U.S. patent application number 10/412694 was filed with the patent office on 2004-02-12 for combinations of cholesteryl ester transfer protein inhibitors and bile acid sequestering agents for cardiovascular indications.
This patent application is currently assigned to G.D. SEARLE, LLC. Invention is credited to Glenn, Kevin C., Sikorski, James A..
Application Number | 20040028644 10/412694 |
Document ID | / |
Family ID | 26811678 |
Filed Date | 2004-02-12 |
United States Patent
Application |
20040028644 |
Kind Code |
A1 |
Sikorski, James A. ; et
al. |
February 12, 2004 |
Combinations of cholesteryl ester transfer protein inhibitors and
bile acid sequestering agents for cardiovascular indications
Abstract
The present invention provides combinations of cardiovascular
therapeutic compounds for the prophylaxis or treatment of
cardiovascular disease including hypercholesterolemia and
atherosclerosis. Combinations disclosed include an cholesteryl
ester transfer protein inhibitor combined with a bile acid
sequestrant.
Inventors: |
Sikorski, James A.; (Des
Peres, MO) ; Glenn, Kevin C.; (Maryland Heights,
MO) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Assignee: |
G.D. SEARLE, LLC
Chicago
IL
|
Family ID: |
26811678 |
Appl. No.: |
10/412694 |
Filed: |
April 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10412694 |
Apr 14, 2003 |
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09466469 |
Dec 17, 1999 |
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6569905 |
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60142682 |
Jul 7, 1999 |
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60113955 |
Dec 23, 1998 |
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Current U.S.
Class: |
424/78.11 |
Current CPC
Class: |
A61K 31/785 20130101;
A61K 31/785 20130101; A61K 45/06 20130101; A61P 9/10 20180101; A61P
9/00 20180101; A61K 2300/00 20130101; A61P 3/06 20180101 |
Class at
Publication: |
424/78.11 |
International
Class: |
A61K 031/74 |
Claims
What is claimed is:
1. A-therapeutic combination comprising a first amount of an
cholesteryl ester transfer protein inhibiting compound and a second
amount of a bile acid sequestering compound wherein the first
amount and the second amount together comprise an
anti-hyperlipidemic condition effective amount, an
anti-atherosclerotic condition effective amount, or an
anti-hypercholesterolemic condition effective amount of the
compounds.
2. The therapeutic combination of claim 1 wherein the bile acid
sequestering compound comprises cholestyramine.
3. The therapeutic combination of claim 1 wherein the bile acid
sequestering compound comprises colestipol.
4. The therapeutic combination of claim 1 wherein the bile acid
sequestering compound comprises an amphiphilic copolymer having a
crosslinked shell domain and an interior core domain.
5. The therapeutic combination of claim 1 wherein the bile acid
sequestering compound comprises a polyallylamine polymer.
6. The therapeutic combination of claim 6 wherein the
polyallylamine polymer comprises CholestaGel.
7. The therapeutic combination of claim 6 wherein the
polyallylamine polymer comprises OmegaGel.
8. The therapeutic combination of claim 1 wherein the combination
comprises a composition comprising a cholesteryl ester transfer
protein inhibiting compound and a bile acid sequestering
compound.
9. A method for the prophylaxis or treatment of a hyperlipidemic
condition comprising administering to a patient in need thereof a
combination in unit dosage form wherein the combination comprises a
first amount of a cholesteryl ester transfer protein inhibiting
compound and a second amount of a bile acid sequestering compound
wherein the first amount and the second amount together comprise an
anti-hyperlipidemic condition effective amount of the
compounds.
10. A method for the prophylaxis or treatment of an atherosclerotic
condition comprising administering to a patient in need thereof a
combination in unit dosage form wherein the combination comprises a
first amount of a cholesteryl ester transfer protein inhibiting
compound and a second amount of a bile acid sequestering compound
wherein the first amount and the second amount together comprise an
anti-atherosclerotic condition effective amount of the
compounds.
11. A method for the prophylaxis or treatment of
hypercholesterolemia comprising administering to a patient in need
thereof a combination in unit dosage form wherein the combination
comprises a first amount of a cholesteryl ester transfer protein
inhibiting compound and a second amount of a bile acid sequestering
compound wherein the first amount and the second amount together
comprise an anti-hypercholesterolemic condition effective amount of
the compounds.
Description
[0001] This application claims priority of U.S. provisional
application Ser. No. 60/142,682 filed Jul. 7, 1999 and of U.S.
provisional application Ser. No. 60/113,955 filed Dec. 23,
1998.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to methods of treating
cardiovascular diseases, and specifically relates to combinations
of compounds, compositions, and methods for their use in medicine,
particularly in the prophylaxis and treatment of hyperlipidemic
conditions such as are associated with atherosclerosis,
hypercholesterolemia, and other coronary artery disease in mammals.
More particularly, the invention relates to cholesteryl ester
transfer protein (CETP) activity inhibiting compounds. The
invention also relates to bile acid sequestering compounds.
[0004] 2. Description of Related Art
[0005] It is well-settled that hyperlipidemic conditions associated
with elevated concentrations of total cholesterol and low-density
lipoprotein (LDL) cholesterol are major risk factors for coronary
heart disease and particularly atherosclerosis. Numerous studies
have demonstrated that a low plasma concentration of high density
lipoprotein (HDL) cholesterol is a powerful risk factor for the
development of atherosclerosis (Barter and Rye, Atherosclerosis,
121, 1-12 (1996). HDL is one of the major classes of lipoproteins
that function in the transport of lipids through the blood. The
major lipids found associated with HDL include cholesterol,
cholesteryl ester, triglycerides, phospholipids and fatty acids.
The other classes of lipoproteins found in the blood are low
density lipoprotein (LDL), intermediate density lipoprotein (IDL),
and very low density lipoprotein (VLDL). Since low levels of HDL
cholesterol increase the risk of atherosclerosis, methods for
elevating plasma HDL cholesterol would be therapeutically
beneficial for the treatment of atherosclerosis and other diseases
associated with accumulation of lipid in the blood vessels. These
diseases include, but are not limited to, coronary heart disease,
peripheral vascular disease, and stroke.
[0006] Atherosclerosis underlies most coronary artery disease
(CAD), a major cause of morbidity and mortality in modern society.
High LDL cholesterol (above about 180 mg/dl) and low HDL
cholesterol (below 35 mg/dl) have been shown to be important
contributors to the development of atherosclerosis. Other diseases
or risk factors, such as peripheral vascular disease, stroke, and
hypercholesterolaemia are negatively affected by adverse HDL/LDL
ratios.
[0007] Interfering with the recirculation of bile acids from the
lumen of the intestinal tract is found to reduce the levels of
serum cholesterol in a causal relationship. Epidemiological data
has accumulated which indicates such reduction leads to an
improvement in the disease state of atherosclerosis. Stedronsky, in
"Interaction of bile acids and cholesterol with nonsystemic agents
having hypocholesterolemic properties," Biochimica et Biophysica
Acta, 1210, 255-287 (1994) discusses the biochemistry, physiology
and known active agents surrounding bile acids and cholesterol.
[0008] Inhibition of cholesteryl ester transfer protein (CETP) has
been shown to effectively modify plasma HDL/LDL ratios, and is
expected to check the progress and/or formation of certain
cardiovascular diseases. CETP is a plasma protein that facilitates
the movement of cholesteryl esters and triglycerides between the
various lipoproteins in the blood (Tall, J. Lipid Res., 34, 1255-74
(1993)). The movement of cholesteryl ester from HDL to LDL by CETP
has the effect of lowering HDL cholesterol. It therefore follows
that inhibition of CETP should lead to elevation of plasma HDL
cholesterol and lowering of plasma LDL cholesterol, thereby
providing a therapeutically beneficial plasma lipid profile.
Evidence of this effect is described in McCarthy, Medicinal Res.
Revs., 13, 139-59 (1993). Further evidence of this effect is
described in Sitori, Pharmac. Ther., 67, 443-47 (1995)). This
phenomenon was first demonstrated by Swenson et al., (J. Biol.
Chem., 264, 14318 (1989)) with the use of a monoclonal antibody
that specifically inhibits CETP. In rabbits, the antibody caused an
elevation of the plasma HDL cholesterol and a decrease in LDL
cholesterol. Son et al. (Biochim. Biophys. Acta, 795, 743-480
(1984)) describe proteins from human plasma that inhibit CETP. U.S.
Pat. No. 5,519,001, herein incorporated by reference, issued to
Kushwaha et al., describes a 36 amino acid peptide derived from
baboon apo C-1 that inhibits CETP activity. Cho et al. (Biochim.
Biophys. Acta 1391, 133-144 (1998)) describe a peptide from hog
plasma that inhibits human CETP. Bonin et al. (J. Peptide Res., 51,
216-225 (1998)) disclose a decapeptide inhibitor of CETP. A
depspeptide fungal metabolite is disclosed as a CETP inhibitor by
Hedge et al. in Bioorg. Med. Chem. Lett., 8, 1277-80 (1998).
[0009] There have been several reports of non-peptidic compounds
that act as CETP inhibitors. Barrett et al. (J. Am. Chem. Soc.,
188, 7863-63 (1996)) describe cyclopropane-containing CETP
inhibitors. Further cyclopropane-containing CETP inhibitors are
described by 4Kuo et al. (J. Am. Chem. Soc., 117, 10629-34 (1995)).
Pietzonka et al. (Bioorg. Med. Chem. Lett., 6, 1951-54 (1996))
describe phosphonate-containing analogs of cholesteryl ester as
CETP inhibitors. Coval et al. (Bioorg. Med. Chem. Lett., 5, 605-610
(1995)) describe Wiedendiol-A and -B, and related sesquiterpene
compounds as CETP inhibitors. Lee et al. (J. Antibiotics, 49,
693-96 (1996)) describe CETP inhibitors derived from an insect
fungus. Busch et al. (Lipids, 25, 216-220, (1990)) describe
cholesteryl acetyl bromide as a CETP inhibitor. Morton and
Zilversmit (J. Lipid Res., 35, 836-47 (1982)) describe that
p-chloromercuriphenyl sulfonate, p-hydroxymercuribenzoate and ethyl
mercurithiosalicylate inhibit CETP. Connolly et al. (Biochem.
Biophys. Res. Comm., 223, 42-47 (1996)) describe other cysteine
modification reagents as CETP inhibitors. Xia et al. describe
1,3,5-triazines as CETP inhibitors (Bioorg. Med. Chem. Lett., 6,
919-22 (1996)). Bisgaier et al. (Lipids, 29, 811-8 (1994)) describe
4-phenyl-5-tridecyl-4H-1,2,4-triazole-thiol as a CETP inhibitor.
Additional triazole CETP inhibitors are described in U.S. patent
application Ser. No. 09/153,360, herein incorporated by reference.
