NZ299704A

NZ299704A - Use of a beta-lactam cholesterol absorption inhibitor and a cholesterol biosynthesis inhibitor (eg lovastatin) in preparation of pharmaceutical compositions for treatment of plasma cholesterol levels

Info

Publication number: NZ299704A
Application number: NZ299704A
Authority: NZ
Inventors: Harry R Davis
Original assignee: Schering Corp
Priority date: 1992-12-23
Filing date: 1993-12-21
Publication date: 1997-06-24

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number £99704 New Zealand No. 299704 International No. PCT/ TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 23.12.1992; International filing date: 21.12.1993 Classification.-^) A61K31/395,365; A61K45/06 Publication date:24 June 1997 Journal No.: 1417 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Use of a combination of a cholesterol biosynthesis inhibitor ana a beta-lactam cholesterol absorption inhibitor, and methods of treatment related thereto Name, address and nationality of applicant(s) as in international application form: SCHERING CORPORATION, a New Jersey corporation of 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States of America 299704 tfmJer ttie provisions "tif lation 23 (1) l!:? .. Sp&C<*K.<*.«•> < TV":.- . r-Ctt'W* «•.. 19 — Inflate NEW ZEALAND PATENTS ACT, 1953 Divided out of NZ No: 259790 Date: 21 December 1993 N.Z PATENT OK • 1 6 - NOV 1996 ? I RECEIVED COMPLETE SPECIFICATION USE OF A COMBINATION OF A CHOLESTEROL BIOSYNTHESIS INHIBITOR AND A B-LACTAM CHOLESTEROL ABSORPTION INHIBITOR, AND METHODS OF TREATMENT RELATED THERETO We, SCHERING CORPORATION, a New Jersey corporation, USA of 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States of America do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: - 1 - (followed by page 1A) V 29 9 7 0 4 -1A- pse of a combihatioh of a cholesterol biosynthesis inhibitor ahp a ft-lactam cholesterol absorption inhibitor, amp methods of treatment related thereto BACKGROUND The present invention relates to the use of a combination of a cholesterol biosynthesis inhibitor and a 0-lactam cholesterol absorption inhibitor in reducing plasna cholesterol levels. Described but not claimed is a method of treating or preventing atherosclerosis comprising administering the claimed combination. Plasma cholesterol levels have been positively correlated with the incidence of clinical events associated with coronary heart disease. The regulation of whole-body cholesterol homeostasis in humans and animals involves modulation of cholesterol biosynthesis, bile acid biosynthesis, and the catabolism of the cholesterol-containing plasma lipoproteins. The liver is the major organ responsible for cholesterol biosynthesis and catabolism and, for this reason, it is a prime determinant of plasma cholesterol levels. The liver is the site of synthesis and secretion of very low density lipoproteins (VLDL) which are subsequently metabolized to low density lipoproteins (LDL) in the circulation. LDL are the predominant cholesterol-carrying lipoproteins in the plasma and an increase in their concentration is correlated with increased atherosclerosis. Another major factor in determining cholesterol homeostasis is the absorption of cholesterol in the small intestine. On a daily basis, the average human consuming a Western diet eats 300 to 500 mg of cholesterol. In addition, 600 to 1000 mg of cholesterol can traverse the intestines each day. This latter cholesterol is a component of bile and is secreted from the liver. The process of cholesterol absorption is complex and multifaceted. St has been reported that approximately 50% of the total cholesterol within the intestinal lumen is absorbed by the cells lining the intestines (/e, enterocytes). This cholesterol includes both diet-derived d bile- or hepatic-derived cholesterol. Much of the newly-absorbed erol in the enterocytes is esterified by the enzyme acyl-A:cholesterol acyttransferase (ACAT). Subsequently, these cholestery! 