Note: Descriptions are shown in the official language in which they were submitted.
)7~
HX24
--1--
COMBINATION OF SOUALENE SYNTHETASE
INHIBITOR AND OTHER TYPE OF SERUM
CHOLESTEROL REDUCING AGENT AND METHOD FOR
LOWERING SERUM CHOLESTEROL USING SUCH COMBINATION
The present invention relates to a
combination of an inhibitor of squalene synthetase
and a pharmaceutical which reduces serum
cholesterol other than by inhib.ting squalene
synthetase, and to a method for lowering serum
cholesterol and/or preventing or treating
atherosclerosis by administering such combination.
There are several different classes of
compounds which have serum cholesterol lowering
properties. Some of these compounds are
inhibitors of the enzyme HMG CoA reductase which
is essential in the production of cholesterol,
such as mevastatin (disclosed in U. S. Patent No.
3,983,140), lovastatin also referred to as -
mevinolin (disclosed in U. S. Patent No.
4,231,938), pravastatin (disclosed in U. S. Patent
No. 4,346,227) and velostatin also referred to as
synvinolin (disclosed in U. S. Patents Nos.
4,448,784 and 4,450,171).
7~
HX24
--2--
Other compounds which lower serum
cholesterol may do so by an entirely different
mechanism than the HMG CoA reductase inhibitors.
For example, serum cholesterol may be lowered
through the use of bile acid seguestrants such as
cholestyramine, colestipol, DEAE-Sephadex and
poly(diallylmethylamine) derivatives (such as
disclosed in U. S. Patents Nos. 4,759,923 and
4,027,009) or through the use of antihyperlipo-
proteinemics such as probucol and gemfibrozilwhich apparently lower serum "low density
lipoproteins" (LDL) and/or convert LDL into high
density lipoproteins (HDL).
U. S. Patent No. 4,759,923 mentioned above
discloses that poly(diallylmethylamine) derivatives
which are bile salt sequestrants may be used in
conjunction with drugs which reduce serum
cholesterol by mechanisms other than sequestration,
such as clofibrate, nicotinic acid, probucol,
neomycin, p-aminosalicylic acid or mevinolin (also
referred to as lovastatin).
Sgualene synthetase is a microsomal enzyme
which catalyzes the reductive dimerization of two
molecules of farnesyl pyrophosphate (FPP) in the
presence of nicotinamide adenine dinucleotide
phosphate ~reduced form) (NADPH) to form squalene
(Poulter, C. D.; Rilling, H. C., in "Biosynthesis
of Isoprenoid Compounds", Vol. I, Chapter 8, pp.
413-441, J. Wiley and Sons, 1981 and references
therein). This enzyme is the first committed step
of the de novo cholesterol biosynthetic pathway.
The selective inhibition of this step should allow
the essential pathways to isopentenyl tRNA,
7~
--3--
ubiquinone, and dolichol to proceed unimpeded.
Squalene synthetase, along with HMG-CoA reductase
has been shown to be down-regulated by receptor
mediated LDL uptake (Faust, J. R.; Goldstein,
J. L.; Brown, M. S. Proc. Nat. Acad. Sci. USA,
1979, 76, 5018-5022), lending credence to the
proposal that inhibiting squalene synthetase will
lead to an up-regulation of LDL receptor levels,
as h~s been demonstrated for HMG-CoA reductase,
and thus ultimately should be useful for the
treatment and prevention of hypercholesterolemia
and atherosclerosis.
One approach to inhibitors of squalene
synthetase is to design analogs of the substrate
FPP. It is clear from the literature that the
pyrophosphate moiety is essential for binding to
the enzyme. However, such pyrophosphates are
unsuitable as components of pharmacological agents
due to their chemical and enzymatic lability
towards allylic C-O cleavage, as well as their
susceptibility to metabolism by phosphatases.
