Method of lowering plasma levels of lipoprotein(a)

The present invention comprises a method of lowering plasma levels of Lp(a) in animals by administering an effective Lp(a)-reducing amount of a microsomal triglyceride transfer protein inhibitor of the structural formula: ##STR1## or a pharmaceutically acceptable salt thereof, wherein R.sup.1 is alkyl, alkenyl alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, cycloalkyl, or cycloalkylalkyl, each of which is independently optionally substituted through available carbon atoms with 1, 2, or 3 groups selected from halo, alkyl, alkenyl, alkoxy, aryloxy, aryl, arylalkyl, alkylmercapto, arylmercapto, cycloalkyl, cycloalkylalkyl, heteroaryl, and heteroarylalkyl. By lowering Lp(a) levels, the animals are protected against developing premature atherosclerosis and consequent cardiovascular and cerebrovascular diseases.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a method for lowering plasma levels of 
lipoprotein known as lipoprotein(a) (Lp(a)) in animals comprising 
administering a compound which inhibits microsomal triglyceride transfer 
protein. 
2. Summary of the Related Art 
Heart disease remains one of the leading causes of death. The high 
incidence of heart disease has led to the identification of various risk 
factors that may be controlled in an effort to reduce such disease. One 
risk factor is hypercholesterolemia which is a condition of high blood 
levels of cholesterol. Cholesterol is a fatty substance that is made by 
the liver and also is present in many foods. Cholesterol circulates in th 
blood and is associated with several forms of lipoproteins. One such 
lipoprotein is known as low-density lipoprotein (LDL). LDL associates with 
cholesterol from the liver to form LDL-cholesterol (LCL-C), which takes 
cholesterol from the liver to cells throughout the body. High levels of 
LDL-C have been shown to cause rapid clogging of coronary arteries with 
fatty deposits, resulting in atherosclerosis, which often leads to heart 
attacks. Levels of LDL-C can be reduced, for example, by modifying diet to 
reduce fat and cholesterol intake and by daily exercise. In contrast, a 
second form of lipoprotein, high density lipoprotein (HDL), associates 
with cholesterol to lower circulating levels of cholesterol by removing it 
from cells and recycling it to the liver for disposal. 
A modified form of LDL is known as lipoprotein(a) (Lp(a)). Lp(a) consists 
of LDL covalently linked to apolipoprotein(a), (apo(a)) via a disulfide 
bond. Elevated levels of Lp(a) are associated with the development of 
atherosclerosis, coronary heart disease, myocardial infarction, cerebral 
infarction, and restenosis following balloon angioplasty. In fact, 
increased Lp(a) levels appear to be an excellent predictor for stroke. 
Accordingly, high concentrations of Lp(a) is one of the major risk factors 
leading to death from heart disease. 
Wetterau et al. (U.S. Pat. No. 5,595,872) describe compounds that inhibit 
the protein known as microsomal triglyceride transfer protein (MTP) and 
have the structural formula: 
##STR2## 
wherein R.sup.1 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, 
heteroarylalkyl, cycloalkyl, or cycloalkylalkyl, each of which is 
optionally substituted through available carbon atoms with 1, 2, or 3 
groups selected from halo, alkyl, alkenyl, alkoxy, aryloxy, aryl, 
arylalkyl, alkylmercapto, arylmercapto, cycloalkyl, cycloalkylalkyl, 
heteroaryl, and heteroarylalkyl. Examples of oxo-substituted groups are 
described in Cortizo, L., J. Med. Chem. 34: 2242-2247 (1991). In vitro, 
MTP catalyzes the transport of lipid molecules between phospholipid 
membranes. Wetterau & Zilversmit, Chem, Phys. Lipids 38, 205-22 (1985). 
The physiological role of MTP has not been demonstrated. Presumably, it 
plays a similar role in vivo, and thus plays some role in lipid 
metabolism. The subcellular (lumen of the microsomal fraction) and tissue 
distribution (liver and intstine) 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. Wetterau & Zilversmit, Biochem, 
Biophys. Acta, 875: 610-7 (1986). Wetterau et al. determined that these 
MTP inhibiting compounds could decrease the MTP-catalyzed transfer of 
triglyceride (TG), cholesteryl ester (CE), and phosphatidylcholine (PC) 
between small unilamellar vesicles (SUV). Presently, however, there are no 
reports that the above reference compound are effective in decreasing the 
plasma levels of Lp(a). We have now discovered that plasma Lp(a) can be 
lowered by administering compounds of formula I, and accordingly an object 
of this invention is to provide a method for lowering Lp(a), and thereby 
treating and preventing coronary artery disease. 
