WO1998003175A1

WO1998003175A1 - Treatment of obesity

Info

Publication number: WO1998003175A1
Application number: PCT/AU1997/000453
Authority: WO
Inventors: Paul Zev Zimmet; Roderick Alan Westerman; Gregory Collier
Original assignee: International Diabetes Institute
Priority date: 1996-07-18
Filing date: 1997-07-18
Publication date: 1998-01-29
Also published as: ZA976333B; AUPO108596A0

Abstract

A method for the treatment of obesity in a patient or for the prevention of obesity in a patient pre-disposed to obesity, comprises administration to the patient of an effective amount of nicotine or a nicotine analogue.

Description

TREATMENT OF OBESITY

Field of the Invention:

The invention relates to the treatment of obesity in all subjects, whether diabetic or nondiabetic, with or without concomitant other therapies, and in particular it relates to a novel method of treatment using nicotinic mechanisms and compounds active at nicotinic binding sites and affecting other relevant sites such as leptin, neuropeptide Y (NPY), galanin, serotonin or dopamine receptors in nerve cells to increase the sensitivity of brain responsiveness to leptin.

Background of the Invention:

The positional cloning of the obese (ob) gene (1) and the subsequent preparation of its encoded product, leptin (2,3), have provided powerful fresh impetus for research relating to the aetiology and control of obesity. Friedman and his coworkers have suggested that leptin, a 16 kilodalton protein, may be involved in a feedback loop as a sensing hormone ie. "iipostat", from adipose tissue to a leptin receptor in the hypothalamus (2), responding to the size of adipose tissue mass. Recently, leptin receptors have been located in the hypothalamus of the diabetic (db) mouse (4) providing further support for the feedback hypothesis that was generated over 20 years ago by Coleman et al from parabiosis experiments in obese (ob/ob) and diabetic (db/db) mice (5).

The present invention provides a method for the treatment of obesity, or for the prevention of obesity in persons pre-disposed to obesity, in which the sensitivity of hypothalamic leptin or other receptors, such as NPY etc, is modified so that leptin synthesis is modulated and as a consequence body weight is controlled. Summary of the Invention:

In one aspect, the present invention relates to a method for the treatment of obesity in a patient, which comprises administration to the patient of an effective amount of nicotine or a nicotine analogue.

In another aspect, the present invention relates to a method for the prevention of obesity in a patient pre-disposed to obesity, which comprises administration to the patient of an effective amount of nicotine or a nicotine analogue.

Preferably, the patient is a human patient, and the methods of the present invention extend to treatment or prevention of obesity in both diabetic and non- diabetic patients.

Nicotine is of course a well known substance. It is to be understood, however, that the present invention also extends to the use of nicotinic analogues such as ABT 418, (+)-2-methylpiperidine, and other agonists or antagonists of nicotine shown to alter the brain and hypothalamic responsiveness to leptin. Various nicotine analogues which are agonists or antagonists of nicotine are disclosed by Williams el a/ (1994) Drug News and Perspectives, Vol. 7, No. 4, 205-223, the contents of which are incorporated herein by reference. The present invention extends to the use of these nicotine analogues, particularly those which are selective for the hypothalamic leptin receptors in humans.

In view of the ethical dilemma of the use of nicotine, the active nicotine component in tobacco smoke, for any therapeutic or prophylactic purpose, the use of nicotine analogues as discussed above, such as ABT 418 and (+)-2-methylpiperidine, in the method of this invention may be preferable. Such nicotine analogues may also have fewer side effects, and may be tailored to the specific hypothalamic nicotine acetylcholine receptor (nAchR) epitope which may be involved in the modulation of leptin receptor sensitivity, either directly or indirectly.

It will be understood that the present invention extends to combination therapy in which more than one active component is used in the therapeutic and prophylactic methods of treatment of obesity. Thus, combinations of nicotine and nicotine analogues may be used in such combination therapy, including a combination such as an agonist together with an antagonist which may be particularly useful in modulating leptin sensitivity and weight gain.

In addition, the use of nicotine and nicotine analogues in accordance with the present invention may be combined with the use of leptin in an synergistic treatment of obesity in order to improve the sensitivity to leptin and to achieve the same therapeutic or prophylactic effect in treating obesity at lower doses and cost. Such a synergistic treatment may involve either co-administration or separate administration of leptin with the active component(s) of the present invention.

