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Weight Loss Patent Abstract
This invention provides a method for inducing weight loss in an
animal by administering to the animal a compound which reduces the
expression and/or secretion of neuropeptide Y (NPY). The effect
may be accomplished directly, indirectly, or humorally. Preferably,
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 .alpha.-methylene-.beta.-carboxyl-.gamma.-butyrolactones,
or inhibitors of malonyl Coenzyme A decarboxylase (MCD). Preferably,
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. In another preferred
embodiment, 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.
In a particular embodiment, 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.
Weight Loss Patent Claims
1. A method for inducing weight loss in an animal, comprising administering
to the animal a compound which reduces the expression and/or secretion
of neuropeptide Y (NPY).
2. The method of claim 1, wherein administration of the compound
increases malonyl CoA levels in the animal.
3. The method of claim 1, wherein the compound is an inhibitor
of fatty acid synthase (FAS) and is administered in an amount sufficient
to reduce the expression and/or secretion of NPY.
4. The method of claim 1, wherein the compound is a substituted
.alpha.-methylene-.beta.-carboxyl-.gamma.-butyrolactone.
5. The method of claim 1, wherein the compound is an inhibitor
of malonyl Coenzyme A decarboxylase (MCD).
6. The method of any one of claims 1 to 5, wherein the compound
is administered in an amount sufficient to reduce expression of
NPY at least to the level observed in fed animals.
7. The method of any one of claims 1 to 5, wherein expression and/or
secretion of NPY is reduced in cells which express FAS.
8. The method of claim 1, wherein administration of the compound
inhibits feeding behavior in the animal.
9. The method of claim 1, wherein the animal is deficient in expression
of leptin or the animal is resistant to leptin.
10. A screening method to aid in identifying weight loss agents
comprising administering a candidate compound to an animal or a
hypothalamic culture; and monitoring expression or secretion of
neuropeptide Y.
11. The screening method according to claim 10, wherein the treated
animal is monitored for reduced frequency or intensity of feeding.
12. The method according to claim 10, wherein the candidate compound
is administered to the animal by injection.
13. The method according to claim 12, wherein the compound is administered
intraperitoneally or intracerebroventricularly.
14. The method according to any one of claims 10-13, wherein the
candidate compound is an inhibitor of the enzyme fatty acid synthase.
15. A screening method for identifying genes whose expression is
associated with control of weight loss comprising administering
a weight loss agent to an animal; and comparing expressed mRNA species
in the animal treated with the weight loss agent to expressed mRNA
species in control animals, wherein mRNA species expressed differentially
are associated with control of weight loss.
16. The method of claim 15, wherein the weight loss agent is an
substituted .alpha.-methylene-.beta.-carboxyl-.gamma.-butyrolactone,
such as C-75.
17. The method of claim 15, wherein comparison of expressed mRNA
species is limited to hypothalamic mRNA.
Weight Loss Patent Description
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention is directed to methods of inducing weight
loss in an animal. In part, this invention concerns methods for
reducing adipocyte mass by controlling the level of neuropeptide
Y in the animal.
[0004] 2. Review of Related Art
[0005] 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. According to 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.
[0006] 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. Lack of leptin, as observed
in the ob/ob mouse (Coleman, D. L., 1978, "Obese and diabetes:
Two mutant genes causing diabetes-obesity syndromes in mice,"
Diabetologia, 14:141-148) and certain human individuals (Montague,
C., et al., 1977, "Congenital leptin deficiency is associated
with severe early-onset obesity," Nature, 387(6636):903-908),
leads to profound early-onset obesity. Insulin, produced by pancreatic
beta cells is also produced in proportion to adiposity and in response
to feeding. While acting to promote energy storage in the periphery,
in the hypothalamus insulin acts in a manner similar to leptin,
inhibiting feeding and promoting increased energy utilization (Chavez,
M., et al., 1996, "Central insulin and macronutrient intake
in the rat," Am J Physiol, 271:R727-731). The gut peptides
(e.g. bombesin and cholecystokinin) 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). Unlike insulin and leptin, which act
by a humoral route, 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.
[0007] 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
(Loftus, T., 1999, "An Adipocyte-central nervous system regulatory
loop in the control of adipose homeostasis," Sem. Cell. Dev.
BioL, 10(1):11-18).
