Note: Descriptions are shown in the official language in which they were submitted.
PC10185CJG CA 02291471 1999-12-02
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LOWERING BLOOD LEVELS OF LIPOPROTEIN(a )
Back4round Of The Invention
The glycoprotein apolipoprotein (a) (apo(a)) is synthesized and secxeted from
hepatic cells and, in humans, drculates largely in assodation with low density
lipoprotein (LDL) in the form of a hybrid lipoprotein referred to as Lp(a).
The
association between apo(a) and the major protein moiety of LDL, namely
apolipoprotein B100 (apo B100), is mediated through covalent linkage of a
single
unpaired cysteine residue in apo(a) to a complimentary unpaired cysteine
residue in
the extreme carboxyl terminus of apo 8100.
Interest in the biology of this lipoprotein species is driven by the
observation
that elevated levels of Lp(a) in humans is assodated with an increased risk
for
atherosderotic heart and vascular disease. The lowering of Lp(a) levels,
however, has
proven problematic since various conventional methods that are effective in
redudng
levels of LDL are not as efficadous or consistent in lowering levels of Lp(a).
For
example, it has been reported that neomycin, alone or in combination with
niadn, is
effective in redudng Lp(a) levels when administered over a period of several
weeks to
years. See Spinier, et al., J. Ann. Phamnacother., 28, 343 (1994). The
administration
of high doses of niacin and neomydn, however, limits the desirability of this
regimen
due to many undesirable dinical side-effects. Alternatively, oral doses of
fosinopril, an
angiotensin-converting enzyme inhibitor, have been demonstrated to lower Lp(a)
levels after 12 weeks of treatment, however, Lp(a) reduction was signficant
only in
patients that showed improvement in renal function and, therefore, the Lp(a)
towering
ability of fosinopril may simply be attributable to the indirect consequence
of improved
kidney function. See Keilani, et al., Ann. Inter. Med., 118, 246 (1993).
Additionally,
certain steroidal hormones, estrogen for example, are known to down-regulate
Lp(a)
levels. See, for example, Frazer, et al., Nature Genet., 9_, 424 (1995).
However,
estrogen therapy alone is assodated with an increased risk of endometrial
cardnoma
and, for this reason, estrogen is nom~any administerea in commnauownntn
progesterone. Although short-term treatment with this estrogeNpmgesterone
combination is an effective therapeutic strategy for redudng Lp(a) levels,
long-term
treatment, i.e. six months or more, does not result in the same degree of
decreased
inhibition as that observed for treatment with estrogen alone. See Soma, et
al., Arch.
CA 02291471 1999-12-02
_2_
Internal. Med., 153, 1462 (1993) and Soma, et al., Chem. Phys. Lipids, 345, 67
(1994). Furthermore, LDL apheresis has been shown to be an effective means for
lowering Lp(a) levels. See Koizumi, et al., Atherosclerosis, 100, 65 (1993).
Mowever,
apheresis is an invasive approach requiring weekly treatments and, therefore,
is not
regarded as a current treatment of choice. Accordingly, improved methods of
inhibiting Lp(a), or formation of the precursors thereof, will have utility in
the treatment
of conditions and diseases arising from hyperlipoproteinemia, including, for
example,
atherosderosis, premature myocardial infarction, stroke, restenosis following
coronary
bypass surgery and so forth.
Microsomal triglyceride transfer protein (MTP) is known to mediate the
transport of triglyceride, cholesteryl ester and phospholipids and has been
implicated
as a mediator in the assembly of apolipoprotein B containing lipoproteins,
chylomicrons and VLDL (very low density lipoprotein). Spedfically, the
subcellular
(lumen of the microsomal fraction) and tissue distribution (liver and
intestine) of MTP
have led to speculation that it plays a role in the assembly of plasma
lipoproteins, as
these are the sites of plasma lipoprotein assembly. The ability of MTP to
catalyze the
transport of triglyceride between membranes is consistent with this
speculation and
suggests that MTP may catalyze the transport of triglyceride from its site of
synthesis
in the endoplasmic reticulum membrane to nascent lipoprotein particles within
the
lumen of the endoplasmic re6culum. Accordingly, compounds that inhibit MTP
and/or
otherwise inhibit apo B secretion are useful in the treatment of
atherosderosis and
other conditions related thereto. Such compounds are also useful in the
treatment of
other diseases or conditions in which, by inhibiting MTP and/or apo B
secretion,
serum cholesterol and triglyceride levels may be reduced. Such conditions may
include, for example, hypercholesterolemia, hypertriglyceridemia,
pancreatitis, obesity
and hypercholesterolemia, hypertriglyceridemia and hyperiipidemia assodated
with
pancrea6tis, obesity and diabetes. For a detailed discussion see, for example,
Wetterau et al., Silence, 258, 999-1001 (1992) and Wetterau et al., Biochem.
