U.S. patent application number 10/917525 was filed with the patent office on 2005-05-19 for stimulation of cpt-1 as a means to reduce weight.
This patent application is currently assigned to JOHNS HOPKINS UNIVERSITY LICENSING AND TECHNOLOGY DEVELOPMENT. Invention is credited to Kuhajda, Francis P., Landree, Leslie E., Ronnett, Gabriele, Thupari, Jagan N..
Application Number | 20050106217 10/917525 |
Document ID | / |
Family ID | 27734382 |
Filed Date | 2005-05-19 |
United States Patent
Application |
20050106217 |
Kind Code |
A1 |
Thupari, Jagan N. ; et
al. |
May 19, 2005 |
Stimulation of CPT-1 as a means to reduce weight
Abstract
This invention provides methods and compositions for inducing
weight loss and maintaining optimum weight comprising administering
an agent that stimulates carnitine palmitoyl transferase-1 (CPT-1)
activity to the patient in need, including human patients. These
methods do not require inhibition of fatty acid synthesis. In
particular, this invention provides methods for development of
therapeutics that selectively enhance fatty acid oxidation,
increase energy production, and reduce adiposity while preserving
lean mass, through the pharmacological stimulation of CPT-1
activity. In a preferred mode, the agent is administered in an
amount sufficient to increase fatty acid oxidation. In another
preferred mode, the agent is administered in an amount sufficient
to antagonize malonyl CoA inhibition of CPT-1. In yet another
preferred mode, the agent is administered in an amount sufficient
to increase malonyl CoA level.
Inventors: |
Thupari, Jagan N.; (Owings
Mills, MD) ; Landree, Leslie E.; (Baltimore, MD)
; Ronnett, Gabriele; (Lutherville, MD) ; Kuhajda,
Francis P.; (Lutherville, MD) |
Correspondence
Address: |
HUNTON & WILLIAMS LLP
INTELLECTUAL PROPERTY DEPARTMENT
1900 K STREET, N.W.
SUITE 1200
WASHINGTON
DC
20006-1109
US
|
Assignee: |
JOHNS HOPKINS UNIVERSITY LICENSING
AND TECHNOLOGY DEVELOPMENT
Baltimore
MD
|
Family ID: |
27734382 |
Appl. No.: |
10/917525 |
Filed: |
August 13, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10917525 |
Aug 13, 2004 |
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10503605 |
Jan 28, 2005 |
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10503605 |
Jan 28, 2005 |
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PCT/US03/03839 |
Feb 10, 2003 |
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60354480 |
Feb 8, 2002 |
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Current U.S.
Class: |
424/439 ;
514/565 |
Current CPC
Class: |
A61K 31/198 20130101;
A23L 33/40 20160801; A61K 31/34 20130101; A61K 31/365 20130101;
A61K 31/381 20130101; A61P 3/04 20180101; A23L 33/175 20160801;
A61P 43/00 20180101; A61K 31/195 20130101; C12Q 1/48 20130101; G01N
2500/04 20130101; A61K 31/205 20130101; A61K 31/00 20130101; A23L
33/10 20160801 |
Class at
Publication: |
424/439 ;
514/565 |
International
Class: |
A61K 031/198; A61K
047/00 |
Claims
1. Method of inducing weight loss comprising administering an agent
that stimulates carnitine palmitoyl transferase-1 (CPT-1)
activity.
2. Method for stabilizing weight comprising chronic administration
of an agent that stimulates CPT-1 activity in an amount that does
not significantly inhibit FAS.
3. Method of screening for agents that induce weight loss,
comprising determining whether a candidate weight loss agent
stimulates CPT-1 activity; and selecting an agent that stimulates
CPT-1 activity.
4. A therapeutic composition comprising an agent that stimulates
CPT-1 activity and L-carnitine.
5. A nutritional composition comprising nutritionally sufficient
amounts of fats, carbohydrates and amino acids, said composition
further comprising L-carnitine and an antagonist of malonyl CoA
inhibition of CPT-1.
Description
BACKGROUND
[0001] 1. Field of the Invention
[0002] This invention is directed to a method for development of
therapeutics that selectively enhance fatty acid oxidation,
increase energy production, and reduce adiposity while preserving
lean mass, through the pharmacological stimulation of CPT-1
activity.