SUMMARY OF INVENTION 
This invention provides a method for lowering plasma levels of Lp(a) in an 
animal (preferably a mammal) by administering an efficacious 
Lp(a)-lowering amount of a compound of the structural formula: 
##STR3## 
or a pharmnaceutically acceptable salt thereof, wherein R.sup.1 is alkyl, 
alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, 
cycloalkyl, or cycloalkylalkyl, each of which is independently optionally 
substituted through available carbon atoms with 1, 2, or 3 groups selected 
from halo, alkyl, alkenyl, alkoxy, aryloxy, aryl, arylalkyl, 
alkylmercapto, arylmercapto, cycloalkyl, cycloalkylalkyl, heteroaryl, and 
heteroarylalkyl. By lowering Lp(a) levels, the animals are protected 
against developing premature atherosclerosis and consequent coronary 
artery disease. 
In particular, the present invention provides a method for lowering plasma 
levels of Lp(a) in an animal (preferably a mammal) by administering an 
efficacious Lp(a)-lowering amount of a compound of the formula: 
##STR4## 
or a pharmaceutically acceptable salt thereof. 
The foregoing merely summarizes certain aspects of the present invention 
and is not intended, nor should be construed, as limiting the invention in 
any manner. All patents and publications cited herein establish the state 
of the art and are hereby incorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION 
This invention provides a method for lowering plasma levels of Lp(a) in an 
animal (preferably a mammal) by administering an efficacious 
Lp(a)-lowering amount of a compound of the structural formula: 
##STR5## 
or a pharmaceutically acceptable salt thereof, wherein R.sup.1 is alkyl, 
alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, 
cycloalkyl, or cycloalkylalkyl, each of which is independently optionally 
substituted through available carbon atoms with 1, 2, or 3 groups selected 
from halo, alkyl, alkenyl, alkoxy, aryloxy, aryl, arylalkyl, 
alkylmercapto, arylmercapto, cycloalkyl, cycloalkylalkyl, heteroaryl, and 
heteroarylalkyl. By lowering Lp(a) levels, the animals are protected 
against developing premature atherosclerosis and consequent cardiovascular 
and cerebrovascular diseases. All of the compounds to be utilized are 
either known or are readily prepared as described by Wetterau et al., U.S. 
Pat. No. 5,595,872 which is hereby expressly incorporated by reference. 
As used herein, the terms "alkyl" and "alk," whether used alone or when 
used as a part of another group, mean a straight or branched chain 
hydrocarbyl groups having from 1 to 20 carbon atoms, with 1 to 12 
preferred and 1 to 8 most preferred. Exemplary alkyl groups are methyl, 
ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, 
isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, 
decyl, undecyl, dodecyl, the various branched chain isomers thereof and 
the like. 
The term "alkenyl" refers to both straight and branched chain hydrocarbyl 
groups of 1 to 20 carbon atoms, with 1 to 12 carbon atoms preferred and 1 
to 8 most preferred, and having at least one double bond. 
The term "alkynyl" refers to both straight and branched chain hydrocarbyl 
groups of 1 to 20 carbon atoms, with 1 to 12 carbon atoms preferred and 1 
to 8 most preferred, and having at least one triple bond. 
The term "cycloalkyl" means a saturated cyclic hydrocarbyl having from 3 to 
20 and preferably 3-13 carbon atoms. Exemplary cycloalkyl groups include 
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 
cyclodecyl, and cyclododecyl. 
The terms "aryl" and "ar" mean a monocyclic or bicyclic aromatic group 
containing from 6 to 10 carbon atoms in the ring portion, such as phenyl 
or napthyl, and may be optionally substituted. 
The term "heteroaryl" means a 5 or 6 membered aromatic ring having 1 or 2 
heteroatoms (i.e., N, S, or O) that may be located at any position within 
the ring as well as such rings fused to an aryl (e.g., benzothiophenyl, 
indolyl). Exemplary heteroaryl groups include pyrrolyl, furanyl, 
thiophenyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridyl, 
pyrimidinyl, and the link, and may be optionally substituted and/or fused 
to an aryl such as indolyl and benzothiophenyl. 
In a preferred embodiment, the present invention provides a method for 
lowering plasma levels of Lp(a) in an animal (preferably a mammal) by 
administering an efficacious amount of a compound II having the structural 
formula: 
##STR6## 
or a pharmaceutically acceptable salt thereof. Particularly preferred is 
2-(1-(3,3-diphenylpropyl)-4-piperidyl)isoindolin-1-one hydrochloride. 
