EP1259121A2 - Perte de poids induite par la reduction du niveau de neuropeptide y - Google Patents

Perte de poids induite par la reduction du niveau de neuropeptide y

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Publication number
EP1259121A2
EP1259121A2 EP01910959A EP01910959A EP1259121A2 EP 1259121 A2 EP1259121 A2 EP 1259121A2 EP 01910959 A EP01910959 A EP 01910959A EP 01910959 A EP01910959 A EP 01910959A EP 1259121 A2 EP1259121 A2 EP 1259121A2
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European Patent Office
Prior art keywords
animal
weight loss
compound
feeding
expression
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EP01910959A
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German (de)
English (en)
Inventor
Thomas M. Loftus
Craig A. Townsend
Gabriele Ronnett
M. Daniel Lane
Francis P. Kuhajda
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Johns Hopkins University
School of Medicine of Johns Hopkins University
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Johns Hopkins University
School of Medicine of Johns Hopkins University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/5755—Neuropeptide Y

Definitions

  • This invention is directed to methods of inducing weight loss in an animal.
  • this invention concerns methods for reducing adipocyte mass by controlling the level of neuropeptide Y in the animal.
  • Body fat mass is controlled by a complex group of feedback pathways that monitor fat mass and feeding status and regulate feeding and energy utilization.
  • the lipostat model originally set forth by Kennedy (Kennedy, G., 1953 "The role of depot fat in the hypothalamic control of food intake in the rat," Proc. Royal Soc. London (Biol), 140:579-592), peripheral signals from adipose tissue, gut and liver and pancreas act on neurons in the hypothalamus to modulate energy homeostasis.
  • a number of the regulatory pathways involved have recently been identified.
  • the best known of the peripheral signals of feeding and adiposity include leptin, insulin, and the gut satiety peptides.
  • Leptin a cytokine-related hormone produced primarily by adipocytes, is released in proportion to adipose mass. Thus it acts as a signal of adipose mass, both peripherally and in the feeding control centers of the hypothalamus, to inhibit feeding and promote weight loss (Hwang, C, et al., 1997, "Adipocyte differentiation and leptin expression,” Annual Review of Cell & Developmental Biology, 13:231-259). Leptin levels are also elevated by feeding, reflecting feeding status as well as adiposity.
  • the gut peptides e.g. bombesin and cholecystokinin
  • the gut peptides are released in response to feeding and act as a signal of meal size (Laburthe, M., et al., 1994, "Receptors for gut regulatory peptides," Baill Clin Endocinol Metab., 8:77-110).
  • these signals are carried to the brain primarily by afferent sensory neurons of the parasympathetic peripheral nervous system, (i.e. the vagus nerves). Other abdominal signals of feeding status are similarly transmitted.
  • the regulation of feeding and energy utilization in the brain is controlled primarily through integration of feeding signals in the hypothalamus.
  • Two distinct groups of regulatory neurotransmitters/neuropeptides are coordinately counterregulated depending on the energy status of the individual. Under conditions of energy deficit, signalled by such things as low leptin levels, anabolic signals are activated that stimulate feeding and reduce energy utilization while catabolic signals, which inhibit feeding and increase energy utilization are downregulated. Conversely, under conditions of energy surplus, anabolic signals are downregulated while catabolic signals are upregulated (Lof us, T., 1999, "An Adipocyte-central nervous system regulatory loop in the control of adipose homeostasis," Sem. Cell. Dev. Biol., 1 (1):11-1S).
  • neuropeptide Y The best known anabolic signal is neuropeptide Y (NPY).
  • This neuropeptide is produced in the hypothalamus in response to fasting (Schwartz, M., et al., 1998, "Effect of fasting and leptin deficiency on hypothalamic neuropeptide Y gene transcription in vivo revealed by expression of a lacZ reporter gene," Endocrinology, 139(5): 2629-2635) and strongly stimulates feeding (O'Shea, D., et al., 1997, "Neuropeptide Y induced feeding in the rat is mediated by a novel receptor," Endocrinology, 138(l):196-202).
  • anabolic signals include inhibition of NPY signalling among their mechanisms of action.