Conveniently, in both therapeutic or prophylactic use in treatment of obesity, the nicotine or nicotine analogue will be administered in the form of a pharmaceutical composition. The formulation of such pharmaceutical compositions is well known to persons skilled in this field. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions. It is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the human subjects to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and/or diluent. The specifications for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active ingredient for the particular treatment.

A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practised using any mode of administration that is medically acceptable, meaning any mode that produces therapeutic levels of the active component of the invention without causing clinically unacceptable adverse effects. Such modes of administration include oral, topical, transmucosal, transdermal, rectal, nasal or parenteral (e.g. subcutaneous, intramuscular and intravenous) routes. Continuous transdermal administration by means of a skin patch or the like is a known, medically acceptable method of administration of nicotine and may be used in the therapeutic and prophylactic methods of the present invention.

The compositions may conveniently be prepared by any of the methods well known in the art of pharmacy. Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active component, in liposomes or as a suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion. Such preparations may also include a fixed- or metered-dose nasal spray. Alternatively, the active component may be formulated as a chewing gum for intermittent oral administration.

Other delivery systems can include sustained or slow release delivery systems. Preferred sustained or slow release delivery systems are those which can provide for release of the active component of the invention in sustained or slow release lozenges, tablets, pellets or capsules. Many types of sustained release delivery systems are available. These include, but are not limited to: (a) erosional systems in which the active component is contained within a matrix, and (b) diffusional systems in which the active component permeates at a controlled rate through a polymer.

Oral administration for many conditions will be preferred because of the convenience to the patient, although topical and localised sustained or slow delivery may be even more desirable for certain treatment regimens.

The active component is administered in an effective amount. Reference herein to an effective amount means that amount necessary at least partly to attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular condition being treated. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition and individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgement. It will be understood by those of ordinary skill in the art, however, that a lower dose or tolerable dose may be administered for medical reasons, psychological reasons or for virtually any other reasons.

Preferably, the effective amount will be selected so that administration of the active component will be free of adverse or undesired side effects. Generally, daily oral doses of active component will be from about 0.01 mg/kg per day to 10 mg/kg per day. Small doses (0.01-1 mg/kg per day) may be administered initially, followed by increasing doses up to about 5-10 mg/kg per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localised delivery route) may be employed to the extent patient tolerance and side effects permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds. In the treatment of obesity in morbidly obese individuals, daily doses of 2-20 mg of active component may be required.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Detailed Description of the Invention:

Section A: EPIDEMIOLOGY, OBESITY, NON-INSULIN DIABETES MELLITUS AND LEPTIN

Leptin: Importance in diabetes.

Unlike the ob/ob mouse (1), there is no current evidence for a major role of ob gene mutations and leptin deficiency in human obesity (6,7). Hyperleptinaemia has been demonstrated in animal models such as Psammomys obesus (8) normal mice made obese by a high fat diet (9) and human obese subjects (10,11) suggesting that leptin receptors in the central nervous system are either downregulated or defective, if one assumes the brain is the location of the apparent resistance to leptin. A defect in the feedback mechanism has been demonstrated in the (db/db) mouse (12), and a similar scenario could lead to human obesity, or some forms of it. The human ob gene is normal in obese humans (13, 14) and in these subjects there is over- rather than under-expression , with increased amounts of ob messenger RNA produced (15,16) which correlated with body weight and fat mass. However, leptin receptors and hypothalamic feedback mechanisms in humans have not yet been defined.

An alternative hypothesis is proposed by Schwartz et al (17) who measured leptin levels in both the cerebrospinal fluid and plasma. They demonstrated that the CSF.plasma leptin ratio was lower among those in the highest as compared with the lowest plasma leptin quintile - a 5.4 fold difference! They explained this disparity in terms of a saturable mechanism which mediates the CSF leptin transport, and that a reduced efficiency of brain leptin delivery among obese individuals with high plasma leptin levels results in apparent leptin resistance (17). Thus the answer to the question is leptin important in diabetes lies at least partly in the manifold unsatisfactory methods attempting to treat obesity in diabetes (18).

Evidence for a Leptin role in body weight regulation,

Animal studies published so far suggest that leptin may have a role in appetite regulation, energy expenditure and possibly modulation of insulin sensitivity (2,3). A major question is whether these observations apply to humans, or whether the leptin concentration in human blood is solely a reflection of the amount of adipose tissue. To examine the importance of leptin in human obesity and non-insulin dependent diabetes mellitus (NIDDM), population based studies serve as a useful tool. In the South Pacific island, Western Samoa, the prevalence of obesity and NIDDM prevalence have escalated as a consequence of modernisation of lifestyle during the latter half of the 20th century (19-21). Epidemiology of leptin levels.