[0008] 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(1):196-202). Several
of the feeding inhibitory catabolic 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, "Hypothalamic 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 .alpha.-MSH receptor
(see below), melanin concentrating hormone (MCH) (Ludwig, D., et
al., 1998, "Melanin-concentrating hormone: a functional melanocortin
antagonist in the hypothalamus," Am. J: Physiol., 274:(E627-633))
and 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.
[0009] Among catabolic signals, the most central is (.alpha.-melanocyte
stimulating hormone (.alpha.-MSH). This peptide is elevated in response
to energy surplus and inhibits feeding and promotes catabolic activity.
Mice carrying a deletion in the .alpha.-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).
Similarly, 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). Two additional
hypothalamic signals, cocaine and amphetamine regulated transcript:
(CART) (Lambert, P., 1998, "CART peptides in the central control
of feeding and interactions with neuropeptide Y," Synapse,.
29(4):293-298) and corticotropin releasing hormone (CRH) (Raber
et al. 1997), respond to high levels of feeding signals such as
leptin and inhibit feeding. Other signals known to inhibit feeding
signals in the brain include neurotensin (Sahu, A., 1998, "Evidence
suggesting that galanin (GAL), melanin-concentrating hormone (MCH),
neurotensin (NT), proopiomelanocortin (POMC) and neuropeptide Y
(NPY) are targets of leptin signaling in the hypothalamus,"
Endocrinology, 139(2):795-798), glucagon-like peptide (Turton, M.,
et al., 1996, "A role for glucagon-like peptide-1 in the central
regulation of feeding," Nature, 379(6560):69-72) and serotonin
(Currie, P., et al., 1997, "Stimulation of 5-HT(2A/2C) receptors
within specific hypothalamic," Neuroreport, 8(17):3759-3762).
Serotonin has been linked to the appetite suppression observed in
anorexia and is the target of the recently withdrawn weight-loss
therapy, phen fen.
[0010] C-75 is a specific inhibitor of fatty acid synthase (FAS)
as disclosed in U.S. Pat. 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.
[0011] Excess body weight is a major health problem in developed
nations, affecting over 50% of the U.S. population (Must, et al.,
1999, J. Amer. Med. Assoc., 282:1523), and is increasing both in
prevalence and severity. This condition is associated with increased
risk of type II diabetes, cardiovascular and cerebrovascular disease
among other disorders as well as significantly increased mortality
(Must, et al., 1999). The magnitude of this health problem and the
recent difficulties with several weight-loss therapies emphasize
the need for, a novel approach to weight loss therapy.
SUMMARY OF THE INVENTION
[0012] It is a object of this invention to promote weight loss
by inhibiting feeding behavior. This and other objects are met by
one or more of the following embodiments.
[0013] In one embodiment, 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. Preferably, 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
.alpha.-methylene-.beta.-carboxyl-.gamma.-butyrolactones, or inhibitors
of malonyl Coenzyme A decarboxylase (MCD). Preferably, 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. In another preferred embodiment,
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.
[0014] In a particular embodiment, 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.
[0015] In another embodiment, this invention provides a screening
method for identifying genes whose expression is associated with
control of weight loss. This method comprises 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.
Preferably, the treated animal is treated with an FAS inhibitor,
more preferably the FAS inhibitor is an substituted .alpha.-methylene-.beta.-carboxyl-.gamma.-butyrolactone,
such as C-75. In a preferred embodiment of this method, the expressed
mRNA is mRNA expressed in hypothalamic tissues. By comparing mRNA
expression between treated and control animals, mRNA species associated
with genes whose expression is either up-regulated or down-regulated
by the weight loss agent ma be identified.
[0016] 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.
[0017] It has been discovered that FAS inhibitors, such as the
.alpha.-methylene-.beta.-carboxy-.gamma.-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
[0018] 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.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 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.
[0020] FIG. 1.2 shows body weight (Panel A) and food intake (Panel
B) for mice treated with C-75 or RPMI vehicle.
[0021] FIG. 2 depicts mice with or without C-75 treatment compared
to fasting mice. Panels show (A) body weight and (B) neuropeptide
Y mRNA. FIG. 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.
[0022] FIG. 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.
[0023] FIG. 4 shows the effect of C-75 on serum glucose in (A)
ob/ob mice and (B) wildtype mice.
[0024] FIG. 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 intracerebroventricular
injection of C-75.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0025] The role of metabolism in controlling feeding is well established.