  • Other anabolic signals include agouti related peptide (AGRP) (Shutter, G.M., et al, 1997, "Hypotlialamic expression of ART, a novel gene related to agouti, is up-regulated in obese and diabetic mutant mice, Genes and Development, 11:593-602) which antagonises the ⁇ -MSH receptor (see below), melanin concentrating hormone (MCH) (Ludwig, D., et al., 1998, "Melanin-concentrating hormone: a functional melanocortin antagonist in the hypothalamus," Am.
  • AGRP agouti related peptide
  • MCH melanin concentrating hormone
  • Orexins A, and B (Sakurai, T., et al., 1998, "Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior," Cell, 92(4):573-585), also known as hypocretins 1 and 2.
  • ⁇ -melanocyte stimulating hormone ⁇ -MSH
  • This peptide is elevated in response to energy surplus and inhibits feeding and promotes catabolic activity.
  • Mice carrying a deletion in the ⁇ - MSH MC4 receptor develop obesity (Huszar, D., et al., 1997, "Targeted disruption of the melanocortin-4 receptor results in obesity, Cell, 88(1): 131-141).
  • mice overexpressing an antagonist of this receptor such as agouti or AGRP also develop late-onset obesity (Graham M., S.J., et al., 1997, “Overexpression of Agrt leads to obesity in transgenic mice,” Nat. Genetics, 17:273-274).
  • CART cocaine and amphetamine regulated transcript
  • CSH corticotropin releasing hormone
  • GAL galanin
  • MCH melanin-concentrating hormone
  • NT neurotensin
  • POMC proopiomelanocortin
  • NPY neuropeptide Y
  • C-75 is a specific inhibitor of fatty acid synthase (FAS) as disclosed in U.S. Patent No. 5,981,575, incorporated herein by reference.
  • FAS fatty acid synthase
  • U.S. Patent No. 5,981,575 incorporated herein by reference.
  • FAS is one of the primary biosynthetic enzymes of fatty acid synthesis in humans and other mammals ( Wakil, 1989, "Fatty acid synthase, a proficient multifunctional enzyme," Biochemistry, 28:4523-4530).
  • Administration of C-75 to BALB/c mice leads to loss of 10-20% of total body weight within a 24 hour period, lasting for several days with total duration depending of dose. Following this period, body weight returns to normal with no obvious long term effect on the animal.
  • this invention provides a method for inducing weight loss in an animal, the method comprising administering to the animal a compound which reduces the expression and/or secretion of neuropeptide Y (NPY) directly or humorally.
  • administration of this compound has the effect of increasing malonyl CoA levels in the animal.
  • Compounds administered according to this invention may be inhibitors of fatty acid synthase (FAS), including substituted ⁇ - methylene- ⁇ -carboxyl- ⁇ -butyrolactones, or inhibitors of malonyl Coenzyme A decarboxylase (MCD).
  • FAS fatty acid synthase
  • MCD malonyl Coenzyme A decarboxylase
  • the compound is administered in an amount sufficient to reduce the amount and/or duration of expression and/or secretion of NPY to levels at or below those observed for lean animals.
  • the administration will reduce expression and/or secretion to levels observed for fed or satiated animals; more preferably, administration will reduce the level of NPY below that of fed animals.
  • this invention provides a method for inducing weight loss in an animal by administering a compound which inhibits feeding behavior in the animal. The method is particularly useful for inducing weight loss in animals deficient in expression of the hormone leptin or animals resistant to the action of leptin.
  • this invention provides a screening method for identifying genes whose expression is associated with control of weight loss.
  • This method comprises comparing RNA species expressed in tissues of an animal treated with a weight loss agent to mRNA species expressed in corresponding tissues of control animals.
  • the treated animal is treated with an FAS inhibitor, more preferably the FAS inhibitor is an substituted ⁇ -methylene- ⁇ - carboxyl- ⁇ -butyrolactone, such as C-75.
  • the expressed mRNA is mRNA expressed in hypothalamic tissues.
