Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
COMPOSITIONS AND METHODS FOR TREATING ELEVATED BLOOD
CHOLESTEROL
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority ofU.S. provisional application
Serial No. 60/090,527, filed June 24, 1998.
BRIEF DESCRIPTION OF THE INVENTION
The present invention relates to compositions and methods for treating
elevated blood cholesterol in a mammal while counteracting potential adverse
side
effects such as myopathy. The compositions useful herein comprise the
combination
of a pharmaceutically effective amount of a 3-hydroxy-3-methylglutaryl
coenzyme A
reductase inhibitor (hereafter "HMG-CoA reductase inhibitor") and a
geranylgeraniol
compound to a mammal in need thereof.
BACKGROUND OF THE INVENTION
It has been clear for several decades that elevated blood cholesterol is a
major risk factor for coronary heart disease, and many studies have shown that
the risk
of coronary heart disease (CHD) events can be reduced by lipid-lowering
therapy.
Prior to 1987, the lipid-lowering armamentarium was limited essentially to a
low
saturated fat and cholesterol diet, the bile acid sequestrants (cholestyramine
and
colestipol), nicotinic acid {niacin), the fibrates and probucol.
Unfortunately, all of
these treatments have limited efficacy or tolerability, or both. Substantial
reductions in
LDL (low density lipoprotein) cholesterol accompanied by increases in HDL
(high
density lipoprotein) cholesterol could be achieved by the combination of a
lipid-
lowering diet and a bile acid sequestrant, with or without the addition of
nicotinic acid.
However, this therapy is not easy to administer or tolerate and was therefore
often
unsuccessful except in specialist lipid clinics. The fibrates produce a
moderate
reduction in LDL cholesterol accompanied by increased HDL cholesterol and a
substantial reduction in triglycerides, and because they are well tolerated
these drugs
have been more widely used. Probucol produces only a small reduction in LDL
cholesterol and also reduces HDL cholesterol, which, because of the strong
inverse
relationship between HDL cholesterol level and CHD risk, is generally
considered
undesirable. With the introduction of lovastatin, the first inhibitor of HMG-
CoA
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reductase to become available for prescription in 1987, for the first time
physicians
were able to obtain large reductions in plasma cholesterol with very few
adverse
effects.
Recent studies have unequivocally demonstrated that lovastatin,
simvastatin and pravastatin, all members of the HMG-CoA reductase inhibitor
class,
slow the progression of atherosclerotic lesions in the coronary and carotid
arteries.
Simvastatin and pravastatin have also been shown to reduce the risk of
coronary heart
disease events, and in the case of simvastatin a highly significant reduction
in the risk
of coronary death and total mortality has been shown by the Scandinavian
Simvastatin
Survival Study. This study also provided some evidence for a reduction in
cerebrovascular events.
However, along with their benefits, HMG-CoA reductase inhibitors can
cause potentially adverse side effects such as myopathy and related disorders
in a small
percentage of patients. Myopathy is characterized by muscle pain and weakness.
The
Physician's Desk Reference, 42nd Ed., 1366 (1988), which is incorporated by
reference herein in its entirety, states that myaglia, i.e. muscle pain, has
been
associated with lovastatin. Tobert, N.E..J.Med. , 48 (January 7, 1988), which
is
incorporated by reference herein in its entirety, states that in a very small
number of
patients (0.5 percent) myopathy appeared to be associated with lovastatin
therapy.
Concommitant therapy with immunosuppressant drugs, including cyclosproine,
with
gemfibrozil, or niacin, or a combination, appears to increase the risk of
myopathy. See
J.A. Tobert, Am.J. Cardiol. , 1988, 62: 28J-34J, which is incorporated by
reference
herein in its entirety. The myopathy is reversible upon discontinuation of
lovastatin
therapy. See U.S. Patent 4, 933, 165, to Brown, issued June 12, 1990, which is
incorporated by reference herein in its entirety. It is seen that it would be
of
ocnsiderable benefit to counteract the myopathy observed in the small
percentage of
patients. Therefore, improved therapies for treating, preventing, and reducing
the risk
of developing atherosclerosis, and cardiovascular and cerebrovascular events
and
related disorders are currently being sought which minimize the potential for
adverse
effects such as myopathy.
Geranylgeraniol and its derivatives belong to a class of naturally-
occurring compounds known as terpenes. Terpenes are constructed of multiples
of
five-carbon isoprene units. See Lehrunger, A.L., Biochemistry, 1975, pp. 296
and
682-683, which is incorporated by reference herein in its entirety.
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For example, geranylgeraniol is a linear terpene containing four
isoprene units, corresponding to the following chemical structure.
OH
The geranylgeraniol derivative, geranylgeranyl pyrophosphate is an
intermediate in the cholesterol biosynthetic pathway and is a substrate in the
prenylation of proteins. See J.A. Glomset et 1., Geranylgeranylated proteins,
Biochem-Soc-Trans., 1992 May, 20(2): 479-484, which is incorporated by
reference
herein in its entirety. Certain of these proteins, for example the small
GTPases Rac,
Rho, and Cdc42, regulate cytoskeletal function.