The infusion of physiological fuels such as glucose (Grossman, et
al., 1997, Physiol. Behav., 61:169) or fatty acids (Scharrer, 1999,
Nutrition, 15:704) has long been known to inhibit feeding. Furthermore,
anti-metabolites of these substrates also lead to stimulation of
feeding, as observed following ICV administration of 2-deoxyglucose,
a non-metabolizable glucose analog (Grossman, et al., 1997). There
is also a precedent for the control of feeding by alteration of
lipid metabolism, as inhibitors of fatty acid oxidation in the liver
lead to increased feeding (Scharrer, 1999). However, inhibition
of FAS differs from these other metabolic feeding control mechanisms
in that it induces a feeding-inhibitory signal in the absence of
an added physiological fuel.
[0026] A linkage between feeding-inhibition and fatty acid synthesis
is consistent with the fact that fatty acid synthesis occurs only
during energy surplus, when excess physiological fuels are being
channeled into energy storage. A well-characterized regulatory mechanism
has been described through which fatty acid synthesis regulates
fatty acid oxidation (Rasmussen, et al., 1999, Ann. Rev. Nutri.,
19:463). In this paradigm, malonyl-CoA, a substrate for FAS, is
elevated during fatty acid synthesis and inhibits carnitine palmitoyl
transferase-mediated uptake of fatty acids into the mitochondrion.
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
No. 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.
[0027] It is unlikely that inhibition of fatty acid synthesis per
se leads to feeding inhibition. Previous studies involving administration
of TOFA (Halvorson, et al., 1984, Lipids, 19:851), an inhibitor
of acetyl CoA carboxylase (ACC), the enzyme preceding FAS in the
fatty acid synthetic pathway, led to inhibition of fatty acid synthesis,
but did not inhibit feeding (Malewiak, et al., 1985, Metabolism,
34:604). TOFA administration would be expected to block malonyl-CoA
production and thus would not be expected to inhibit feeding. In
contrast, inhibition of FAS by C-75 leads to dramatic elevation
of malonyl-CoA levels (see U.S. patent application No. 60/164,765)
that may mimic active fatty acid synthesis and thus, the fed state.
[0028] 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.
[0029] The mechanism through which FAS inhibition leads to suppression
of NPY signaling is unlikely to be related to the mechanism of feeding
control by fatty acid oxidation, as feeding control by fatty acid
oxidation is mediated by parasympathetic sensory neurons in a process
independent of hypothalamic control (Scharrer, 1999). Such sensory
neurons have also been reported to play a role in signaling by gut
satiety peptides and by leptin (Niijima 1998, J. Auton. Nerv. Syst.,
73:19). The gut peptides are also unlikely mediators of this effect
as they typically lead to decreased meal size, but not to an overall
decrease in food intake or body weight (West, et al., 1984, Am.
J. Physiol., 246:R776). However, mediation of FAS effects on feeding
by afferent peripheral neurons remains a possible mechanism of such
a feeding signal, as these neurons innervate the major sites of
fatty acid synthesis, notably the liver and adipose tissue.
[0030] Substantial expression of FAS, ACC, and MCD have been observed
in selective neuronal populations within the brain such as: the
arcuate nucleus, cerebellum, brainstem, hippocampus, and cortex.
It is unclear what role these enzymes play as neurons are not thought
to carry out significant levels of fatty acid synthesis; however,
these neurons possess the machinery to undergo elevation of malonyl-CoA
in the presence of C-75 or cerulenin. Studies with [5-.sup.3H]-C-75
indicate that the drug enters the brain. Thus, these inhibitors
may act directly on the brain to control the feeding centers, either
in neurons of the arcuate nucleus itself or in neurons that act
on them. The efficacy of C-75 in animals depleted of serotonin by
pretreatment with the tryptophan hydroxylase inhibitor, para-Chloro-phenylalanine
(Yang, et al., 1995, Am. J. Physiol., 268:E389), argues against
that neurotransmitter as a mediator of this effect.
[0031] Alternatively, 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-.alpha. and interleukin-1.beta..
Nor is it reversed by administration of 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. In addition, C-75-induced
weight loss was observed in mice lacking IL-1r and TNF.alpha.rla
receptors suggesting that the weight loss is not mediated by an
inflammatory response.
[0032] In addition to NPY, several other regulatory molecules combine
in the hypothalamus to control feeding (Loftus, 1999). The expression
of these signals are coordinately regulated either in concert with
NPY (e.g. agouti-related peptide) or in opposition to NPY (e.g.