Sikorski et al. disclosed further novel CETP inhibitors in PCT
Patent Application No. WO 9914204.
[0010] Substituted 2-mercaptoaniline amide compounds can be used as
CETP inhibitors and such therapeutic compounds are described by H.
Shinkai et al. in PCT Patent Application No. WO 98/35937.
[0011] Some substituted heteroalkylamine compounds are known as
CETP inhibitors. In European Patent Application No. 796846, Schmidt
et al. describe 2-aryl-substituted pyridines as cholesterol ester
transfer protein inhibitors useful as cardiovascular agents. One
substituent at C.sub.3 of the pyridine ring can be an hydroxyalkyl
group. In European Patent Application No. 801060, Dow and Wright
describe heterocyclic derivatives substituted with an aldehyde
addition product of an alkylamine to afford 1-hydroxy-1-amines.
These are reported to be .beta.3-adrenergic receptor agonists
useful for treating diabetes and other disorders. In Great Britain
Patent Application No. 2305665, Fisher et al. disclose 3-agonist
secondary amino alcohol substituted pyridine derivatives useful for
treating several disorders including cholesterol levels and
atherosclerotic diseases. In European Patent Application No. 818448
(herein incorporated by reference), Schmidt et al. describe
tetrahydroquinoline derivatives as cholesterol ester transfer
protein inhibitors. European Patent Application No. 818197, Schmek
et al. describe pyridines with fused heterocycles as cholesterol
ester transfer protein inhibitors. Brandes et al. in German Patent
Application No. 19627430 describe bicyclic condensed pyridine
derivatives as cholesterol ester transfer protein inhibitors. In
PCT Patent Application No. WO 9839299, Muller-Gliemann et al.
describe quinoline derivatives as cholesteryl ester transfer
protein inhibitors.
[0012] Polycyclic compounds that are useful as CETP inhibitors are
also disclosed by A. Oomura et al. in Japanese Patent No. 10287662.
For example, therapeutic compounds having the structures C-1 and
C-8 were prepared by culturing Penicillium spp.
[0013] Cycloalkylpyridines useful as CETP inhibitors are disclosed
by Schmidt et al. in European Patent No. EP 818448. For example,
the therapeutic compound having the structure C-9 is disclosed as
being particularly effective as a CETP inhibitor.
[0014] Substituted tetrahydronaphthalene compounds useful as CETP
inhibitors are described in PCT Patent Application No. WO 9914174.
Specifically described in that disclosure as a useful CETP
inhibitor is
(8S)-3-cyclopentyl-1-(4-fluorophenyl)-2-[(S)-fluoro(4-trifluoromethylphen-
yl)
methyl]-8-hydroxy-6-spirocclobutyl-5,6,7,8-tetrahydronaphthalene.
[0015] Some 4-heteroaryl-tetrahydroquinolines useful as CETP
inhibitors are described in PCT Patent Application No. WO 9914215.
For example, that disclosure describes
3-(4-trifluoromethylbenzoyl)-5,6,7,8-tetrahydroquino- lin-5-one as
a useful CETP inhibitor.
[0016] Some combination therapies for the treatment of
cardiovascular disease have been described in the literature.
Combinations of IBAT inhibitors with HMG CoA reductase inhibitors
useful for the treatment of cardiovascular disease are disclosed in
U.S. patent application Ser. No. 09/037,308.
[0017] A combination therapy of fluvastatin and niceritrol is
described by J. Sasaki et al. (Id.). Those researchers conclude
that the combination of fluvastatin with niceritrol "at a dose of
750 mg/day dose does not appear to augment or attenuate beneficial
effects of fluvastatin."
[0018] L. Cashin-Hemphill et al. (J. Am. Med. Assoc., 264 (23),
3013-17 (1990)) describe beneficial effects of a combination
therapy of colestipol and niacin on coronary atherosclerosis. The
described effects include nonprogression and regression in native
coronary artery lesions.
[0019] A class of materials which operates by another mechanism to
lower LDL cholesterol comprises bile acid sequestering agents
("bile acid sequestrants" or "bile acid sequestering compounds").
Such agents are typically anion exchange polymers administered
orally to a patient. As the agent passes through the gut, anions of
bile acids are sequestered by the agent and excreted. Such
sequestering has been speculated to prevent reabsorption by the
gut, for example the ileum, causing the body to increase conversion
of cholesterol into bile acids, and thereby decreasing serum
cholesterol levels. One such bile acid sequestering agent is
cholestyramine, a styrene-divinylbenzene copolymer containing
quaternary ammonium cationic groups capable of binding bile acids.
It is believed that cholestyramine binds the bile acids in the
intestinal tract, thereby interfering with their normal
enterohepatic circulation. This effect is described by Reihnr et
al., in "Regulation of hepatic cholesterol metabolism in humans:
stimulatory effects of cholestyramine on HMG-CoA reductase activity
and low density lipoprotein receptor expression in gallstone
patients", Journal of Lipid Research, 31, 2219-2226 (1990). Further
description of this effect is found in Suckling et al. in
"Cholesterol Lowering and bile acid excretion in the hamster with
cholestyramine treatment", Atherosclerosis, 89, 183-90 (1991). This
results in an increase in liver bile acid synthesis because of the
liver using cholesterol as well as an upregulation of the liver LDL
receptors which enhances clearance of cholesterol and decreases
serum LDL cholesterol levels.
[0020] Another bile acid sequestering agent is colestipol, a
copolymer of diethylenetriamine and 1-chloro-2,3-epoxypropane.
Colestipol is described in U.S. Pat. No. 3,692,895. A frequent side
effect of colestipol and of cholestyramine is gastric distress.
[0021] Additional bile acid sequestering agents are described in
U.S. Pat. No. 5,703,188, assigned to Geltex Pharmaceuticals, Inc.
For example, one such bile acid sequestering agent is
3-methacrylamidopropyltrimethyl-ammo- nium chloride copolymerized
with ethylene glycol dimethacrylate to yield a copolymer.
[0022] Further bile acid sequestering agents are described in PCT
Patent Application No. WO 98/57652, assigned to Geltex
Pharmaceuticals, Inc. The WO 98/57652 application describes
polyallylamine polymers.
[0023] An example of a bile acid sequestering agent is CholestaGel,
CAS Registry No. 182815-44-7. CholestaGel is
N,N,N-trimethyl-6-(2-propenylami- no)-1-hexanaminium chloride
polymer with (chloromethyl)oxirane, 2-propen-1-amine and
N-2-propenyl-1-decanamine hydrochloride.
[0024] Yet another class materials proposed as bile acid
sequestering agents comprises particles comprising amphiphilic
copolymers having a crosslinked shell domain and an interior core
domain (Patent application no. PCT/US 97/11610). Structures and
preparation of such crosslinked amphiphilic copolymers are
described in PCT/US97/11345. Such particles have been given the
common name of "knedels" (K. B. Thurmond et al., J. Am. Chem. Soc.,
118 (30), 7239-40 (1996)).
[0025] Some combination therapies for the treatment of
cardiovascular disease have been described in the literature.
Combinations of IBAT inhibitors with HMG CoA reductase inhibitors
useful for the treatment of cardiovascular disease are disclosed in
U.S. patent application Ser. No. 09/037,308, herein incorporated by
reference.
[0026] A combination therapy of fluvastatin and niceritrol is
described by J. Sasaki et al. (Id.). Those researchers conclude
that the combination of fluvastatin with niceritrol "at a dose of
750 mg/day dose does not appear to augment or attenuate beneficial
effects of fluvastatin."
[0027] L. Cashin-Hemphill et al. (J. Am. Med. Assoc., 264 (23),
3013-17 (1990), herein incorporated by reference) describe
beneficial effects of a combination therapy of colestipol and
niacin on coronary atherosclerosis. The described effects include
nonprogression and regression in native coronary artery
lesions.
[0028] A combination therapy of acipimox and simvastatin shows
beneficial HDL effects in patients having high triglyceride levels
(N. Hoogerbrugge et al., J. Internal Med., 241, 151-55 (1997),
herein incorporated by reference).
[0029] Sitostanol ester margarine and pravastatin combination
therapy is described by H. Gylling et al. (J. Lipid Res., 37,
1776-85 (1996), herein incorporated by reference). That therapy is
reported to simultaneously inhibit cholesterol absorption and lower
LDL cholesterol significantly in non-insulin-dependent diabetic
men.
[0030] Brown et al. (New Eng. J. Med., 323 (19), 1289-1339 (1990),
herein incorporated by reference) describe a combination therapy of
lovastatin and colestipol which reduces atherosclerotic lesion
progression and increase lesion regression relative to lovastatin
alone.
[0031] A combination therapy of an apoB secretion inhibitor with a
CETP inhibitor was disclosed by Chang et al. in PCT Patent
Application No. WO9823593, herein incorporated by reference.
[0032] Buch et al. (PCT Patent Application No. WO 9911263, herein
incorporated by reference) describe a combination therapy
comprising amlodipine and a statin compound for treating subjects
suffering from angina pectoris, atherosclerosis, combined
hypertension and hyperlipidemia, and to treat symptoms of cardiac
arrest. Buch et al. describe in PCT Patent Application No. WO
9911259 a combination therapy comprising amlodipine and
atorvastatin.
[0033] Scott et al. (PCT Patent Application No. WO 9911260)
describe a combination therapy comprising atorvastatin and an
antihypertensive agent.
[0034] Dettmar and Gibson (UK Patent Application No. GB 2329334 A)
claim a therapeutic composition useful for reducing plasma low
density lipoprotein and cholesterol levels, wherein the composition
comprises an HMG CoA reductase inhibitor and a bile complexing
agent.
[0035] The above references show continuing need to find safe,
effective agents for the prophylaxis or treatment of cardiovascular
diseases.
SUMMARY OF THE INVENTION
[0036] To address the continuing need to find safe and effective
agents for the prophylaxis and treatment of cardiovascular
diseases, combination therapies of cardiovascular drugs are now
reported.
[0037] Among its several embodiments, the present invention
provides a combination therapy comprising the use of a first amount
of an CETP inhibitor and a second amount of another cardiovascular
therapeutic useful in the prophylaxis or treatment of
hyperlipidemia, atherosclerosis, or hypercholesterolemia, wherein
said first and second amounts together comprise an
anti-hyperlipidemic condition effective amount, an
anti-atherosclerotic condition effective amount, or an
anti-hypercholesterolemic condition effective amount of the
compounds. For example one of the many embodiments of the present
invention is a combination therapy comprising therapeutic dosages
of an CETP inhibitor and a bile acid sequestering agent.