For use in the method of this invention, the compounds of formula I, or, 
preferably II, are preferably combined with one or more pharmaceutically 
acceptable diluents, carriers, excipients, or the like, for convenient 
oral, parenteral, and topical administration to animals, preferably 
humans. The compounds of formula I are ideally suited to formulation for 
oral administration in the form of tablets, capsules, dispersible powders, 
granules, suspensions, elixirs, buccal seals, and the like. The 
formulations typically will contain from about 1% to about 90% by weight 
of active compounds of formula I, and more commonly from about 5% to about 
60% by weight. 
Oral formulations can contain, for suspensions, from about 0.05% to about 
5% by weight of a suspending agent, such as talc or the like, and syrups 
will contain from about 10% to about 50% by weight of a sugar such as 
dextrose. Tablets may contain normal amounts of binders, stabilizers, and 
common diluents such as corn starch and sugars. Parenteral formulations, 
for instance, solutions for IV injection, will be made by dissolving or 
suspending the compounds of formula I in a solvent such as isotonic saline 
or 5% glucose in sterile water. 
The dose of compounds of formula I to be administered is that amount which 
is effective for lowering plasma levels of Lp(a) in an animal. 
Formulations utilizing one or more of the compounds if formula I are 
contemplated in the present invention. 
The effective dosage of active ingredient employed may vary depending on 
the particular compound employed, the mode of administration, and the 
severity of the condition being treated. However, in general, satisfactory 
results are obtained when the compounds of formula I are administered at a 
daily dosage of from about 0.5 to about 500 mg/kg of animal body weight, 
preferably given in divided doses two to four times a day, or in 
sustained-release form. For most large mammals, such as humans, the total 
daily dosage is form about 1 to 100 mg, preferably from about 2 to 80 mg. 
Dosage forms suitable for internal use comprise from about 0.5 to 500 mg 
of the active compound in intimate admixture with a solid or liquid 
pharmaceutically acceptable carrier. This dosage regimen may be adjusted 
to provide the optimal therapeutic response. For example, several divided 
doses may be administered daily or the dose may be proportionally reduced 
as indicated by the exigencies of the therapeutic situation. 
The compounds of formula I may be administered orally as well as by 
intravenous, intramuscular, or subcutaneous routes. Solid carriers include 
starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose, 
and kaolin, while liquid carriers include sterile water, polyethlene 
glycols, nonoinic surfactants, and edible oils such as corn, peanut, and 
sesame oils, as are appropriate to the nature of the active ingredient and 
the particular form of administration desired. Adjuvants customarily 
employed in the preparation of pharmaceutical compositions may be 
advantageously included, such as flavoring agents, coloring agents, 
preserving agents, and antioxidants, for example, vitamin E, ascorbic 
acid, BHT, and BHA. 
The preferred pharmaceutical compositions from the stand point of ease of 
preparation and administration are solid compositions, particularly 
tablets and hard-filled or liquid-filled capsules. Oral administration of 
the compounds of formula I is preferred. 
These active compounds may also be administered parenterally or 
intraperitoneally Solutions or suspensions of these active compounds as a 
free base or pharmacologically acceptable salt can be prepared in water 
suitable mixed with a surfactant such as hydroxy-propylcellulose. 
Dispersions can also be prepared in glycerol, liquid polyethylene glycols, 
and mixtures thereof in oils. Under ordinary conditions of storage and 
use, these preparations contain a preservative to prevent the growth of 
microorganisms. 
The pharmaceutical forms suitable for injectable use include sterile 
aqueous solutions or dispersions and sterile powder for the extemporaneous 
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 the contaminating 
action of microorganisms such as bacterial and fungi. The carrier can be a 
solvent or dispersion medium containing, for example, water, ethanol, 
polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol), 
suitable mixtures thereof, and vegetable oils. 
Suitable cream, lotion, gel, stick, ointment, spray aerosol formulations 
that may be used for compounds of formula I and, preferably, II (or, if 
appropriate, a pharmaceutically acceptable salt thereof) are conventional 
formulations well known in the art, for example, as described in standard 
text books such as Harry's Cosmeticology published by Leonard Hill Books, 
Remington's Pharmaceutical Sciences. and the British and US 
Pharmacopoeias. 