.alpha.-melanocyte stimulating hormone), depending on feeding status
and adiposity. Control of NPY by C-75 may also extend to these co-regulated
molecules.
[0033] 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
[0034] With the escalation of obesity-related disease, mechanisms
for the control of adipose balance are becoming a more crucial health
issue. Taken together, the present studies provide evidence of a
role for FAS in the control of feeding. As demonstrated by two distinct
inhibitors of FAS, C-75 and cerulenin, this enzyme represents a
potential therapeutic target for the control of appetite and body
weight.
Weight Loss Agents
[0035] Weight loss agents according to this invention 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. (2000), "Brain-derived neurotrophic factor but not neurotrophin-3
enhances differentiation of somatostatin neurons in hypothalamic
cultures," Neuroendocrinology. 72(3):144-53, incorporated herein
by reference). As an alternative or supplemental test, 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.
[0036] 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 application Nos. 60/164,749, 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 Inhibitors
[0037] A wide variety of compounds have been shown to inhibit fatty
acid synthase (FAS), and selection of a suitable FAS inhibitor for
use in this invention is within the skill of the ordinary worker
in this art 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. Pat. No. 5.759,837, and methods of synthesizing preferred
FAS inhibitors, the .alpha.-methylene-.beta.-carboxy-.gamma.-butyrolactones,
are described in U.S. Pat. No. 5,981,575, both of which are incorporated
herein by reference.
[0038] Suitable FAS inhibitors may be identified by a simple test
exemplified in Example 7 of U.S. Pat. No. 5,981,575, and in U.S.
Pat. 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.
[0039] Alternatively, 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.sub.50 or ID.sub.50). FAS inhibitors with high
therapeutic index will inhibit fatty acid synthesis at a lower concentration
(as measured by IC.sub.50) than the IC.sub.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.sub.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.sub.50
determined for cell growth in the presence of exogenous fatty acid.
Therapy
[0040] Human therapy according to this invention will lead to decreased
intracellular fat storage and a reduction in adipocyte mass. This
may be expected to have the primary and/or secondary effects listed
in the Table. 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). Similarly, individuals with fatty livers (e.g.,
type II diabetics or obese persons) 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. In patients with. elevated low density lipoproteins (LDLs),
this method may be used to reduce the LDL level. Thus, 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 osteoartritis, and the method of
this invention may reduce the effects of the disease or delay the
onset.
Table Effects of decreased intracellular fat storage and reduction
in adipocyte mass
[0041] Weight loss without muscle loss [0042] Reduction in hepatic
fat [0043] Increased insulin responsiveness (especially in Type
II diabetes mellitus) [0044] Decreased blood pressure [0045] Decreased
arterial vascular disease [0046] Decreased susceptibility to liver
injury associated with fatty change, including endotoxin mediated
liver injury
[0047] 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. Similarly, 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.
[0048] Treatment according to this invention involves administering
a compound according to this invention (for example, an FAS inhibitor
such as an .alpha.-methylene-.beta.-carboxy-.gamma.-butyrolactone)
to the subject of treatment. 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.
[0049] 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. The 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).
[0050] 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 .mu.g/ml, preferably 10 ng/ml to 10 .mu.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.
[0051] Preferably, the therapeutic compounds of this invention,
such as FAS inhibitors, are administered based in the level necessary
to control secretion of neuropeptide Y. In particular, 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.
[0052] The 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. Preferably 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. On the other hand, administration of inhibitors at a level
that kills some cells which will subsequently be regenerated (e.g.,
endometrial cells) is not necessarily excluded.
[0053] In addition to identifying neuropeptide Y as a key component
in the pathway responsible for weight control, 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 In addition,
subtraction suppression hybridization, microarray or chip technology
can be used to screen for differentially-expressed mRNAs (see also,
Lockhart, et al. (2000), "Genomics, gene expression and DNA
arrays." Nature 405:827-836, incorporated herein by reference).
In a preferred embodiment of this method, the expressed mRNA is
mRNA expressed in control and treated hypothalamic tissues. Weight
loss agents which are substituted .alpha.-methylene-.beta.-carboxyl-.gamma.-butyrolactones,
such as C-75, are preferred agents for treatment of animals for
comparisons according to this method. By comparing mRNA expression
between treated and control animals, mRNA species associated with
genes. whose expression is either up-regulated or down-regulated
by the weight loss agent may be identified.