[0003] 2. Review of Related Art
[0004] Cerulenin treatment of MCF-7 human breast cancer cells in
vitro significantly inhibits fatty acid oxidation, probably through
increased levels of malonyl-CoA (Loftus, et al. (2000) Science,
288:2379-2381). C75 is a member of a family of
.alpha.-methylene-.gamma.-butyrolactones which are known inhibitors
of fatty acid synthase (FAS) (Kuhajda, et al. (2000) Proc. Natl.
Acad Sci USA, 97:3450-3454). Treatment of mice with C75 leads to
inhibition of hepatic fatty acid synthesis and high levels of
malonyl-CoA (Loftus, et al. (2000); Pizer, et al. (2000) Cancer
Res., 60:213-218). In the brain, C75 reduces the expression of
hypothalamic neuropeptide-Y (NPY) leading to reversible inanition
(Loftus, et al, 2000). During in vivo treatment of ob/ob mice with
C75 there was profound loss of fat in the liver and peripheral
tissues despite the increased levels of hepatic malonyl-CoA
(Loftus, et al., 2000).
[0005] Malonyl-CoA is a potent inhibitor of fatty acid oxidation
through its action as an inhibitor of
carnitine-palmitoyl-transferase-1 (CPT-1) (Witters, et al. (1992)
J. Biol. Chem., 267:2864-2867). CPT-1 enables the entry of
long-chain acyl-CoA's into the mitochondria for fatty acid
oxidation. When treated with FAS inhibitors, genetically and
diet-induced obese mice undergo a selective and significant loss of
adipose tissue despite the high levels of malonyl-CoA induced by
FAS inhibition. Since malonyl-CoA is a potent inhibitor of fatty
acid oxidation through its inhibition of carnitine
palmitoyltransferase-1 (CPT-1, E.C. 2.3.1.21), the rapid and
selective loss of adipose tissue was surprising. High systemic
levels of malonyl-CoA would be expected to inhibit fatty acid
oxidation leading instead to a selective loss of lean mass during
C75 induced inanition.
SUMMARY OF THE INVENTION
[0006] It is an object of this invention to provide methods and
compositions for inducing weight loss and maintaining optimum
weight which do not require inhibition of fatty acid synthesis.
This and other objects are met by one or more of the following
embodiments.
[0007] In one embodiment, this invention provides a method of
inducing weight loss comprising administering an agent that
stimulates carnitine palmitoyl transferase-1 (CPT-1) activity to
the patient in need, including human patients. In a preferred mode,
the agent is administered in an amount sufficient to increase fatty
acid oxidation. In another preferred mode, the agent is
administered in an amount sufficient to antagonize malonyl CoA
inhibition of CPT-1. In yet another preferred mode, the agent is
administered in an amount sufficient to increase malonyl CoA level.
In still another preferred mode, upon administration of the agent,
malonyl CoA level is not substantially increased. Substantial
increase in malonyl CoA level as contemplated herein is equivalent
to about one-half the K.sub.i for malonyl CoA inhibition of CPT-1.
In yet another preferred mode, the agent which stimulates CPT-1
activity also inhibits fatty acid synthase (FAS). In an alternative
mode, FAS is not significantly inhibited. Insignificant inhibition
as contemplated herein is less that 15%, preferably less than 10%,
and more preferably less than 5% inhibition. Methods for assay of
FAS activity are disclosed in U.S. Pat. No. 5,981,575, incorporated
herein by reference. In preferred modes of the above embodiments,
the agent which stimulates CPT-1 activity is not a compound of
formula: 1
[0008] wherein R is a substitute selected from the group consisting
of:
[0009] (a) saturated linear or branched alkyl groups having 3-18
carbon atoms,
[0010] (b) unsaturated linear or branched alkyl groups having 3-18
carbon atoms, 2
[0011] wherein:
[0012] R.sup.1 and R.sup.2, the same or different, are H, CH.sub.3,
C.sub.2H.sub.5, C.sub.3H.sub.7, C.sub.4H.sub.9, CF.sub.3,
OCH.sub.3, F, Cl, or Br;
[0013] R.sup.3 is H, CH.sub.3, C.sub.2H.sub.5, C.sub.2H.sub.5,
C.sub.4H.sub.9, COOH, COOCH.sub.3, COOC.sub.2H.sub.5,
COOC.sub.2H.sub.5, or COOC.sub.4H.sub.9;
[0014] R.sup.4 is H, CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, or
C.sub.4H.sub.9;
[0015] X is NH, S, or O;
[0016] Z is CH.sub.2, O, NH, or S;
[0017] i is 1 to 5;
[0018] j is 0 to 10;
[0019] k is 1 to 10;
[0020] m is 1-13; and
[0021] n is 1 to 15.