299704 4 -2- esters are packaged along with triglycerides and other components (ie, phospholipids, apoproteins) into another lipoprotein class, chylomicrons. Chylomicrons are secreted by intestinal cells into the lymph where they can then be transported to the blood. Virtually all of the 5 cholesterol absorbed in the intestines is delivered to the liver by this route. When cholesterol absorption in the intestines is reduced, by whatever means, less cholesterol is delivered to the liver. The consequence of this action is a decreased hepatic lipoprotein (VLDL) production and an increase in the hepatic clearance of plasma cholesterol, mostly as LDL 10 Thus, the net effect of an inhibition of intestinal cholesterol absorption is a decrease in plasma cholesterol levels. Beta-lactams such as (3R-4S)-1,4-bis-(4-methoxyphenyl)-3-(3-phenylpropyl)-2-azetidinone disclosed in PCT/US92/05972 are potent inhibitors of intestinal cholesterol absorption, leading to decreased 15 plasma cholesterol levels in several animal species (hamsters, rats, rabbits, monkeys). The inhibition of cholesterol biosynthesis by 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC1.1.1.34) inhibitors has been shown to be an effective way to reduce plasma cholesterol (Witzum, 1989) 20 and reduce atherosclerosis. Combination therapy of an HMG CoA reductase inhibitor and a bile acid sequestrant has been demonstrated to be more effective in human hypolipidemic patients than either agent in monotherapy (lllingworth, 1988). We have unexpectedly found that a combination of a beta 25 lactam cholesterol absorption inhibitor and the HMG CoA reductase inhibitor lovastatin (MEVACOR™) results in a greater decrease in plasma cholep^rol than either agent alone in chow-fed dogs and rhesus monkeys, and in cholesterol-fed hamsters and rabbits. These findings were unexpected because HMG CoA reductase inhibitors alone do not 30 lower plasma cholesterol levels in hamsters and monkeys. SUMMARY OF THE INVENTION The present invention relates to the use of a p-lactam cholesterol absorption inhibitor for the manufacture of a medicament for 35 the combined use with a cholesterol biosynthesis inhibitor in the treatment or prevention of atherosclerosis, or for the reduction of plasma cholesterol levels. In a further aspect, the present invention relates to the use « 10 15 20 25 30 35 29 9 70 4 - 3 - of a cholesterol biosynthesis inhibitor for the manufacture of a medicament for the combined use with a p-lactam cholesterol absorption inhibitor in the treatment or prevention of atherosclerosis, or for the reduction of plasma cholesterol levels. Described but; not claimed is a method of treating or preventing atherosclerosis comprising administering an effective -amount of a combination of a cholesterol biosynthesis inhibitor arid a p-lactam cholesterol absorption inhibitor to a mammal in need of such treatment. That is, the use of a (i-lacts.m cholesterol absorption inhibitor for combined use with a cholesterol biosynthesis inhibitor (and, similarly, use of a cholesterol biosynthesis inhibitor for combined use with a p-lactam cholesterol absorption inhibitor) to treat or prevent athersclerosis or to reduce plasma cholesterol levels Also described but not claimed is a pharmaceutical composition comprising an effective amount of a cholesterol biosynthesis inhibitor, a p-lactam cholesterol absorption inhibitor and a pharmaceutically acceptable carrier. a kit comprising in one container an effective amount of a cholesterol biosynthesis inhibitor in a pharmaceutically acceptable carrier, and in a separate container, an effective amount of a p-lactam cholesterol absorption inhibitor in a pharmaceutically acceptable carrier, iS also described but not claimed. DETAILED DESCRIPTION Cholesterol biosynthesis inhibitors for use in the combination of the present invention include HMG