Biophys. Ada., 875, 610-617 (1986).
While the preclse mechanisms governing blood levels of Lp(a) are presently
unknown, there is evidence to suggest that Lp(a) levels are regulated at the
level of
synthesis rather than catabolism. Accordingly, because it is known
thatzinhibition. of _ ~ -
hepatic secretion of VLDL and apolipoprotein B (apo B) results in the pre-
secretory
degradation of apo B and concomitant decrease in hepatic apo B levels and
because
CA 02291471 1999-12-02
72222-394
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each Lp(a) particle contains one copy of apo(a) bound to apo
B, it is believed that decreasing the concentration of
hepatic apo B, by the administration of an apo B secretion/-
MTP inhibitor, will result in a lowering of Lp(a) secreted
and, thereby, a lowering of blood Lp(a) levels.
Summary of the Invention
The instant invention is directed to pharmaceutical
compositions for lowering blood levels of lipoprotein(a)
(Lp (a) ) in a mammal, which comprise (i) an effective Lp (a)
blood level-lowering amount of an apolipoprotein B secretion/-
microsomal triglyceride transfer protein inhibitor and (ii) a
pharmaceutically acceptable carrier or diluent.
Another aspect of the present invention provides
commercial packages comprising the above-described pharma
ceutical compositions and written matters including
indications that the pharmaceutical compositions can or should
be used for lowering blood levels of Lp(a) in a mammal.
In a preferred embodiment of the invention, the apo
B secretion/MTP inhibitor is a compound selected from the
group consisting of 4'-trifluoromethylbiphenyl-2-carboxylic
acid-[2-(1H-[1,2,4]triazol-3-ylmethyl)-1,2,3,4-tetrahydro-
isoquinolin-6-yl]-amide., 4'-trifluoromethylbiphenyl-2-
carboxylic acid-[2-(2-acetylaminoethyl)-1,2,3,4-tetrahydro-
isoquinolin-6-yl]-amide, 9H-(4-~4-[4'-trifluoromethylbiphenyl-
2-carbonyl)amino]-piperidin-1-yl}butyl)-9H-fluorene-9-
carboxylic acid-(2,2,2-trifluoroethyl)-amide 9-{4-[4-(2-
benzothiazol-2-ylbenzoylamino)piperidin-1-yl]butyl}-9H-
fluorene-9-carboxylic acid-(2,2,2-trifluoroethyl)amide,
[lla-R]-8-[(4-cyanophenyl)methoxy]-2-cyclopentyl-7-(prop-2-
enyl)-2,3,11,11a-tetrahydro-6H-pyrazino[1,2b]isoquinoline-
1,4-dione, [lla-R]-cyclopentyl-7-(prop-2-enyl)-8-[(pyridin-
2-yl)methoxy]-2,3,ll,lla-tetrahydro-6H-pyrazino[1,2b]iso-
quinoline-1,4-dione, 2-cyclopentyl-2-[4-(2,4-dimethylpyrido-
[2,3b]indol-9-ylmethyl)phenyl]-N-(2-hydroxy-1-phenylethyl)-
acetamide and 2-cyclopentyl-N-(2-hydroxy-1-phenylethyl)-2-
[4-(quinolin-2-ylmethoxy)-phenyl]acetamide, a hydrate or
CA 02291471 1999-12-02
72222-394
-4-
stereoisomer thereof, or a pharmaceutically acceptable salt
of the compound, hydrate or stereoisomer.