[0022] In another embodiment, this invention provides a method for
stabilizing weight comprising chronic administration of an agent
that stimulates CPT-1 activity in an amount that does not
significantly inhibit FAS. In a preferred mode, the agent is
administered in an amount sufficient to increase fatty acid
oxidation. In another preferred mode, the agent is administered in
an amount sufficient to antagonize malonyl CoA inhibition of CPT-1.
In yet another preferred mode, the agent is administered in an
amount sufficient to increase malonyl CoA level. In still another
preferred mode, upon administration of the agent, malonyl CoA level
is not substantially increased. Substantial increase in malonyl CoA
level as contemplated herein is equivalent to about one-half the
K.sub.i for malonyl CoA inhibition of CPT-1.
[0023] In still another embodiment, this invention provides a
method of screening for agents that induce weight loss, comprising
determining whether a candidate weight loss agent stimulates CPT-1
activity; and selecting an agent that stimulates CPT-1 activity.
Preferably, this method further comprises determining whether the
candidate weight loss agent is an antagonist of malonyl CoA
inhibition of CPT-1, and candidate weight loss agents are selected
that obviate malonyl CoA inhibition of CPT-1.
[0024] In yet another embodiment, this invention provides a
therapeutic composition comprising an agent that stimulates CPT-1
activity and L-carnitine. Preferably, the therapeutic composition
comprises an antagonist of malonyl CoA inhibition of CPT-1.
[0025] In still another embodiment, this invention provides a
nutritional composition comprising nutritionally sufficient amounts
of fats, carbohydrates and amino acids, said composition further
comprising an antagonist of malonyl CoA inhibition of CPT-1 and
L-carnitine. In one mode, the nutritional composition is adapted
for parenteral administration.
[0026] To investigate the mechanism of action leading to the
paradoxical reduction of fatty liver in the setting of high hepatic
levels of malonyl-CoA during C75 treatment, the effect of C75 on
CPT-1 activity was studied. Surprisingly, C75 and related compounds
concomitantly stimulated CPT-1 activity and fatty acid oxidation in
vitro while inhibiting FAS. In addition to its overall allosteric
activation of CPT-1, C75 abrogated the inhibitory effect of
malonyl-CoA on CPT-1 activity in vitro. As a consequence of
increased fatty acid oxidation, C75 increased cellular ATP
levels.
[0027] To test the effect of C75 on fatty acid oxidation in vivo,
whole animal calorimetry was utilized to measure the respiratory
exchange ratio (RER) in mice treated with C75. Following C75
therapy, the RER dropped within 2 h to the range of 0.7, indicative
of fatty acid oxidation. This rate of RER decline was similar to
food withdrawal from animals fed ad libitum with mouse chow. These
studies indicate that, despite high hepatic levels of malonyl-CoA,
C75 treated animals freely oxidized fatty acids.
[0028] These data suggest that C75 blocks the inhibitory action of
malonyl-CoA on CPT-1 activity in vivo leading to a reduction in
fatty liver and adipose mass during FAS inhibition. This invention
describes a method to develop therapeutics that selectively reduce
adiposity while preserving lean mass through the pharmacological
stimulation of CPT-1 activity.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1 shows the effect of C75 on fatty acid oxidation in
MCF-7 cells, compared to the effect of Etomoxir.
[0030] FIG. 2 shows concentration dependent stimulation of CPT-1
activity by C75 and inhibition by malonyl CoA.
[0031] FIG. 3 shows reversible stimulation of CPT-1 by C75.
[0032] FIG. 4 shows stimulation of CPT-1 by various C75
analogs.
[0033] FIG. 5 shows concentration dependent enhancement of cellular
ATP levels by C75 in MCF-7 cells.
[0034] FIG. 6 shows concentration dependent stimulation of fatty
acid oxidation by C75 in mouse adipocytes.
[0035] FIG. 7 shows concentration dependent enhancement of cellular
ATP levels by C75 in mouse adipocytes.