In cell cultures, geranylgeraniol is found to block apoptosis, i.e.
programmed cell death that can be induced by an HMG-CoA reductase inhibitor.
However, geranylgeraniol and its derivatives have not previously been
investigated
either in vitro or in vivo for their ability to mitigate the potentially
adverse myopathy
side effects that can be associated with HMG-CoA reductase inhibitor therapy
for
treating or preventing elevated blood cholesterol.
In the present invention, it is found that the combination of an HMG-
CoA reductase inhibitor and a geranylgeraniol compound is effective for
treating or
preventing elevated blood cholesterol while mitigating the potentially adverse
myopathy side effects that can be associated with the therapy. The combination
has
the advantage of providing increased safety and better patient compliance,
which
should maximize therapeutic efficacy. Without being limited by theory it is
believed
that the geranylgeraniol compound blocks the potentially harmful effect of the
HMG-
CoA reductase inhibitor on muscle cells. In other words, the geranylgeraruol
compound is believed to interfere with apoptosis, or functional impair due to
reduced
geranylgeranylation, which can potentially be induced in muscle cells by the
HMG-
CoA reductase inhibitor.
It is an object of the present invention to provide compositions
comprising the combination of an HMG-CoA reductase inhibitor and a
geranylgeraniol
compound.
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It is another object of the present invention to provide methods for
treating or preventing elevated blood cholesterol in a mammal, particularly
wherein
said mammal is a human.
It is another object of the present invention to provide such methods
while counteracting potential adverse myopathy effects.
It is another object of the present invention to provide such methods
wherein the dosing is maintained until the desired therapeutic effect is
achieved and/or
maintained.
These and other objects will become readily apparent from the detailed
description which follows.
SUMMARY OF THE INVENTION
The present invention relates to a pharmaceutical composition
comprising an HMG-CoA reductase inhibitor and a geranylgeraniol compound.
In further embodiments the present invention relates to a
pharmaceutical composition comprising a pharmaceutically-effective amount of
an
HMG-CoA reductase inhibitor and an amount of a geranylgeraniol compound
effective
to counteract HMG-CoA reductase inhibitor-associated myopathy.
In further embodiments, the present invention relates to a method for
treating or preventing elevated blood cholesterol in a mammal in need thereof
comprising administering an HMG-CoA reductase inhibitor and a geranylgeraniol
compound.
In further embodiments, the present invention relates to a method for
treating or preventing elevated blood cholesterol in a mammal in need thereof
comprising sequentially administering a geranylgeraniol compound and an HMG-
CoA
reductase inhibitors.
In further embodiments, the present invention relates to the use of a
composition in the manufacture of a medicament for treating or preventing
elevated
blood cholesterol in a mammal in need thereof, said composition comprising an
HMG-
CoA reductase inhibitor and a geranylgeraniol compound.
In further embodiments, the present invention relates to the use of a
composition comprising an HMG-CoA reductase inhibitor and a geranylgeraniol
compound for treating or preventing elevated blood cholesterol in a mammal in
need
thereof.
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All percentages and ratios used herein, unless otherwise indicated, are
by weight. The invention hereof can comprise, consist of, or consist
essentially of the
essential as well as optional ingredients, components, and methods described
herein.
BRIEF DESCRIPTION OF THE FIGURE
Figure 1 shows that activation of Mstl cleavage by 10 p,M lovastatin is
blocked by geranylgeraniol. Osteoclast-like cells are purified from cocultures
by
sequential treatment of culture dishes with collagenase and EDTA. Cells are
then
treated for 17 hours with lovastatin. Cell lysates are made and then analyzed
by an in-
gel kinase assay using myelin basic protein as a substrate. Lane 1 is a no-
treatment
control. Lane 2 shows treatment with 10 N.M lovastatin. Lane 3 shows treatment
with
the combination of 1.0 N.M Lovastatin and 10 p.M geranylgeraruol.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to compositions and methods for treating
or preventing elevated blood cholesterol in a mammal in need of such
treatment, while
counteracting the occurence of adverse myopathy effects. The compositions
comprise
a pharmaceutically effective amount of an HMG-CoA reductase inhibitor and a
pharmaceutically effective amount of a geranylgeraniol compound.
The term "pharmaceutically effective amount", as used herein, means
that amount of the HMG-CoA reductase inhibitor or geranylgeraniol compound
that
will elicit the desired therapeutic effect or response or provide the desired
benefit when
administered in accordance with the desired treatment regimen. A prefered
pharmaceutically effective amount of the HMG-CoA reductase inhibitor is an
amount
that is effective for treating or preventing elevated blood cholesterol. A
preferred
pharmaceutically effective amount of the geranylgeraruol compound is an amount
that
will block or mitigate the occurrence of adverse myopathy effects, while not
blocking,
or only minimally blocking, the therapeutic blood cholesterol effects of the
HMG-CoA
reductase inhibitor.
The term "counteracting the occurence of adverse myopathy effects",
as used herein, means preventing, decreasing, or lessening the occurrence of
unwanted
side effects in the muscular effects, relative to treatment with a HMG-CoA
reductase
inhibitor alone.