A recent study by Zimmet et al (22) reports the first epidemiological associations between leptin concentrations in humans and anthropometric, demographic, behavioural and metabolic risk factors in a Western Samoan population, using data collected in 1991 (20,21). Among these relatively obese individuals, leptin was strongly correlated with measures of obesity in both men and women. These data agree with other recent reports (10,11) and are consistent with leptin concentration being directly related to the adipose tissue mass. However, there appears to be considerable variability in the absolute level of leptin between individuals with similar degrees of obesity in this and other studies (10,11). This observation indicates the potential importance of other variables that may regulate blood leptin concentration, including physical activity, nutritional factors, genotype, fat distribution and insulin or other hormones. The independent effects of body mass index (BMI) and waist circumference on leptin levels suggest that the distribution of body fat may also be an important determinant of leptin concentration.

Animal models suggest ob RNA levels are higher in adipose tissue from central fat deposits than other regional adipose sites (23). Zimmet et al's finding that waist circumference was, independently of BMI, associated with leptin is consistent with this observation. The weaker association of WHR with leptin, and its lack of association independent of BMI may reflect the fact that WHR does not measure the absolute amount of intraabdominal fat.

Gender differences in leptin concentrations.

Leptin levels were found to be significantly higher in women than in men at all

BMI levels. However, it has been shown that these differences disappeared when leptin was compared across similar body fat percentages (10,11). This is consistent with a higher body fat content of women at any BMI. Women also had higher concentrations of leptin than men for the same waist circumference, which may reflect the overall different patterns of fat distribution between the sexes. At any level of waist women would be expected to have a greater overall body fat content contributing to leptin production.

Rural : urban differences in leptin concentration.

Zimmet et al. have already reported on the rural-urban differences in obesity in Western Samoa with lower BMI in rural Polynesians (21). The present results suggest that this factor explains to a large extent, the trend for leptin to be lower in rural subjects.

Physical activity and leptin.

Increased energy or fat intake and reduced physical activity in urban, as compared with rural subjects, may contribute to increased obesity and hence indirectly to higher leptin levels. Certainly there was no effect of physical activity independent of obesity, on leptin levels in Polynesians. A more direct effect of physical activity on leptin levels could be postulated, either by activity reducing leptin resistance as is the case with insulin resistance, or via improvements in insulin sensitivity brought about by physical activity (24). Leptin and fasting insulin were strongly correlated in Western Samoans and studies in animals suggest that insulin may directly affect leptin levels (25-27), or even vice versa, that leptin reduces insulin levels (28).

Physical activity is difficult to measure accurately in epidemiological studies (29) and, especially among the Samoan women, there was only a narrow range of activity levels with 79% having an activity score of 4. Perhaps with a better estimate of activity level, an independent association with leptin could be found. Insulin and leptin concentration.

While there appears to be no association between glucose tolerance status and serum leptin amongst the Samoan subjects, there is clearly an association between insulin and leptin levels, even after correcting for obesity. Previously it has been shown in animal models that ob gene expression is increased by insulin (27) and reduced when insulin deficiency is induced by streptozotocin (25). Other studies have also highlighted the association between insulin and leptin (2,27,28,30). In the study of Considine et al. (11), however, the relationship between insulin and leptin in humans was not independent of body fat percentage. It seems unlikely that this inconsistency is explained by the use of body fat percentage rather than the less direct measures of BMI or waist circumference as used in Samoa. The correlation of BMI with serum leptin levels in Samoans was as strong as the correlation of body fat percentage with leptin reported by Considine et al. (11). It is possible that the range of fasting insulin concentrations in our study was wider allowing the detection of statistically significant associations between insulin and leptin.

The leptin/insulin relationship held even in subjects with diabetes, supporting the decision to combine data from diabetic and non-diabetic individuals. The duration of diabetes was nevertheless relatively short. Among the 17 subjects known to have diabetes, only 5 had an established duration longer than 3 years. In subjects with longer duration of NIDDM and impaired insulin secretion, the linear relationship between insulin and leptin may be weakened. Nevertheless, insulin has been shown to have an important effect on leptin production in adipocytes (31).