P. Ortiz de Montellano et al. in
J. Med. Chem., 1977, 20, 243-249 describe the
preparation of a series of substituted terpenoid
pyrophosphates (Table A), and have shown these to
be competitive inhibitors of the squalene
synthetase enzyme. These substances retain the
unstable allylic pyrophosphate moiety of FPP.
6~
HX24
-4-
Table A
'"j , \~ o
10 N X Y Z_
1 CH3 CH3 H
2 H H
3 C2H5 H H
4 I H H
15 5 H I H
6 CH3 H SCH3
Corey and Volante, J. Am. Chem. Soc. 1976,
98, 1291-3, have prepared FPP analog A and
presqualene pyrophosphate (PSQ-PP) analog B as
inhibitors of squalene biosynthesis. (Presqualene
pyrophosphate i8 an intermediate in the conversion
of FPP to squalene). These inhibitors possess
methylene groups in place of the allylic oxygen
moiety of FPP and PSQ-PP, but still retain the
chemically and enzymatically unstable
pyrophosphate linkage.
2~Q'7~
HX24
_5_
~ ~ r ~/\~--
A X = CH2
FPP X = O
/ ~\ O/ ~~
B X = CH2
PSQ-PP X = O
Poulter and co-workers have prepared
cyclopropane C (Sandifer, R. M., et al.,
J. Am. Chem. Soc. 1982, 104, 7376-8) which in the
presence of inorganic pyrophosphate is an
intermediate analog inhibitor of the enzyme
squalene synthetase.
;~Gi7~
HX24
-6-
Me
10 ~
Altman and co-workers, Bertolino, A.,
et al., Biochim. BioPhys. Acta. 1978, 530, 17-23,
lS reported that farnesyl amine and related
derivatives D inhibit squalene synthetase, but
provide evidence that this inhibition is
non-specific and probably related to membrane
disruption.
NH-R
R = H, CH2CH2OH, CH2CH2OCH3
Poulter, C.D., et al, J. Org. Chem., 1986,
51, 4768, prepared compound ~ in a demonstration of
a synthetic method, but did not report any
biological data.
HX24
-7-
~ O_~ Q-OH
~ ~H
3~ E
Poulter, C.D., Stremler, K.~., J.A.C.S.,
1987 109 5542 describes the synthesis and
biological evaluation of compounds having structure
F. These compounds were evaluated as alternative
substrates for avian liver farnesyl diphosphate and
lemon peel cyclase.
~ ~O-~-X-~-OH
2 0 F X=CH2 CF2
McClard, R. W. and Poulter, C. D., et al.,
J A C S 1987 109 5544 reported that
phosphinylphosphonates G and H were competitive
inhibitors of the 1'-4-condensation between
isopentenyl diphosphate and geranyl diphosphate
catalyzed by avian liver farnesyl diphosphate
synthetase. Phosphinylphosphonates G and H had
Ki's of l9~M and 71~M, respectively. They also
reported the speculative isolation of the farnesyl
phosphinylphosphonate I, and the geranyl
phosphinylphosphonate J from the enzymatic reaction
of G with geranyl pyrophosphate or dimethylallyl
~Q7~
HX24
--8--
pyrophosphate, respectively. The structures of I
and J were tentatively assigned based on relative
TLC mobilities. They hypothesized that I could be
a potentlal inhibitor of squalene synthetase.
s
~ - '
-- o--
G
~ O-
~ o
~ o_
b
~37~4~
HX24
_g_
Capson, T.L., PhD dissertation, June 1987,
Dept. of Medicinal Chemistry, the University of
Utah, Abstract, Table of Contents, pp. 16, 17,
40-43, 48-S1, Summary, discloses cyclopropanes of
the structure
0_ '
o~ 1-
as intermediate analog inhibitors of squalene
synthetase.
S. A. Biller et al., Journal of Medicinal
Chemistry, 1988, Vol. 31, No. 10, pp 1869 to 1871
disclose that isoprenoid (phosphinylmethyl)
phosphonates (PMPs) inhibit squalene synthetase.