CoA reductase inhibitors such as iovastatin, pravastatin, fluvastatin, simvastatin and CI-981; HMG CoA synthetase inhibitors, for example L-659,699 ((E,E-11-[3'R-(hydroxy-methyl)-4'-oxo-2,R-oxetanyll-3,5,7R-trimethyl-2,4-undecadienoic acid); squalene synthesis inhibitors, for example squaiestatin 1; and squalene epoxidase inhibitors, for example, NB-598 ((E)-N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[(3,3'-bithiophen-5-yl)methoxy]benzene-methanamine hydrochloride). Preferred HMG CoA reductase inhibitors are Iovastatin, pravastatin and simvastatin. ^lactam cholesterol absorption inhibitors include those identified as novel compounds in formula I of New Zealand Patent No. 243669 and W093/02048 published on February 4, 1993 as well as those identified in formula II for use in lowering cholesterol. Said New Zealand Patent and W093/02048 are incorpo ated herein by-fgfQjpncp; formulas I and II are shown herein as follows] 10 APR 1997 RECEIVED 299704 4- R d" cr R* i wherein A is -CHeCH-B; -C-C-B; 5 -(CH2)p-X-B, wherein p is 0,1 or 2 and X is a bond, -NH- or -S(0)o-2-: heteroaryl, benzofused heteroaryl, W-substituted heteroaryl or W-substituted benzofused heteroaryl, wherein heteroaryl is selected from the group consisting of pyrrolyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, 10 imidazolyl, thiazolyl, pyrazolyl, thienyl, oxazolyl and furanyl, and for % nitrogen-containing heteroaryls, the N-oxides thereof, and wherein W is 1-3 substituents on the ring carbon atoms selected from the group consisting of lower alkyl, hydroxy lowr - alkyl, lower alkoxy, alkoxyalkyl, alkoxyaikoxy, alkoxycarbonylalkoxy, (lower alkoxy-imino)lower alkyl, lower alkanedioyl, 15 lower alkyl lower alkanedioyl, allyloxy, -CF3, -OCF3, benzyl, Ri4-benzyl, benzyloxy, R-u-benzyloxy, phenoxy, Ri4-phenoxy, dioxolanyl, NO2, -NR10R11, NRioRii(lower alkyl)-, NRioRn(lower alkoxy)-, OH, halogeno, -NHC(0)0Rs, -NHC(0)R5, R6O2SNH-, (R602S)2N-, -S(0)2NH2, -S(0)o-2Rio, tert-butyldimethylsilyloxymethyl, -C(0)Ri2 and -CHa-N R„ 20 >—/ , and wherein the substituents on the substituted heteroaryl ring nitrogen atoms, when present, are selected from the group consisting of tower alkyl, lower alkoxy, -C(0)0Rs, -C(0)Rs, OH, NR10R11 (lower alkyl)-, NR10R11 (lower alkoxy)-, -S(0)2NH2 and 2-(trimethylsilyl)ethoxymethyl; 25 -C(0)-B; or -<ch2)k-n r18 / , wherein k is 1 or 2; D Is B'-(CH2)mC(0)«, wherein m is 1, 2, 3,4 or 5; B'-(CH2)q-, wherein q is 2,3,4,5 or 6; B'-(CH2)e-Z-(CH2)r. wherein Z is -O-, -C(O)-, phenylene, 30 -NRs- or -S(0)o.2-, e is 0,1,2, 3, 4 or 5 and r is 1,2, 3, 4 or 5, provided that the sum of e and r is 1, 2, 3, 4, 5 or 6; B'-(C2-C6 alkenylene)-; B'-(C4-C6 alkadienylene)-; 299704 -5- B'-(CH2)t-Z-(C2-C6 alkenylene)-, wherein Z is as defined above, and wherein t is 0,1,2 or 3, provided that the sum of t and the number of carbon atoms in the alkenylene chain is 2, 3, 4, 5 or 6; B,-(CH2)f-V-(CH2)g-. wherein V is C3-C6 cycloalkylene, f is 1,2, 3,4 or 5 and g is 0,1,2,3,4 or 5, provided that the sum of f and g is 1,2, 3,4,5 or 6; B*-(CH2)t"V-(C2-C6 alkenylene)- or B'-(C2-C6 alkenylene)-V-(CH2>t-, wherein V and t are as defined above, provided that the sum of t and the number of carbon atoms in the alkenylene chain is 2,3,4, 5 or 6; B'-(CH2)a-Z-(CH2)b-V-(CH2)d-. wherein Z and V are as defined above and a ,b and d are independently 0,1,2, 3,4, 5 or 6, provided that the sum of a, b and d is 0,1,2,3,4,5 or 6; T-(CH2)s-. wherein T is cycloaikyl of 3-6 carbon atoms and s is 1,2,3,4,5or6; or naphthylmethyl, heteroarylmethyl, or W-substituted heteroarylmethyl, wherein heteroaryl and W are as defined above; B' is naphthyl, heteroaryl or W-substituted heteroaryl, wherein heteroaryl is as defined above, or Rv R is hydrogen, fluoro, C1-C15 alkyl, C1-C15 alkenyl, C1-C15 alkynyl, or B-(CH2)h -• wherein h is 0,1,2, or 3; R-j, R2 and R3 are independently selected from the group consisting of H and