[0036] FIG. 8 shows respiratory exchange ratio (RER) measured by
indirect calorimetry for mice in the absence (A) and presence (B,
C) of C75.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0037] Inhibition of fatty acid synthase (FAS) in vivo has been
shown to cause rapid and profound weight loss. Both cerulenin, a
natural product, and C-75, a synthetic small-molecule, cause
similar weight loss when administered intracerebroventricularly
(i.c.v.) to rats. When treated systemically (e.g.,
intraperitoneally), C-75 causes more profound weight loss, even
weight loss greater than starved animals. These data demonstrate a
greater peripheral (non-CNS) effect on weight loss for C-75 than
for cerulenin.
[0038] While studying the mechanism of action of this profound
peripheral effect of C-75, the inventors have recently found that
in addition to inhibition of FAS, C-75 and its family of
.alpha.-methylene-.gamma.-butyr- olactones, directly stimulates
carnitine palmitoyltransferase-1 (CPT-1) leading to increased
mitochondrial fatty acid oxidation. Cerulenin, in contrast, leads
to reduced CPT-1 activity and reduced fatty acid oxidation through
generation of high malonyl-CoA levels from FAS inhibition.
[0039] C75 treatment of MCF-7 cells in vitro stimulated CPT-1
activity from 150-160%. There was also a concomitant increase in
fatty acid oxidation. Among the C75 analogs, a carbon chain length
of C6-C16 was optimum for CPT-1 stimulatory activity. In the
presence of C75, malonyl-CoA is no longer able to inhibit CPT-1
activity, suggesting that in addition to its stimulatory effect,
C75 also prevents malonyl-CoA inhibition of CPT-1. There is no
detectable covalent interaction between CPT-1 and C75.
[0040] Thus, the peripheral (non-CNS) weight loss effect of C-75 is
at least in part due to CPT-1 stimulation and increased fatty acid
oxidation with concomitant fatty acid synthesis inhibition. These
data identify a family of .alpha.-methylene-.gamma.-butyrolactones
as malonate or malonyl-CoA mimetics and CPT-1 as a target for
weight loss therapeutics and. More broadly, our data suggest that
other malonate or malonyl-CoA mimetics can be designed and
synthesized to function as effective weight loss agents.
[0041] Data demonstrate that C-75 and its family of
.alpha.-methylene-.gamma.-butyrolactones directly interact with
CPT-1 leading to increased CPT-1 enzymatic activity and fatty acid
oxidation. The chemical structure of C75 and numerous analogs, as
well as methods of synthesizing these analogs, are disclosed in
U.S. Pat. No. 5,981,575, which is incorporated herein by reference.
The stimulatory effect of C75 is related to the length of the
saturated carbon side chain, with the optimum length between 6-18
carbon atoms. With regard to the discussion of the present
invention, C75 is the prototype agent for stimulation of CPT-1;
reference to C75 hereinafter includes other suitable agents which
stimulate CPT-1 activity, except where indicated otherwise by
context. Other suitable agents which stimulate CPT-1 activity
include a variety of gamma-butyrolactones which can be readily
identified by testing the effect on CPT-1 activity of
gamma-substituted-alpha-methylene-butyrolacto- nes, such as those
described in International Patent Publication WO 2004/006835,
incorporated herein by reference, substituted thiotetronic acids,
such as those described in International Patent Publication WO
2004/005277, incorporated herein by reference, and substituted
thiophene diones described in U.S. Provisional Patent Application
60/574,639, incorporated herein by reference.
[0042] In addition to its direct effect upon CPT-1, C-75 abolishes
the inhibitory effect of malonyl-CoA on CPT-1 activity. Although
C75 exhibits kinetic features of a slow-binding inhibitor with
purified FAS (1), its interaction with CPT-1 appears rapid and
competitive. Thus, the stimulatory effect of C75 upon fatty acid
oxidation may be due to either its direct stimulation of CPT-1
activity, its interference of malonyl-CoA inhibition of CPT-1, or
both. Interestingly, the effects of C75 are not restricted to
murine CPT-1, as human CPT-1 was similarly affected. As a
consequence of increased fatty acid oxidation, C75 also increased
ATP levels in both the human and murine cells.
[0043] The effect of C75 on fatty acid metabolism in vivo mirrored
the alterations seen on a cellular level. C75 treatment of lean
mice led to a profound and rapid increase in fatty acid oxidation,
despite the high levels of malonyl-CoA generated by C75 in vivo.
Thus, C75 and its family of
.alpha.-methylene-.gamma.-butyrolactones, appear to act as
competitive agonists of CPT-1. This agonist activity of C75 appears
to overcome inhibitory effects of malonyl CoA on the same enzyme.