[0038] A further embodiment of the instant invention comprises the
use of any of the cardiovascular combination therapies described
herein for the prophylaxis or treatment of hypercholesterolemia,
atherosclerosis, or hyperlipidemia. Therefore, in one embodiment
the present invention provides a method for the prophylaxis or
treatment of a hyperlipidemic condition comprising administering to
a patient in need thereof a combination in unit dosage form wherein
the combination comprises a first amount of an bile acid
sequestering compound and a second amount of a CETP inhibiting
compound wherein the first amount and the second amount together
comprise an anti-hyperlipidemic condition effective amount of the
compounds.
[0039] In another embodiment, the present invention provides a
method for the prophylaxis or treatment of an atherosclerotic
condition comprising administering to a patient in need thereof a
combination in unit dosage form wherein the combination comprises a
first amount of an bile acid sequestering compound and a second
amount of a CETP inhibiting compound wherein the first amount and
the second amount together comprise an anti-atherosclerotic
condition effective amount of the compounds.
[0040] In still another embodiment, the present invention provides
method for the prophylaxis or treatment of hypercholesterolemia
comprising administering to a patient in need thereof a combination
in unit dosage form wherein the combination comprises a first
amount of an bile acid sequestering compound and a second amount of
a CETP inhibiting compound wherein the first amount and the second
amount together comprise an anti-hypercholesterolemic condition
effective amount of the compounds.
[0041] Further scope of the applicability of the present invention
will become apparent from the detailed description provided below.
However, it should be understood that the following detailed
description and examples, while indicating preferred embodiments of
the invention, are given by way of illustration only since various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from
this detailed description.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0042] The following detailed description is provided to aid those
skilled in the art in practicing the present invention. Even so,
this detailed description should not be construed to unduly limit
the present invention as modifications and variations in the
embodiments discussed herein can be made by those of ordinary skill
in the art without departing from the spirit or scope of the
present inventive discovery.
[0043] The contents of each of the references cited herein,
including the contents of the references cited within these primary
references, are herein incorporated by reference in their
entirety.
[0044] a. Definitions
[0045] The following definitions are provided in order to aid the
reader in understanding the detailed description of the present
invention:
[0046] "Combination therapy" means the administration of two or
more therapeutic agents to treat a hyperlipidemic condition, for
example atherosclerosis and hypercholesterolemia. Such
administration encompasses co-administration of these therapeutic
agents in a substantially simultaneous manner, such as in a single
capsule having a fixed ratio of active ingredients or in multiple,
separate capsules for each inhibitor agent. In addition, such
administration also encompasses use of each type of therapeutic
agent in a sequential manner. In either case, the treatment regimen
will provide beneficial effects of the drug combination in treating
the hyperlipidemic condition.
[0047] The phrase "therapeutically effective" is intended to
qualify the combined amount of inhibitors in the combination
therapy. This combined amount will achieve the goal of reducing or
eliminating the hyperlipidemic condition.
[0048] "Therapeutic compound" means a compound useful in the
prophylaxis or treatment of a hyperlipidemic condition, including
atherosclerosis and hypercholesterolemia.
[0049] b. Combinations
[0050] The combinations of the present invention will have a number
of uses. For example, through dosage adjustment and medical
monitoring, the individual dosages of the therapeutic compounds
used in the combinations of the present invention will be lower
than are typical for dosages of the therapeutic compounds when used
in monotherapy. The dosage lowering will provide advantages
including reduction of side effects of the individual therapeutic
compounds when compared to the monotherapy. In addition, fewer side
effects of the combination therapy compared with the monotherapies
will lead to greater patient compliance with therapy regimens.
[0051] Another use of the present invention will be in combinations
having complementary effects or complementary modes of action. For
example, bile acid sequestering agents control blood serum
cholesterol levels by inhibiting the reabsorption of bile acids in
the ileum. In contrast, CETP inhibitors inhibit the movement of
cholesteryl esters and triglycerides between the various
lipoproteins in the blood.
[0052] Compounds useful in the present invention encompass a wide
range of therapeutic compounds. Some individual CETP Inhibitor
compounds useful in the present invention are separately described
in the following individual patent applications, each of which is
herein incorporated by reference.
[0053] R9. U.S. Patent Application Serial No. 60/101,661.
[0054] R10. U.S. Patent Application Serial No. 60/101,711.
[0055] R11. U.S. Patent Application Serial No. 60/101,660.
[0056] R12. U.S. Patent Application Serial No. 60/101,664.
[0057] R13. U.S. Patent Application Serial No. 60/101,668.
[0058] R14. U.S. Patent Application Serial No. 60/101,662.
[0059] R15. U.S. Patent Application Serial No. 60/101,663.
[0060] R16. U.S. Patent Application Serial No. 60/101,669.
[0061] R17. U.S. Patent Application Serial No. 60/101,667.
[0062] R18. U.S. patent application Ser. No. 09/401,916.
[0063] R19. U.S. patent application Ser. No. 09/405,524.
[0064] R20. U.S. patent application Ser. No. 09/404,638.
[0065] R21. U.S. patent application Ser. No. 09/404,638.
[0066] R22. U.S. patent application Ser. No. 09/400,915.
[0067] R23. U.S. Pat. No. 5,932,587.
[0068] R24. U.S. Pat. No. 5,925,645.
[0069] CETP inhibitor compounds of particular interest in the
present invention include those shown in Table 1, as well as the
diastereomers, enantiomers, racemates, salts, and tautomers of the
CETP inhibitors of Table 1.
1TABLE 1 Compound Number Structure C-1 1 C-2 2 C-3 3 C-4 4 C-5 5
C-6 6 C-7 7 C-8 8 C-9 9 C-10 10 C-11 11 C-12 12 C-13 13 C-14 14
C-15 15 C-16 16 C-17 17 C-18 18 C-19 19 C-20 20
[0070] Bile acid sequestrants useful in the combinations and
methods of the present invention comprise a wide variety of
structures and functionalities. Preferred bile acid sequestrants
for the present invention are described in Table 2. The therapeutic
compounds of Table 2 can be used in the present invention in a
variety of forms, including acid form, salt form, racemates,
enantiomers, zwitterions, and tautomers. The individual patent
documents referenced in Table 2 are each herein incorporated by
reference. Additional bile acid sequestrants useful herein are
particles comprising amphiphilic copolymers having a crosslinked
shell domain and an interior core domain (knedels, Patent
application No. PCT/US 97/11610, herein incorporated by reference).
Knedels of particular interest in the present invention comprise
polystyrene-b-poly(acrylic acid) (PS-b-PAA) crosslinked with one or
more polyamine. Especially preferred knedels comprise PS-b-PAA
crosslinked with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
methiodide and triethylenetetramine ("knedel A") or
1,7-diaza-4,10-diazonium-4,4,10,10-t- etramethylundecane diiodide
("knedel P").
2TABLE 2 CAS Patent Compound Registry Document Number Common Name
Number Reference G-35 cholestyramine 11041-12-6 G-46 colestipol
50925-79-6 U.S. Pat. No. 3,692,895 S-1 knedel A PCT/US97/11345 S-2
knedel B PCT/US97/11345 S-3 3- U.S. Pat. No. 5,703,188
methacrylamido- propyltrimethyl- ammonium chloride copolymerized
with ethylene glycol dimethacrylate S-4 CholestaGel 152751-57-0 WO
98/57652 S-5 OmegaGel WO 98/57652
[0071] The compounds (for example, CETP inhibiting compounds or
bile acid sequestering compounds) useful in the present invention
can have no asymmetric carbon atoms, or, alternatively, the useful
compounds can have one or more asymmetric carbon atoms. When the
useful compounds have one or more asymmetric carbon atoms, they
therefore include racemates and stereoisomers, such as
diastereomers and enantiomers, in both pure form and in admixture.
Such stereoisomers can be prepared using conventional techniques,
either by reacting enantiomeric starting materials, or by
separating isomers of compounds of the present invention.
[0072] Isomers may include geometric isomers, for example
cis-isomers or trans-isomers across a double bond. All such isomers
are contemplated among the compounds useful in the present
invention.
[0073] The compounds useful in the present invention also include
tautomers.
[0074] The compounds useful in the present invention as discussed
below include their salts, solvates and prodrugs.
Dosages, Formulations, and Routes of Administration
[0075] The compositions of the present invention can be
administered for the prophylaxis and treatment of hyperlipidemic
diseases or conditions by any means, preferably oral, that produce
contact of these compounds with their site of action in the body,
for example in the ileum, plasma, or liver of a mammal, e.g., a
human.
[0076] For the prophylaxis or treatment of the conditions referred
to above, the compounds useful in the compositions and methods of
the present invention can be used as the compound per se.
Pharmaceutically acceptable salts are particularly suitable for
medical applications because of their greater aqueous solubility
relative to the parent compound. Such salts must clearly have a
pharmaceutically acceptable anion or cation. Suitable
pharmaceutically acceptable acid addition salts of the compounds of
the present invention when possible include those derived from
inorganic acids, such as hydrochloric, hydrobromic, phosphoric,
metaphosphoric, nitric, sulfonic, and sulfuric acids, and organic
acids such as acetic, benzenesulfonic, benzoic, citric,
ethanesulfonic, fumaric, gluconic, glycolic, isothionic, lactic,
lactobionic, maleic, malic, methanesulfonic, succinic,
toluenesulfonic, tartaric, and trifluoroacetic acids. The chloride
salt is particularly preferred for medical purposes. Suitable
pharmaceutically acceptable base salts include ammonium salts,
alkali metal salts such as sodium and potassium salts, and alkaline
earth salts such as magnesium and calcium salts.
[0077] The anions useful in the present invention are, of course,
also required to be pharmaceutically acceptable and are also
selected from the above list.
[0078] The compounds useful in the present invention can be
presented with an acceptable carrier in the form of a
pharmaceutical composition. The carrier must, of course, be
acceptable in the sense of being compatible with the other
ingredients of the composition and must not be deleterious to the
recipient. The carrier can be a solid or a liquid, or both, and is
preferably formulated with the compound as a unit-dose composition,
for example, a tablet, which can contain from 0.05% to 95% by
weight of the active compound. Other pharmacologically active
substances can also be present, including other compounds of the
present invention. The pharmaceutical compositions of the invention
can be prepared by any of the well known techniques of pharmacy,
consisting essentially of admixing the components.
[0079] Optionally, the combination of the present invention can
comprise a composition comprising an ileal bile acid transport
inhibiting compound and a bile acid sequestering compound. In such
a composition, the CETP inhibiting compound and the bile acid
sequestering compound can be present in a single dosage form, for
example a pill, a capsule, or a liquid which contains both of the
compounds.
[0080] These compounds can be administered by any conventional
means available for use in conjunction with pharmaceuticals, either
as individual therapeutic compounds or as a combination of
therapeutic compounds.
[0081] The amount of compound which is required to achieve the
desired biological effect will, of course, depend on a number of
factors such as the specific compound chosen, the use for which it
is intended, the mode of administration, and the clinical condition
of the recipient.