These phosphonates have the structures
Rl-~-CH2-~-O- Rl-~-CF2-~-O-
2a-d 3a,b
Rl
2 ~ HX24
--10--
10 c
In accordance with the present invention, a
pharmaceutical combination is provided for use in
reducing serum cholesterol and in inhibiting
formation of,or treating atherosclerosis, which
combination is formed of an inhibitor of the enzyme
squalene synthetase and a pharmaceutical (also
referred to as other serum cholesterol lowering
agent) which reduces serum cholesterol and/or
inhibits cholesterol biosynthesis by a mechanism
other than by inhibiting production of the enzyme
squalene synthetase or 3-hydroxy-3-methyl-glutaryl
coenzyme A (HMG CoA~ reductase, such as a bile salt
sequestrant or antihyperlipoproteinemic agent which
inhibits formation of LDL or converts LDL to HDL.
The squalene synthetase inhibitor will be employed
in a weight ratio to the "pharmaceutical" of within
the range of from about 0.001:1 to about 1000:1 and
preferably from about 0.05:1 to about 100:1.
In addition, in accordance with the present
invention, a method is provided for lowering serum
cholesterol or inhibiting formation of or treating
~)7~
HX24
--11--
atherosclerosis wherein a therapeutically effective
amount of the above combination is systemically,
such as orally or parenterally, administered over a
prolonged period.
The combination of the squalene synthetase
inhibitor and other serum cholesterol lowering
agent, as described above, is a surprising and
unique concept in inhibiting or treating elevated
cholesterol and/or atherosclerosis in that it may
provide additional anticholesterolemic effects over
that which may be obtained using each of the
components of the combination alone. In addition,
the combination of the invention which includes
compounds with different mechanisms of action, may
be used to effectively treat cholesterol-related
diseases of multiple etiology.
The squalene synthetase inhibitors suitable
for use herein include, but are not limited to,
those disclosed by Biller et al., supra, including
isoprenoid (phosphinylmethyl)phosphonates such as
those of the formula
R1_p-CH2-P-0 R1-P-CF2-P-0
O O O O
I II
HX24
-12-
Rl :
~1 ~~
c
d ~
including the triacids thereof, triesters thereof
and tripotassium and trisodium salts thereof as
well a8 other squalene synthetase inhibitors
disclosed in pending U.S. patent application
Serial No. 141,744, filed January 11, 1988.
In addition, other squalene synthetase
inhibitors suitable for use herein include the
terpenoid pyrophosphates disclosed by P. Ortiz de
Montellano et al., J. Med. Chem.; 1977, 20,
243-249, the farnesyl diphosphate analog A and
presgualene pyrophosphate (PSQ-PP) analogs as
disclo8ed by Corey and Volante, J. Am. Chem. Soc.
1976, 98, 1291-1293, phosphinylphosphonates
reported by McClard, R. W. et al., J.A.C.S., 1987,
109, 5544 and cyclopropanes reported by Capson,
T.L., PhD dissertation, June, 1987, Dept. Med.
Chem. U. of Utah, abstract, Table of Contents,
pp. 16, 17, 40-43, 48-51, Summary, as well as other
known squalene synthetase inhibitors.