The increased fatty acid oxidation induced by C75 is an important
mechanism accounting for marked reduction in adiposity seen during
C75 treatment of mice.
[0044] In summary, this invention describes a method to develop
therapeutics that selectively enhance fatty acid oxidation,
increase energy production, and reduce adiposity while preserving
lean mass, through the pharmacological stimulation of CPT-1
activity.
[0045] Formulation of therapeutic compositions containing C75
and/or other agents that stimulate CPT-1, and methods of
administering such agents, are within the skill of the art,
particularly in view of the description in U.S. Pat. No. 5,981,575,
the text of which is incorporated herein by reference.
[0046] Use of CPT-1 stimulating agents to increase energy
production by administering the agents contemporaneously with fatty
acids or compounds containing fatty acid residues is also within
the skill of the art, particularly in view of the nutritional
compositions disclosed in U.S. Pat. No. 4,434,160, the text of
which is incorporated herein by reference.
EXAMPLES
[0047] 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
Paradoxical Effects of a Fatty Acid Synthase Inhibitor
[0048] Cerulenin, an FAS inhibitor, increases malonyl-CoA amount in
MCF-7 cells (3). As a consequence of the massive increase in
malonyl-CoA, cerulenin causes inhibition of fatty acid oxidation
through the malonyl-CoA inhibition of CPT-1 (Thupari, et al. (2001)
Biochem. Biophys. Res. Comm., 285:217-223). Previously, it was
shown that C75 treatment of MCF-7 cells resulted in a >5-fold
increase in malonyl-CoA through C75 inhibition of fatty acid
synthase (FAS) (3). The effect of C-75 on fatty acid oxidation was
tested as follows.
[0049] Human breast cancer cell line MCF-7 was obtained from the
American Type Culture Collection. 1.times.10.sup.6 MCF-7 cells were
plated in T-25 flasks in triplicate and incubated overnight at
37.degree. C. Drugs were then added as indicated diluted from 5
mg/ml stock in DMSO. After 2 hours, medium with drugs was removed
and cells were preincubated for 30 min. with 1.5 ml of the
following buffer: 114 mM NaCl, 4.7 mM KCl, 1.2 mM KH.sub.2PO.sub.4,
1.2 mM MgSO.sub.4, glucose 11 mM. After preincubation, 200 .mu.l of
assay buffer was added containing: 114 mM NaCl, 4.7 mM KCl, 1.2 mM
KH.sub.2PO.sub.4, 1.2 mM MgSO.sub.4, glucose 11 mM, 2.5 mM
palmitate (containing with 10 .mu.Ci of [1-.sup.14C]palmitate)
bound to albumin, 0.8 mM L-carnitine, and cells were incubated at
37.degree. C. for 2 h. Following the incubation, 400 .mu.l of
benzethonium hydrochloride was added to the center well to collect
released .sup.14CO.sub.2. Immediately, the reaction was stopped by
adding 500 .mu.l of 7% perchloric acid to the cells. The flasks
with wells were then incubated for 2 h at 37.degree. C. after which
the benzothonium hydrochloride was removed and counted for
.sup.14C. Blanks were prepared by adding 500 .mu.l of 7% perchloric
acid to the cells prior to the incubation with the assay buffer for
2 h.
[0050] When cells were treated with C75 2 hours before fatty acid
oxidation was measured, C75 treatment resulted in a 156% increase
in fatty acid oxidation compared to the control (see FIG. 1;
p=0.0012, two-tailed t-test, Prism 3.0). In contrast, Etomoxir, a
known inhibitor of fatty acid oxidation and non-competitive
inhibitor of CPT-1, decreased fatty acid oxidation to 32% of
control (p=0.0006, two-tailed t-test, Prism 3.0). C-75 treatment of
MCF-7 cells repeatedly resulted in increased fatty acid oxidation
with doses from 5-20 .mu.g/ml.
[0051] Paradoxically, despite an increase in malonyl-CoA similar to
that induced by cerulenin, C75 treatment increased rather than
decreased fatty acid oxidation in MCF-7 cells. This implies a
direct effect of C75 upon carnitine palmitoyltransferase-1
(CPT-1).
Example 2
C75 Stimulates Activity of Human CPT-1
[0052] CPT-1 activity was assayed in MCF-7 cells by the following
published procedure: MCF-7 cells were plated in DMEM with 10% fetal
bovine serum at 10.sup.6 cells in 24-well plates in triplicate.