Especially preferred apo B secretion/MTP inhibitors
useful in the practice of invention are the compounds
4'-trifluoromethylbiphenyl-2-carboxylic acid-[2-(1H-[1,2,4]-
triazol-3-ylmethyl)-1,2,3,4-tetrahydroisoquinolin-6-yl]-amide
and 4'-trifluoromethylbiphenyl-2-carboxylic acid-[2-(2-
acetylaminoethyl)-1,2,3,4-tetrahydroisoquinolin-6-yl]-amide,
hydrates thereof or the pharmaceutically acceptable salts of
the compounds or hydrates.
Detailed Description of the Invention
This invention provides pharmaceutical compositions
for lowering blood levels of lipoprotein (a) (Lp (a) ) in a
mammal, which comprise an effective Lp(a) blood level-lowering
amount of an apolipoprotein B secretion/microsomal triglyceride
transfer protein inhibitor.
As employed throughout the instant specification
including appendant claims, the term "blood", when employed
in relation to the term "blood levels", includes whole blood,
plasma, i. e. the fluidic portion of the blood in which
particulate components are suspended and serum, i. e. the
fluidic portion of the blood obtained following removal of
the fibrin clot and corpuscles.
As employed throughout the instant specification
including appendant claims, the term "apo B secretion/MTP
inhibitor" means a compound having the ability to inhibit
the secretion of apolipoprotein B and/or the ability to
inhibit the transfer action of microsomal triglyceride
transfer protein.
A variety of apo B secretion/MTP inhibitors are,
or will be, known to one of ordinary skill in the art.
Although any apo B secretion/MTP inhibitor may be used in
the practice of the instant invention, generally preferred
apo B secretion/MTP inhibitors include those compounds,
hydrates or stereoisomers thereof, or the pharmaceutically
acceptable salts of the compounds, hydrates or stereoisomers
CA 02291471 1999-12-02
72222-394
-4a-
which are disclosed in, for example, European Patent
Publication Nos. EP 643057, EP 719763, EP 753,517, EP 764647,
EP 765878, EP 779276, EP 779279, EP 799828, EP 799829, EP
802186, EP 802188, EP 802192 and EP 802197; PCT Patent
Publication Nos. WO 96/13499, WO 96/33193, WO 96/40640, WO
97/26240, WO 97/43255, WO 97/43257, WO 98/16526 and WO
98/23593; and U. S. Pat. Nos. 5,595,872; 5,646,162,
5,684,014; 5,712,279; 5,739,135 and 5,789,197.
Especially preferred apo B secretion/MTP inhibitors
are those biphenyl-2-carboxylic acid-tetrahydroisoquinolin-6-
yl amide derivatives, and the stereoisomers, hydrates,
prodrugs and pharmaceutically acceptable salts thereof
disclosed in commonly assigned PCT Patent Publication Nos.
WO 96/40640 and WO 98/23593, each of which designate, inter
alia, the United States. Especially preferred apo B
secretion/MTP inhibitors disclosed in PCT Patent Publication
Nos. WO 96/40640 and WO 98/23593, and useful in the present
invention, are
CA 02291471 1999-12-02
-5-
the compounds illustrated hereinbelow: 4'-trifluoromethyl-biphenyl-2-
carboxylic acid-
[2-(1 H-[1,2,4]triazol-3-ylmethyl)-1,2,3,4-tetrahydroisoquin-6-yl]-amide and
4'-
trifluoromethyl-biphenyl-2-carboxylic acid-[2-(acetylaminoethyl)-1,2,3,4-
tetrahydroisoquinolin-6-ylJ-amide, or the hydrates thereof, or the
pharmaceutically
acceptable salts of the compounds or the hydrates.
CF3 CF3
H
/ O I \ N~N'NH / O
/ N~ ~ _ / _ O
H I H
/ /
Another especially preferred Gass of apo B secretioNMTP inhibitors is
disGosed in U.S. Pat. Nos. 5,595,872; 5,712,279; 5,739,135 and 5,789,197 and
includes compounds having the structural formula
Q p N
Rs' N-R~ and Rs'
R R6
Especially preferred apo B secretionMITP inhibitors disclosed in U.S. Pat.