The compounds may also be encapsulated in liposomes to allow an intravenous 
administration of the drug. The liposomes suitable for use in the 
intention are lipid vesicles and may include plurilamellar lipid vesicles, 
small sonicated multimellar vesicles, reverse phase evaporation vesicles, 
large multilamellular vesicles, and the like, wherein the lipid vesicles 
are formed by one or more phospholipids such as phosphotidylcholine, 
phosphatidylglycerol, sphingomyelin, phospholactic acid, and the like. In 
addition, the liposomes may also comprise a sterol component such as 
cholesterol. 
Some typical formulations which can be administered to humans are as 
follows: 
Tablet Formulation 
2-(1-(3,3-diphenylpropyl)-4-piperidyl)isoindolin-1-one hydrochloride (250 
mg) is blended to uniformity with 100 mg of corn starch and 50 mg of 
lactose. The mixture is compressed into a tablet. Such tablets are 
administered orally at the rate of one to three times a day. 
______________________________________ 
Preparation of Oral Suspension 
Ingredient Amount 
______________________________________ 
2-(1-(3,3-diphenylpropyl)4-piperidyl) 
500 mg 
isoindolin-1-one hydrochloride 
Sorbitol solution (70% NF) 
40 mL 
Sodium benzoate 150 mg 
Saccharin 10 mg 
Red dye 10 mg 
Cherry flavor 50 mg 
Distilled water qs OD 100 mL 
______________________________________ 
The sorbitol solution is added to 40 mL of distilled water and the retinoid 
is suspended thereon. The saccharin, sodium benzoate, flavor, and dye are 
added and dissolved. The volume is adjusted to 100 mL with distilled 
water. Each milliliter of syrup contains 5 mg of 
2-(1-(3,3-diphenylpropyl)-4-piperidyl)isoindolin-1-one hydrochloride. 
Suppositories 
A mixture of 400 mg 2-(1-(3,3-diphenylpropyl)-4-piperidyl)isoindolin-1-one 
hydrochloride and 600 mg of theobroma oil is stirred at 60.degree. C. to 
uniformity. The mixture is cooled and allowed to harden in a tapered mold 
to provide a 1-g suppository. 
Parenteral Solution 
In a solution of 700 mL of propylene glycol and 200 mL of sterile water is 
suspended 20.9 g of 2-(1-(3,3-diphenylpropyl)-4-piperidyl)isoindolin-1-one 
hydrochloride. The pH is adjusted to pH 6.5 with dilute sodium hydroxide, 
and the volume is made up to 100 mL with water for injection. The 
formulation is sterilized, filled into 5.0 mL ampoules each containing 2.0 
mL (representing 40 mg of drug), and sealed under nitrogen. 
Preferred formulations are those incorporating any of the preferred 
compounds of formula I to be utilized to lower Lp(a). Specifically 
preferred is 2-(1-(3,3-diphenylpropyl)-4-piperidyl)isoindolin-1-one 
hydrochloride. 
Those skilled in the art will recognize that the compounds of formula I can 
be prepared according to the above teachings and examples below and 
combined with a wide variety of other chemicals for use in a composition 
that is effective in lowering plasma levels of Lp(a) in animals. 
The following Example is provided for illustrative purposes only and is not 
intended, nor should it be construed, as limiting the invention in any 
manner. Those skilled in the art will appreciate that variations on and 
modifications of the following can be made without exceeding the spirit or 
scope of the invention. 
EXAMPLE 
Evaluation of 
2-1-3,3-diphenyl-2-propenyl)-4-piperidinyl-2,3-dihydro-1H-isoindol-1-one 
hydrochloride for Lowering Lp(a) 
2-(1-(3,3-diphenylpropyl)-4-piperidyl)isoindolin-1-one hydro-chloride was 
evaluated for lowering Lp(a) levels in animals. This compounds was 
evaluated according to the following protocol. 
HepG2 cells were obtained from the American Type Culture Collection (ATCC 
accession no. HB-8065) and permanently transfected with a human apo(a) 
cDNA containing 17-kringles. The 17-kringle apo(a) cDNA contained 24 
nucleotides of 5'-untranslated sequence; leader sequence; kringle IV (KIV) 
type-1; eight copies of KIV type-2; KIV types 3-10; kringle V; protease 
domain; and 67 nuclcotides of 3'-untranslated region. The 17-kringle cDNA 
was cloned into the eukarycltic expression vector pcDNA-Amp (In Vitrogen, 
Carlsbad, Calif.) that contains the cytomegalovius (CMV) immeidate early 
gene enhancer/propter and SV40 transcription termination and 
polyadenylation signals. This expression construct was co-transfected with 
pRc/CMV (In Vitrogen), which contains the neomycin resistance gene, into 
50% confluent HepG2 cells using the calcium phosphate method (Madison, 
Wis.). Cells permanently transfected with the later constructs were 
selected by their ability to form colonies in the presence of the neomycin 
analogue G418. Selected colonies expressing Lp(a) as a result of the 
covalent association of recombinant apo(a) with edogenous apoB100-LDL were 
screened by ELISA as described below. Of these, the colony expressing the 
highest relative level of Lp(a) (K-17, 145.9) was used for all 
experiments. 