Following overnight incubation at 37.degree. C., the medium was
removed and replaced with 700 .mu.l of assay medium consisting of:
50 mM imidazole, 70 mM KCl, 80 mM sucrose, 1 mM EGTA, 2 mM
MgCl.sub.2, 1 mM DTT, 1 mM KCN, 1 mM ATP, 0.1% fatty acid free
bovine serum albumin, 70 .mu.M palmitoyl-CoA, 0.25 .mu.Ci
[methyl-.sup.14C]L-carnitine, 40 .mu.g digitonin with or without 20
.mu.M malonyl-CoA. After incubation for 6 minutes at 37.degree. C.,
the reaction was stopped by the addition of 500 .mu.l of ice-cold 4
M perchloric acid. Cells were then harvested and centrifuged at
13,000.times.g for 5 min. The pellet was washed with 500 .mu.l ice
cold 2 mM perchloric acid and centrifuged again. The resulting
pellet was resuspended in 800 .mu.l dH.sub.2O and extracted with
150 .mu.l of butanol. The butanol phase was counted by liquid
scintillation and represents the acylcarnitine derivative.
[0053] MCF-7 cells were treated with C75 at 10 or 20 .mu.g/mL for 1
hr before CPT-1 activity was assayed. The assay was performed with
the C75 and malonyl-CoA concentrations indicated ("M" indicates
malonyl-CoA at 20 uM). Malonyl-CoA treatment alone caused a 46%
reduction in CPT-1 activity similar to the previous experiment (see
FIG. 2; p=0.02, two-tailed t-test, Prism 3.0). The level of
malonyl-CoA inhibition of the CPT-1 activity is consistent with the
activity of the liver isoform of CPT-1 in MCF-7 cells. The K.sub.i
of malonyl-CoA for the liver isoform of CPT-1 is .about.7 .mu.M
while the K.sub.i for the muscle isoform of CPT-1 is 0.07 .mu.M.
Thus, MCF-7 cells express predominantly the liver isoform of CPT-1
(consistent with the immunoblot analysis).
[0054] There was no statistically significant difference in CPT-1
activity between cells treated with C75 or C75 and malonyl-CoA
(FIG. 2). Thus, in the presence of C75, malonyl-CoA lost its
inhibitory effect on CPT-1; conversely, C75 stimulation of CPT-1
occurred regardless of the presence of malonyl-CoA. Thus, in the
presence of C75, the normal inhibitory activity of malonyl-CoA is
lost. Malonyl-CoA inhibition of CPT-1 activity demonstrated that
C75 and related compounds were activating CPT-1 rather than CPT-2
activity which is not inhibitable by malonyl-CoA.
[0055] In a subsequent experiment (data in FIG. 3), MCF-7 cells
were untreated (left bar) or treated with C75 at 20 .mu.g/ml for
one hour before CPT-1 activity was measured (middle and right
bars). During the 6 minutes of the CPT-1 assay, C75 was removed
from the assay buffer and replaced with buffer (middle bar) or
malonyl-CoA 20 .mu.M was added (left & right bars). Malonyl-CoA
treatment alone during the assay resulted in a .about.70%
inhibition of CPT-1 activity (left bar) (p=0.0045, two-tailed
t-test, Prism 3.0). Prior C75 treatment with no C75 in the assay
buffer resulted in CPT-1 activity of 158% of control (p=0.028,
two-tailed t-test, Prism 3.0), similar to the results when C75 is
kept in the assay buffer (see above experiment). However, when C75
is removed from the reaction buffer and malonyl-CoA is replaced,
C75 stimulatory activity is lost (right bar). Thus, C-75 does not
detectably bind covalently to CPT-1, and it is likely a competitive
antagonist with malonyl-CoA. These data also suggest that C-75
interacts with CPT-1 at the malonyl-CoA binding site.