The term "until the desired therapeutic effect is achieved and/or
3 S maintained", as used herein, means that the therapeutic agent or agents
are
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continuously administered, according to the dosing schedule chosen, up to the
time
that the clinical or medical effect sought for the disease or condition being
treated is
observed by the clinician or researcher. For methods of treatment of the
present
invention, the pharmaceutical composition is continuously administered until
the
desired change in blood cholesterol is observed. In such instances, achieving
a
decrease in blood cholesterol is a desried objective. For methods of
prevention of the
present invention, the pharmaceutical composition is continuously administered
for as
long as necessary to prevent the undesired condition. In such instances,
maintenance
of blood cholesterol level is often an objective as well as prevention of or
reducing the
risk of developing atherosclerotic disease or cardiovascular disorders such as
heart
attack and stroke.
Compositions of the present invention
The pharmaceutical compositions of the present invention comprise a
pharmaceutically effective amount of an HMG-CoA reductase inhibitor and a
pharmaceutically effective amount of a geranylgeraniol compound. These
compositions are useful for treating or preventing elevated blood cholesterol
in a
mammal in need thereof while counteracting the potentially adverse effects,
such as
myopathy, that can be associated with the administration of the HMG-CoA
reductase
inhibitor.
HMG-CoA Reductase Inhibitor
The compositions herein comprise a compound which inhibits the
enzyme, HMG-CoA reductase. Compounds which have inhibitory activity for HMG-
CoA reductase can be readily identified by using assays well-known in the art.
See
U.S. Patent No. 4,231,938, to Monoghan et al., issued November 4, 1980 and
U.S.
Patent No. 5,354,772, to Kathawal, issued October 11, 1994, both of which are
incorporated by reference herein in their entirety.
Examples of HMG-CoA reductase inhibitors that are useful herein
include but are not limited to lovastatin (MEVACOR~; see U.S. Patent No.
4,231,938, already cited above and incorporated by reference herein),
simvastatin
(ZOCOR~; see U.S. Patent No. 4,444,784, to Hofflnan et al., issued April 24,
1984),
pravastatin (PRAVACHOL~; see U.S. Patent No. 4,346,227, to Terahara et al.,
issued August 24, 1982), fluvastatin (LESCOL~; see U.S. Patent No. 5,354,772,
already cited above and incorporated by reference herein), atorvastatin
(LIPITOR~;
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see U.S. Patent No. 5,273,995, to Roth, issued December 28, 1993) and
cerivastatin
(also known as rivastatin; see U.S. Patent No. 5,177,080, to Angerbauer et
al., issued
January 5, 1993); and mevastatin (compactin, see U.S. Patent No. 3,983,140, to
Endo
et al, issued September 28, 1976. The patents cited in the previous sentence
not
already incorporated by reference are also incorporated by reference herein in
their
entirety. The structural formulas of these and additional HMG-CoA reductase
inhibitors that can be used in the present invention are described at page 87
of M.
Yalpani, "Cholesterol Lowering Drugs", Chemistry & Industry, pp. 85-89 (5
February
1996), which is incorporated by reference herein in its entirety. The term HMG-
CoA
reductase inhibitor is intended to include all pharmaceutically acceptable
lactone and
open acid (that is where the lactone ring is opened to form the free acid), as
well as
salt and ester forms of compounds which have HMG-CoA reductase inhibitory
activity, and therefor the use of such lactone, open acid, salt, and ester
forms is
included within the scope of this invention. Preferably, the HMG-CoA reductase
inhibitor is selected from the group consisting of Iovastatin, simvastatin,
pravastatin,
fluvastatin, atorvastatin, cerivastatin, mevastatin, and the pharmaceutically
acceptable
lactones, open acids, salts, and esters thereof, and mixtures thereof. More
preferably,
the HMG-CoA reductase inhibitor is selected from the group consisting of
lovastatin,
simvastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, and the
pharmaceutically
acceptable lactones, open acids, salts, and esters thereof, and mixtures
thereof. More
preferably, the HMG-CoA reductase inhibitor is selected from the group
consisting of
lovastatin, simvastatin, and the pharmaceutically acceptable lactones, open
acids, salts,
and esters thereof, and mixtures thereof.
Preferred HMG-CoA reductase inhibitors can be represented by the
chemical formula
HO O
O
Z (I)
wherein Z is selected from the group consisting of
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a)
O
R~ Q
CH3
_a b~.. c d.l ~R2)n
R3
wherein R 1 i s C 1-C l 0 alkyl,
R2 is selected from the group consisting of C1-C3 alkyl, hydroxy, oxo, and C1-
C3
hydroxy substituted alkyl,
R3 is selected from the group consisting of hydrogen, hydroxy, C 1-C3 alkyl,
and C 1-
C3 hydroxy substituted alkyl,
a, b, c, and d are all single bonds, or a and c are double bonds, or b and d
are double
bonds, or one of a, b, c, and d is a double bond, and
n is 0, 1, or 2;
b)
F
X
wherein X is selected from the group consisting of N[CH(CH3)2] and CH(CH2)3CH3
c)
~ Me
/ I N\-.