EXAMPLES
[0054] In order to facilitate a more complete understanding of
the invention, a number of Examples are provided below. However,
the scope of the invention is not limited to specific embodiments
disclosed in these Examples, which are for purposes of illustration
only.
Example 1
Inhibitors of FAS and Fatty Acid Synthesis
[0055] FIG. 1.1 (Panel A) shows the chemical structures of C-75
and Cerulenin. The inhibitory effects of these compounds were demonstrated
on BALB/c mice.
[0056] Female BALB/c mice were treated with 0.6 mg of C-75 in 200
.mu.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-.sup.14C]-acetate, lipids were extracted and
counted, [A. Rashid et al., Am. J. Pathol. 150 (1997)]. The results
are shown in FIG. 1.1 (Panel B). C-75 markedly inhibited adipose
fatty acid synthesis compared to vehicle control. Values represent
mean +/- SEM (* P<05).
[0057] Male BALB/c mice (4 per group) were given 2 g/kg dextrose
by oral gavage. After 15 min mice were injected IP with 20 mg/kg
C-75 or RPMI vehicle. One hour post-treatment, livers were rapidly
removed, frozen and pulverized in liquid nitrogen, HClO4 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 FIG. 1.1
(Panel C). Intraperitoneal injection of mice with C-75 leads to
a 95% reduction in .sup.14C-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
[0058] The effect of C-75 treatment on feeding behavior and body
weight in mice is both rapid and dramatic. A single treatment leads
to the loss of as much as 20% of total body weight within 24 hours
(FIG. 1.2A). This weight loss occurs in a dose dependent manner
and persists for a duration that increases with dose. In all cases,
treated animals recover lost body weight after the effect of the
drug has dissipated, arguing against induction of a persistent wasting.
The treatment is well tolerated by the mice, the only evident effect
being excessive weight loss. Histological analysis of tissues from
treated mice revealed no indication of adverse pathology (not shown).
[0059] Male BALB/c mice 19-22 g were weighed, treated by a single
intra peritoneal (I.P.) injection and housed in metabolic cages.
Body weight (FIG. 1.2A) and food intake (FIG. 1.2B) were monitored
at 24 hour intervals. FIG. 1.2A shows mean change from initial body
weight in mice treated with 7.5 (.DELTA.), 15 (.omicron.) or 30
(.quadrature.)mg/kg of C-75 or RPMI vehicle (.cndot.) is expressed
+/- SEM. FIG. 1.2B shows total food intake for mice treated with
RPMI vehicle (black bars) or 15 mg/kg C-75 (grey bars) per day following
treatment.
[0060] 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. Administration of 15 mg/kg
body weight led to a greater than 90% reduction in food intake over
the first 24 hours (FIG. 1.2B). Feeding behavior then returned to
normal progressively over a 48-72 hour period as the drug effect
dissipated. The role of feeding inhibition in C-75 induced weight
loss was confirmed by studies in which forced feeding of the drug
treated animals largely reversed the observed weight loss.
[0061] In concert with the feeding inhibition, there was a modest
reduction in water intake, mirrored by a similar reduction in urinary
output (not shown). Rather than a direct inhibition of water intake,
this is consistent with a change in osmotic balance resulting from
decreased intake of salts and other solutes in the diet. However,
it is possible some component of the observed weight loss is due
to water.
Example 2
Regulation of Feeding by C-75 in Fed and Fasted States: Role of
NPY
[0062] To determine whether the weight loss is attributable entirely
to suppression of feeding, treatment with a dose of C-75 that completely
suppresses feeding was compared with fasting. Both fasting and C-75
led to significant weight loss relative to control; however, in
many experiments the C-75 treated mice lost more weight than did
the fasted animals (FIG. 2A). The normal response to fasting is
to reduce energy utilization to limit depletion of energy stores
(Loftus, 1999). If C-75 treatment results in a "perceived fed
state", it may allow maintenance of a normal metabolic rate
as well as inhibition of feeding.
[0063] Male BALB/c mice 19-21 g were preweighed and treated with
vehicle or 30 mg/kg C-75 and allowed free access to food, or were
denied all access to food (fasted). After 24 hours, mice were weighed.
Change from initial body weight is shown in FIG. 2A, expressed as
mean +/- SEM (n=7). C-75 treated mice lost 45% more weight than
did the fasted animals.