The "pharmaceutical" or other serum
cholesterol lowering agents which function other
7 ~ ~
HX24
-13-
than by inhibiting the enzyme HMG CoA reductase
or squalene synthetase suitable for use herein
include, but are not limited to, antihyper-
lipoproteinemic agents such as probucol, and
gemfibrozil and related compounds as disclosed in
U. S. Patent No. 3,674,836, probucol and
gemfibrozil being preferred, bile acid sequestrants
such as cholestyramine, colestipol and DEAE-
Sephadex (Secholex~, Polidexide~), as well as
clofibrate, lipostabil (Rhone-Poulenc), Eisai
E-5050 (an N-substituted ethanolamine derivative),
imanixil (HOE-402) tetrahydrolipstatin (THL),
istigmastanyl-phosphorylcholine (SPC, Roche),
aminocyclodextrin ~Tanabe Seiyoku), Ajinomoto
AJ-814 (azulene derivative), melinamide (Sumitomo),
Sandoz 58-035, American Cyanamid CL-277,082 and
CL-283,546 (di-substituted urea derivatives),
nicotinic acid, neomycin, p-aminosalicylic acid,
aspirin, poly(diallylmethylamine) derivatives such
as disclosed in U. S. Patent No. 4,759,923,
quaternary amine poly(diallyldimethylammonium
chloride) and ionenes such as disclosed in U. S.
Patent No. 4,027,009, and other known serum
cholesterol lowering agents which lower cholesterol
through a mechanism other than by the inhibition of
the enzyme HMG CoA reductase or squalene
synthetase.
Preferred are combinations of any of the
isoprenoid (phosphinylmethyl) phosphonates
disclosed by Biller et al., supra with probucol or
gemfibrozil.
The disclosure of the above-mentioned
patents and patent applications are incorporated
herein by reference.
X~3~)7~1
HX24
-14-
In carrying out the method of the present
invention, the combination of the invention may be
administered to mammalian species, such as monkeys,
dogs, cats, rats, humans, etc. and as such may be
incorporated in a conventional systemic dosage
form, such as a tablet, capsule, elixir or
injectable. The above dosage forms will also
include the necessary carrier material, excipient,
lubricant, buffer, antibacterial, bulking agent
(such as mannitol), anti-oxidants (ascorbic acid or
sodium bisulfite) or the like. Oral dosage forms
are preferred, although parenteral forms are quite
satisfactory as well.
The dose administered must be carefully
adjusted according to age, weight and condition of
the patient, as well as the route of
admini~tration, dosage form and regimen and the
desired result.
Thus, for oral administration, a satis-
factory result may be obtained employing the
squalene synthetase inhibitor in dosages in an
amount within the range of from about 10 to about
2000 mg and preferably from about 25 to about 200
mg in combination with the other serum cholesterol
lowering agent in dosages normally employed as
indicated in the Physician's Desk Reference, for
each of such agents such as in an amount within the
range of from about 2 mg to about 7500 mg and
preferably from about 2 mg to about 4000 mg with
the squalene synthetase inhibitor and other serum
cholesterol lowering agent being employed together
in the same oral dosage form or in separate oral
dosage forms taken at the same time.
2~36Ji7~
HX24
-15-
A preferred oral dosage form, such as
tablets or capsules, will contain the squalene
synthetase inhibitor in an amount of from about 10
to about 500 mg, preferably from about 25 to about
200 mg, and the other serum cholesterol lowering
agent in an amount of from about 2 to about 3000
mg, preferably from about 2 to about 2000 mg.
The composition described above may be
administered in the dosage forms as described above
in single or divided doses of one to four times
daily. It may be advisable to start a patient on a
low dose combination and work up gradually to a
high dose combination.
Tablets of various sizes can be prepared,
e.g., of about 2 to 2000 mg in total weight,
containing one or both of the active substances in
the ranges described above, with the remainder
being a physiologically acceptable carrier of other
materials according to accepted pharmaceutical
practice. These tablets can, of course, be scored
to provide for fractional doses. Gelatin capsules
can be similarly formulated.
Liquid formulations can also be prepared by
dissolving or suspending one or the combination of
active substances in a conventional liquid vehicle
acceptable for pharmaceutical administration so as
to provide the desired dosage in one to four
teaspoonsful.
Such dosage forms can be administered to the
patient on a regimen of one to four doses per day.
According to another modification, in order
to more finely regulate the dosage schedule, the
active substances may be administered separately in
. . .
.
7~
B24
--16--
individual dosage units at the same time or
carefully coordinated times. Since blood levels
are built up and maintained by a regulated schedule
of administration, the same result is achieved by
S the simultaneous presence of the two substances.