W, provided that when W is halogeno, it is o-halogeno or m-haolgeno; or R1 is hydrogen and R2 and R3, together with adjacent carbon atoms to which they are attached, form a dioxolanyl ring; Rr. R2' and R3' are independently selected from the group consisting of H and W; or Rr is hydrogen and R2' and R3, together with adjacent carbon atoms to which they are attached, form a dioxolanyl ring; Bis «V 299704 - 6 - R4 is Nssf , wherein n is 0,1,2 or 3, indanyl, benzofuranyl, benzodioxolyl, tetranydronaphthyl, pyridyl, pyrazinyl, pyrimidinyl or quinolyi; Rs is lower alkyl, phenyl, Ri4-phenyl, benzyl or R-u-benzyl; Re is OH, lower alkyl, phenyl, benzyl, Ri4-phenyl or R14-benzyl; R7 is lower alkyl, lower alkoxy, OH, halogeno, -NR10R11, -NHC(0)0Rs, -NHC(0)R5, N02, -CN, -N3, -SH, •S(O)0.2-(iower alkyl), -COORg, -CONR10R11, -COR12. phenoxy, benzyloxy, -OCF3, or tert-butyldimethyisilyioxy, and where n is 2 or 3, the R7 groups can be the same or different; Re is H, lower alkyl, phenyl lower alkyl, or -C(0)Rg; Rg is H, tower alkyl, phenyl or phenyl lower alkyl; R10 and R11 are independently selected from H and lower alkyl; -N R13 R12 is H, OH, alkoxy, phenoxy, benzyloxy, >—' , -NR10R11, lower alkyl, phenyl or R-u-phenyl; R13 is -O-, -CH2-, -NH- or -N(lower alkyl)-; and R14 is 1-3 groups independently selected from the group consisting of lower alkyl, lower alkoxy, -COOH, NO2, -NR10R11, OH or halogeno; or a pharmaceutically acceptable salt thereof. R22 I Rgg F G' h-TT ^—K II Rao wherein R20 is phenyl, W-substituted phenyl, naphthyl, W-substituted naphthyl, benzodioxolyl, heteroaryl, W-substituted heteroaryl, benzofused heteroaryl and W-substituted benzofused heteroaryl, wherein heteroaryl is as defined above; R21, R22 and R23 are independently selected from H or R20; W is 1 to 3 substituents independently selected as defined above; E, F and G are independently a bond; C3-C6 cycloalkylene; C1-C10 alkylene; C1-C10 alkenylene; C1-C10 alkynylene; an alkylene, alkenylene or alkynylene chain as defined substituted by one or more 299704 -7- substituents independently selected from the group consisting of phenyl, W-substituted phenyl, heteroaryl and W-substituted heteroaryl, wherein heteroaryl is as defined above; an alkyiene, alkenylene or alkynylene chain as defined interrupted by one or more groups independently 5 selected from the group consisting of -O-, -S-, -SO-, -SO2-, -NRs, -C(O)-, C3-C6 cycloalkylene, phenylene, W-substituted phenylene, heteroarylene and W-substituted heteroarylene; or an interrupted alkylene, alkenylene or alkynylene chain as defined substituted by one or more substituents independently selected from the group consisting of phenyl, W-substituted 10 phenyl, heteroaryl and W-substituted heteroaryl; or one of R21-E and R22-F is selected from the group consisting of halogeno. OH, lower alkoxy, -0C(0)R5, -NR10R11, -SH or -S(lower alkyl); and wherein R5, Re, and Rs-R-u are as defined above; provided that when G is a-bond, R23 is not H, and provided that 15 when R23 is W-substituted phenyl, W is not p-halogeno; or a pharmaceutically acceptable salt thereof. Preferred are compounds of formula I wherein R is H. Another group of preferred compounds of formula I is that wherein D is 20 B'-(CH2)q-, B,-(CH2)e-Z-(CH2)r. B'-^Ce alkenylene)-, or B'-(CH2)f-V-(CH2)g-, wherein B't Z, V, q, e, r, f, and g are as defined above. A third group of preferred compounds of formula I is that wherein R4 is phenyl, R7-substituted phenyl or indanyl. Still another group of preferred compounds of formula I is that wherein A is -(CH2 )P-X-B, wherein X, B and 25 p are as defined above. Especially preferred are compounds of formula I wherein D is: B'-(CH2)q-, wherein B' is phenyl and q is 3 or 4; B'-(CH2)e-Z-(CH2)r. wherein B1 is p-fluorophenyl or p-methoxyphenyl, e is zero, Z is -O-, and r is 2; B'-^-Ce alkenylene)- is 3-phenyH-propenyl; or B'-(CH2)f-V-30 (CH2)o-, wherein B' is phenyl, f is 1, V is cyclopropylene, and g is zero. Also especially preferred are compounds of formula i wherein A is -(CH2 )p-X-B wherein p is zero and X is a bond. Preferably Ri, R2 and R3 in formula I are selected from H, OH, -N02, lower alkoxy, alkoxyaikoxy, lower alkyl tower alkandioyi, rn-halogeno, NR10R11 (lower alkoxy)-, 35 allyloxy, phenoxy, alkoxycarbonyialkoxy and -C(0)Ri2. Compounds of formula I wherein Ri and R3 are each H, and R2 is in the para-position are more preferred. 