[0064] 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). In the arcuate nucleus, 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). To ascertain whether C-75 might
alter NPY regulation in the hypothalamus, the expression of NPY
was examined by northern blot analysis of hypothalamic tissue microdissected
from the brains of the fed, fasted and C-75-treated mice shown in
FIG. 2A.
[0065] The hypothalamic region was microdissected from the brains
of mice in FIG. 2A and total RNA was isolated. RNA was subjected
to northern blot analysis using random primed probes (Feinberg,
et al., 1983, Anal. Biochem., 132:6) for NPY and S26 (as a loading
control). Tissue was extracted for total RNA as described, P. Chomczynski
and N. Sacchi, Anal. Biochem. 162, 156 (1987). 15 .mu.g of total
RNA was subjected to Northern blot analysis as described, T. Brown,
K. Mackey, in Current Protocols in Molecular Biology, F. Ausubel,
et al., Eds. (John Wiley and Sons, New York, 1997) pp. 4.9.1-4.9.16.
As expected, fasting markedly up-regulated NPY mRNA expression (FIG.
2B). However, the level of hypothalamic NPY mRNA in C-75-treated
mice was even lower than that of the fed controls, although they
had not eaten and represented the fasted state. This suggests that
C-75 inhibits feeding, at least in part, by blocking the prophagic
NPY signal.
[0066] To confirm this finding, the capacity of NPY to reverse
C-75-induced inhibition of feeding was examined. Mice were pretreated
with 30 mg/kg of C-75 by I.P. injection. After 4 hours, mice were
anaesthetized by inhaled metofane and given a direct intracerebroventricular
injection of 500 ng NPY (2.5 .mu.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 FIG. 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.
[0067] Intracerebroventricular (ICV) injection of 500 ng of NPY
into mice pretreated with either vehicle or C-75 rapidly led to
voracious feeding, while ICV injection of vehicle had no effect
on feeding. Although the feeding effects of this dose of NPY had
completely subsided in less than an hour, it was sufficient to substantially
elevate the total food intake in C-75-treated mice (FIG. 2C). These
results confirm both that the feeding control pathways downstream
of NPY are intact in C-75-treated mice, and that C-75 acts upstream
of NPY release, as anticipated from the northern blot analysis.
[0068] The effect of C-75 on feeding was also examined with fasted
mice which exhibit up-regulated NPY levels, and feed voraciously.
Mice were fasted for 24 hours to induce voracious feeding. Initial
feeding interval (time in seconds between food presentation and
initiation of feeding) was measured in naive mice (pretreat). Mice
were then treated by I.P. injection of 30 mg/kg C-75 or RPMI vehicle
and feeding interval determined at 20, 40 and 60 minutes post-injection.
The results are shown in FIG. 2D. Observation was terminated if
no feeding was initiated within 1000 sec (experimental cut off).
Times represent mean +/- SEM, (n=4).
[0069] Prior to treatment, all animals fed ravenously within 3
minutes of being offered food. However, within 20 minutes of C-75
treatment, the mice lost all interest in feeding, while vehicle
treated mice continued to initiate feeding within 3 minutes of food
presentation (FIG. 2D). The fact that these animals had already
up-regulated their NPY message levels indicates that C-75 must have
additional. actions, either on NPY release, or on other regulators
of feeding behavior.
Example 3
Leptin Independence of C-75 Action and Treatment of ob/ob Mice
[0070] 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. BALB/c mice treated
with RPMI vehicle (.omicron.) or 30 mg/kg C-75 (.box-solid.) I.P.
and free fed, or fasted (.cndot.) 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 (FIG. 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
FIG. 3A). This is consistent with the normal regulation of leptin
levels during weight loss (Boden, et al., 1996, J. Clin. Endocrinol.
Metab., 81:3419) and indicates that leptin does not mediate the
C-75 signal. Northern blot analysis of leptin message levels in
white adipose tissue from the same animals (performed as described
above) supports this observation (data not shown).