N '~N
F
_g_
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d)
e)
F
and
f)
R~
Rs~J N~N,CH3
R4 1 /
N=N
wherein R4 and RS are each independently selected from the group consisting of
hydrogen, fluorine, chlorine, bromine, iodine, C1-C4 alkyl, Cl-C4 alkoxy, and
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trifluoromethyl, and R.6, R7, Rg, and R9 are each independently selected from
the
group consisting of hydrogen, fluorine, chlorine, bromine, iodine, C1-C4
alkyl, and
C1-C4 alkoxy. See U.S. Patent No. 5,650,523, to Decamp et al., issued July 22,
1997, which is incorporated by reference herein in its entirety. The
pharmaceutically
acceptable lactone, open acid, salt, and ester forms of the compounds depicted
by the
preceding chemical formulas are intended to be within the scope of the present
invention.
The term "pharmaceutically acceptable salts" as used herein in referring
to the HMG-CoA reductase inhibitors shall mean non-toxic salts of the
compounds
employed in this invention which are generally prepared by reacting the free
acid with a
suitable organic or inorganic base. Examples of salt forms of HMG-CoA
reductase
inhibitors include, but are not limited to, acetate, benzenesulfonate,
benzoate,
bicarbonate, bisulfate, bitartrate, borate, bromide, calcium, calcium edetate,
camsylate,
carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate,
edisylate, estolate,
esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate,
hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynapthoate,
iodide, isothionate, lactate, lactobionate, laurate, malate, maleate,
mandelate, mesylate,
methylbromide, methylnitrate, methylsulfate, mutate, napsylate, nitrate,
oleate,
oxalate, pamaote, palmitate, panthothenate, phosphate/diphosphate,
polygalacturonate,
potassium, salicylate, sodium, stearate, subacetate, succinate, tannate,
tartrate,
teoclate, tosylate, triethiodide, valerate, and mixtures thereof.
The term "esters" as used herein in referring to the HMG-CoA
reductase inhibitors is used in its standard meaning to denote the
condensation product
of a carboxylic acid and an alcohol. Ester derivatives of the described
compounds can
function as prodrugs which, when absorbed into the bloodstream of a warm-
blooded
animal, can cleave in such a manner as to release the drug form and permit the
drug to
afford improved therapeutic efficacy.
The term "lactones" is used herein in referring to the HMG-CoA
reductase inhibitors is used in its standard meaning to denote a cyclic
condensation
product of a carboxylic acid and an alcohol, i.e. a cyclic ester.
The term "open acid" is used herein in referring to the HMG-CoA
reductase inhibitors to denote that the lactone ring is open, i.e. uncyclized,
to form the
free acid.
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It is recognized that mixtures of two or more HMG-CoA reductase
inhibitors can be utilized.
The dosage regimen utilizing a HMG-CoA reductase inhibitor is
selected in accordance with a variety of factors including type, species, age,
weight,
sex and medical condition of the patient; the severity of the condition to be
treated; the
route of administration; the renal and hepatic function of the patient; and
the particular
compound or salt or ester thereof employed. A consideration of these factors
is well
within the purview of the ordinarily skilled clinician for the purpose of
determining the
therapeutically effective or prophylactically effective dosage amounts needed
to
prevent, counter, or arrest the progress of the condition. The term "patient"
includes
mammals, especially humans, who take an HMG-CoA reductase inhibitor or
combination for any of the uses described herein. Administering of the drug or
drugs
to the patient includes both self administration and administration to the
patient by
another person.
The precise dosage of the HMG-CoA reductase inhibitor will vary with
the dosing schedule, the particular compound chosen, the age, size, sex and
condition
of the mammal or human, the nature and severity of the disorder to be treated,
and
other relevant medical and physical factors. Thus, a precise pharmaceutically
effective
amount cannot be specified in advance and can be readily determined by the
caregiver
or clinician. Appropriate amounts can be determined by routine experimentation
from
animal models and human clinical studies.
In particular, for daily dosing, the amounts of the HMG-CoA reductase
inhibitor can be the same or similar to those amounts which are employed for
anti-
hypercholesterolemic treatment and which are described in the Physicians' Desk
Reference (PDR), 52nd Ed. of the PDR, 1998 (Medical Economics Co), which is
incorporated by reference herein in its entirety. For the additional active
agents, the
doses can be the same or similar to those amounts which are known in the art.
The HMG-CoA reductase inhibitors can be administered via a wide
variety of routes including oral administration, intravenous administration,
intranasal
administration, injections, ocular administration, and the like.
A preferred route of delivery is oral administration.
Oral dosage amounts of the HMG-CoA reductase inhibitor are from
about 1 to 200 mg/day, and more preferably from about 5 to 160 mg/day.
However,
dosage amounts will vary depending on the potency of the specific HMG-CoA
reductase inhibitor used as well as other factors as noted above. An HMG-CoA
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reductase inhibitor which has su~ciently greater potency may be given in sub-
milligram daily dosages. The HMG-CoA reductase inhibitor may be administered
from
1 to 4 times per day, and preferably once per day.