  • a combination of anabolic and catabolic signals control the body's perception of feeding status. By altering the control of these signals, it is possible to create the perception of the fed or fasted state regardless of the dietary status of the individual. By inhibiting the anabolic signals and activating the catabolic signals, it is possible to induce weight loss, not only through the suppression of feeding, but also by maintaining a normal rate of metabolism, in contrast to the lowered metabolic rate that normally accompanies weight loss.
  • FAS inhibitors such as the ⁇ -methylene- ⁇ - carboxy- ⁇ -butyrolactone C-75, induce weight loss primarily by an inhibition of feeding (see Example 1). At a sufficient dose, C-75 will completely block all feeding behavior. Furthermore, the observed weight loss can be largely reversed by forced feeding of drug treated animals. C-75 inhibited expression of the prophagic signal neuropeptide Y in the hypothalamus and acted in a leptin- independent manner that appears to be mediated by malonyl-CoA. There may also be an effect on metabolic rate. C-75 treatment leads to greater weight loss than total food restriction alone (see Example 2). The normal response to fasting in mammals is to reduce the metabolic rate in order to conserve energy. Agents that signal a fed state to the body not only inhibit feeding, but also maintain an elevated metabolic rate, resulting in greater weight loss than lack of feeding alone. This elevation of metabolic rate may also account for the incomplete reversal of weight loss by feeding alone.
  • Figure 1.1 shows the structures for cerulenin and C-75 (Panel A), as well as fatty acid synthesis (Panel B) and hepatic malonyl-CoA level (Panel C) in control and C-75-treated mice.
  • Figure 1.2 shows body weight (Panel A) and food intake (Panel B) for mice treated with C-75 or RPMI vehicle.
  • Figure 2 depicts mice with or without C-75 treatment compared to fasting mice. Panels show (A) body weight and (B) neuropeptide Y mRNA. Figure 2C shows reversal of the feeding-inhibitory effects of C-75 by intracerebroventricular administration of NPY, thus demonstrating that the animals are capable of responding to NPY if they were not prevented from making it. Panel D shows the effect of C-75 on feeding interval.
  • Figure 3 shows leptin independence of the C-75 effects in ob/ob (leptin. deficient) mice.
  • Various panels show (A) leptin levels, (B) weight change, (C) representative individuals, and (D) photomicrographs of control and treated liver.
  • Figure 4 shows the effect of C-75 on serum glucose in (A) ob/ob mice and (B) wildtype mice.
  • Figure 5 (A) shows a model of feeding regulation by inhibitors of FAS via malonyl-CoA.
  • Panel B shows the interaction of inhibitors of ACC and FAS.
  • Panel C shows the effect of intracerebro ventricular injection of C-75.
  • malonyl-CoA a substrate for FAS
  • This regulatory mechanism prevents fatty acid synthesis and oxidation of fatty acids from occurring simultaneously.
  • Elevated malonyl-CoA associated with fatty acid synthesis See U.S. Patent Application 60/164,765 "Modulation of Cellular Malonyl-CoA Levels as a Means to Selectively Kill Cancer Cells," incorporated herein by reference) may similarly be linked to feeding control.
  • Fatty acid synthesis regulates fatty acid oxidation via rising malonyl-CoA levels during fatty acid synthesis, which results in inhibition of carnitine palmitoyl transferase-1-mediated uptake of fatty acids into the mitochondrion. This results in elevation of cytoplasmic long-chain fatty acyl-CoA's and diacylglycerol, molecules that may play a signaling role, leading to the proposal that malonyl-CoA levels act as a signal of the availability of physiological fuels.
  • the signal from the FAS target tissue to the hypothalamus may be mediated by a humoral signal.
  • This FAS-associated signal appears to be independent of the systemic release of the known feeding inhibitory hormones leptin and insulin, and the pro-inflammatory cytokines tumor necrosis factor- ⁇ and interleukin-l ⁇ .
  • dexamethasone a synthetic glucocorticoid.
  • Necropsy and histological analysis of all major organs in treated mice revealed no adverse pathology and plasma alanine aminotransferase activity was unchanged.
  • C-75-induced weight loss was observed in mice lacking IL-lr and TNF ⁇ rla receptors suggesting that the weight loss is not mediated by an inflammatory response.