Nos.
5,595,872; 5,712,279; 5,739,135 and 5,789,197, and useful in the methods of
the
present invention, are 9H-(4-{4-[4'trifluoromethyl-biphenyl-2-carbonyl}-amino]-
piperidin-1-yl}-butyl-9H-fluorene-9-carboxylic acid-(2,2,2-trifluoroethyl)-
amide and 9-
{4-[4-(2-benzothiazol-2-yl-benzoylamino)-piperidin-1-ylj-butyl}-9H-fluorene-9-
carboxylic acid-(2,2,2-trifluoroethyl)-amide.
Another Gass of espeaally preferred apo B secretioNMTP inhibitors is
disGosed in PCT Application Publication No. WO 98/16526 and includes compounds
having the structural formula
i~
-- ~ - _
Especially preferred Apo-B secretionMITP inhibitors disGosed in PCT
Application
Publication No. WO 98/16526, and useful in the methods of the present
invention, are
CA 02291471 1999-12-02
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[11 a-R]-8-[(4-cyanophenyl)methoxyJ-2-cyclopentyl-7-(prop-2-enyl)-2,3,11,11 a-
tetrahydro-6H-pyrazino[1,2b]isoquinoline-1,4-dione and [11a-R]-cyclopentyl-7-
(prop-
2-enyl)-8-[(pyridin-2-yl)methoxyJ-2,3,11,11 a-tetrahydro-6H-
pyrazino[1,2b]isoquinoline-
1,4-dione.
Another especlally preferred lass of apo B secretion/MTP inhibitors is
disclosed in U.S. Pat. No. 5,684,014 and includes compounds having the
structural
formula
R3 R~
N R2
HyC
Rs
E~
L Rg
R~
D
An especially prefer-ed apo B secretioNMTP inhibitor disclosed in U.S. Pat.
No.
5,684,014, and useful in the methods of the present invention, is 2-cydopentyl-
2-[4-
(2,4-dimethyl-pyrido[2,3-b]indol-9-ylmethyl)-phenyl]-N-(2-hydroxy-1-phenyl-
ethyl)-
acetamide.
Yet another class of especially preferred apo B secretioNMTP inhibitors is
disclosed in U.S. Pat. No. 5,646,162 and includes compounds having the
structural
formula
E
A~O
O
~ OH
~L~N
H
An especially preferred apo B seaetioNMTP inhibitor disclosed in U.S. Pat. No.
5,646,162, and useful in the methods of the present invention, is 2-cydopentyl-
N-(2-
hydroxy-1-phenyl-ethyl)-2-[4-(quinolin-2-ylmethoxy)-phenyl]-acetamide.
The apo B secretioNMTP inhibitors, including the prefer-ed and espedally
preferred embodiments and the pharmaceutically acceptable salts, hydrates or .
stereoisomers thereof, or the pharmaceutically acceptable salts of the
hydrates or
stereoisomers employed in the methods of the instant invention may be prepared
CA 02291471 1999-12-02
_7_
according to the disclosures of the aforementioned European, PCT and U.S.
patents
and application publication documents.
The apo B secretion/MTP inhibitors, the pharmaceutically acceptable salts,
hydrates or stereoisomers thereof, or the phamnaceutically acceptable salts of
the
hydrates or stereoisomers employed in the methods of the invention, are
preferably
administered in the form of a pharmaceutical composition comprising a
pharmaceutically acceptable carrier or diluent. Accordingly, the apo B
secretion/MTP
inhibitors, the pharmaceutically acceptable salts; hydrates or stereoisomers
thereof, or
the pharmaceutically acceptable salts of the hydrates or stereoisomers of this
invention, can be administered individually or together in any conventional
oral,
parenteral or transdemial dosage form.
Suitable pharmaceutically-acceptable carriers indude inert solid fillers or
diluents and sterile aqueous or organic solutions. The apo B secretion/MTP
inhibitors,
the pharmaceutically acceptable salts, hydrates or stereoisomers thereof, or
the
pharmaceutically acceptable salts of the hydrates or stereoisomers of this
invention
will be present in such pharmaceutical compositions in amounts sufficient to
provide
the desired dosage amount in the range described hereinbelow. Thus, for oral
administration, the compounds can be combined with a suitable solid or liquid
carrier
or diluent to form capsules, tablets, powders, syrups, solutions, suspensions
and the
like. The pharmaceutical compositions may, if desired, contain additional
components
such as flavorants, sweeteners, excipients and the like.