Ethnic differences in leptin levels?

Leptin levels previously reported in humans appear to be high relative to the concentrations observed in Western Samoans. Considine et al.(11) report mean levels of 31.4 ng/ml in obese subjects, and 7.5 ng/ml in normal-weight subjects with men and women combined (11). Using a similar BMI criterion for obesity, geometric mean leptin levels in Samoans were 15.0 ng/ml and 2.1 ng/ml in obese and normal-weight subjects respectively. However, our population contained approximately equal proportions of men and women (48:52), whereas the subjects studied by Considine et al. (11) were predominantly women (71% of obese subjects). Given that women have consistently been observed to have higher leptin levels than men of the same BMI (11 ,12), this bias towards women could increase mean leptin levels. Although not specified, it is likely that these subjects were also mainly Caucasian. Swinbum et al. (32) have suggested that Polynesians have a lower body fat percentage than Caucasians at any BMI, based on bioimpedance measures. If this finding is confirmed it could explain the low leptin levels in Polynesians relative to Caucasians with a similar BMI.

In conclusion, the data reported here showing large differences between leptin levels in normal weight and obese subjects, a progressive increase in leptin with increasing BMI, and significant independent correlations of leptin with BMI, waist circumference and fasting insulin, strongly support an important role for leptin in human metabolism and obesity. Since the publication of evidence against a mutation in leptin, or a leptin deficiency in obese humans (6), subsequent authors have reported consistently elevated leptin (10,11) or ob mRNA (14,15) in obese subjects. However, apart from one study where Pima Indians and other Americans were compared (10), ethnicity has not been specified. Otherwise, no ethnic differences in leptin levels have been demonstrated, and by confirming previous results in a very different population, specifically one with a high prevalence of obesity and NIDDM, the Zimmet et al. study (22) also provides important evidence against a form of human obesity analogous to the ob/ob mouse model, ie. with leptin deficiency.

Are leptin and insulin sensitivity related?.

Zimmet and Alberti speculate that the leptin receptor may be synonymous with Neel's thrifty gene and lead to Syndrome X (18). Possible mechanisms by which leptin may affect insulin sensitivity are only speculative, but include: (a) Down regulation of hypothalamic leptin receptors leads to increased leptin levels. This causes further leptin synthesis and release with further downregulation of hypothalamic receptors and this vicious cycle results in hyperinsulinaemia, adiposity and dyslipidaemia, precursors to NIDDM; (b) Leptin itself could cause insulin resistance or increased food-energy intake, leading to increased adipocyte mass and decreased insulin sensitivity. This primary increase in central adipose tissue mass in NIDDM increases leptin production, loss of appetite control, weight gain, and further insulin resistance and hyperleptinaemia.

Thus by virtue of the relationship of leptin to insulin suggested by Zimmet et al (22) and de Courten et al (33) the nicotine analogues will be useful as insulin sensitisers in the therapy of all insulin resistant states such as diabetes.

SECTION B: SMOKING AND BODY WEIGHT

An important deterent to quitting smoking is the well recognised subsequent weight gain (34), the explanation of which has yet to be determined. Equally recognised is the negative relationship between cigarette smoking and body weight which has been confirmed in a large epidemiological study (NHANES II) by Klesges and Klesges(35). Carboxyhaemoglobin measurements, rather than self report, were used to quantify smoking exposure among the subjects. Willliamson et al (36) related weight changes to changes in smoking status in American adults examined in NHANES I (1971-76) and the Follow-UP Study (1982-84). After adjusting for other factors the mean weight gain attributable to smoking cessation was 2.8 kg in men and 3.8 kg in women. This weight gain discourages many smokers from trying to quit and is a reason why many resume (34). It appears that increased energy intake is the major cause of the weight gain, although there is also evidence that nicotine increases metabolic rate (34,37). It has been suggested that the inverse relationship between nicotine and body weight may be mediated to some extent by reduced insulin levels as seen in animal models (38).

Thus smokers have lower weights than non-smokers (39) , because of a smoking-related increase in basal metabolism (40) or energy expenditure (41) or because of an effect of smoking on caloric intake (40,42). Data from the Collaborative Perinatal Project indicate lower interpregnancy weight gains for smokers than for non- smokers for both black women and white women(43). National Health Interview Survey and NHANES data (34) indicate that among women who smoke, blacks are more likely than whites to be light smokers (less than 10 or 15 cigarettes per day) (44, 45). Although the dose-response effects of smoking on weight are not well specified (46,47), it is possible that lower cigarette use by black women smokers influences their weight status upward.