299704 -8- R7 in formula I is preferably selected from lower alkyl, lower alkoxy, halogeno, -OCF3, lower alkylthio, -NR10R11, -CN, OH, and -COR12. More preferred are compounds of formula I are those wherein n is 1 and R7 is in the para-position. A preferred p-lactam cholesterol absorption inhibitor is (3R-4S)-1,4-bis-(4-methoxyphenyl)-3-(3-phenylpropyl)-2-azetidinone. The effectiveness of the combinations of this invention for the reduction of plasma cholesterol levels is demonstrated by the following 10 test procedures. In the procedures, the p-lactam cholesterol absorption inhibitor is (3R-4S)-1,4-bis-(4-methoxyphenyl)-3-(3-phenylpropyl)-2-azetidinone (hereinafter Compound A) and the HMG CoA reductase inhibitor is Iovastatin. 15 Experiment 1 - Hypocholestdrolemic effect of the combination of Compound A and Iovastatin in the cholesterol-fed hamster: Method; Male Golden Syrian hamsters (Charles River Labs, Wilmington, MA.) weighing between 100 and 125g were fed Wayne 20 rodent chow until study onset. At study onset (Day 1), animals were separated into groups (n=4-6/group) and fed Purina Chow #5001 supplemented with 0.5% by weight of cholesterol (Research Diets Inc., New Brunswick, NJ). Compound A at 3 mg/kg and Iovastatin at 10 mg/kg were administered once daily for 7 days, starting on Day 1 via oral gavage 25 in 0.2 ml corn oil. On Day 7, animals were sacrificed by decapitation, biood was collected into tubes containing ethylenediaminetetraacetic acid (EDTA), and plasma was prepared by low speed centrifugation at 4°C. Nonfasted plasma cholesterol levels were determined by a modification of the cholesterol oxidase method of Allain et al. (Clin. Cham.. 30 2Q (1974) p. 470-475), in which the reagents were available in a kit form from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan). Ten \i\ of serum was assayed for total cholesterol in 1 ml of 0.15 M tris buffer, pH 7.0, containing p-chlorophenol (0.1%), cholesterol oxidase (0.13 U/ml), cholesterol ester hydrolase (0.13 U/ml), peroxidase (2.4 U/ml) and 4-35 aminoantipyrine (0.015%). Assays were carried out at 37°C for 10 min, along with cholesterol standards, and the absorbance of the resultant red quinone pigment was determined spectrophotometrically at 505 nm. 29 97 0 4 10 -9- Results; Hamsters fed a 0.5% cholesterol-containing diet for 7 days showed a 2-fold increase in plasma cholesterol. The increase in plasma cholesterol is primarily in VLDL and LDL (Schnitzer- Polokoff et al, Comp Biochem. Physiol.. 99A (1991) p. 665-670). Compound A at 3 mg/kg/day resulted in a 15% reduction in plasma cholesterol levels, while Iovastatin had no effect at 10 mg/kg/day (Table 1). When Compound A and Iovastatin were given in combination, a reduction in plasma cholesterol levels of 31% was found, which was significantly greater than either treatment alone iTable 1). Table 1 Group Dose (mo/kg/day) N Hamster Plasma cholesterol (mq/dl) Control m 6 227+6 Compound A 3 4 192±6a Lovastatin 10 4 223±14 Compound A+ Lovastatin 3 10 4 156±11®.b Values are Means±SEM. ap<0.05 compared to control group. *>p<0.05 compared to either Compound A alone or Iovastatin alone. 15 Experiment 2 - Hypocholesterolemlc effect of Compound A in combination with iovastatin in cholesterol-fed raobits: Methods; Male New Zealand White rabbits weighing 2.4 - 2.6 kg were challenged for one week with a diet containing 1% cholesterol and 6% 20 peanut oil. Hyper- and hypo-responding rabbits with serum cholesterol levels more than one standard deviation from the mean were excluded and four groups of rabbits with equivalent serum cholesterol levels were formed (n«8/group). The rabbits were then fed a diet containing 0. 