Example 3
Structure of Effective CPT-1 Stimulators
[0056] Analogs of .alpha.-methylene-.gamma.-butyrolactones
differing only in the length of their saturated carbon `tail` were
prepared as described in U.S. Pat. No. 5,981,575, incorporated
herein by reference. C75 has an eight-carbon tail, C12 and C16 have
tails of 12 and 16 carbons respectively. Cells were treated with
C75 and C75 analogs at 20 .mu.g/ml 1 hr before CPT-1 activity was
measured. Malonyl-CoA was added only to the reaction buffer since
the whole cell is impermeable to malonyl-CoA. C75 stimulated CPT-1
activity to 166% of control at a dose of 20 .mu.g/ml (see FIG. 4;
p=0.0092, two-tailed t-test, Prism 3.0). C12 analog stimulated to
186% (p=0.0099, two-tailed t-test, Prism 3.0) and C16 analog
stimulated to 138% of control (p=0.055, two-tailed t-test, Prism
3.0). Malonyl-CoA, an intracellular competitive inhibitor of CPT-1,
reduced CPT-1 activity to 64% of control at 20 .mu.M (p=0.023,
two-tailed t-test, Prism 3.0). The optimum carbon chain length for
CPT-1 activation is between 6 and 16 carbons.
Example 4
Enhanced Fatty Acid Oxidation from CPT-1 Stimulation Produces
ATP
[0057] As a consequence of increased fatty acid oxidation, ATP was
elevated in MCF-7 cells following C75 treatment. 1.times.10.sup.5
MCF-7 cells were plated in 96 well plates. Cells were treated with
C75 or vehicle. After 2 hours, ATP was measured using a luciferase
assay using the ATP Bioluminescence Kit CLS II (Roche) following
the manufacturer's protocol. Plates were read by a Perkin Elmer
Wallac Victor.sup.2 1420 luminometer. C75 treatment at 10 .mu.g/ml
and 20 .mu.g/ml both resulted in a significant increase in total
cellular ATP (see FIG. 5; p=0.0001; p<0.0001 compared to
control, two-tailed t-test, Prism 3.0). Similar results were
obtained after 1 hr incubation with C75. Thus, cellular energy
production increased as a result of C75 increasing fatty acid
oxidation.
Example 5
C75 Stimulates Activity of Muscle Form CPT-1
[0058] To expand the study of effects of C75 on fatty acid
metabolism beyond cancer cell lines to normal adipocytes,
differentiated (non-transformed) mouse NIH 3T3-L1 adipocytes were
used in assays similar to those performed with the MCF-7 cells.
3T3-L1 cells were obtained from the American Type Culture
Collection, and cells were cultured in DMEM with high glucose (4.5
g/l) (Gibco 12100-046) with 10% fetal calf serum and Biotin (Sigma
B-4639) 0.008 g/L. Differentiation was initiated three days after
the cells were confluent, when the standard culture medium was
replaced with differentiation medium. The differentiation medium
contained standard culture medium to which the following were added
to achieve the final concentrations: methylisobutylxanthine (MIX)
0.5 mM, dexamethasone (DEX) 1 .mu.M, and insulin 10 .mu.g/ml. After
48 hrs, the differentiation medium was replaced with
post-differentiation medium which contained insulin at the above
concentration, without MIX and DEX. The differentiated cells were
ready to be used for experiments in 7-10 days.
[0059] C75 increased CPT-1 activity and fatty acid metabolism in
the NIH 3T3-L1 cells differentiated into adipocytes. One week post
differentiation, cells were treated with either vehicle control or
C75 for 2 hours at doses indicated below. CPT-1 activity, fatty
acid oxidation, and total cellular ATP were measured as described
in Examples 2, 1, and 4. C75 treatment of 3T3-L1 adipocytes led to
a 377% increase in CPT-1 activity above control (p<0.0001,
two-tailed t-test, Prism 3.0). As a consequence of increased CPT-1
activity, C75 at doses of 20 .mu.g/ml or greater, significantly
increased fatty acid oxidation (see FIG. 6; 20 .mu.g/ml, p=0.007;
<20 .mu.g/ml, p<0.0001; two-tailed t-test, Prism 3.0).
Moreover, the increase in fatty acid oxidation led to significantly
increased levels of ATP at C75 doses of 20 .mu.g/ml or greater (see
FIG. 7; 20 .mu.g/ml, p=0.05; 30 .mu.g/ml, p<0.01; 40 .mu.g/ml,
p<0.0001; two-tailed t-test, Prism 3.0). The enhanced fatty acid
oxidation induced by C75 is likely responsible for the marked
reduction in adipose tissue mass seen with C75 administration in
vivo.