  • NPY neuropeptide-like protein
  • C-75 Control of NPY by C-75 may also extend to these co-regulated molecules.
  • malonyl-CoA One role proposed for malonyl-CoA is the mediation of nutrient-stimulated insulin secretion in the beta cell. Glucose-sensing neurons that regulate feeding in the hypothalamus share many features with the beta cell including expression of glucokinase and the ATP-sensitive potassium channel (20). The data reported herein support the prediction that malonyl-CoA may signal fuel status in hypothalamic neurons
  • Weight loss agents are agents that interfere with Neuropeptide Y expression and/or secretion and that block or reduce feeding activity.
  • Candidate agents may be tested for their ability to reduce NPY expression by administering the agent to an animal and measuring NPY levels in the brain of the treated animal (for example as described in Example 2 for mouse brain) or by measuring NPY expression in hypothalamic cultures (see culture procedure in, e.g., Loudes, et al. (1999), "Distinct populations of hypothalamic dopaminergic neurons exhibit differential responses to brain-derived neurotrophic factor (BNDF) and neurotrophin-2 (NT3).” European Journal of Neuroscience, 11:617-624; Loudes, et al.
  • BNDF brain-derived neurotrophic factor
  • NT3 neurotrophin-2
  • the weight loss agent may be injected intracerebroventriclularly in a test animal, and the feeding behavior of the test animal monitored (see Example 2).
  • Preferred weight loss agents of this invention would be expected to inhibit feeding behavior.
  • FAS inhibitors are preferred as weight loss agents according to this invention; more preferred are FAS inhibitors that induce a reduction in expression and/or secretion of Neuropeptide Y.
  • Therapeutic compounds are preferably compounds that inhibit FAS activity and/or raise the level of malonyl CoA without any significant (direct) effect on other cellular activities, at least at comparable concentrations. Suitable compounds for increasing malonyl CoA may be obtained as described in U.S. patent applications 601164,749, 60/164,765, and 60/164,768, incorporated herein by reference. Particularly preferred therapeutic compounds are compounds that directly reduce the activity of FAS in animal cells without any significant (direct) effect on other cellular activities, at least at comparable concentrations. As discussed above, compounds which reduce FAS activity will generally tend to increase the level of malonyl CoA.
  • FAS fatty acid synthase
  • Compounds which inhibit FAS can be identified by testing the ability of a compound to inhibit fatty acid synthase activity using purified enzyme. Fatty acid synthase activity can be measured spectrophotometrically based on the oxidation of NADPH, or radioactively by measuring the incorporation of radiolabeled acetyl- or malonyl-CoA. (Dils, et al, Methods Enzymol, 35:74-83).
  • FAS inhibitors are exemplified in U.S. Patent No.
  • Suitable FAS inhibitors may be identified by a simple test exemplified in Example 7 of U.S. Patent No. 5,981,575, and in U.S. Patent No. 5,759,837, both of which are incorporated herein by reference. Generally, this test uses a tumor cell line in which an FAS inhibitor, typically cerulenin, is cytotoxic. Such cell lines include SKBR-3, ZR-75-1, and preferably HL60. Suitable FAS inhibitors will inhibit growth of such cell lines, but the cells are rescued by exogenous supply of the product of the FAS enzyme (fatty acid). When cell growth is measured in the presence and absence of exogenous fatty acid (e.g., palmitate or oleate), inhibition by specific FAS inhibitors is relieved by the fatty acid.
  • exogenous fatty acid e.g., palmitate or oleate
  • suitable FAS inhibitors can be characterized by a high therapeutic index.
  • Inhibitors can be characterized by the concentration required to inhibit fatty acid synthesis in cell culture by 50%> (IC 50 or ID 50 ).
  • FAS inhibitors with high therapeutic index will inhibit fatty acid synthesis at a lower concentration (as measured by IC 50 ) than the IC 50 for inhibition of cell growth in the presence of exogenous fatty acid. Inhibitors whose effects on these two cellular activities show greater differences are more preferred.
  • Preferred inhibitors of fatty acid synthesis will have IC 50 for fatty acid synthetic activity that is at least 1 log lower, more preferably at least 2 logs lower, and even more preferably at least 3 logs lower than the inhibitor's IC 50 determined for cell growth in the presence of exogenous fatty acid.