For example, the daily dosage amount for simvastatin can be selected
from 5 mg, 10 mg, 20 mg, 40 mg, and 80 mg; for lovastatin, 10 mg, 20 mg, 40 mg
and
80 mg; for fluvastatin sodium, 20 mg, 40 mg and 80 mg; for pravastatin sodium,
10
mg, 20 mg, and 40 mg; and for atorvastatin calcium, 10 mg, 20 mg, and 40 mg.
Geran~lsteraniol Compounds
The compositions of the present invention comprise a pharmaceutically
effective amount of a geranylgeraniol compound.
The geranylgeraniol compounds useful herein correspond to the
chemical formula
~ ~ ~ ~ OR~o
wherein R10 is selected from the group consisting of H (i.e. geranylgeraniol),
C1-C30
alkyl (including straight, branched, and cyclic alkyl), C2-C30 alkenyl
(including
straight, branched, and cyclic alkenyl), C2-C30 alkynyl (including straight,
branched,
and cyclic alkynyl), CS-C14 aryl, P03H2 (i.e. geranylgeranyl phosphate),
P20~H3
(i.e. geranylgeranyl pyrophosphate), C=O-R11 (i.e. esters), wherein R11 is
selected
from the group consisting of H, C 1-C 10 alkyl (including straight, branched,
and cyclic
alkyl), C2-C 10 alkenyl (including straight, branched, and cyclic alkenyl), C2-
C 10
alkynyl (including straight, branched, and cyclic alkynyl), C2-C10 hydroxy-
substituted
alkyl (including straight, branched, and cyclic), C2-C 10 amino-substituted
alkyl
(including straight, branched, and cyclic), C2-C10 carbonylhydroxy-substituted
alkyl
(including straight, branched, and cyclic), and CS-C14 aryl, and n is an
integer from 0
to 3.
Preferably R10 is selected from the group consisting of H, P03H2,
P20~H3 , and C=O-R11, wherein R11 is selected from the group consisting ofH,
C1-
C 10 alkyl, C2-C 10 hydroxy-substituted alkyl, C2-C 10 amino-substituted
alkyl, C2-
C10 carbonylhydroxy-substituted alkyl, and CS-C14 aryl, and n is an integer
from 2 to
3.
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More preferably R10 is selected from the group consisting of H,
P03H2, P20~H3 , and C=O-R11, wherein R11 is selected from the group consisting
of H, C 1-C 10 alkyl, C2-C 10 hydroxy-substituted alkyl, C2-C 10 amino-
substituted
alkyl, C2-C 10 carbonylhydroxy-substituted alkyl, and CS-C 14 aryl, and n is
3.
The term "aryl," as used herein, refers to a monocyclic or polycyclic
system comprising at least one aromatic ring, wherein the monocyclic or
polycyclic
system contains 0, 1, 2, 3, or 4 heteroatoms chosen from N, O, or S, and
wherein the
monocyclic or polycyclic system is either unsubstituted or substituted with
one or
more groups independently selected from hydrogen, halogen, C 1 _ 10 alkyl,
C3_8
cycloalkyl, aryl, aryl C 1 _g alkyl, amino, amino C 1 _g alkyl, C 1 _3
acylamino, C 1 _3
acylamino C 1 _g alkyl, C 1 _b alkylamino, C 1 _6 alkylamino C 1 _ g alkyl, C
1 _6
dialkylamino, C 1 _6 dialkylamino-C 1 _g alkyl, C 1 _4 alkoxy, C 1 _4 alkoxy C
1 _6 alkyl,
hydroxycarbonyl, hydroxycarbonyl C 1 _6 alkyl, C 1 _5 alkoxycarbonyl, C 1-3
alkoxycarbony! C 1 _6 alkyl, hydroxycarbonyl C 1 _6 alkyloxy, hydroxy, hydroxy
C 1 _6
alkyl, cyano, trifluoromethyl, oxo or C 1 _5 alkylcarbonyloxy. Examples of
aryl include,
but are not limited to, phenyl, naphthyl, pyridyl, pyrazinyl, pyrimidinyl,
imidazolyl,
benzimidazolyl, indolyl, thienyl, furyl, dihydrobenzofuryl, benzo(1,3)
dioxolane,
oxazolyl, isoxazolyl and thiazolyl.
The esters are also intended to encompass esters of substituted acids
such as lactic acid, amino acids, and other complex acids, and mono and higher
esters
of di and higher carboxylic acids such as succinic acid and glutaric acid.
Preferred geranylgerniol compounds are selected from the group
consisting of geranylgeraniol, geranylgeranyl ethyl ether, geranylgeranyl
phosphate,
geranylgeranyl pyrophosphate, geranylgeranyl acetate, geranylgeranyl
propionate,
geranylgeranyl benzoate, geranylgeranyl lactate, geranylgeranyl succinate,
geranylgeranyl glutarate, and mixtures thereof.