Example 6
In Vivo Stimulation of CPT-1 Shifts Metabolism to Fatty Acid
Oxidation
[0060] In keeping with the C75 effect on both human and murine
CPT-1 and fatty acid metabolism, C75 induces a profound and rapid
stimulation of fatty acid oxidation in vivo. Mice were maintained
in the Oxymax calorimeter (Oxymax Equal Flow System.RTM., Columbus
Instruments, Columbus, Ohio). Oxygen consumption and CO.sub.2
production was measured in up to four mice simultaneously using the
indirect calorimeter. Measurements were recorded every 15 minutes
over the entire course of the experiments. The respiratory exchange
ratio (RER) was calculated by the Oxymax.RTM. software version 5.9.
RER is defined as the ratio of CO.sub.2 to O.sub.2 at any given
time irrespective if equilibrium was reached. Mice were maintained
on water and mouse chow ad libitum. In the control mice (FIG. 8A),
note the diurnal variation of RER indicating feeding and resting
cycles of the animals. An RER of 1 is consistent with oxidation of
carbohydrates while 0.7 indicates oxidation of fatty acids. Mice
treated with C75 and maintained in the Oxymax calorimeter showed a
rapid decrease in the respiratory exchange ratio (RER) to
.about.0.7 (FIG. 8B). C75 treatment at 30 mg/kg disrupts the
diurnal pattern of the control mice, showing a rapid drop in RER to
complete oxidation of fatty acids within about 2 hours. Similarly,
C75 treatment at 20 mg/kg shows a similar rate of drop of RER but
without the prolonged effect (FIG. 8C). Importantly, the rate of
decline of RER was similar to that observed for animals deprived of
food (data not shown).
[0061] Despite the elevated levels of malonyl-CoA generated by C75
in vivo, C75 treatment led to a rapid, profound increase in fatty
acid oxidation as measured by RER. Thus, C75 treated animals are
able to significantly reduce adipose mass and reverse fatty liver,
by allowing fatty acid oxidation to occur despite the high levels
of malonyl-CoA generated during FAS inhibition in vivo.
Example 7
Enzyme Effector Activity of Various Substituted
Gamma-Butyrolactones
[0062] Various gamma-butyrolactone analogs were prepared and tested
for their effect on FAS activity, CPT-1 activity and fatty acid
oxidation. The compounds included C-75, a
gamma-substituted-alpha-methylene-butyrola- ctone synthesized as
described in U.S. Pat. No. 5,981,575, FAS231 and FAS65,
gamma-substituted-alpha-methylene-beta-amido-butyrolactones,
synthesized as described in International Patent Publication WO
2004/006835, FAS115, a 5,5-disubstituted thiotetronic ethyl
carbonate synthesized as described in International Patent
Publication WO 2004/005277, and FAS89B, an 3,3,5,5-tetrasubstitued
thiophene dione, synthesized as described in U.S. Provisional
Patent Application 60/574,639. These compounds were assayed for
inhibitory effect on FAS or stimulatory effect on CPT-1 and fatty
acid oxidation, and the results of the assays are shown in the
following table.
[0063] FAS activity was measured by monitoring the malonyl-CoA
dependent oxidation of NADPH spectrophotometrically at OD.sub.340
in 96-well plates, as described in International Patent Publication
WO 2004/005277. The IC.sub.50 for the compounds against FAS was
determined by plotting the change in OD.sub.340 against time for
each inhibitor concentration tested and determining the rate of
change by linear regression. The concentration of a particular
compound yielding 50% inhibition of the rate for FAS in the absence
of the compound is the IC.sub.50. Stimulation of CPT-1 activity was
measured as described in Example 2, except the cells were
preincubated with the compound for 2 hours at the concentrations
indicated in the table. Stimulation of Fatty acid oxidation was
determined as described in Example 1 for cells preincubated with
the compounds at the concentrations indicated in the table.
1TABLE Effect of Selected Compounds on Enzyme Activities FAS
Inhibition (IC.sub.50) CPT-1 Stimulation Fatty Acid Oxidation
Compound .mu.g/mL .mu.g/mL (% Control) .mu.g/mL (% Control) C75 55
20 125 10 400 3 4.6 20 150 10 140 FAS 115 4 47.9 10 400 0.625 400
FAS 231 5 52.1 80 500 40 500 FAS 65 6 n/a* 20 175 10 175 FAS 89B
*Slow binding assays not yet completed
[0064] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
claims. Modifications of the above-described modes for carrying out
the invention that are obvious to persons of skill in medicine,
immunology, hybridoma technology, pharmacology, and/or related
fields are intended to be within the scope of the following
claims.
[0065] 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 such 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.
* * * * *