Evidence that these effects of smoking on body weight are likely to be nicotine- mediated is the report of Hajek et al (48) who showed a significantly lower body weight in users of nicotine chewing gum than non-users. Furthermore, after smoking cessation with consequent nicotine withdrawal, Curtsiter (49) has reported significant weight gain. Epidemiological data (50) from populations in Nauru, Mauritius and Western Samoa have shown a significant association between smoking and leptin levels, matched for weight, body mass index (BMI) and within gender groups - see also Wei et al. (50a). The mechanisms of nicotine action may be central, as already proposed, or peripheral involving nicotinic ACh receptors on adipocytes because alterations of lipolysis and lipoprotein lipase have been demonstrated in chronically nicotine-treated rats (51). In support of the proposed actions of nicotine on leptin and body weight are the tabulated data from these three populations described by Hodge et al (50) incorporated as Tables 1 and 2 below, and in Figure 1. SECTION C: NICOTINIC MECHANISMS IN THE CENTRAL NERVOUS SYSTEM

Receptor classification

Acetylcholine (Ach) receptors in the mammalian central nervous system (CNS) have been divided into muscarinic (mAChR) and nicotinic (nAChR) subtypes, based on the relative agonist activities of the natural alkaloids muscarine and nicotine (52).

Muscarinic agonists

ACh replacement strategies with choline precursors, cholinesterase inhibitors like tacrine and velnacrine, Ach-releasing agents like linopirdine, and directly acting Ach receptor agonists have all been used to replace or potentiate the actions of ACh at the synaptic cleft. These have predominantly targeted agonists active at mAChRs but the majority of mAChR ligands tested to date have been disappointing in terms of the goal of centrally active agents devoid of peripheral side effects (52).

Nicotinic receptor subtypes and agonists

This trend has now changed, with recent preclinical and clinical studies indicating that nAChRs play a significant role in mediating the molecular events related to cognitive performance, affect modulation, and enhancement of brain function. So far, at least 7 putative neuronal nAChR subtypes have been identified in transfected oocyte preparations (53,54). The wide distribution of the a2, a3,a4 and b2 transcripts in human brain indicates that neuronal nAChRs are a major neurotransmitter receptor superfamily, structurally related to the other ligand-gated ion channel families that include GABA_A N-methyl-D-aspartate(NMDA) and glycine (55,56). However, apart from the classic agonist nicotine, pharmacological tools for the characterization of nAChRs are only just emerging. The subtypes of nAChRs are presently characterised by radioligand binding techniques: those that have a high affinity (0.5-5nM) for [³H]-acetylcholine (57), [³HJ- nicotine (58), [³H]-cytisine (59) and³ [ H]-methylcarbamylcholine (60); those that recognise a-BgT with high affinity (0.5nM) (58) and a population of nAChRs that display marked selectivity for n-BgT (61).

Another alkaloid, dihydro-β- erythroidine (DHβE) also has nanomolar affinity for neuronal nAChRs and a regional distribution similar to that seen with [³H]-nicotine (62).

Two additional sites distinct from those that bind [³H]-nicotine or the snake toxins have been identified. The noncompetitive antagonist mecamylamine does not bind at the same site as nicotine (63). Similarly, [³H]-pempidine does not label a nicotine sensitive site in mammalian brain.

Finally, another site located on the a-subunit of both nicotine-sensitive and a- BgT-sensitive nAChRs is labelled by [³H]-1-methyl-physostigmine (63), displaced by physostigmine, benzoquinonium and galanthium but not displaced by competitive neuronal nAChR antagonists.

Modulation of nAChR complex protein structure

In muscle nAChRs, transitions may occur between states that have distinct binding ion channel opening characteristics (64). nAChR function can also be modulated in a more persistent manner by phosphorylation of receptor proteins (65). At least four protein kinases - a cAMP-dependent kinase, PKA, protein kinase C, a tyrosine kinase and a Ca++ -calmodulin kinase differentially phosphorylate muscle, neuronal and Torpedo nAChR subunits. This provides potential for the indirect modulation of nAChR equilibrium transitional states by neuropeptides (66). Ligand binding sites on the nAChR

The combination of neuronal nAChR subunits present will determine the nAChR pharmacological and functional properties. The ACh binding site has been localised at the interfaces between the a- and b- subunits and the a- and g- subunits (67).

