagonist and sibutramine:

[0210] Individuals having a BMI of greater than 25 are identified. Each individual is instructed to take nalmefene, naltrexone, or naloxone in the dosage setforth in Examples 1-3. In addition, each individual is instructed to take 10 mg of sibutramine oraily once a day.

[0211] The individuals are monitored for a period of months. It is recommended that the dosage be adjusted so that each individual loses weight at a rate of 10% of initial weight every 6 months. However, the rate of weigh loss for each individual may be adjusted by the treating physician based on the individual’s partieuiar needs.

[0212] If the initial dosage is nőt effective, then the sibutramine dosage can be increased 15 mg per day. Dosages of sibutramine in excess of 15 mg per day are nőt recommended. If the initial dosage results in a more rapid weight loss than the above rate, the dosage of each of sibutramine, nalmefene, naltrexone, or naloxone can be reduced.

Example 5: Combination of opioid antagonist and bupropion:

[0213] Individuals having a BMI of greater than 25 are identified. Each individual is instructed to take nalmefene, naltrexone, or naloxone in the dosage setforth in Examples 1-3. In addition, each individual is instructed to take bupropion. The usual aduit does is 300 mg per day, given three times daily. Dosing should begin at 200 mg per day, given as 100 mg twice daily. Based on clinical response, this dose may be increased to 300 mg per day, given as 100 mg three times daily. No single dose is to exceed 150 mg.

[0214] The individuals are monitored for a period of months. It is recommended that the dosage be adjusted so that each individual loses weight at a rate of 10% of initial weight every 6 months. However, the rate of weigh loss for each individual may be adjusted by the treating physician based on the individual’s partieuiar needs.

Example 6: Combination of opioid antagonist and phentermine:

[0215] Individuals having a BMI of greater than 25 are identified. Each individual is instructed to take nalmefene, naltrexone, or naloxone in the dosage set forth in Examples 1-3. In addition, each individual is instructed to take 37.5 mg of phentermine oraily once a day.

[0216] The individuals are monitored for a period of months. It is recommended that the dosage be adjusted so that each individual loses weight at a rate of 10% of initial weight every 6 months. However, the rate of weigh loss for each individual may be adjusted by the treating physician based on the individual’s partieuiar needs.

Example 7: Combinations with naltrexone:

[0217] In a multicenter, randomized, blinded, placebo-controlled clinical trial with 6 groups, the following drug combinations are tested:

• Group 1 • Group 2 • Group 3 • Group 4 • Group 5 • Group 6

Fluoxetine 60 mg po QD plus Naltrexone 50 mg po QD Fluoxetine 60 mg po QD plus N-placebo po QD Bupropion-SR 150 mg po BID plus Naltrexone 50 mg po QD Bupropion-SR 150 mg po BID plus N-placebo po QD P-placebo po BID plus Naltrexone 50 mg po QD P-placebo po BID plus N-placebo po QD [0218] In any ofthe above groups, the dosage offluoxetine may be in the rangé between 6 mg and 60 mg, for example, 6 mg, 10 mg, 12 mg, 18 mg, 20 mg, 24 mg, 30 mg, 36 mg, 40 mg, 42 mg, 45 mg, 48 mg, 54 mg, and 60 mg. Bupropion may be administered in doses in the rangé between 30 mg and 300 mg, for example, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, and 300 mg. Naltrexone may be administered in doses in the rangé between 5 mg and 50 mg, for example, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, and 50 mg.

[0219] Subjects are evaluated as out-patients during this study. All subjects in this trial récéivé diet instruction, behavior modification advice and instruction to increase their activity, a régimén shown to give weight loss. Subjects are randomized to récéivé study drugs in various combinations.

[0220] Subjects in groups 5 and 6 cross-over to treatment with fluoxetine plus naltrexone or bupropion SR plus naltrexone after week 16 for the extension treatment period which provide additional data on safety of the combination therapies.

[0221] The primary endpoint is percent and absolute change from baseline in body weight at 16 weeks. Secondary

ΕΡ 2 316 456 Β1 endpoints include weight loss at 24, 36, and 48 weeks, number and proportion of subjects who achieve at least a 5% weight loss and a 10% weight loss (responder analysis), changes in obesity-associated cardiovascular risk factors (totál cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, glucose and insulin) and waist circumference, and safety and tolerability. Adverse events, laboratory parameters, vitai signs, and the Hospital Anxiety and Depression (HAD) Scale are used to monitor safety and tolerability.