5% cholesterol and 6% peanut oil, alone or with 0.03% Compound A; 25 0.015% Iovastatin; or 0.03% Compound A and 0.015% Iovastatin. Non-fasting serum samples were obtained weekly for 4 weeks and serum cholesterol levels were determined as described in Experiment 1. 29 9 7 0 -10- Results. The one week challenge with the 1% cholesterol/6% peanut oil diet resulted in average serum cholesterol levels of approximately 1000 mg/dl (Table 2). Similar food consumption and weight gains were 5 found amonjj the four groups of rabbits over the 4 week study period. The dose of Coir pound A at 0.03% of the diet was calculated to be 14 mg/kg/day and the dose of lovastatin at 0.015% was 7 mg/kg/day. Serum cholesterol levels continued to rise in the control group from 1015 to 1358 mg/dl at the 4 week time point (Table 2). Compound A alone caused a 10 29% reduction in serum cholesterol at week 4 compared to week 0, while lovastatin alone caused a 33% reduction over the 4 week period, but these reductions over time were not statistically significant by ANOVA. The combination of Compound A with lovastatin caused statistically significant reductions in plasma cholesterol levels at all timepoints, with a 15 61% decrease at week 4 compared to week 0 (Table 2). The relative reductions in serum cholesterol levels were even greater when the 4 week values were compared to the control group, with a 47% decrease with Compound A alone, a 51% decrease with lovastatin alone, and a 72% reduction with the combined Compound A and lovastatin therapy. 20 Table 2 Rabb t Serum Cholesterol Levels (mg/dl) Group Week 0 Week 1 Week 2 Week 3 Week 4 Control 1015 ±90 1138 ±170 1316 ±164 1437 ±211 1358 ±193 Compound A (0.03% in diet) 1005 ±89 781 ±122 879s ±109 808® ±121 713° ±112 Lovastatin (0.015% in diet) 993 ±95 895 ±150 839® ±80 767a ±87 667® ±81 Compound A+ Lovastatin (0.03% + 0.015% in diet) 986 ±93 552a-b ±76 506a-b ±58 427a»b ±62 382a-b ±66 Values represent means±SEM with 8 rabbits/group. ap<0.05 compared to control group; bp<0.05 compared to Week 0 value by ANOVA over time for each treatment. 30 299704 -11 Experiment 3 - Hypcu ulesterolemic effect of Compound A in combination with lov \statin in rhesus monkeys fed a choiesterol-free diet Methods; 5 'TYv ity rhesus monkeys (17 male, 3 female) weighing 4.4 • 8.5 kg were f ei a fat-free monkey chow (Purina #5038-7) containing 5% com oil for 2 weeks. Four groups of monkeys were formed with equivalent serum cholesterol levels and body weights (n=5/group). The monkeys were then continued on the fat-free chow containing 5% corn oil, alone or 10 with 3 mg/kg/day Compound A; 20 mg/kg/day lovastatin; or Compound A (3 mg/kg/day) and lovastatin (20 mg/kg/day). Fasting serum samples were obtained weekly for 3 weeks and serum cholesterol levels were measured as described in Experiment 1. Statistical differences were determined by ANOVA and Dunnett t tests on the change in serum cholesterol levels. A 15 probability level of p<0.05 was considered significant. Results; Control monkeys fed the fat-free chow containing 5% com oil maintained a constant level of serum cholesterol over the three week 20 study period (Table 3). Individually, Compound A at 3 mg/kg/day and lovastatin at 20 mg/kg/day caused slight reductions in serum cholesterol levels at 3 weeks, but these changes were not statistically significant compared to the 3-week control group. The combination of Compound A &nd lovastatin caused a significantly greater reduction of plasma 25 cholesterol than either treatment alone at all timepoints and reached a 25% reduction at Week 3 (Table 3). 