Apo(a) cDNA transfected HepG2 cells (K-17, 145.9) were plated at a density 
of 75,000 cell/well using 96-well plates in Dulbecco's Modified Eagle 
Medium (DMEM), supplemented with 5.5 mM glucose, 6.4 mM L-glutamine, 1 mM 
sodium pyruvate, 19.5 nM pyridoxine hydrochloride, 20 mM 
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10% 
heat-inactivated fetal bovine serum (FBS), 100 units penicillin and 100 
.mu.g streptomycin/ml. Cells were grown in a humidified incubator 
maintained at 37.degree. C. and 5% CO.sub.2 '95% air. The next morning, 
fresh DMEM (without FBS) containing 
2-(1-(3,3-diphenylpropyl)-4-piperidyl)isoindolin-1-one hydrochloride at 
various concentrations (0.03, 0.1, 0.3; 1.0; 3.0, 10.0, 30.0, and 74 
.mu.M) in 0.74% DMSO was added to the cells and incubated for 8 hours. The 
media was harvested from the wells, transferred to polypropylene 96-well 
plate, sealed with 96-well caps and frozen immediately at -80.degree. C. 
until needed for analysis. The cells left in the original plate were 
digested by adding 100 .mu.L of 0.5 N NaOH to each well, wrapped tightly 
in Saran Wrap and allowed to incubate at room temperature overnight. 
Lp(a) was measured in the culture media using the Apo-Tek ELISA kit as 
previously described (Taddei-Peters W C, Butman B T, Jones G R, Venetta T 
M, Macomber P F, Ransom J H: Quantification of lipoprotein(a) particles 
containing various apolipoprotein(a) isofroms by a monoclonal anti-apo(a) 
capture antibody and a polyclonal anti-apolipoprotein B detection antibody 
sandwich enzyme immunoassay. Clin. Chem. 1993; 39:1382-1389). Briefly, 
samples were incubated for one hour at 37.degree. C. in wells coated with 
a monoclonal antibody specific for the apo(a) moiety of Lp(a). Unbound 
material was washed away, then a polyclonal antibody that recognizes apoB 
and conjugated to horseradish peroxidase was added and the plates 
incubated for 1 hour at 37.degree. C. After washing, the plates were then 
incubated with the chromogenic substrate, tetramethybenzidine and hydrogen 
peroxide. During the substrate incubation a blue color forms that turns 
yellow when 2N sulfuric acid was added to stop the reaction. The 
absorbance of each well was read in a Organon Teknika 96-well plate reader 
at a wavelength of 450 mn. 
Total cellular protein determination was done using the DC Bio-Rad Assay 
(Bio-Rad Laboratories, Richmond Calif.). In this assay, protein reacts 
with an alkaline copper tartrate solution and Folin reagent. The reaction 
reaches 95% maximum in 15 minutes but the color is stable for 1 hour. An 
aliquot of 10 .mu.L from each well was transferred to a polystyrene 
96-well plate then 25 .mu.L of the copper tartrate solution was added 
followed by 200 .mu.L of the Folin reagent. The assay incubates at room 
temperature for 20 minutes before reading the absorbance in each well with 
the 96-well plate reader at a wavelength of 690 nm. 
The data is presented in Table 1 and is expressed as percent inhibition at 
various concentrations relative to cells treated with vehicle only. 
TABLE 1 
______________________________________ 
Concentration of 2-(1-(3,3- 
diphenylpropyl)-4-piperidyl)isoindolin- 
1-one hydrochloride (.mu.M) 
Percent Lowering of Lp(a) 
______________________________________ 
0.03 5.0 .+-. 1.3 
0.10 3.9 .+-. 1.9 
0.3 2.0 .+-. 0.6 
1.0 34.3 .+-. 3.0 
3.0 57.6 .+-. 1.6 
10.0 71.7 .+-. 0.6 
30.0 78.7 .+-. 0.3 
74.0 84.8 .+-. 0.6 
______________________________________