Example 8: Dose-response experiments:

[0222] Seventy, four week old, male C57/B16J mice (Jackson Laboratory), 22-30 g were sham injected daily with 0.1 mL 0.9% saline (pH 7.4) fór 1 week prior to the experiments. Animals were weighed and randomized to 1 of 7 weightmatched dose groups (0, 1.5, 3, 5.5, 10, 18, and 30 mg/kg; n=10/group fór fluoxetine; 0, 1.5, 3, 5.5, 10, 18, and 30 mg/kg; n=3/group fór naltrexone) the day before experiments began. Food was removed between 4:30-5:30 pm the day before the experiment. Animals received a 0.3 mL bolus (fluoxetine) or0.1 mL bolus (naltrexone) intraperitoneal injection between 9-10:30 am, and food was provided immediately following injection. 3 animals/group received injections on each testing day (i.e., 3 runs of 3/group; 1 run of 1 /group). Food was weighed 1,2,4, 8, and 24 h post-injection. Cumulative food intake ± SEM was calculated and analyzed using Prizm. The SEM fór these numbers was found to be between 0.0041 and 0.26. Doses were lóg transformed and fit to a sigmoidal curve, food intake was expressed as a proportion ofthe food intake in saline treated animals. From the curve, the EC₅₀ at each time point fór each drug was determined. [0223] Similar procedures as described above were followed using fluvoxamine and nalmefene, and bupropion and naltrexone. The results are set forth in the table below.

<td rowspan="2"></td><td> Hour 1</td><td> Hour 2</td><td> Hour 4</td><td> Hour 8</td><td> Hour24</td>

<td> MEAN</td><td> MEAN</td><td> MEAN</td><td> MEAN</td><td> MEAN</td>

<td> Saline</td><td> 1.00</td><td> 1.00</td><td> 1.00</td><td> 1.00</td><td> 1.00</td>

<td> Fluvoxamine</td><td> 0.77</td><td> 0.85</td><td> 0.95</td><td> 0.91</td><td> 0.92</td>

<td> Nalmefene</td><td> 0.0083</td><td> 0.11</td><td> 0.57</td><td> 0.81</td><td> 0.98</td>

<td> Fluvoxamine + Nalmefene</td><td> 0.0041</td><td> 0.019</td><td> 0.42</td><td> 0.79</td><td> 0.99</td>

<td> Bupropion</td><td> 0.32</td><td> 0.64</td><td> 0.97</td><td> 0.96</td><td> 0.99</td>

<td> Naltrexone</td><td> 0.41</td><td> 0.77</td><td> 0.99</td><td> 1.1</td><td> 0.98</td>

<td> Naltrexone + Bupropion</td><td> 0.042</td><td> 0.34</td><td> 0.89</td><td> 0.97</td><td> 0.95</td>

<td> Naltrexone</td><td> 0.30</td><td> 0.56</td><td> 0.83</td><td> 0.98</td><td> 1.01</td>

<td> Fluoxetine</td><td> 0.36</td><td> 0.57</td><td> 0.68</td><td> 0.76</td><td> 1.05</td>

<td> Naltrexone + Fluoxetine</td><td> 0.070</td><td> 0.26</td><td> 0.72</td><td> 0.95</td><td> 1.04</td>

Example 9: Electrophysiology Data:

[0224] To test the hypothesis that drugs selectively activate POMC neurons, we used a strain of transgenic mice expressing green fluorescent protein (EGFP, Clontech), under the transcriptional control of mouse Pomc genomic sequences that include a region located between -13 kb and -2 kb required fór accurate neuronal expression Bright green fluorescence (509 nm) was seen in the two CNS regions where POMC is produced: the ARC and the nucleus of the solitary tract. Under ultraviolet (450 - 480 nm) excitation, POMC neurons were clearly distinguished from adjacent, nonfluorescent neurons visualized under infrared optics.