[0082] For a CETP inhibitor, a daily dose of about 0.01 to about
100 mg/kg body weight/day, and preferably between about 0.5 to
about 20 mg/kg body weight/day, may generally be appropriate.
[0083] For a bile acid sequestrant, a total daily dose can be in
the range of from about 1,000 to about 30,000 mg/day, and
preferably from about 5,000 to about 15,000 mg/day in a single or a
divided dose.
[0084] The daily doses described in the preceding paragraphs for
the various therapeutic compounds can be administered to the
patient in a single dose, or in proportionate multiple subdoses.
Subdoses can be administered 2 to 6 times per day. Doses can be in
sustained release form effective to obtain desired results.
[0085] In the case of pharmaceutically acceptable salts, the
weights indicated above refer to the weight of the acid equivalent
or the base equivalent of the therapeutic compound derived from the
salt.
[0086] Oral delivery of the combinations of the present invention
can include formulations, as are well known in the art, to provide
prolonged or sustained delivery of the drug to the gastrointestinal
tract by any number of mechanisms. These include, but are not
limited to, pH sensitive release from the dosage form based on the
changing pH of the small intestine, slow erosion of a tablet or
capsule, retention in the stomach based on the physical properties
of the formulation, bioadhesion of the dosage form to the mucosal
lining of the intestinal tract, or enzymatic release of the active
drug from the dosage form. For some of the therapeutic compounds
useful in the present invention the intended effect is to extend
the time period over which the active drug molecule is delivered to
the site of action by manipulation of the dosage form. Thus,
enteric-coated and enteric-coated controlled release formulations
are within the scope of the present invention. Suitable enteric
coatings include cellulose acetate phthalate, polyvinylacetate
phthalate, hydroxypropylmethylcellulose phthalate and anionic
polymers of methacrylic acid and methacrylic acid methyl ester. In
another embodiment, when a bile acid sequestrant is used in a
combination of the present invention, the bile acid sequestrant can
be provided in the form of a liquid, a solid dispersed in a liquid,
or in a capsule.
[0087] The combinations of the present invention can be delivered
orally either in a solid, in a semi-solid, or in a liquid form.
When in a liquid or in a semi-solid form, the combinations of the
present invention can, for example, be in the form of a liquid,
syrup, or contained in a gel capsule (e.g., a gel cap). In one
embodiment, when a CETP inhibitor is used in a combination of the
present invention, the CETP inhibitor can be provided in the form
of a liquid, syrup, or contained in a gel capsule.
[0088] For a CETP inhibitor the intravenously administered dose
can, for example, be in the range of from about 0.003 mg/kg body
weight to about 1.0 mg/kg body weight, preferably from about 0.01
mg/kg body weight to about 0.75 mg/kg body weight, more preferably
from about 0.1 mg/kg body weight to about 0.6 mg/kg body
weight.
[0089] The dose of any of these therapeutic compounds can be
conveniently administered as an infusion of from about 10 ng/kg
body weight to about 100 ng/kg body weight per minute. Infusion
fluids suitable for this purpose can contain, for example, from
about 0.1 ng to about 10 mg, preferably from about 1 ng to about 10
mg per milliliter. Unit doses can contain, for example, from about
1 mg to about 10 g of the compound of the present invention. Thus,
ampoules for injection can contain, for example, from about 1 mg to
about 100 mg.
[0090] Pharmaceutical compositions according to the present
invention include those suitable for oral, rectal, topical, buccal
(e.g., sublingual), and parenteral (e.g., subcutaneous,
intramuscular, intradermal, or intravenous) administration,
although the most suitable route in any given case will depend on
the nature and severity of the condition being treated and on the
nature of the particular compound which is being used. In most
cases, the preferred route of administration is oral. Typically a
bile acid sequestering agent is delivered orally.
[0091] Pharmaceutical compositions suitable for oral administration
can be presented in discrete units, such as capsules, cachets,
lozenges, or tablets, each containing a predetermined amount of at
least one therapeutic compound useful in the present invention; as
a powder or granules; as a solution or a suspension in an aqueous
or non-aqueous liquid; or as an oil-in-water or water-in-oil
emulsion. As indicated, such compositions can be prepared by any
suitable method of pharmacy which includes the step of bringing
into association the active compound(s) and the carrier (which can
constitute one or more accessory ingredients). In general, the
compositions are prepared by uniformly and intimately admixing the
active compound with a liquid or finely divided solid carrier, or
both, and then, if necessary, shaping the product. For example, a
tablet can be prepared by compressing or molding a powder or
granules of the compound, optionally with one or more assessory
ingredients. Compressed tablets can be prepared by compressing, in
a suitable machine, the compound in a free-flowing form, such as a
powder or granules optionally mixed with a binder, lubricant, inert
diluent and/or surface active/dispersing agent(s). Molded tablets
can be made by molding, in a suitable machine, the powdered
compound moistened with an inert liquid diluent.
[0092] Pharmaceutical compositions suitable for buccal (sublingual)
administration include lozenges comprising a compound of the
present invention in a flavored base, usually sucrose, and acacia
or tragacanth, and pastilles comprising the compound in an inert
base such as gelatin and glycerin or sucrose and acacia.
[0093] Pharmaceutical compositions suitable for parenteral
administration conveniently comprise sterile aqueous preparations
of a compound of the present invention. These preparations are
preferably administered intravenously, although administration can
also be effected by means of subcutaneous, intramuscular, or
intradermal injection. Such preparations can conveniently be
prepared by admixing the compound with water and rendering the
resulting solution sterile and isotonic with the blood. Injectable
compositions according to the invention will generally contain from
0.1 to 5% w/w of a compound disclosed herein.
[0094] Pharmaceutical compositions suitable for rectal
administration are preferably presented as unit-dose suppositories.
These can be prepared by admixing a compound of the present
invention with one or more conventional solid carriers, for
example, cocoa butter, and then shaping the resulting mixture.
[0095] Pharmaceutical compositions suitable for topical application
to the skin preferably take the form of an ointment, cream, lotion,
paste, gel, spray, aerosol, or oil. Carriers which can be used
include petroleum jelly (e.g., Vaseline), lanolin, polyethylene
glycols, alcohols, and combinations of two or more thereof. The
active compound is generally present at a concentration of from 0.1
to 50% w/w of the composition, for example, from 0.5 to 2%.
[0096] Transdermal administration is also possible. Pharmaceutical
compositions suitable for transdermal administration can be
presented as discrete patches adapted to remain in intimate contact
with the epidermis of the recipient for a prolonged period of time.
Such patches suitably contain a compound of the present invention
in an optionally buffered, aqueous solution, dissolved and/or
dispersed in an adhesive, or dispersed in a polymer. A suitable
concentration of the active compound is about 1% to 35%, preferably
about 3% to 15%. As one particular possibility, the compound can be
delivered from the patch by electrotransport or iontophoresis, for
example, as described in Pharmaceutical Research, 3(6), 318
(1986).
[0097] In any case, the amount of active ingredient that can be
combined with carrier materials to produce a single dosage form to
be administered will vary depending upon the host treated and the
particular mode of administration.
[0098] The solid dosage forms for oral administration including
capsules, tablets, pills, powders, gel caps, and granules noted
above comprise one or more compounds useful in the present
invention admixed with at least one inert diluent such as sucrose,
lactose, or starch. Such dosage forms may also comprise, as in
normal practice, additional substances other than inert diluents,
e.g., lubricating agents such as magnesium stearate or solubilizing
agents such as cyclodextrins. In the case of capsules, tablets,
powders, granules, gel caps, and pills, the dosage forms may also
comprise buffering agents. Tablets and pills can additionally be
prepared with enteric coatings.
[0099] Liquid dosage forms for oral administration can include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups, and elixirs containing inert diluents commonly used in the
art, such as water. Such compositions may also comprise adjuvants,
such as wetting agents, emulsifying and suspending agents, and
sweetening, flavoring, and perfuming agents.
[0100] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or setting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose any bland fixed oil may be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid find use in the preparation of injectables.
[0101] Pharmaceutically acceptable carriers encompass all the
foregoing and the like.
[0102] In combination therapy, administration of two or more of the
therapeutic agents useful in the present invention may take place
sequentially in separate formulations, or may be accomplished by
simultaneous administration in a single formulation or separate
formulations. Administration may be accomplished by oral route, or
by intravenous, intramuscular, or subcutaneous injections. The
formulation may be in the form of a bolus, or in the form of
aqueous or non-aqueous isotonic sterile injection solutions or
suspensions. These solutions and suspensions may be prepared from
sterile powders or granules having one or more
pharmaceutically-acceptable carriers or diluents, or a binder such
as gelatin or hydroxypropylmethyl cellulose, together with one or
more of a lubricant, preservative, surface active or dispersing
agent.
[0103] For oral administration, the pharmaceutical composition may
be in the form of, for example, a tablet, capsule, suspension, or
liquid. Capsules, tablets, etc., can be prepared by conventional
methods well known in the art. The pharmaceutical composition is
preferably made in the form of a dosage unit containing a
particular amount of the active ingredient or ingredients. Examples
of dosage units are tablets or capsules. These may with advantage
contain one or more therapeutic compound in an amount described
above. For example, in the case of a CETP inhibitor, the dose range
may be from about 0.01 mg to about 500 mg or any other dose,
dependent upon the specific inhibitor, as is known in the art. In
the case of an bile acid sequestrant the dose range can be from
about 1,000 mg to about 30,000 mg or any other dose, dependent upon
the specific bile acid sequestrant, as is known in the art.
[0104] The active ingredients may also be administered by injection
as a composition wherein, for example, saline, dextrose, or water
may be used as a suitable carrier. A suitable daily dose of each
active therapeutic compound is one that achieves the same blood
serum level as produced by oral administration as described
above.
[0105] The therapeutic compounds may further be administered by any
combination of oral/oral, oral/parenteral, or parenteral/parenteral
route.
[0106] Pharmaceutical compositions for use in the treatment methods
of the present invention may be administered in oral form or by
intravenous administration. Oral administration of the combination
therapy is preferred. Dosing for oral administration may be with a
regimen calling for single daily dose, or for a single dose every
other day, or for multiple, spaced doses throughout the day. The
therapeutic compounds which make up the combination therapy may be
administered simultaneously, either in a combined dosage form or in
separate dosage forms intended for substantially simultaneous oral
administration. The therapeutic compounds which make up the
combination therapy may also be administered sequentially, with
either therapeutic compound being administered by a regimen calling
for two-step ingestion. Thus, a regimen may call for sequential
administration of the therapeutic compounds with spaced-apart
ingestion of the separate, active agents. The time period between
the multiple ingestion steps may range from a few minutes to
several hours, depending upon the properties of each therapeutic
compound such as potency, solubility, bioavailability, plasma
half-life and kinetic profile of the therapeutic compound, as well
as depending upon the effect of food ingestion and the age and
condition of the patient. Circadian variation of the target
molecule concentration may also determine the optimal dose
interval. The therapeutic compounds of the combined therapy whether
administered simultaneously, substantially simultaneously, or
sequentially, may involve a regimen calling for administration of
one therapeutic compound by oral route and another therapeutic
compound by intravenous route. Whether the therapeutic compounds of
the combined therapy are administered by oral or intravenous route,
separately or together, each such therapeutic compound will be
contained in a suitable pharmaceutical formulation of
pharmaceutically-acceptable excipients, diluents or other
formulations components. Examples of suitable
pharmaceutically-acceptable formulations containing the therapeutic
compounds for oral administration are given above.