More preferred are geranylgeraniol, geranylgeranyl phosphate,
geranylgeranyl pyrophosphate, geranylgeranyl acetate, geranylgeranyl
propionate,
geranylgeranyl benzoate, geranylgeranyl lactate, geranylgeranyl succinate,
geranylgeranyl glutarate, and mixtures thereof.
Even more preferred herein are geranyigeraniol, geranylgeranyl
pyrophosphate, and mixtures thereof.
It is recognized that mixtures of two or more of the geranylgeraniol
compounds can be utilized.
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The precise dosage of the geranylgeraniol compounds will also vary
with the dosing schedule, the particular compound chosen, the age, size, sex
and
condition of the mammal or human, the nature and severity of the disorder to
be
treated, and other relevant medical and physical factors. Thus, a precise
pharmaceutically effective amount cannot be specified in advance and can be
readily
determined by the caregiver or clinician. Appropriate amounts can be
determined by
routine experimentation from animal models and human clinical studies.
Generally, an
appropriate amount is chosen to counteract the potential adverse myopathy
effects of
the HMG-CoA reductase inhibitor. The amount should be below that level which
will
inhibit the desired cholesterol lowering effect of the HMG-CoA reductase
inhibitor.
For humans, an effective oral dose of the geranylgernaiol compound is
typically chosen
so as to provide a concentration in the blood stream from about I pM to about
100
~M, preferably about IO p.M, although other ranges can be used. Nonlimiting
exemplary doses are about 1 ug/kg to about 100 ug/kg, preferably about 10
ug/kg, for
a human subject.
For the geranylgeraniol compound, human doses which can be
administered are generally in the range of about 0.1 mg/day to about 10
mg/day,
preferably from about 0.25 mg/day to about 5 mg/day, and more preferably from
about
0.5 mg/day to about 1.5 mg/day, based on a geranylgeraniol active weight
basis. A
typical nonlimiting dosage amount would be about 0.75 mg/day. The
pharmaceutical
compositions herein comprise from about 0.1 mg to about 10 mg, preferably from
about 0.25 mg to about 5 mg, and more preferably from about 0.5 mg to about
1.5 mg
of.the geranylgeraniol compound. A typical nonlimiting amount for is about
0.75 mg.
Other components of the pharmaceutical compositions
The HMG-CoA reductase inhibitor and the geranylgeraniol compound
are typically administered in admixture with suitable pharmaceutical diluents,
excipients, or carriers, collectively referred to herein as "carner
materials", suitably
selected with respect to oral administration, i.e. tablets, capsules, elixirs,
syrups,
powders, and the like, and consistent with conventional pharmaceutical
practices. For
example, for oral administration in the form of a tablet, capsule, or powder,
the active
ingredient can be combined with an oral, non-toxic, pharmaceutically
acceptable inert
carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium
stearate,
mannitol, sorbitol, croscarmellose sodium and the like; for oral
administration in liquid
form, e.g., elixirs and syrups, the oral drug components can be combined with
any
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oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol,
glycerol,
water and the like. Moreover, when desired or necessary, suitable binders,
lubricants,
disintegrating agents and coloring agents can also be incorporated. Suitable
binders
can include starch, gelatin, natural sugars such a glucose, anhydrous lactose,
free-flow
lactose, beta-lactose, and corn sweeteners, natural and synthetic gums, such
as acacia,
guar, tragacanth or sodium alginate, carboxymethyl cellulose, polyethylene
glycol,
waxes, and the like. Lubricants used in these dosage forms include sodium
oleate,
sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium
chloride and the like. The compounds used in the present method can also be
coupled
with soluble polymers as targetable drug carriers. Such polymers can include
polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropyl-methacrylamide, and
the
like.
Methods of the Present Invention
The present invention comprises methods for treating or preventing
elevated blood cholesterol in mammals. In preferred embodiments of the present
invention, the mammal is a human.
The compositions and methods of the present invention are
administered and caned out until the desired therapeutic effect is achieved.
In the methods of the present invention the HMG-CoA reductase
inhibitor and the geranylgeraniol compound are generally administered
concurrently.
In alternate embodiments, the HMG-CoA reductase inhibitor and the
geranylgeraniol
compound can be adminsitered sequentially. Preferably, the gernaylgeraniol
compound is administered first.
The following Examples are presented to better illustrate the invention.
EXAMPLE 1
Method for Evaluating the Effect of a HMG-CoA Reductase Inhibitor and a
Geranvlszeraniol Compound on Kinase Activities in Cultured Osteoclasts
Murine co-cultures of osteoblasts and marrow cells are prepared using
the methods of Wesolowski, et al., Exp Cell Res, (1995), 219, pp. 679-686,
which is
incorporated by reference herein in its entirety. Bone marrow cells are
harvested from
6-week-old male Balb/C mice by flushing marrow spaces of freshly isolated long
bones
(tibiae and femora) with a-MEM (minimal essential media) containing
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penicillin/streptomycin (100 LU./ml of each and 20 mM Hepes buffer). The bone
marrow cells are suspended in a-MEM and the cells are filtered through an
approximately 70 p,m cell strainer. The filtrate is centrifuged at about 300 x
g for
about 7 minutes. The resulting pellet is resuspended in a-MEM supplemented
with
fetal calf serum (10 % v/v) and 10 nM 1, 25-(OH)2 vitamin D3. These bone
marrow
isolates are added to sub-confluent monolayers of osteoblastic MB 1.8 cells in
cell
culture plates and cultured for 7 days at 370C in the presence of 5% C02.