Alternative channel activator sites (64) are distinct from the sites at which Ach and nicotine bind, and include cholinesterase inhibitors physostigmine and galanthium (64). (+)-2-Methyl-piperidine enhances receptor interactions with the endogenous ligand, Ach, facilitating only ongoing or evoked cholinergic neurotransmission, thereby limiting the potential for side-effect liability (68). This type of compound resembles glycine antagonists acting at NMDA receptors or the various allosteric modulators of the GABA/benzodiazepine receptor complex (55,56).

Non-competitive blockers(NCBs)

Alternative allosteric ligand binding sites exist. Many diverse molecules such as histrionicotoxin, chlorpromazine, phencyclidine(PCP), MK-801 , local anaesthetics, lipophilic agents, such as detergents, fatty acids, barbiturates, volatile anaesthetics and n-alcohols can modify the properties of the nAChRs without acting at the Ach binding site or affecting directly the binding of Ach (66,69). These NCBs are an important class of compounds affecting nAChRs.

Steroid binding sites

Steroids can allosterically desensitize the nAChR by occupying their binding sites on neuronal nAChRs. In vitro progesterone, testosterone, dexamethasone, hydrocortisone and prednisolone can act as noncompetitive inhibitors of nAChRs (67). In vivo there is an association between circulating corticosteroids, [^{1 5}l]-a-BgT binding proteins and behavioural sensitivity to nicotine (71). At high micromolar concentrations corticosterone inhibited binding of [¹²⁵l]-a-BgT to rat brain membranes and reduced the affinity of nicotine for this site, consistent with a negative allosteric interaction.

Dihydro-pyridine (DHP) binding site

Low micromolar concentrations of DHPs like nimodipine interact with neuronal nAChR at clinically relevant concentrations (72) and may limit vasoconstrictor effects of excess circulating catecholamines (evoked by increased sympathetic outflow). Findings suggest that chromaffin neuronal nAChRs contain a DHP site whose occupation blocks ligand-gated Na+ entry through the ionophore, limiting the ensuing membrane depolarization, firing of APs, recruitment of Ca++ channels, Ca++ entry.

Direct binding of Ca++ and other divalent cations to sites within the nAChR channel can decrease the single-channel conductance in a voltage-dependent way and enhance the desensitization of muscle nAChRs. By contrast, extracellular Ca++ affects some neuronal nAChRs in the opposite direction, with Ca++ potentiating the response to agonists at both positive and negative membrane potentials at sites located outside of the ion channel (67). In the habenula nucleus Ca++ in physiological concentrations increases the frequency but not the duration of channel opening (73).

Neuronal nicotine pharmacology

Nicotine modulates brain function by enhancing ion flux and neuronal transmitter release, leading to a facilitation or gating of a number of neuronal systems, and thus eliciting a number of behavioural states (74). The only proven site of nicotinic AChR neurotransmission is the motor neuron-Renshaw cell synapse in spinal cord (75). However, nicotine responses can also be observed in retina, spinal cord, hippocampus, brainstem respiratory nuclei, cerebral and cerebellar cortex, thalamus, hypothalamus, interpeduncular nucleus, septal nucleus, substantia nigra, striatum and locus coeruleus (76). In general, activation of presynaptic nAChRs leads to facilitation of the release of Ach, dopamine, noradrenaline, serotonin, GABA, and glutamate (76,77).

A number of CNS functions are regulated via the basal forebrain cholinergic system, including aspects of attention (78) cognitive performance (79), cerebral blood flow (80), cerebral glucose utilization (69), and neocortical electrical activity (81). Nicotinic agonists augment these activities, and they are reduced by mecamylamine (82), age-related decrements of the basal forebrain cholinergic system, or abolished by excitotoxin-evoked destruction of the system (69).

Therapeutic potential of nicotine

Surgically induced and age-associated deficits in central acetylcholinergic systems can produce an impairment of performance in various models of cognitive function (83, 84,85,86) his raises the possibility of a beneficial effect in Alzheimer's disease and nicotine has been shown to improve cognitive performance in animal (87,88 ) and human studies (82). Again, it would be of interest to have epidemiological studies to determine whether there is an inverse relationship between a history of smoking and the development of Alzheimer's disease, and prospective studies are in progress to determine whether nicotine analogues are of therapeutic value.