  • Treatment with compounds according to this invention will lead to reduction in hepatic fat, and this in turn can lead to reduction in the rate or incidence of cirrhosis in alcoholics (see, e.g., French, 1989, Clinical Biochemistry, 22:41-9; Clements, et al., 1995, Am. J. Respir. Crit. Care Med., 151:780-784, incorporated herein by reference).
  • individuals with fatty livers may benefit from administration of the agents of this invention to reduce hepatic fat (which may be detected by liver biopsy).
  • Increased insulin responsiveness is a direct consequence of decreased adipocyte mass.
  • Reduced adipocyte mass will reduce the risk of arterial vascular disease, stroke, etc.
  • LDLs low density lipoproteins
  • the method of this invention is particularly applicable to overweight individuals, diabetics, and alcoholics.
  • the method is generally useful as part of a program to treat obesity and complications thereof. For example, obese individuals are prone to osteoarthritis, and the method of this invention may reduce the effects of the disease or delay the onset.
  • the method of the present invention for inducing weight loss is applicable to animals, including vertebrates, especially mammals.
  • Animals particularly contemplated include food animals such as poultry, swine, cattle, sheep, and other animals where reduction in fat accumulation without reduction in muscle mass may be desirable for veterinary health or economic reasons.
  • therapeutic compounds according to this invention such as FAS inhibitors, may be administered according to the method of this invention to dogs, cats, horses and other animals for veterinary health reasons, particularly reasons analogous to the reasons given herein for medical therapeutic use of this invention.
  • Dosing protocols for the compounds according to this method may be adapted to various animals from the medical procedures and the in vitro and in vivo data provided herein, in view of standard veterinary pharmacological principles. Generally, this method will not be applied to lactating animals.
  • Treatment according to this invention involves administering a compound according to this invention (for example, an FAS inhibitor such as an ⁇ -methylene- ⁇ -carboxy- ⁇ -butyrolactone) to the subject of treatment.
  • a compound according to this invention for example, an FAS inhibitor such as an ⁇ -methylene- ⁇ -carboxy- ⁇ -butyrolactone
  • the pharmaceutical compositions containing any of the compounds of this invention may be administered by parenteral (subcutaneously, intramuscularly, intravenously, intraperitoneally, intrapleurally, intravesicularly or intrathecally), topical, oral, rectal, or nasal route, as necessitated by choice of drug and disease.
  • Therapeutic compounds according to this invention are preferably formulated in pharmaceutical compositions containing the compound and a pharmaceutically acceptable carrier.
  • Therapeutic compounds may be formulated in liposomes or for administration in aerosol form.
  • concentrations of the active agent in pharmaceutically acceptable carriers will depend on solubilities.
  • the dose used in a particular formulation or application will be determined by the requirements of the particular type of disease and the constraints imposed by the characteristics and capacities of the carrier materials.
  • the pharmaceutical composition may contain other components so long as the other components do not reduce the effectiveness of the compound according to this invention so much that the therapy is negated.
  • Pharmaceutically acceptable carriers are well known, and one skilled in the pharmaceutical art can easily select carriers suitable for particular routes of administration (see, e.g., "Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, 1985).
  • Dose and duration of therapy will depend on a variety of factors, including the therapeutic index of the drugs, disease type, patient age, patient weight, and tolerance of toxicity. Dose will generally be chosen to achieve serum concentrations from about 1 ng to about 100 ⁇ g/ml, preferably 10 ng/ml to lO ⁇ g/ml. Preferably, initial dose levels will be selected based on their ability to achieve ambient concentrations shown to be effective in in-vitro models, such as that used to determine therapeutic index, and in-vivo models and in clinical trials, up to maximum tolerated levels. Typical doses approach 100 ng/ml in blood. Standard clinical procedure prefers that chemotherapy be tailored to the individual patient and the systemic concentration of the therapeutic agent be monitored regularly.
  • the dose of a particular drug and duration of therapy for a particular patient can be determined by the skilled clinician using standard pharmacological approaches in view of the above factors.