Culture
media is replenished ever other day. Fusion of the osteoclast precursor cells
from bone
marrow (with each other) to form multinucleated osteoclast-like cells
typically occurs
after about 7 days. Osteoclast-like cells are enriched by sequential treatment
with
collagenase (1 mg/mL in phosphate buffered saline) for one hour at 37°C
and EDTA
(0.2 g/L in phosphate buffered saline) for 20 min at 37°C. Non-adherent
cells are
rinsed away by washing with phosphate buffered saline. Osteoclast-like cells
which are
resistant to the sequential treatments are present at about 95% purity and are
maintained in a-MEM supplemented with fetal calf serum {10 % v/v), 10 nM 1,25-
(OH)2 vitamin D3, macrophage-colony-stimulating factor (5 ng/mL).
The compounds to be evaluated are prepared as a solution of the
desired concentration in a-MEM. Examples of compounds that can be evaluated
include HMG-CoA reductase inhibitors such lovastatin, simvastatin,
pravastatin,
fluvastatin, atorvastatin, cerivastsin, mevastatin, and the pharmaceutically
acceptable
salts, esters, and lactones thereof, as well as compounds that block the
effects of these
HMG-CoA reductase inhibitors, such as geranylgeraniol compounds, for example,
geranylgeraniol, geranylgeranyl ethyl ether, geranylgeranyl phosphate,
geranylgeranyl
pyrophosphate, geranylgeranyl acetate, geranylgeranyl propionate,
geranylgeranyl
benzoate, geranylgeranyl lactate, geranylgeranyl succinate, geranylgeranyl
glutarate.
Combinations of compounds can also be evaluated. The solutions of the
compounds
to be evaluated are added to the cultures for a time period of 17-24 hours. No
treatment controls (controls not treated with comcpounds) are prepared by
adding
equivalent volumes of a-MEM to the control dishes.
Cells are then harvested and lysed in a HEPES (N-(2-
hydroxyethyl)piperazine-N'-(2-ethansulfonic acid) or Tris buffer containing
the
following: ~i-glycerophosphate (50 mM); Na3V04 (1mM); NaF (1mM); Microcystin
LR (1 p.M); leupeptin (10 pg/ml); aprotinin (10 p,g/ml); phenylmethyl
sulfonylfluoride
(1 mM). Protein concentrations are determined for each lysate and S-20 p,g are
loaded
into each lane of a SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel
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electrophoresis) gel containing Myelin Basic Protein, or another kinase
substrate,
which has been polymerized into the gel at a concentration between 50-400
p,g/ml.
Molecular weight standards are also loaded into one or more lanes of the gels.
In-gel
kinase assays are run according to a standard procedure based on Kameshita and
S Fujisawa, 1989 (Anal. Biochem. 183:139-143) and of Gotoh et al., 1990 (Eur.
J.
Biochem. 193: 661-669), both references being incorporated by reference herein
in
their entirety. The proteins are electrophoresed in the above gels. The gels
are then
successively soaked in 50 mM HEPES, pH 7.6; S mM 2-mercaptoethanol and each of
the following (for each wash): (a) 20% isopropanol; (b) no additions; (c) urea
(6 M);
(d) Urea (3 M); (e) Urea (0.75 M); and Tween 20 (0.05% vol:vol). Kinase
reactions
are then run by first soaking the gels in 20 mM HEPES, pH 7.6; 20 mM MgCl2; 2
mM
DTT and then in the same buffer containing 0.02 M ATP (non-radioactive) with
ca.
1000 cpm/pmol 32P-y-ATP. The gels are then washed six times with 5%
trichloroacetic acid and 1% pyrophosphate. The gels are then stained with
Coomassie
brilliant blue dye (0.125%) in 50% methanol, 10% acetic acid; destained with
30%
methanol, 10% acetic acid; soaked in 2% glycerol; and dried using a gel dryer.
The
gels are then exposed to autoradiography film for times ranging from several
hours to
weeks. The bands observed in the autoradiographs representing the gels reflect
kinase
activities. Mst 1 (apparent molecular weight about 59 kDa), Mst 2 (apparent
molecular weight about 60 kDa), and a 34 kDa Mst kinase fragment are observed
and
identified by their migration as compared to the migration of molecular weight
standards. The band intensities on the autoradiography film are quantitated by
densitometry and comparisons between bands from untreated controls and bands
from
echistatin-treated cells provide the basis for the analyses.