The lower prevalence of ulcerative colitis and Parkinson's disease in smokers suggests that a component of tobacco smoke has a prophylactic effect (89) and this hypothesis has the scientific appeal of being testable by prospective studies. Nicotine does appear to be of benefit in preliminary clinical studies on patients with ulcerative colitis (89) and in animal models of Parkinson's disease (90).

In Tourette's syndrome, the beneficial effect of nicotine in ameliorating the manifestations were rapid and dramatic (91). Regulation of gene expression by nicotinic cholinergic ligands

This has been less extensively studied in neural tissue than at neuromuscular junction and motor endplate (92). The main evidence for a contribution of nicotinic receptors in the regulation of the number of surface receptor molecules is the up- regulation of the nicotine, acetylcholine, and cytisine binding sites (93). in rodent brain after chronic systemic administration of nicotine (92,93,94) or in human brain after tobacco smoking (95). However, the enhanced nicotine binding in mouse brain is not accompanied by changed levels of RNA encoding a-2,-3, -4, -5, nor the b-2 subunits (96) suggesting that the regulation of receptor number takes place at a post- transcriptional level.

Another effect of membrane depolarization by nicotinic agonists is rapid transient stimulation of transcription of the c-fos proto-oncogenes and actin genes . In addition to immediate early genes, nicotinic receptors appear to regulate the late onset genes, and nicotine stimulates late transcription of proencephlin A and tyrosine hydroxylase genes in bovine chromaffin cells (97).

Furthermore, there are sustained effects of chronic nicotine administration on dopamine synthesis in hippocampus (98). The competitive antagonist a-BgT binds to intemeurons from CA1 and CA3 regions and increases mRNA levels for brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in CA3 and dentate neurons by 2-20 fold (99), suggesting that regulation of gene expression by nicotinic agents might be either positive or negative. SUMMARY

Altogether, the large diversity of neuronalnicotinic receptors is consonant with the multiple actions mediated by cholinergic ligands in the nervous system . Besides its strong addictive effects (76), nicotine has been described as a cognitive enhancer, increasing attention and improving learning and memory, and also having neuroprotective effects (100). Nicotinic nAChR binding sites are present in spinal cord and cerebral cortex on capsaicin- sensitive neurons (101) many of which are involved in nociception and consonant with nicotine having analgetic and anxiolytic actions(100). Chronic nicotine does not upregulate nAChR binding in spinal cord but does, in a dose-dependent manner in cerebral cortex (102,103) emphasizing the regional and even site-specificity of its actions. Its potential therapeutic uses have been mentioned in a short section above, and are further discussed in a review by Levin and Rosecrans (88). Nicotine and its analogues have been administered by mouth eg as chewing gum, applied as transdermal patches (104) in attempts to assist smoking cessation. Combined therapy using the antagonist mecamylamine together with the agonist nicotine has proven very helpful. Novel nicotinic cholinergic ligands with ion- channel-selective enhancing or blocking properties are now being examined for their therapeutic efficacy in various state (105). Although this field is now extremely active, no specific actions on hypothalamic leptin receptors have been proposed or foreshadowed apart from the proposals by Hodge et al (50).

Table 1 Characteristics of male smokers and non-smokers in three populations.

Non-smokers Smokers P

NAURU 1987 n 106 135

Age (years) 37.1 (11.3)* 35.5 (10.2) 0.284

Body mass index (kg/m²) 35.3 (7.6) 33.7 (6.0) 0.059

Waist girth (cm) 101.9 (14.0) 98.2 (11.3) 0.024

Waist/hip ratio 0.91 (0.05) 0.90 (0.05) 0.261

Diabetes (%) 24.5 23.4 0.850

Physically active 34.0 36.1 0.732

MAURITIUS 1987 n 146 170

Age (years) 45.1 (12.4) 45.9 (11.8) 0.579

Body mass index (kg/m²) 25.3 (3.3) 23.8 (3.6) <0.001

Waist girth (cm) 83.1 (7.8) 81.1 (8.3) 0.028

Waist/hip ratio 0.91 (0.05) 0.91 (0.06) 0.413

Diabetes (%) NONE NONE

Physically active 49.3 51.2 0.741

WESTERN SAMOA 1991 n 54 60

Age (years) 45.7 (12.3) 44.0 (11.8) 0.475

Body mass index (kg/m²) 31.7 (6.5) 30.1 (7.0) 0.198

Waist girth (cm) 102.1 (16.1) 97.6 (16.7) 0.145

Waist/hip ratio 0.92 (0.06) 0.91 (0.06) 0.444

Diabetes (%) 27.8 21.7 0.449

Physically active 50.0 56.7 0.476

* Standard deviations. Table 2 Geometric means of leptin and fasting insulin, unadjusted and adjusted for body mass index (BMI), in male smokers and non-smokers from three populations.