  • the response to treatment may be monitored by analysis of blood or body fluid levels of the compound according to this invention, measurement of activity if the compound or its levels in relevant tissues or monitoring disease state in the patient.
  • the skilled clinician will adjust the dose and duration of therapy based on the response to treatment revealed by these measurements.
  • the therapeutic compounds of this invention are administered based in the level necessary to control secretion of neuropeptide Y.
  • the skilled worker is encouraged to administer FAS inhibitors to a subject so that NPY levels in the subject are at or below the level subsequent to normal feeding. Maintaining; effective NPY levels at or below the level observed following feeding will inhibit feeding behavior, and this will lead to weight loss and reduction in adipose tissue mass.
  • compositions described above may be combined or used together or in coordination with another therapeutic substance.
  • the inhibitor of fatty acid synthesis, or the synergistic combination of inhibitors will of course be administered at a level (based on dose and duration of therapy) below the level that would kill the animal being treated.
  • administration will be at a level that will not irreversibly injure vital organs, or will not lead to a permanent reduction in liver function, kidney function, cardiopulmonary function, gastrointestinal function, genitourinary function, integumentary function, musculoskeletal function, or neurologic function.
  • administration of inhibitors at a level that kills some cells which will subsequently be regenerated is not necessarily excluded.
  • the present invention also provides a screening method for identifying other genes whose expression is associated with control of weight loss. Such screening can be done by comparing mRNA species expressed in tissues of an animal treated with a weight loss agent to mRNA species expressed in corresponding tissues of control animals. Procedures for obtaining total mRNA from selected tissues of treated animals are described in Example 2 for mice treated with exogenous NPY. The skilled artisan can readily provide other suitable procedures to obtain and compare mRNA expressed under treatment and control conditions, for example by adapting known techniques from the human genome project.
  • the expressed mRNA is mRNA expressed in control and treated hypothalamic tissues.
  • Weight loss agents which are substituted ⁇ -methylene- ⁇ - carboxyl- ⁇ -butyrolactones, such as C-75, are preferred agents for treatment of animals for comparisons according to this method.
  • Example 1 Inhibitors of FAS and fatty acid synthesis Figure 1.1 shows the chemical structures of C-75 and Cerulenin.
  • mice Female BALB/c mice were treated with 0.6 mg of C-75 in 200 ⁇ l RPMI, or vehicle control IP (3 per group). After 3 hours, the animals were killed and approximately 5 mg of adipose tissue was labeled with [U- 1 C]-acetate, lipids were extracted and counted, [A. Rashid et al., Am. J. Pathol 150 (1997)]. The results are shown in Figure 1.1 (Panel B). C-75 markedly inhibited adipose fatty acid synthesis compared to vehicle control. Values represent mean +/- SEM (* P ⁇ .05).
  • mice Male BALB/c mice (4 per group) were given 2g/kg dextrose by oral gavage. After 15 min mice were injected IP with 20mg/kg C-75 or RPMI vehicle. One hour post-treatment, livers were rapidly removed, frozen and pulverized in liquid nitrogen, HC1O4 extracted and assayed for malonyl-CoA [J.D. McGarry, M.J. Stark, D.W. Foster., J. Biol.Chem 253, 8291 (1978)]. The results are shown in Figure 1.1 (Panel C).
  • mice with C-75 leads to a 95%> reduction in 14 C-acetate incorporation into fatty acids and to a 110% increase in the level of hepatic malonyl-CoA, the principal substrate of FAS. Experiments described in panels B and C were repeated twice.
  • Example 1A Effect of C-75 on body weight and food intake in mice
  • mice 19-22g were weighed, treated by a single intra peritoneal (I.P.) injection and housed in metabolic cages.
  • Body weight ( Figure 1.2A) and food intake ( Figure 1.2B) were monitored at 24 hour intervals.
  • Figure 1.2A shows mean change from initial body weight in mice treated with 7.5 ( ⁇ ), 15 (o) or 30 ( ⁇ )mg/kg of C-75 or RPMI vehicle (•) is expressed +/- SEM.
  • Figure 1.2B shows total food intake for mice treated with RPMI vehicle (black bars) or 15mg/kg C-75 (grey bars) per day following treatment.