Treatment Regimen
[0107] The dosage regimen to prevent, give relief from, or
ameliorate a disease condition having hyperlipidemia as an element
of the disease, e.g., atherosclerosis, or to protect against or
treat further high cholesterol plasma or blood levels with the
compounds and/or compositions of the present invention is selected
in accordance with a variety of factors. These include the type,
age, weight, sex, diet, and medical condition of the patient, the
severity of the disease, the route of administration,
pharmacological considerations such as the activity, efficacy,
pharmacokinetics and toxicology profiles of the particular compound
employed, whether a drug delivery system is utilized, and whether
the compound is administered as part of a drug combination. Thus,
the dosage regimen actually employed may vary widely and therefore
deviate from the preferred dosage regimen set forth above.
[0108] Initial treatment of a patient suffering from a
hyperlipidemic condition can begin with the dosages indicated
above. Treatment should generally be continued as necessary over a
period of several weeks to several months or years until the
hyperlipidemic disease condition has been controlled or eliminated.
Patients undergoing treatment with the compounds or compositions
disclosed herein can be routinely monitored by, for example,
measuring serum LDL and total cholesterol levels by any of the
methods well known in the art, to determine the effectiveness of
the combination therapy. Continuous analysis of such data permits
modification of the treatment regimen during therapy so that
optimal effective amounts of each type of therapeutic compound are
administered at any point in time, and so that the duration of
treatment can be determined as well. In this way, the treatment
regimen/dosing schedule can be rationally modified over the course
of therapy so that the lowest amount of the therapeutic compounds
which together exhibit satisfactory effectiveness is administered,
and so that administration is continued only so long as is
necessary to successfully treat the hyperlipidemic condition.
[0109] A potential advantage of the combination therapy disclosed
herein may be reduction of the amount of any individual therapeutic
compound, or all therapeutic compounds, effective in treating
hyperlipidemic conditions such as atherosclerosis and
hypercholesterolemia.
[0110] One of the several embodiments of the present invention
comprises a combination therapy comprising the use of a first
amount of an CETP inhibitor and a second amount of another
cardiovascular therapeutic useful in the prophylaxis or treatment
of hyperlipidemia or atherosclerosis, wherein said first and second
amounts together comprise an anti-hyperlipidemic condition
effective amount or an anti-atherosclerotic condition effective
amount of said compounds. For example one of the many embodiments
of the present invention is a combination therapy comprising
therapeutic dosages of an CETP inhibitor and a bile acid
sequestering agent.
[0111] The following non-limiting examples serve to illustrate
various aspects of the present invention.
c. EXAMPLES
[0112] Table 3 illustrates examples of some combinations of the
present invention wherein the combination comprises a first amount
of a CETP inhibitor and a second amount of a bile acid
sequestration agent, wherein said first and second amounts together
comprise an anti-hyperlipidemic condition effective amount or an
anti-atherosclerotic condition effective amount of said
compounds.
3TABLE 3 Example Number Component 1 Component 2 5501 C-1
cholestyramine 5502 C-2 cholestyramine 5503 C-3 cholestyramine 5504
C-4 cholestyramine 5505 C-5 cholestyramine 5506 C-6 cholestyramine
5507 C-7 cholestyramine 5508 C-8 cholestyramine 5509 C-9
cholestyramine 5510 C-10 cholestyramine 5511 C-11 cholestyramine
5512 C-12 cholestyramine 5513 C-13 cholestyramine 5514 C-14
cholestyramine 5515 C-15 cholestyramine 5516 C-16 cholestyramine
5517 C-17 cholestyramine 5518 C-18 cholestyramine 5519 C-19
cholestyramine 5520 C-20 cholestyramine 5521 C-1 colestipol 5522
C-2 colestipol 5523 C-3 colestipol 5524 C-4 colestipol 5525 C-5
colestipol 5526 C-6 colestipol 5527 C-7 colestipol 5528 C-8
colestipol 5529 C-9 colestipol 5530 C-10 colestipol 5531 C-11
colestipol 5532 C-12 colestipol 5533 C-13 colestipol 5534 C-14
colestipol 5535 C-15 colestipol 5536 C-16 colestipol 5537 C-17
colestipol 5538 C-18 colestipol 5539 C-19 colestipol 5540 C-20
colestipol 5541 C-1 knedel A 5542 C-2 knedel A 5543 C-3 knedel A
5544 C-4 knedel A 5545 C-5 knedel A 5546 C-6 knedel A 5547 C-7
knedel A 5548 C-8 knedel A 5549 C-9 knedel A 5550 C-10 knedel A
5551 C-11 knedel A 5552 C-12 knedel A 5553 C-13 knedel A 5554 C-14
knedel A 5555 C-15 knedel A 5556 C-16 knedel A 5557 C-17 knedel A
5558 C-18 knedel A 5559 C-19 knedel A 5560 C-20 knedel A 5561 C-1
knedel B 5562 C-2 knedel B 5563 C-3 knedel B 5564 C-4 knedel B 5565
C-5 knedel B 5566 C-6 knedel B 5567 C-7 knedel B 5568 C-8 knedel B
5569 C-9 knedel B 5570 C-10 knedel B 5571 C-11 knedel B 5572 C-12
knedel B 5573 C-13 knedel B 5574 C-14 knedel B 5575 C-15 knedel B
5576 C-16 knedel B 5577 C-17 knedel B 5578 C-18 knedel B 5579 C-19
knedel B 5580 C-20 knedel B 5580.1 C-1 S-3 5580.2 C-2 S-3 5580.3
C-3 S-3 5580.4 C-4 S-3 5580.5 C-5 S-3 5580.6 C-6 S-3 5580.7 C-7 S-3
5580.8 C-8 S-3 5580.9 C-9 S-3 5580.10 C-10 S-3 5580.11 C-11 S-3
5580.12 C-12 S-3 5580.13 C-13 S-3 5580.14 C-14 S-3 5580.15 C-15 S-3
5580.16 C-16 S-3 5580.17 C-17 S-3 5580.18 C-18 S-3 5580.19 C-19 S-3
5580.20 C-20 S-3 5580.21 C-1 CholestaGel 5580.22 C-2 CholestaGel
5580.23 C-3 CholestaGel 5580.24 C-4 CholestaGel 5580.25 C-5
CholestaGel 5580.26 C-6 CholestaGel 5580.27 C-7 CholestaGel 5580.28
C-8 CholestaGel 5580.29 C-9 CholestaGel 5580.30 C-10 CholestaGel
5580.31 C-11 CholestaGel 5580.32 C-12 CholestaGel 5580.33 C-13
CholestaGel 5580.34 C-14 CholestaGel 5580.35 C-15 CholestaGel
5580.36 C-16 CholestaGel 5580.37 C-17 CholestaGel 5580.38 C-18
CholestaGel 5580.39 C-19 CholestaGel 5580.40 C-20 CholestaGel
5580.41 C-1 OmegaGel 5580.42 C-2 OmegaGel 5580.43 C-3 OmegaGel
5580.44 C-4 OmegaGel 5580.45 C-5 OmegaGel 5580.46 C-6 OmegaGel
5580.47 C-7 OmegaGel 5580.48 C-8 OmegaGel 5580.49 C-9 OmegaGel
5580.50 C-10 OmegaGel 5580.51 C-11 OmegaGel 5580.52 C-12 OmegaGel
5580.53 C-13 OmegaGel 5580.54 C-14 OmegaGel 5580.55 C-15 OmegaGel
5580.56 C-16 OmegaGel 5580.57 C-17 OmegaGel 5580.58 C-18 OmegaGel
5580.59 C-19 OmegaGel 5580.60 C-20 OmegaGel
Biological Assays
[0113] The utility of the combinations of the present invention can
be shown by the following assays. These assays are performed in
vitro and in animal models essentially using procedures recognized
to show the utility of the present invention.
[0114] In Vitro Assay of Compounds that Inhibit IBAT-Mediated
Uptake of [.sup.14C]-Taurocholate (TC) in H.sub.14Cells
[0115] Baby hamster kidney cells (BHK) transfected with the cDNA of
human IBAT (H14 cells) are to be seeded at 60,000 cells/well in 96
well Top-Count tissue culture plates for assays run within in 24
hours of seeding, 30,000 cells/well for assays run within 48 hours,
and 10,000 cells/well for assays run within 72 hours.
[0116] On the day of assay, the cell monolayer is gently washed
once with 100 .mu.l assay buffer (Dulbecco's Modified Eagle's
medium with 4.5 g/L glucose+0.2% (w/v) fatty acid free bovine serum
albumin--(FAF)BSA). To each well 50 .mu.l of a two-fold concentrate
of test compound in assay buffer is added along with 50 .mu.l of 6
.mu.M [.sup.14C]-taurocholate in assay buffer (final concentration
of 3 .mu.M [.sup.14C]-taurocholate). The cell culture plates are
incubated 2 hours at 37.degree. C. prior to gently washing each
well twice with 100 .mu.l 4.degree. C. Dulbecco's
phosphate-buffered saline (PBS) containing 0.2% (w/v) (FAF)BSA. The
wells are then to be gently washed once with 100 .mu.l 4.degree. C.
PBS without (FAF)BSA. To each 200 .mu.l of liquid scintillation
counting fluid is to be added, the plates are heat sealed and
shaken for 30 minutes at room temperature prior to measuring the
amount of radioactivity in each well on a Packard Top-Count
instrument.
[0117] In Vitro Assay of Compounds that Inhibit Uptake of
[.sup.14C]-Alanine
[0118] The alanine uptake assay can be performed in an identical
fashion to the taurocholate assay, with the exception that labeled
alanine is to be substituted for the labeled taurocholate.
[0119] In Vivo Assay of Compounds that Inhibit Rat Ileal Uptake of
[.sup.14C]-Taurocholate into Bile
[0120] (See "Metabolism of
3.alpha.,7.beta.-dihydroxy-7.alpha.-methyl-5.be- ta.-cholanoic acid
and 3.alpha.,7.beta.-dihydroxy-7.alpha.-methyl-5.beta.-- cholanoic
acid in hamsters" in Biochimica et Biophysica Acta, 833, 196-202
(1985) by Une et al., herein incorporated by reference.)