The respective substances can be individually
formulated in separate unit dosage forms in a
manner similar to that described above.
Fixed combinations of squalene synthetase
inhibitor and other serum cholesterol lowering
agent are more convenient and are preferred,
especially in tablet or capsule form for oral
administration.
In formulating the compositions, the active
substances, in the amounts described above, are
compounded according to accepted pharmaceutical
practice with a physiologically acceptable vehicle,
carrier, excipient, binder, preservative,
stabilizer, flavor, etc., in the particular type of
unit dosage form.
Illustrative of the adjuvants which may be
incorporated in tablets are the following: a binder
such as gum tragacanth, acacia, corn starch or
gelatin; an excipient such as dicalcium phosphate
or cellulose; a disintegrating agent such as corn
starch, potato starch, alginic acid or the like; a
lubricant such as stearic acid or magnesium
stearate; a sweetening agent such as sucrose,
aspartame, lactose or saccharin; a flavoring agent
such as orange, peppermint, oil of wintergreen or
cherry. When the dosage unit form is a capsule, it
may contain in addition to materials of the above
type a liquid carrier such as a fatty oil. Various
;~07~i4~
HX24
-17-
other materials may be present as coatings or to
otherwise modify the physical form of the dosage
unit. For instance, tablets or capsules may be
coated with shellac, sugar or both. A syrup of
elixir may contain the active compound~ water,
alcohol or the like as the carrier, glycerol as
solubilizer, sucrose as sweetening agent, methyl
and propyl parabens as preservatives, a dye and a
flavoring such as cherry or orange.
Some of the active substances described:
above form commonly known, pharmaceutically
acceptable salts such as alkali metal and other
common basic salts or acid addition salts, etc.
References to the base substances are therefore
intended to include those common salts known to be
substantially equivalent to the parent compound.
The formulations as described above will be
administered for a prolonged period, that i6, for
as long as the potential for elevated serum
cholesterol and atherosclerosis remains or the
symptoms continue. Sustained release forms of such
formulations which may provide such amounts
biweekly, weekly, monthly and the like may also be
employed. A dosing period of at least one to two
weeks are required to achieve minimal benefit.
;~Q7~i4:
HX24
-18-
The following Examples represent preferred
embodiments of the present invention. A11
temperatures are expressed in degrees Centigrade
unless otherwise indicated and all mesh sizes are
S U.S. Standard ASTME.
Exam~le 1
A squalene synthetase inhibitor formulation
in the form of tablets having the following
composition was prepared as described below.
In~redient Weiqht (mg)
(E,E)-[[hydroxy(4,8,12-trimethyl-100 mg
3,7,11-tridecatrienyl~phosphinyl]-
methyl]phosphonic acid tripotassium
salt (squalene synthetase inhibitor
prepared as described by
Biller et al. supra)
Cornstarch 50 mg
20 Gelatin 7.5 mg
Avicel (microcrystalline 25 mg
cellulose)
Magnesium stearate 2.5 mg
185 mg
The tablets are prepared from sufficient
bulk quantities by mixing the tripotassium salt
and cornstarch with an aqueous solution of the
gelatin. The mixture is dried and ground to a
fine powder. The Avicel and then the magnesium
stearate are admixed with granulation. The
mixture is compressed in a tablet press to form
tablets each containing lO0 mg of active
ingredient.
f~ 7~
HX24
-19-
Probucol tablets containing 25C mg probucol
are prepared employing conventional procedures
containing the following additional ingredients as
set out in the 1988 PDR: corn starch, ethyl
S cellulose, glycerin, hydroxypropyl cellulose,
hydroxypropylmethyl cellulose 2910, iron oxide,
lactose, magnesium stearate, microcrystalline
cellulose, polysorbate 80, talc and titanium
dioxide.