EXAMPLE 2
Tablet com osp ition
In redient Amount per tablet
Simvastatin 10.0 mg
Geranylgeraniol 0.75 mg
B~ 0.02mg
Ascorbic acid 2.50 mg
Citric acid 1.25 mg
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Microcrystalline cellulose 5.0 mg
Pregel starch 10.0 mg
Magnesium stearate 0.5 mg
Lactose 74.73 mg
All the ingredients except magnesium stearate are blended together in a
suitable mixer. The powder mixture is then granulated with adequate quantities
of
granulating solvent(s), e.g. water. The wet granulated mass is dried in a
suitable dryer.
The dried granulation is sized through a suitable screen. The sized
granulation is mixed
with magnesium stearate before tableting. The tablets may be coated if deemed
necessary. Additional ingredients that may be added to the above include
suitable
color and mixtures of colors.
The composition is useful for treating or preventing elevated blood
cholesterol.
In alternative formulations, the simvastatin is replaced by an HMG-CoA
reductase inhibitor selected from lovastatin, pravastatin, fluvastatin,
atorvastatin,
cerivastatin, or mevastatin, and the geranylgeraniol is replaced by
geranylgeranyl
phosphate, geranylgeranyl pyrophosphate, geranylgeranyl acetate,
geranylgeranyl
propionate, geranylgeranyl benzoate, geranylgeranyl lactate, geranylgeranyl
succinate,
or geranylgeranyl glutarate
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EXAMPLE 3
Directly compressed tablet com osition
Amount per tablet Ingredient
mg Lovastatin
0.75 mg Geranylgeraniol
116.9 mg Microcrystalline cellulose
116.9 mg Lactose anhydrite
7.5 mg ~ Crosmeiiose sodium
3.7 mg Magnesium stearate
5 The ingredients are combined and blended together and are compressed
using conventional tableting techniques.
The composition is useful for treating or preventing elevated blood
cholesterol.
In alternative formulations, the simvastatin is replaced by an HMG-CoA
10 reductase inhibitor selected from lovastatin, pravastatin, fluvastatin,
atorvastatin,
cerivastatin, or mevastatin, and the geranylgeraniol is replaced by
geranylgeranyl
phosphate, geranylgeranyl pyrophosphate, geranylgeranyl acetate,
geranylgeranyl
propionate, geranylgeranyl benzoate, geranylgeranyl lactate, geranylgeranyl
succinate,
or geranylgeranyl glutarate
EXAMPLE 4
Hard elatin c~sule composition
Amount per capsule In edient
10 mg Simvastatin
0.75 mg Geranylgeraniol
47 mg Microcrystalline cellulose
47 mg Lactose anhydrite
1 mg Magnesium stearate
1 capsule Hard gelatin capsule
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The dry ingredients are combined and blended together and
encapsulated in a gelatin coating using standard manufacturing techniques.
The composition is useful for treating or preventing elevated blood
cholesterol.
In alternative formulations, the simvastatin is replaced by an HMG-CoA
reductase inhubitor selected from lovastatin, pravastatin, fluvastatin,
atorvastatin,
cerivastatin, or mevastatin, and the geranylgeraniol is replaced by
geranylgeranyl
phosphate, geranylgeranyl pyrophosphate, geranylgeranyl acetate,
geranylgeranyl
propionate, geranylgeranyl benzoate, geranylgeranyl lactate, geranylgeranyl
succinate,
or geranylgeranyl glutarate
EXAMPLE 5
Oral suspension composition
Amount,~er 5 mL dose Ingredient
10 mg Lovastatin
0.75 mg Geranylgeraniol
150 mg Polyvinylpyrrolidone
2. S mg Poly oxyethylene sorbitan monolaurate
10 mg Benzoic acid
to 5 mL with aqueous sorbitol solution
(70%)
An oral suspension is prepared by combining the ingredients using
standard formulation techniques.
The composition is useful for treating or preventing elevated blood
cholesterol.
In alternative formulations, the simvastatin is replaced by an HMG-CoA
reductase inhubitor selected from lovastatin, pravastatin, fluvastatin,
atorvastatin,
cerivastatin, or mevastatin, and the geranylgeraniol is replaced by
geranylgeranyl
phosphate, geranylgeranyl pyrophosphate, geranylgeranyl acetate,
geranylgeranyl
propionate, geranylgeranyl benzoate, geranylgeranyl lactate, geranylgeranyl
succinate,
or geranylgeranyl glutarate
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EXAMPLE 6
Intravenous infusion composition
Amount per 200mL doseIngredient
mg Simvastatin
0.75 mg Geranylgeraniol
0.2 mg Polyethylene oxide
400
1.8 mg Sodium chloride
to 200mL Purified water
The ingredients are combined using standard formulation techniques.
The composition is useful for treating or preventing elevated blood
cholesterol.
In alternative formulations, the simvastatin is replaced by an HMG-CoA
reductase inhibitor selected from lovastatin, pravastatin, fluvastatin,
atorvastatin,
10 cerivastatin, or mevastatin, and the geranylgeraniol is replaced by
geranylgeranyl
phosphate, geranylgeranyl pyrophosphate, geranyigeranyl acetate,
geranylgeranyl
propionate, geranylgeranyl benzoate, geranylgeranyl lactate, geranylgeranyl
succinate,
or geranylgeranyl glutarate
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