[0121] Male wistar rats (200-300 g) are to be anesthetized with
inactin @100 mg/kg. Bile ducts are cannulated with a 10" length of
PE10 tubing. The small intestine is exposed and laid out on a gauze
pad. A canulae (1/8" luer lock, tapered female adapter) is inserted
at 12 cm from the junction of the small intestine and the cecum. A
slit is cut at 4 cm from this same junction (utilizing a 8 cm
length of ileum). 20 ml of warm Dulbecco's phosphate buffered
saline, pH 6.5 (PBS) is used to flush out the intestine segment.
The distal opening is cannulated with a 20 cm length of silicone
tubing (0.02"I.D..times.0.037" O.D.). The proximal cannulae is
hooked up to a peristaltic pump and the intestine is washed for 20
min with warm PBS at 0.25 ml/min. Temperature of the gut segment is
to be monitored continuously. At the start of the experiment, 2.0
ml of control sample ([.sup.14C]-taurocholate @ 0.05 mCi/ml with 5
mM non-radiolabeled taurocholate) is loaded into the gut segment
with a 3 ml syringe and bile sample collection is begun. Control
sample is infused at a rate of 0.25 ml/min for 21 min. Bile samples
fractions will be collected every 3 minute for the first 27 minutes
of the procedure. After the 21 min of sample infusion, the ileal
loop is washed out with 20 ml of warm PBS (using a 30 ml syringe),
and then the loop is washed out for 21 min with warm PBS at 0.25
ml/min. A second perfusion is to be initiated as described above
but with test compound being administered as well (21 min
administration followed by 21 min of wash out) and bile to be
sampled every 3 min for the first 27 min. If necessary, a third
perfusion will be performed as above that typically contains the
control sample.
[0122] Measurement of Hepatic Cholesterol Concentration (HEPATIC
CHOL)
[0123] Liver tissue is to be weighed and homogenized in
chloroform:methanol (2:1). After homogenization and centrifugation
the supernatant is separated and dried under nitrogen. The residue
is to be dissolved in isopropanol and the cholesterol content will
be measured enzymatically, using a combination of cholesterol
oxidase and peroxidase, as described by Allain, C. A. et al., Clin.
Chem., 20, 470 (1974) (herein incorporated by reference).
[0124] Measurement of Hepatic HMG CoA-Reductase Activity (HMG
COA)
[0125] Hepatic microsomes are to be prepared by homogenizing liver
samples in a phosphate/sucrose buffer, followed by centrifugal
separation. The final pelleted material is resuspended in buffer
and an aliquot will be assayed for HMG CoA reductase activity by
incubating for 60 minutes at 37.degree. C. in the presence of
.sup.14C-HMG-CoA (Dupont-NEN). The reaction is stopped by adding 6N
HCl followed by centrifugation. An aliquot of the supernatant is
separated, by thin-layer chromatography, and the spot corresponding
to the enzyme product is scraped off the plate, extracted and
radioactivity is determined by scintillation counting. (Reference:
Akerlund, J. and Bjorkhem, I. (1990) J. Lipid Res. 31, 2159).
[0126] Determination of Serum Cholesterol (SER.CHOL, HDL-CHOL, TGI
and VLDL+LDL)
[0127] Total serum cholesterol (SER.CHOL) are to be measured
enzymatically using a commercial kit from Wako Fine Chemicals
(Richmond, Va.); Cholesterol C11, Catalog No. 276-64909. HDL
cholesterol (HDL-CHOL) will be assayed using this same kit after
precipitation of VLDL and LDL with Sigma Chemical Co. HDL
Cholesterol reagent, Catalog No. 352-3 (dextran sulfate method).
Total serum triglycerides (blanked) (TGI) will be assayed
enzymatically with Sigma Chemical Co. GPO-Trinder, Catalog No.
337-B. VLDL and LDL (VLDL+LDL) cholesterol concentrations will be
calculated as the difference between total and HDL cholesterol.
[0128] Measurement of Hepatic Cholesterol 7-.alpha.-Hydroxylase
Activity (7a-OHase)
[0129] Hepatic microsomes are to be prepared by homogenizing liver
samples in a phosphate/sucrose buffer, followed by centrifugal
separation. The final pelleted material is resuspended in buffer
and an aliquot will be assayed for cholesterol
7-.alpha.-hydroxylase activity by incubating for 5 minutes at
37.degree. C. in the presence of NADPH. Following extraction into
petroleum ether, the organic solvent is evaporated and the residue
is dissolved in acetonitrile/methanol. The enzymatic product will
be separated by injecting an aliquot of the extract onto a C.sub.18
reversed phase HPLC column and quantitating the eluted material
using UV detection at 240 nm. (Reference: Horton, J. D., et al.
(1994) J. Clin. Invest. 93, 2084).
[0130] Rat Gavage Assay
[0131] Male Wister rats (275-300 g) are to be administered IBAT
inhibitors using an oral gavage procedure. Drug or vehicle (0.2%
TWEEN 80 in water) is administered once a day (9:00-10:0 a.m.) for
4 days at varying dosages in a final volume of 2 mL per kilogram of
body weight. (TWEEN 80 is a 20 molar polyethyleneoxide sorbitan
monooleate surfactant manufactured by ICI Specialty Chemicals,
Wilmington, Del., U.S.A.) Total fecal samples are collected during
the final 48 hours of the treatment period and analyzed for bile
acid content using an enzymatic assay as described below. Compound
efficacy will be determined by comparison of the increase in fecal
bile acid (FBA) concentration in treated rats to the mean FBA
concentration of rats in the vehicle group.
[0132] Measurement of Fecal Bile Acid Concentration (FBA)
[0133] Total fecal output from individually housed rats is to be
collected for 24 or 48 hours, dried under a stream of nitrogen,
pulverized and weighed. Approximately 0.1 gram is weighed out and
extracted into an organic solvent (butanol/water). Following
separation and drying, the residue is dissolved in methanol and the
amount of bile acid present will be measured enzymatically using
the 3.alpha.-hydroxysteroid steroid dehydrogenase reaction with
bile acids to reduce NAD. (see Mashige, F. et al. Clin. Chem., 27,
1352 (1981), herein incorporated by reference).
[0134] [.sup.3H]taurocholate Uptake in Rabbit Brush Border Membrane
Vesicles (BBMV)
[0135] Rabbit Ileal brush border membranes are to be prepared from
frozen ileal mucosa by the calcium precipitation method describe by
Malathi et al. (Biochimica Biophysica Acta, 554, 259 (1979), herein
incorporated by reference). The method for measuring taurocholate
is essentially as described by Kramer et al. (Biochimica Biophysica
Acta, 1111, 93 (1992), herein incorporated by reference) except the
assay volume will be 200 .mu.l instead of 100 .mu.l. Briefly, at
room temperature a 190 .mu.l solution containing 2 .mu.M
[.sup.3H]-taurocholate(0.75 .mu.Ci), 20 mM tris, 100 mM NaCl, 100
mM mannitol pH 7.4 is incubated for 5 sec with 10 .mu.l of brush
border membrane vesicles (60-120 .mu.g protein) The incubation is
initiated by the addition of the BBMV while vortexing and the
reaction is to be stopped by the addition of 5 ml of ice cold
buffer (20 mM Hepes-tris, 150 mM KCl) followed immediately by
filtration through a nylon filter (0.2 .mu.m pore) and an
additional 5 ml wash with stop buffer.
[0136] Acyl-CoA; Cholesterol Acyl Transferase (ACAT)
[0137] Hamster liver and rat intestinal microsomes are to be
prepared from tissue as described previously (J. Biol. Chem., 255,
9098 (1980), herein incorporated by reference) and used as a source
of ACAT enzyme. The assay will consist of a 2.0 ml incubation
containing 24 .mu.M Oleoyl-CoA (0.05 .mu.Ci) in a 50 mM sodium
phosphate, 2 mM DTT ph 7.4 buffer containing 0.25% BSA and 200
.mu.g of microsomal protein. The assay will be initiated by the
addition of oleoyl-CoA. The reaction proceeds for 5 min at
37.degree. C. and will be terminated by the addition of 8.0 ml of
chloroform/methanol (2:1). To the extraction is added 125 .mu.g of
cholesterol oleate in chloroform methanol to act as a carrier and
the organic and aqueous phases of the extraction are separated by
centrifugation after thorough vortexing. The chloroform phase is to
be taken to dryness and then spotted on a silica gel 60 TLC plate
and developed in hexane/ethyl ether (9:1). The amount of
cholesterol ester formed will be determined by measuring the amount
of radioactivity incorporated into the cholesterol oleate spot on
the TLC plate with a Packard Instaimager.
[0138] Dog Model for Evaluating Lipid Lowering Drugs
[0139] Male beagle dogs, obtained from a vendor such as Marshall
farms and weighing 6-12 kg are fed once a day for two hours and
given water ad libitum. Dogs may be randomly assigned to a dosing
groups consisting of 6 to 12 dogs each, such as: vehicle, i.g.; 1
mg/kg, i.g.; 2 mg/kg, i.g.; 4 mg/kg, i.g.; 2 mg/kg, p.o. (powder in
capsule). Intragastric dosing of a therapeutic material dissolved
in aqueous solution (for example, 0.2% Tween 80 solution
[polyoxyethylene mono-oleate, Sigma Chemical Co., St. Louis, Mo.])
may be done using a gavage tube. Prior to initiating dosing, blood
samples may be drawn from the cephalic vein in the morning before
feeding in order to evaluate serum cholesterol (total and HDL) and
triglycerides. For several consecutive days animals are dosed in
the morning, prior to feeding. Animals are to be allowed 2 hours to
eat before any remaining food is removed. Feces are to be collected
over a 2 day period at the end of the study and may be analyzed for
bile acid or lipid content. Blood samples are also to be taken, at
the end of the treatment period, for comparison with pre-study
serum lipid levels. Statistical significance will be determined
using the standard student's T-test with p<0.05.
[0140] Dog Serum Lipid Measurement
[0141] Blood is to be collected from the cephalic vein of fasted
dogs in serum separator tubes (Vacutainer SST, Becton Dickinson and
Co., Franklin Lakes, N.J.). The blood is centrifuged at 2000 rpm
for 20 minutes and the serum decanted.
[0142] Total cholesterol may be measured in a 96 well format using
a Wako enzymatic diagnostic kit (Cholesterol CII) (Wako Chemicals,
Richmond, Va.), utilizing the cholesterol oxidase reaction to
produce hydrogen peroxide which is measured calorimetrically. A
standard curve from 0.5 to 10 .mu.g cholesterol is to be prepared
in the first 2 columns of the plate. The serum samples (20-40
.mu.l, depending on the expected lipid concentration) or known
serum control samples are added to separate wells in duplicate.