  • Inhibitors of fatty acid synthesis would be expected to prevent triglyceride accumulation due to inhibition of de novo fatty acid synthesis and impact body weight in this manner. Indeed, C-75 markedly reduces cytoplasmic triglyceride accumulation by 3T3-L1 adipocytes in cell culture (not shown). However, the dramatic C-75-induced weight loss cannot be accounted for by a blockade of fatty acid/triglyceride biosynthesis. Rather, the weight loss observed in response to C-75 treatment results primarily from an inhibition of feeding. The loss of adipose mass was accompanied by a reduction of lean body mass typical of that observed in fasting.
  • NPY neuropeptide Y
  • the control of body weight is integrated in the hypothalamus by a coordinated group of neuropeptides that monitor adiposity and feeding status and regulate feeding and energy utilization.
  • a central regulator in this process is neuropeptide Y (NPY) (loftus, 1999, Sem. Cell. Dev. Biol, 10:11).
  • NPY neuropeptide Y
  • the level of NPY increases in the fasted state (Schwartz, et al., 1998, Endocrinology, 139:2629), acting as a potent stimulus of feeding (O'Shea, et al., 1997, Endocrinology, 138:196-202).
  • mice were anaesthetized by inhaled metofane and given a direct intracerebroventricular injection of 500ng NPY (2.5 ⁇ l total volume) or artificial CSF vehicle. Mice were placed into metabolic cages and observed for feeding behavior and monitored for food intake over 18 hours. The results are shown in Figure 2C. Total food intake within one hour by C-75/NPY treated mice was similar to that by mice treated with NPY alone and was 9 times greater than that by C-75-treated mice.
  • ICN injection of vehicle had no effect on feeding.
  • the feeding effects of this dose of ⁇ PY had completely subsided in less than an hour, it was sufficient to substantially elevate the total food intake in C-75-treated mice ( Figure 2C).
  • leptin One of the primary signals modulating NPY function in feeding control is leptin. This hormone is elevated in the fed state and inhibits NPY production and feeding (Schwartz, et al., 1996, Diabetes, 45:531) in a manner similar to that observed with C-75 treatment. Leptin was an attractive candidate as its primary site of production, white adipose tissue (Zhang, et al., 1994, Nature, 372:425), is a site of fatty acid synthesis and expresses high levels of FAS. To test for increased leptin release as the signal mediating C-75 regulation of NPY, serum leptin levels were assessed in fed (end of light cycle) fasted and C-75-treated mice.
  • mice treated with RPMI vehicle (o) or 30mg/kg C-75 ( ⁇ ) I.P. and free fed, or fasted (•) for 24 hours were weighed, decapitated and exsanguinated.
  • Serum leptin levels were determined using a Quantikine murine leptin ELISA (R&D Systems) and plotted against total body weight (Figure 3A). Rather than elevation, a reduction in leptin levels was observed. This reduction correlates with the reduction in body weight, presumably body fat, resulting from C-75 treatment Figure 3A). This is consistent with the normal regulation of leptin levels during weight loss (Boden, et al, 1996, J. Gin.
  • Example 5A shows a model of feeding regulation by inhibitors of FAS via malonyl-CoA. This model predicts that feeding inhibition by FAS inhibitors should be attenuated by inhibitors of ACC's.
  • mice were pretreated with the ACC inhibitor TOFA or vehicle by ICN injection and examined the ability of C-75, administered IP, to inhibit feeding.
  • BALB/c mice were anesthetized with metofane and injected ICN with 2 ⁇ g of TOFA or DMSO vehicle. After 2 hours recovery, mice were injected IP with 15 mg C-75/kg or RPMI vehicle and monitored for total food intake over 2 hours.
  • Antibodies specific for the enzymes fatty acid synthase, acetyl-CoA carboxylase alpha isoform, and malonyl-CoA decarboxylase may be used to detect the presence of the respective enzymes in neural tissue.
  • Fatty acid synthase, acetyl- CoA carboxylase alpha isoform, and malonyl-CoA decarboxylase all co-localize to the arcuate nucleus of the hypothalamus in mice by standard methods of immunohistochemical detection using these antibodies.