The tablets, pills, capsules, and the like may also contain a binder such as
gum tragacanth, acacia, com starch or gelatin; exdpients such as dicaldum
phosphate; a disintegrating agent such as com starch, potato starch, alginic
add; a
lubricant such as magnesium stearate; and a sweetening agent such as sucrose,
lactose or saccharin. When a 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 other materials may be present as coatings or to modify the physical
form of the dosage unit. For instance, tablets may be coated with shellac,
sugar or
both. A syrup or elixir may contain, in addition to the active ingredient,
sucrose as a
sweetening agent, methyl and propylparabens as preservatives, a dye and a
flavoring
such as cherry or orange flavor. ~ - -
The pharmaceutical compositions of this invention may also be administered
parenteraliy. For parenteral administration the pharmaceutical compositions
can be
CA 02291471 1999-12-02
_g_
combined with sterile aqueous or organic media to form injectable solutions or
suspensions. Solutions or suspensions of these pharmaceutical compositions can
be
prepared in water suitably mixed with a surfactant such as
hydroxypropylcellulose.
Dispersions can also be prepared in sesame or peanut oil, ethanol, water,
polyol (e.g.,
glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures
thereof,
vegetable oils, N-methyl glucamine, polyvinylpyrrolidone and mixtures thereof
in oils
as well as aqueous solutions of water-soluble pharmaceutically acceptable
salts of the
compounds. Under ordinary conditions of storage and use, these preparations
contain a preservative to prevent the growth of microorganisms. The injectable
solutions prepared in this manner can then be administered intravenously,
intraperitoneally, subcutaneously, or intramuscularly, with intramuscular
administration
being the preferred parenteral route in humans. Solutions prepared for
intravenous
administration are preferably rendered isotonic prior to usage.
The pharmaceutical forms suitable for injectable use include sterile aqueous
solutions or dispersions and sterile powders for the preparation of sterile
injectable
solutions or dispersions. In all cases, the form must be sterile and must be
fluid to the
extent that easy syringability exists. It must be stable under the conditions
of
manufacture and storage and must be preserved against contamination by
microorganisms such as bacteria and fungi.
The pharmaceutical compositions may also be administered transdemially.
Suitable formulations for transdermal application include an effective
lipoprotein(a)
blood level-lowering amount of a compound or pharmaceutical composition of the
invention with a suitable transdermal carrier. Preferred transdemial carriers
include
absorbable pharmacologically acceptable solvents to promote and assist passage
through the skin of the subject being treated. Characteristically, transdemial
devices
comprise the form of a bandage having a backing member, a reservoir containing
the
compound, optionally with carriers, optionally a rate-controlling barrier to
deliver the
compound to the skin of the subject being treated at a controlled and
predetermined
rate over a prolonged period of time and means to secure the device to the
skin of the
subject being treated.
Methods of preparing the various pharmaceutical compositions with a desired
amount of ari active ingredient are known, or will be apparent in light of
this_disclosu~e, . - - _
to one of ordinary skill in the art. See, for example, Remington's
Pharmaceutical
Sciences, Mack Publishing Company, Easton, PA, 15th Edition (1975).
CA 02291471 1999-12-02
_g_
The dosage of the apo B secretioNMTP inhibitor or the pharmaceutically
acceptable salt, hydrate or stereoisomer thereof, or the pharmaceutically
acceptable
salt of the hydrate or stereoisomer necessary to achieve the desired
therapeutic effect
according to this invention is within the ordinary skill of those who practice
in the art of
having the benefit of the disclosure herein.
In general, dosage ranges for certain apo B secretioNMTP inhibitors have
been reported with representative effective ranges being from about 0.01
mg/kg/day
to about 15.0 mg/kg/day. Generally preferable dosages range from about 0.1 to
about
5.0 mg/kg/day and especially preferred dosages range from about 0.1 to about
1.0
mg/kg/day. Dosage ranges for the particular compounds described hereinabove
are
set forth in the above-referenced patents and patent application publications.