[0071] A leptin independent mechanism suggested that C-75 should
be effective in reducing the obesity of ob/ob mice which do not
express functional leptin (Schwartz, et al., 1996). This was confirmed
over a two week course of treatment which led to a substantial reduction
in the body weight of C-75-treated animals while vehicle treated
mice continued to gain weight (FIG. 3B). Male ob/ob (C57BL/6OlaHsd-Lep.sup.ob,
Harlan) mice were treated with RPMI vehicle (.omicron.) or 22 mg/kg
C-75 (.cndot.) I.P. every third day and body weight monitored change
in body weight is displayed as mean +/- SEM. The magnitude of this
effect is readily evident by inspection of representative C-75 and
control treated ob/ob mice. (See FIG. 3C, which shows representative
vehicle and C-75 treated mice from FIG. 3B at the termination of
treatment (14 days)).
[0072] C-75 treatment not only led to weight loss, but also corrected
many of the pathological consequences that result from the extreme
obesity of ob/ob mice. Liver samples from vehicle and C-75 treated
mice (from FIG. 3B) were fixed in formalin and paraffin embedded.
Tissue sections (4 .mu.m) were stained with hematoxylin and eosin.
Histological examination of the liver from C-75 treated animals
showed a marked reduction in the hepatomegaly and fatty liver observed
in control ob/ob mice (FIG. 3D, scale bar=50 .mu.). Analysis of
white adipose tissue demonstrated a dramatic reduction in mean adipocyte
size (not shown). There was no evidence of histological. abnormality
resulting from chronic treatment of the animals even in these primary
tissues of fatty acid synthesis. The observation that C-75 acts
through a leptin independent mechanism is particularly promising
in that the majority of obese individuals appear to be relatively
resistant to leptin's effects (Caro, et al., 1996, Lancet, 348:159).
Example 4
C-75 Treatment Corrects Hyperglycemia in ob/ob Mice
[0073] In addition to obesity, ob/ob mice also develop overt diabetes
with significant elevation of blood glucose. C-75 corrected the
hyperglycemia observed in vehicle treated mice with a nearly 3-fold
reduction in mean serum glucose (FIG. 4A). Male ob/ob mice (n=3)
were treated with C-75 or vehicle for 2 weeks (FIGS. 3B and C) and
compared with age matched, untreated c57BL/6j mice (+/+) 24 hour
IP treatment of wild-type mice had no effect on serum glucose beyond
that attributable to fasting. The normalization of blood glucose
occurred from the profound weight loss in the ob/ob mice as acute
treatment of normal mice with C-75 had no effect on serum other
than that resulting from inhibition of feeding (FIG. 4B). Male BALB/c
mice (n=7) were fasted for 24 hours or injected IP with 30 mg/kg
C-75 or RPMI vehicle and allowed free access to food for 24 hours.
In both cases, serum was collected at death and assayed for glucose:
Ref Lab.TM. GLU (Medical Analysis Systems, Inc., Camarillo, Calif.).
These data highlight the importance of C-75's independence from
leptin, since over 75% of obese humans appear to be resistant to
leptin's effects. Both panels are representative of 2 experiments.
Example 5
Regulation of Feeding by Malonyl CoA
[0074] FIG. 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. To
test this, mice were pretreated with the ACC inhibitor TOFA or vehicle
by ICV injection and examined the ability of C-75, administered
IP, to inhibit feeding. BALB/c mice were anesthetized with metofane
and injected ICV with 2 .mu.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. TOFA largely restored
food intake in C-75-treated mice (FIG. 5B), supporting the hypothesis
that malonyl-CoA mediates feeding inhibition. In addition, mice
were anesthetized and injected ICV with 2 .mu.l of RPMI or C-75
at 2.5 or 5 .mu.g/.mu.l and food intake monitored over 2 (shaded)
and 4 (solid) hours. The efficacy of centrally administered TOFA
argues for a central (CNS) mechanism of action. ICV administration
of C-75 inhibited feeding by 82% (FIG. 5C), supporting the central
target action of C-75. FIG. 5 (B) and (C) combine results from 3
experiments with N=3 for each (9 total).
Example 6
Immunohistochemical Localization of Malonyl CoA Metabolism
[0075] 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.
[0076] For purposes of clarity of understanding, the foregoing
invention has been described in some detail by way of illustration
and example in conjunction with specific embodiments, although other
aspects, advantages and modifications will be apparent to those
skilled in the art to which the invention pertains. The foregoing
description and examples are intended to illustrate, but not limit
the scope of the invention. Modifications of the above-described
modes for carrying out the invention that are apparent to persons
of skill in clinical medicine, physiology, pharmacology, and/or
related fields are intended to be within the scope of the invention,
which is limited only by the appended claims.
[0077] All publications and patent applications mentioned in this
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
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