Water is added to bring the volume to 100 .mu.l in each well. A 100
.mu.l aliquot of color reagent is added to each well and the plates
will be read at 500 nm after a 15 minute incubation at 37 degrees
centigrade.
[0143] HDL cholesterol may be assayed using Sigma kit No. 352-3
(Sigma Chemical Co., St. Louis, Mo.) which utilizes dextran sulfate
and Mg ions to selectively precipitate LDL and VLDL. A volume of
150 .mu.l of each serum sample is to be added to individual
microfuge tubes, followed by 15 .mu.l of HDL cholesterol reagent
(Sigma 352-3). Samples are to be mixed and centrifuged at 5000 rpm
for 5 minutes. A 50 .mu.l aliquot of the supernatant is to be then
mixed with 200 .mu.l of saline and assayed using the same procedure
as for total cholesterol measurement.
[0144] Triglycerides are to be measured using Sigma kit No. 337 in
a 96 well plate format. This procedure will measure glycerol,
following its release by reaction of triglycerides with lipoprotein
lipase. Standard solutions of glycerol (Sigma 339-11) ranging from
1 to 24 .mu.g are to be used to generate the standard curve. Serum
samples (20-40 .mu.l, depending on the expected lipid
concentration) are added to wells in duplicate. Water is added to
bring the volume to 100 .mu.l in each well and 100 .mu.l of color
reagent is also added to each well. After mixing and a 15 minute
incubation, the plates will be read at 540 nm and the triglyceride
values calculated from the standard curve. A replicate plate is
also to be run using a blank enzyme reagent to correct for any
endogenous glycerol in the serum samples.
[0145] Dog Fecal Bile Acid Measurement
[0146] Fecal samples may be collected to determine the fecal bile
acid (FBA) concentration for each animal. Fecal collections may be
made during the final 48 hours of the study, for two consecutive 24
hour periods between 9:00 am and 10:00 am each day, prior to dosing
and feeding. The separate two day collections from each animal are
to be weighed, combined and homogenized with distilled water in a
processor (Cuisinart) to generate a homogeneous slurry. About 1.4 g
of the homogenate is to be extracted in a final concentration of
50% tertiary butanol/distilled water (2:0.6) for 45 minutes in a
37.degree. C. water bath and centrifuged for 13 minutes at
2000.times.g. The concentration of bile acids (mmoles/day) may be
determined using a 96-well enzymatic assay system (1,2). A 20 .mu.l
aliquot of the fecal extract is to be added to two sets each of
triplicate wells in a 96-well assay plate. A standardized sodium
taurocholate solution and a standardized fecal extract solution
(previously made from pooled samples and characterized for its bile
acid concentration) will also analyzed for assay quality control.
Twenty-microliter aliquots of sodium taurocholate, serially diluted
to generate a standard curve are similarly to be added to two sets
of triplicate wells. A 230 .mu.l reaction mixture containing 1M
hydrazine hydrate, 0.1 M pyrophosphate and 0.46 mg/ml NAD is to be
added to each well. A 50 .mu.l aliquot of 3a-hydroxysteroid
dehydrogenase enzyme (HSD; 0.8 units/ml) or assay buffer (0.1 M
sodium pyrophosphate) are then added to one of the two sets of
triplicates. All reagents may be obtained from Sigma Chemical Co.,
St. Louis, Mo. Following 60 minutes of incubation at room
temperature, the optical density at 340 nm will be measured and the
mean of each set of triplicate samples will be calculated. The
difference in optical density.+-.HSD enzyme is to be used to
determine the bile acid concentration (mM) of each sample based on
the sodium taurocholate standard curve. The bile acid concentration
of the extract, the weight of the fecal homogenate (grams) and the
body weight of the animal are to be used to calculate the
corresponding FBA concentration in mmoles/kg/day for each animal.
The mean FBA concentration (mmoles/kg/day) of the vehicle group is
to be subtracted from the FBA concentration of each treatment group
to determine the increase (delta value) in FBA concentration as a
result of the treatment.
[0147] CETP Activity Assay in Human Plasma (Tritiated Cholesteryl
Ester)
[0148] Blood is to be obtained from healthy volunteers. Blood is
collected in tubes containing EDTA (EDTA plasma pool). The EDTA
human plasma pool previously stored at -20.degree. C., is to be
thawed at room temperature, and centrifuged for 5 minutes to remove
any particulate matter. Tritiated HDL, radiolabeled in the
cholesteryl ester moiety ([.sup.3H]CE-HDL) as described by Morton
and Zilversmit (J. Biol. Chem., 256, 11992-95 (1981)), is to be
added to the plasma to a final concentration of (25 .mu.g/ml
cholesterol). Inhibitor compounds are to be added to the plasma as
follows: Equal volumes of the plasma containing the [.sup.3H]CE-HDL
(396 .mu.l) are added by pipette into micro tubes (Titertube.RTM.,
Bio-Rad laboratories, Hercules, Calif.). Compounds, usually
dissolved as 20-50 mM stock solutions in DMSO, are to be serially
diluted in DMSO (or an alternative solvent in some cases, such as
dimethylformamide or ethanol). Four .mu.l of each of the serial
dilutions of inhibitor compounds or DMSO alone are then added to
each of the plasma tubes. The tubes are immediately mixed.
Triplicate aliquots (100 .mu.l) from each plasma tube are then
transferred to wells of 96-well round-bottomed polystyrene
microtiter plates (Corning, Corning, N.Y.). Plates are sealed with
plastic film and incubated at 37.degree. C. for 4 hours. Test wells
are to contain plasma with dilutions of inhibitor compounds.
Control wells are to contain plasma with DMSO alone. Blank wells
are to contain plasma with DMSO alone that are left in the micro
tubes at 4.degree. C. for the 4 hour incubation and are added to
the microtiter wells at the end of the incubation period. VLDL and
LDL are precipitated by the addition of 10 .mu.l of precipitating
reagent (1% (w/v) dextran sulfate (Dextralip50)/0.5 M magnesium
chloride, pH 7.4) to all wells. The wells are mixed on a plate
mixer and then incubated at ambient temperature for 10 min. The
plates are then centrifuged at 1000.times.g for 30 min at
10.degree. C. The supernatants (50 .mu.l) from each well are then
transferred to Picoplate.TM. 96 plate wells (Packard, Meriden,
Conn.) containing 250:1 Microscint.TM.-40 (Packard, Meriden,
Conn.). The plates are heat-sealed (TopSeal.TM.-P, Packard,
Meriden, Conn.) according to the manufacturer's directions and
mixed for 30 min. Radioactivity will be measured on a microplate
scintillation counter (TopCount, Packard, Meriden, Conn.).
IC.sub.50 values will be determined as the concentration of
inhibitor compound inhibiting transfer of [.sup.3H]CE from the
supernatant [.sup.3H]CE-HDL to the precipitated VLDL and LDL by 50%
compared to the transfer obtained in the control wells. The maximum
percentage transfer (in the control wells) will be determined using
the following equation: 1 % Transfer = [ dpm blank - dpm control ]
.times. 100 dpm blank
[0149] The percentage of control transfer determined in the wells
containing inhibitor compounds is determined as follows: 2 %
Control = [ dpm blank - dpm test ] .times. 100 dpm blank - dpm
control
[0150] IC.sub.50 values will be calculated from plots of % control
versus concentration of inhibitor compound.
[0151] CETP Activity In Vitro
[0152] The ability of compounds to inhibit CETP activity are
assessed using an in vitro assay that measures the rate of transfer
of radiolabeled cholesteryl ester ([.sup.3H]CE) from HDL donor
particles to LDL acceptor particles. Details of the assay are
provided by Glenn et al. (Glenn and Melton, "Quantification of
Cholesteryl Ester Transfer Protein (CETP): A) CETP Activity and B)
Immunochemical Assay of CETP Protein," Meth. Enzymol., 263, 339-351
(1996)). CETP can be obtained from the serum-free conditioned
medium of CHO cells transfected with a cDNA for CETP (Wang, S. et
al. J. Biol. Chem. 267, 17487-17490 (1992)). To measure CETP
activity, [.sup.3H]CE-labeled HDL, LDL, CETP and assay buffer (50
mM tris(hydroxymethyl)aminomethane, pH 7.4; 150 mM sodium chloride;
2 mM ethylenediamine-tetraacetic acid; 1% bovine serum albumin) are
incubated in a volume of 200 .mu.l, for 2 hours at 37.degree. C. in
96 well plates. LDL is differentially precipitated by the addition
of 50 .mu.l of 1% (w/v) dextran sulfate/0.5 M magnesium chloride,
mixed by vortex, and incubated at room temperature for 10 minutes.
The solution (200 .mu.l) is transferred to a filter plate
(Millipore). After filtration, the radioactivity present in the
precipitated LDL is measured by liquid scintillation counting.
Correction for non-specific transfer or precipitation is made by
including samples that do not contain CETP. The rate of [.sup.3H]CE
transfer using this assay is linear with respect to time and CETP
concentration, up to 25-30% of [.sup.3H]CE transferred.
[0153] The potency of test compounds can be determined by
performing the above described assay in the presence of varying
concentrations of the test compounds and determining the
concentration required for 50% inhibition of transfer of
[.sup.3H]CE from HDL to LDL. This value is defined as the
IC.sub.50. The IC.sub.50 values determined from this assay will be
accurate when the IC.sub.50 is greater than 10 nM. In the case
where compounds have greater inhibitory potency, accurate
measurements of IC.sub.50 may be determined using longer incubation
times (up to 18 hours) and lower final concentrations of CETP
(<50 nM)
[0154] Inhibition of CETP Activity In Vivo.
[0155] Inhibition of CETP activity by a test compound can be
determined by administering the compound to an animal by
intravenous injection or oral gavage, measuring the amount of
transfer of tritium-labeled cholesteryl ester ([.sup.3H]CE) from
HDL to VLDL and LDL particles, and comparing this amount of
transfer with the amount of transfer observed in control
animals.
[0156] Male golden Syrian hamsters are to be maintained on a diet
of chow containing 0.24% cholesterol for at least two weeks prior
to the study. For animals receiving intravenous dosing, immediately
before the experiment, animals are anesthetized with pentobarbital.
Anesthesia is maintained throughout the experiment. In-dwelling
catheters are to be inserted into the jugular vein and carotid
artery. At the start of the experiment all animals will receive 0.2
ml of a solution containing [.sup.3H]CE-HDL into the jugular vein.