Unadjusted Adjusted for BMI

Non- Smokers P Non- Smokers P smokers smokers

NAURU 1987

Leptin (ng/ml) 9.83 7.38 <0.001 9.27 7.87 0.005

Fasting insulin (μU/ml) 28.9 28.6 0.875 28.4 29.0 0.748

MAURITIUS 1987

Leptin (ng/ml) 5.46 4.29 O.001 5.10 4.62 0.020

Fasting insulin (μU/ml) 8.08 5.78 <0.001 7.21 6.44 0.147

Western SAMOA 1991

Leptin (ng/ml) 4.56 2.31 <0.001 4.00 2.63 0.007

Fasting insulin (μU/ml) 10.69 7.10 0.031 9.98 7.62 0.085

Further features of the present invention are more fully described in the following Example(s). It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the present invention, and should not be understood in any way as a restriction on the broad description of the invention as set out above.

EXAMPLE

The following study has been carried out in the Psammamys obesus (Israel sand rat) animal model (8) to determine the effect of nicotine on body weight, food intake and glucose levels. Study Protocol

Animals were followed during a run-in period of approximately 7 days. Animals were then implanted with Alzet mini-osmotic pumps containing either saline (control) or nicotine. The pumps delivered nicotine at a dosage of 12 mg/day. After 6 days, the animals were sacrificed and a number of tissues (including adipose stores and muscle) removed for body fat and muscle distribution and future RNA analyses. During the study, body weight, food intake, glucose and insulin were measured on days 0, 2 and 6 from the date of implantation of the mini-osmotic pumps.

Results

The results of the study are summarised in the following Table, and shown graphically in Figure 2.

Control Nicotine (n = 10) (n = 12)

% Body weight change

Day 2 0.7 ± 1.5 -2.7 ± 0.7 Day 6 1.0 ± 1.0 9.0 ± 1.0

% Food intake change

Day 2 -29.8 ± 6.3 -35.6 ± 5.2 Day 6 -7.1 ± 4.0 -55.6 ± 5.8

Glucose (mmol/L)

Day O 5.1 ± 0.5 5.0 ± 0.6 Day 2 4.3 ± 0.2 5.5 ± 0.3 Day 6 4.2 ± 0.2 7.6 ± 0.5

n = number of animals. REFERENCES:

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Claims

CLAIMS:

1. A method for the treatment of obesity in a patient, which comprises administration to said patient of an effective amount of at least one compound selected from the group consisting of nicotine and nicotine analogues.

2. A method for the prevention of obesity in a patient pre-disposed to obesity, which comprises administration to said patient of an effective amount of at least one compound selected from the group consisting of nicotine and nicotine analogues.

3. A method according to claim 1 or claim 2, wherein said patient is a human.

4. A method according to any of claims 1 to 3, wherein said nicotine analogues are agonists or antagonists of nicotine which alter the hypothalamic responsiveness to leptin.

5. A method according to claim 4, wherein said nicotine analogues are compounds which are selected for the hypothalamic leptin receptors in humans.

6. A method according to any of claims 1 to 3, wherein said nicotine analogues are selected from ABT 418 and (+)-2-methylpiperidine.

7. A method according to any of claims 1 to 3, wherein a combination of two or more of said compounds is administered to the patient.

8. A method according to claim 7, wherein said combination comprises (i) nicotine or nicotine analogue which is an agonist of nicotine, and (ii) a nicotine analogue which is an antagonist of nicotine.

9. A method according to any of claims 1 to 3, wherein leptin is also administered to said patient.

10. A method according to claim 9, wherein leptin is co-administered with said compound to said patient.

11. A method according to claim 9, wherein leptin and said compound are separately administered to said patient.

12. Use of at least one compound selected from the group consisting of nicotine and nicotine analogues for, or in the manufacture of a medicament for, the treatment of obesity in a patient or the prevention of obesity in a patient predisposed to obesity.