[0225] 200 μπι thick coronal slices were cut from the ARC of four-week old male POMC-EGFP mice. Slices were maintained in Krebs solution (NaCI (126 mM), KCI (2.5 mM), MgCI₂ 91.2 mM), CaCI₂.2H₂O (2.4 mM), NaH₂PO₄.H₂O (1.2 mM), NaHCO₃ (21.4 mM), glucose (11.1 mM)) at 35 °C and saturated with 95% O₂ and 5% CO₂ fór 1 hr prior to recordings. Recordings were made in Krebs at 35 °C. Slices were visualized on an Axioskop FS2 plus (Zeiss) through standard infra red optics and using epifluorescence through a FITC (longpass) filter set. POMC-EGFP neurons in hypothalamic slices had a resting membráné potential of-40to-45 mV and exhibited frequent spontaneous action potentials. Cell-attached recordings were made from fluorescent neurons using an Axopatch 200B amplifier (Axon Instruments) and Clampex 8 (Axon Instruments). Action potentials frequencies were determined using an event detection program (Mini Analysis; Synaptosoft Inc., Decatur, GA). Drugs were applied to the bath fór 3 min.

[0226] Data were analyzed by determining the average firing rate fór 500 sec prior to drug addition, and analyzing

ΕΡ 2 316 456 Β1 treatments reiative to this frequency (that is, firing rates were normalized to the pre-treatment frequency). The ratio’s listed fór the combinations are the ratio of the effect of naltrexone in combination with the POMC activator, reiative to naltrexone alone (that is the extra effectiveness that naltrexone conferred to the POMC activator). Alsó listed are the mean effects ofthe drugs alone.

Fenfluramine

Fenfluramine + Naltrexone Fluoxetine

Fluoxetine + Naltrexone Dopamine

Dopamine + Naltrexone

2X increase (n=6) 5.2X (n=8)

3X (n=1) 1.2X(n=1)

11X (n=9) 1.5X(n=3) [0227] Naltrexone alone has a potent (7X) bút variable effect. many cells did nőt respond to naltrexone alone, bút gave a significant response to combination treatment. Heisler et al. (Science 297(5581):609-11 (2002)) show that fenfluramine alone causes a 200% effect.

<td> Drug</td><td> Dose</td><td> Effect (%)</td><td> Drug</td><td> Dose</td><td> Effect (%)</td><td> Ratio</td>

<td> Naltrexone</td><td> 1 μΜ</td><td> 29650</td><td> Naltrexone + Fenfluramine</td><td> 1 μΜ + 20μΜ</td><td> 15080</td><td> 0.51</td>

<td> Naltrexone</td><td> 1 μΜ</td><td> 2200</td><td> Naltrexone + Fenfluramine</td><td> 1 μΜ + 20μΜ</td><td> 11440</td><td> 520</td>

<td> Naltrexone</td><td> 1 μΜ</td><td> 2500</td><td> Naltrexone + Fenfluramine</td><td> 1 μΜ + 20μΜ</td><td> 856</td><td> 0.34</td>

<td> Naltrexone</td><td> 1 μΜ</td><td> 417</td><td> Naltrexone + Fenfluramine</td><td> 1 μΜ + 20μΜ</td><td> 5700</td><td> 13.67</td>

<td> Naltrexone</td><td> 1 μΜ</td><td> 177</td><td> Naltrexone + Fenfluramine</td><td> 1 μΜ + 20μΜ</td><td> 430</td><td> 2.43</td>

<td> Naltrexone</td><td> 1 μΜ</td><td> 200</td><td> Naltrexone + Fenfluramine</td><td> 1 μΜ + 20μΜ</td><td> 2933</td><td> 14.67</td>

<td> Naltrexone</td><td> 1 μΜ</td><td> 700</td><td> Naltrexone + Fenfluramine</td><td> 1 μΜ + 20μΜ</td><td></td><td></td>

<td> Naltrexone</td><td> 1 μΜ</td><td> 900</td><td> Naltrexone + Fenfluramine</td><td> 1 μΜ + 20μΜ</td><td> 1831</td><td> 2.03</td>

<td> Naltrexone</td><td> 1 μΜ</td><td> 2273</td><td> Naltrexone + Fenfluramine</td><td> 1 μΜ + 20μΜ</td><td></td><td></td>

<td> Naltrexone</td><td> 1 μΜ</td><td> 300</td><td> Naltrexone + Fenfluramine</td><td> 1 μΜ + 20μΜ</td><td> 920</td><td> 3.07</td>