[.sup.3H]CE-HDL is a preparation of human HDL containing
tritium-labeled cholesteryl ester, and is prepared according to the
method of Glenn et al. (Meth. Enzymol., 263, 339-351 (1996)). Test
compound is dissolved as a 80 mM stock solution in vehicle (2%
ethanol: 98% PEG 400, Sigma Chemical Company, St. Louis, Mo., USA)
and administered either by bolus injection or by continuous
infusion. Two minutes after the [.sup.3H]CE-HDL dose is
administered, animals are to receive 0.1 ml of the test solution
injected into the jugular vein. Control animals are to receive 0.1
ml of the intravenous vehicle solution without test compound. After
5 minutes, the first blood samples (0.5 ml) are taken from the
carotid artery and collected in standard microtainer tubes
containing ethylenediamine tetraacetic acid. Saline (0.5 ml) is
injected to flush the catheter and replace blood volume. Subsequent
blood samples are to be taken at two hours and four hours by the
same method. Blood samples are mixed well and kept on ice until the
completion of the experiment. Plasma is obtained by centrifugation
of the blood samples at 40 C. The plasma (50 .mu.l) is treated with
5 .mu.l of precipitating reagent (dextran sulfate, 10 g/1; 0.5 M
magnesium chloride) to remove VLDL/LDL. After centrifugation, the
resulting supernatant (25 .mu.l) containing the HDL will be
analyzed for radioactivity using a liquid scintillation
counter.
[0157] The percentage [3H]CE transferred from HDL to LDL and VLDL
(% transfer) will be calculated based on the total radioactivity in
equivalent plasma samples before precipitation. Typically, the
amount of transfer from HDL to LDL and VLDL in control animals will
be 20% to 35% after 4 hours.
[0158] Alternatively, conscious, non-anesthetized animals can
receive an oral gavage dose of test compound as a suspension in
0.1% methyl cellulose in water. At a time determined for each
compound at which plasma levels of the test substance reach their
peak (C.sub.max) after oral dosing, the animals are to be
anesthetized with pentobarbital and then dosed with 0.2 ml of a
solution containing [.sup.3H]CE-HDL into the jugular vein as
described above. Control animals are to receive 0.25 ml of the
vehicle solution without test compound by oral gavage. After 4
hours, the animals are to be sacrificed, blood samples are
collected, and the percentage [.sup.3H]CE transferred from HDL to
LDL and VLDL (% transfer) is assayed as described above.
[0159] Alternatively, inhibition of CETP activity by a test
compound can be determined by administering the compound to mice
that have been selected for expression of human CETP (hCETP) by
transgenic manipulation (hCETP mice). Test compounds can be
administered by intravenous injection, or oral gavage and the
amount of transfer of tritium-labeled cholesteryl ester
([.sup.3H]CE) from HDL to VLDL and LDL particles is determined, and
compared to the amount of transfer observed in control animals.
CS7Bl/6 mice that are homozygous for the hCETP gene are to be
maintained on a high fat chow diet, such as TD 88051, as described
by Nishina et al. (J Lipid Res., 31, 859-869 (1990)) for at least
two weeks prior to the study. Mice are to receive an oral gavage
dose of test compound as a suspension in 0.1% methyl cellulose in
water or an intravenous bolus injection of test compound in 10%
ethanol and 90% polyethylene glycol. Control animals are to receive
the vehicle solution without test compound by oral gavage or by an
intravenous bolus injection. At the start of the experiment all
animals will receive 0.05 ml of a solution containing
[.sup.3H]CE-HDL into the tail vein. [.sup.3H]CE-HDL will be a
preparation of human HDL containing tritium-labeled cholesteryl
ester, and is prepared according to the method of Glenn et al.
(Meth. Enzymol., 263, 339-351 (1996)). After 30 minutes, the
animals are exsanguinated and blood collected in standard
microtainer tubes containing ethylenediamine tetraacetic acid.
Blood samples are mixed well and kept on ice until the completion
of the experiment. Plasma will be obtained by centrifugation of the
blood samples at 4.degree. C. The plasma is separated and analyzed
by gel filtration chromatography and the relative proportion of
[.sup.3H]CE in the VLDL, LDL and HDL regions will be
determined.
[0160] The percentage [.sup.3H]CE transferred from HDL to LDL and
VLDL (% transfer) will be calculated based on the total
radioactivity in equivalent plasma samples before precipitation.
Typically, the amount of transfer from HDL to LDL and VLDL in
control animals will be 20% to 35% after 30 min.
[0161] Intestinal Cholesterol Absorption Assay
[0162] A variety of compounds are shown to inhibit cholesterol
absorption from the intestinal tract. These compounds lower serum
cholesterol levels by reducing intestinal absorption of cholesterol
from both exogenous sources (dietary cholesterol) and endogenous
cholesterol (secreted by the gall bladder into the intestinal
tract).
[0163] In hamsters the use of a dual-isotope plasma ratio method to
measure intestinal cholesterol absorption has been refined and
evaluated as described by Turley et al. (J. Lipid Res. 35, 329-339
(1994), herein incorporated by reference).
[0164] Male hamsters weighing 80-100 g are to be given food and
water ad libitum in a room with 12 hour alternating periods of
light and dark. Four hours into the light period, each hamster is
administered first an intravenous dose of 2.5 .mu.Ci of
[1,2-.sup.3H]cholesterol suspended in Intralipid (20%) and then an
oral dose of [4-.sup.14C]cholesterol in an oil of medium chain
triglycerides (MCT). The i.v. dose is given by injecting a 0.4 ml
volume of the Intralipid mixture into the distal femoral vein. The
oral dose is given by gavaging a 0.6 ml volume of the MCT oil
mixture introduced intragastrically via a polyethylene tube. After
72 hours the hamsters are bled and the amount of .sup.3H and
.sup.14C in the plasma and in the original amount of label
administered are determined by liquid scintillation spectrometry.
The cholesterol absorption will be calculated based on the
following equation: 3 Percent cholesterol absorbed = % of oral dose
per ml of 72 hour plasma sample % of i . v . dose per ml of 72 hour
plasma sample .times. 100
[0165] Microsomal Triglyceride Transfer Protein (MTP) Assay:
[0166] MTP can be purified from liver tissue or cultured cells
(e.g. HepG2 cells) using standard methods as described by Ohringer
et al. (Acta Crystallogr. D52, 224-225 (1996), herein incorporated
by reference).
[0167] Subsequent analysis of MTP activity can be performed as
described by Jamil et al. (Proc. Natl. Acad. Sci. 93, 11991-11995
(1996), herein incorporated by reference).
[0168] The basis of this assay is to measure the transfer of
labeled triglycerides from a population of donor vesicles to a
population of acceptor vesicles in the presence of MTP. Inhibitors
of MTP can be evaluated by adding them to the mixture prior to the
introduction of MTP. Donor vesicles are prepared by sonication of
an aqueous mixture of egg phospholipids, cardiolipin,
.sup.3H-labeled phospholipid and .sup.14C-labeled triglycerides.
Acceptor vesicles are prepared by sonication of an aqueous mixture
of egg phospholipids. The vesicle solutions are mixed together,
with or without added MTP inhibitors, and MTP is added to initiate
the transfer reaction. The assay is terminated after 60 minutes by
addition of 0.5 ml of DE-52 cellulose followed by centrifugation to
pellet the donor molecules. The amount of .sup.3H and .sup.14C in
the pellet and in the original amount of label in the mixture are
determined by liquid scintillation spectrometry. The lipid transfer
rate will be calculated based on first order kinetics using the
expression:
[S]=[S].sub.0e.sup.-kt
[0169] where [S].sub.0 and [S] are the fractions of .sup.14C label
in the donor membrane pellet at times 0 and t, respectively, and
the term k is the fraction of label transferred per unit time.
[0170] Plasma Lipids Assay in Rabbits
[0171] Plasma lipids can be assayed using standard methods as
reported by J. R. Schuh et al., J. Clin. Invest., 91, 1453-1458
(1993), herein incorporated by reference. Groups of male, New
Zealand white rabbits are placed on a standard diet (100 g/day)
supplemented with 0.3% cholesterol and 2% corn oil (Zeigler
Bothers, Inc., Gardners, Pa.). Water is available ad lib. Groups of
control and treated animals are killed after 1 and 3 months of
treatment. Tissues are removed for characterization of
atherosclerotic lesions. Blood samples are to be taken for
determination of plasma lipid concentrations.
[0172] Plasma Lipids
[0173] Plasma for lipid analysis is to be obtained by withdrawing
blood from the ear vein into EDTA-containing tubes (Vacutainer;
Becton Dickenson & Co., Rutherford, N.J.), followed by
centrifugal separation of the cells. Total cholesterol is
determined enzymatically, using the cholesterol oxidase reaction
(C. A. Allain et al., Clin. Chem., 20, 470-475 (1974), herein
incorporated by reference). HDL cholesterol is also measured
enzymatically, after selective precipitation of LDL and VLDL by
dextran sulfate with magnesium (G. R. Warnick et al., Clin. Chem.,
28, 1379-1388 (1982), herein incorporated by reference). Plasma
triglyceride levels will be determined by measuring the amount of
glycerol released by lipoprotein lipase through an enzyme-linked
assay (G. Bucolo et al., Clin. Chem., 19, 476-482 (1973), herein
incorporated by reference).
[0174] Atherosclerosis
[0175] Animals are to be killed by pentobarbital injection.
Thoracic aortas are rapidly removed, immersion fixed in 10% neutral
buffered formalin, and stained with oil red 0 (0.3%). After a
single longitudinal incision along the wall opposite the arterial
ostia, the vessels are pinned open for evaluation of the plaque
area. The percent plaque coverage is determined from the values for
the total area examined and the stained area, by threshold analysis
using a true color image analyzer (Videometric 150; American
Innovision, Incl, San Diego, Calif.) interfaced to a color camera
(Toshiba 3CCD) mounted on a dissecting microscope. Tissue
cholesterol will be measured enzymatically as described, after
extraction with a chloroform/methanol mixture (2:1) according to
the method of Folch et al. (J. Biol. Chem., 226, 497-509 (1957),
herein incorporated by reference).
[0176] In Vitro Vascular Response
[0177] The abdominal aortas are rapidly excised, after injection of
sodium pentobarbital, and placed in oxygenated Krebs-bicarbonate
buffer. After removal of perivascular tissue, 3-mm ring segments
are cut, placed in a 37.degree. C. muscle bath containing
Krebs-bicarbonate solution, and suspended between two stainless
steel wires, one of which is attached to a force transducer (Grass
Instrument Co., Quincy, Mass.). Force changes in response to
angiotensin II added to the bath will be recorded on a chart
recorder.
[0178] The examples herein can be performed by substituting the
generically or specifically described therapeutic compounds or
inert ingredients for those used in the preceding examples.
[0179] The invention being thus described, it is apparent that the
same can be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of the present
invention, and all such modifications and equivalents as would be
obvious to one skilled in the art are intended to be included
within the scope of the following claims.
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