However, some variability in the general dosage range may be required
depending
upon the age and weight of the patient, the intended route of administration,
and the
progress and degree of severity of the illness being treated.
It will be appreciated that, when treating a mammal according to the methods
of the instant invention, the actual preferred route of administration and
optimum
dosage utilized will be at the sound professional discretion of the person
responsible
for the treatment and may vary according to the severity of the condition to
be treated,
the intended route of administration and patient characteristics such as age,
weight,
rate of excretion, concurrently administered medications and general physical
condition of the subject. Normally, the optimum dosage for the subject being
treated
will be determined by generally administering smaller doses initially and
thereafter
incrementally modifying the regimen, if required, to determine the most
suitable
dosage. This may vary according to the particular compound employed and with
the
nature of the subject being treated.
EXPERIMENTAL
The utility of apo B secretioNMTP inhibitors in the lowering of blood levels
of
lipoprotein (a) according to the practice of the methods of the invention may
be
demonstrated according to protocols disclosed in Nassir, et al., J. Biological
Chem.,
273, 17793-17800 (1998), which protocols are summarized hereinbelow.
CA 02291471 1999-12-02
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Pulse-Chase Studies
Transfected HepG2 and McA-RH7777 cells are grown to 90% confluence in
T-25 flasks. On the day of the experiment, the cells are washed twice with
phosphate
s buffered saline, preincubated in methionine- and cysteine-free DMEM for 1 hr
without
serum, pulse-labeled for 4 hr in the same medium containing 250 ~Ci/ml Tran35S-
label
and the apo B secretion/MTP inhibitor and then chased in complete medium
containing 3 mM cysteine and 10 mM methionin~ for a predetermined time. The
apo B
secretaon/MTP inhibitor is dissolved in dimethyl sulfoxide at a concentration
of 100
mg/ml and diluted to an appropriate concentration in media just prior to
incubation with
the cells. Dimethyl sulfoxide, at identical final concentrations, is added
alone to control
cells. At pre-determined times following radiolabeling, media are collected on
ice and
adjusted to a final concentration of the following protease inhibitors (100 mM
leupeptin, 450 mM apoprotin, 2 mM pepstatin, 1 mM phenylmethylsulfonyl
fluoride
and 1 mM benzamidine). The cells are washed three times with ice-cold,
phosphate-
buffered saline and subsequently lysed in cold lysis buffer (100 mM Tris, pH
8.0, 100
mM NaCI, 10 mM EDTA, 1 % Triton X-100, 0.1 % SDS) containing protease
inhibitors
and, for HepG2 cells, 100 mM E-aminocaproic acrd. Cell lysates and media are
clarified by centrifugation at 10,000 rpm at 4 C for 5 min to remove cellular
debris and
immunopreapitations are then conducted as described hereinbelow. Incorporation
of
radioactivity into total protein is determined by trichloroacetic acid
precipitation of cell
lysates, in all cases demonstrating comparable values between control and
experimental groups.
Immunooreapita6ons
Both medium and lysates are precleared by incubation with protein G-agarose
for 2-3 hr at 4 C. Aliquots are immunoprecipitated with saturating quantities
of anti
apo(a), anti-apo B, and apoA-I or anti-albumin antisera. After overnight
incubation at
0
4 C, protein G-agarose beads are added and the inarbation continued for
another 2-3
hr at 4 C. The final pellet is washed four times in immunoprecipitation wash
buffer (50
mM Tris, pt~ 7.4, 0.65 M NaCI. 10 mM EDTA, 1 % Triton X-100, 1 % sodium _
deoxycholate, 01.% SDS), two times in water and boiled for 10 min in SDS
sample
buffer (4% SDS, 20% gycerol, 0.001 % bromphenol blue, 125 mM Tris, pH 6.8 and
CA 02291471 1999-12-02
_11_
100 mM dithiothreitol). After centrifugation, the supernatant is analyzed by
SDS-PAGE
and fluorography. Quantitation is then effected by standard techniques.