  • the arcuate nucleus is important in appetite control in the hypothalamus.

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  • Obesity (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Child & Adolescent Psychology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measurement And Recording Of Electrical Phenomena And Electrical Characteristics Of The Living Body (AREA)
  • Diaphragms For Electromechanical Transducers (AREA)
  • Steroid Compounds (AREA)
  • Furan Compounds (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

La présente invention concerne une technique induisant une perte de poids chez l'animal par l'administration à cet animal d'un composé qui réduit l'expression et/ou la sécrétion de neuropeptide Y (NPY). Cette effet peut être réalisé directement, indirectement ou par voie humorale. L'administration de ce composé a, de préférence, pour effet d'augmenter les taux de malonyle CoA chez l'animal. Les composés de cette invention administrés peuvent être des inhibiteurs de synthase d'acide gras (FAS), qui comprennent α-méthylène-β-carboxyl-η-butyrolactones substitués ou des inhibiteurs de décarboxylase de coenzyme de malonyle (MDC). Ce composé est administré, de préférence, en quantité suffisante pour réduire la quantité et/ou la durée de l'expression et/ou de la sécrétion de NPY à des taux inférieurs à ceux que l'on observe chez des animaux maigres. Dans un autre mode préféré de réalisation de l'invention, cette administration réduira l'expression et/ou la sécrétion à des taux observés chez des animaux nourris ou rassasiés, et mieux, cette administration réduira le taux de NPY en dessous de celui des animaux nourris. Dans un mode de réalisation particulier, cette invention concerne une technique induisant une perte de poids chez l'animal par l'administration d'un composé qui inhibe les comportements alimentaires de l'animal. Cette technique convient particulièrement pour réduire la perte de poids chez l'animal chez lequel on observe un déficit de l'expression de la leptine hormonale ou chez des animaux résistant à l'action de la leptine.
EP01910959A 2000-02-16 2001-02-16 Perte de poids induite par la reduction du niveau de neuropeptide y Withdrawn EP1259121A2 (fr)

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US182901P 2000-02-16
US20856000P 2000-06-02 2000-06-02
US208560P 2000-06-02
PCT/US2001/005316 WO2001060174A2 (fr) 2000-02-16 2001-02-16 Perte de poids induite par la reduction du niveau de neuropeptide y

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AU2003215111A1 (en) * 2002-02-08 2003-09-02 The Johns Hopkins University School Of Medicine Stimulation of cpt-1 as a means to reduce weight
EA010484B1 (ru) * 2002-07-01 2008-10-30 Фасджен, Ллс. Новые соединения, фармацевтические композиции, содержащие их, и способы их использования
EP1548131A3 (fr) * 2003-12-22 2005-07-27 F. Hoffmann-La Roche Ag Molécules cibles pour l'obésité dans le muscle squelettique
WO2005089773A1 (fr) * 2004-03-18 2005-09-29 Fasgen, Llc Regulation du comportement alimentaire par modification du bilan energetique neuronal
US8729239B2 (en) 2009-04-09 2014-05-20 Nuclea Biotechnologies, Inc. Antibodies against fatty acid synthase

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US5539132A (en) * 1994-01-24 1996-07-23 Johns Hopkins University Cerulenin compounds for fatty acid synthesis inhibition
US5614551A (en) * 1994-01-24 1997-03-25 The Johns Hopkins University Inhibitors of fatty acid synthesis as antimicrobial agents
US5552411A (en) * 1995-05-26 1996-09-03 Warner-Lambert Company Sulfonylquinolines as central nervous system and cardiovascular agents
US5981575A (en) * 1996-11-15 1999-11-09 Johns Hopkins University, The Inhibition of fatty acid synthase as a means to reduce adipocyte mass
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US6013622A (en) * 1998-04-15 2000-01-11 Nutriceutical Technology Corporation Method of regulating appetite and metabolism

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WO2001060174A2 (fr) 2001-08-23
KR20030016228A (ko) 2003-02-26
WO2001060174A3 (fr) 2002-04-25
CA2400136A1 (fr) 2001-08-23
JP2003528051A (ja) 2003-09-24

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