The squalene synthetase inhibitor and
probucol tablets may be administered as a
combination in accordance with the teachings of the
present invention to lower serum cholesterol and/or
treat atherosclerosis. In addition, the squalene
synthetase inhibitor and probucol tablets may be
ground up into powders and used together in a
single capsule.
Exam~le 2
Tablets each containing the following
ingredients:
. "~ . :
4~
HX24
-20-
Inaredient Wei~ht (mq)
(E,E)-[difluoro[hydroxy(4,8,12-100 mg
trimethyl-3,7,11-tridecatrienyl)-
phosphinyl]methyl]phosphonic acid
tripotassium salt (squalene
synthetase inhibitor prepared as
described by Biller et al. supra)
Avicel 112.5 mg
Lactose 113 mg
10 Cornstarch 17.5 mg
Stearic Acid 7 mq
350 mg
are prepared from sufficient bulk quantities by
slugging the squalene synthetase inhibitor, Avicel,
and a portion of the stearic acid. The slugs are
ground and passed through a #2 screen and then
mixed with the lactose, cornstarch, and the
remainder of stearic acid. The mixture is
compressed into 350 mg capsule shaped tablets in a
tablet press. The tablets are scored for dividing
in half.
Capsules containing 300 mg gemfibrozil are
prepared employing conventional pharmaceutical
techniques containing the following additional
ingredients as described in the 1988 PDR:
polysorbate 80 NF, starch NF and silica gel.
The squalene synthetase inhibitor tablet and
gemfibrozil capsule may be administered as a
combination or the squalene synthetase inhibitor
tablet may be ground into a powder and used in a
single capsule containing gemfibrozil to lower
2BC~7{i41
HX24
-21-
serum cholesterol and/or treat atherosclerosis in
accordance with the teachings of the present
invention.
Exam~les 3
A formulation in the form of tablets having
the following composition was prepared as
described in Example 1.
10 Ingredient Weiqht (mq~
(E,E,E)-[difluoro[hydroxy(4,8,12-100 mg
trimethyl-1,3,7,11-tridecate-
traenyl)phosphinyl]methyl]-
phosphonic acid tripotassium salt
(squalene synthetase inhibitor
prepared as described by
Biller et al. supra)
Cornstarch 50 mg
Gelatin 7.5 mg
20 Avicel (microcrystalline cellulose) 25 mg
Magnesium stearate 2.5 mg
185 mg
The above squalene synthetase inhibitor
tablets may be employed in combination with
clofibrate capsules containing 500 mg clofibrate
and inactive ingredients including color, and
gelatin as described in the 1988 PDR. The squalene
synthetase inhibitor and clofibrate may be employed
in separate dosage forms or combined in a single
capsule form to lower elevated serum cholesterol or
treat atherosclerosis in accordance with the
present invention.
Z~7~
HX24
-22-
Examples 4 to 6
Sgualene synthetase inhibitor tablets
described in Examples 1, 2 and 3, respectively,
may be employed in combination with cholestyramine
resin containing 4 g cholestyramine, acacia, citric
acid, color, flavor, polysorbate 80, propylene
glycol alginate and sucrose as described in the
1988 PDR. The squalene synthetase inhibitor and
cholestyramine may be employed in separate dosage
forms or combined in a single capsule form to lower
serum cholesterol or treat atherosclerosis in
accordance with the present invention..
Exam~les 7 to 10
Squalene synthetase inhibitor tablets,
prepared as described in Examples l, 2 and 3,
respectively, may be employed in combination with
nicotinic acid, colestipol, dextrothyroxine or
other serum cholesterol lowering agent in
accordance with the teaching of the present
invention to lower cholesterol or treat
atherosclerosis.
It will also be appreciated that any of the
sgualene synthetase inhibitors disclosed in the
Biller et al. paper and in pending U.S. patent
application Serial No. 141,744 may be employed in
combination with any of the serum cholesterol
lowering agents disclosed herein in accordance
wi~h the present invention.