35 -12-Table 3 Rhesus Monkey Seru (mQ m Cholesterol Levels /dl) Group Week 0 Week 1 Week 2 Week 3 Control 131 ±1 129 ±7 125 ±8 132 ±8 Compound A (3 mg/kg/day) 140 ±10 122 ±11 117 ±7 125 ±9 Lovastatin (20 mg/kg/day) 139 ±7 127 ±6 117 ±5 120 ±6 Compound A + Lovastatin (3 -i- 20 mg/kg/day) 136 ±8 108* ±7 101* ±7 102* ±8 Values represent means±SEM with 5 monkeys/group. *p<0.05 compared to control group. Experiment 4 - Hypocholesterolemic effect of Compound A In combination with lovastatin in dogs fed a chow diet Methods; Fifteen male beagles were divided into three groups with equivalent serum cholesterol levels and body weights (n=5/group). The dogs were fed Purina Dog Chow (#5006) containing maltodextrin and either 0.0234% Compound A; or 0.0234% lovastatin; or the combination of Compound A (0.0234%) and lovastatin (0.0234%) tor seven days. Serum samples were obtained at Day 0, 3 and 7. and serum total cholesterol levels were measured as described in Experiment 1. Statistical differences were determined by ANOVA and a probability level of p<0.05 was considered significant. Results: Dogs fed the chow diet containing either Compound A at 0.0234% (5 mg/kg/day) or lovastatin at 0.0234% (5 mg/kg/day) resulted in serum cholesterol levels which were unchanged from baseline ievels (Day 0) at Day 3 or Day 7 (Table 4). The combination of Compound A at 29 9 7 0 4 -13- 5 mg/kg/day and lovastatin at 5 mg/kg/day caused a 33% reduction in serum cholesterol levels at Day 7 compared to baseline at Day 0 (Table 4). The serum cholesterol levels in the combination group were also significsr^y lower than levels in either group administered Compound A or lovas^nin alone at Day 7. (Table 4) Table 4 Dog Serum Cholesterol Levels (mg/dl) Group Day 0 Day 3 Day7 Compound A (S mo/ko/day) 114 ±5 106 ±13 109 ±10 Lovastatin (5 mg/kg/day) 107 ±10 107 ±8 114 ±9 Compound A + Lovastatin (5 mg/kg/day each) 109 ±8 89 ±4 77a«b ±3 Values represent means±SEM with 5 dogs/group. a p<0.05 compared to Day 0. 3 p<0.05 compared to Day 7 values of either Compound A alone or iovastatin alone. A method of treatment is described herein comprising the administration of a combination of two components, the components can be co-administered simultaneously or sequentially, or a single pharmaceutical composition comprising a cholesterol biosynthesis inhibitor and a ^-lactam cholesterol absorption inhibitor in a pharmaceutically acceptable carrier can be administered. The components of the combination can be administered individually or together in any conventional oral or parenteral dosage form such as a capsule, tablet, powder, cachet, suspension or solution. The formulations can be prepared using conventional pharmaceutical excipients and additives using conventional techniques. Such pharmaceutically acceptable excipients and additives include non-toxic compatible fillers, binders, disintegrants, buffers, preservatives, anti-oxidants, lubricants, flavorings, thickeners, coloring agents, emulsifiers and the Hke. Representative formulations comprising p-lactam cholesterol absorption inhibitor are disclosed in New Zealand Patent No. 243669 and W093/02048 cited above. Representative formulations comprising a cholesterol biosynthesis inhibitor are well known in the art. It is contemplated that where the two </div>

Claims

<div class="application article clearfix printTableText" id="claims"> 29 9 7 0 4 -14- active ingredients are administered as a single composition, the dosage forms as disclosed in the aforementioned PCT application may readily be modified using the knowledge of one skilled in the art. The daily doses of the compounds in the combination 5 for reducing plasma cholesterol levels are as follows: for cholesterol biosynthesisjnhibitors, the typical dosage is 0.1 to 80 mg/kg of mammalian weight per day administered in single or divided dosages, usually once or twice a day; for the ^-lactam cholesterol absorption inhibitor, the typical dosage is 0.1 to 10 mg/kg mammalian weight per day 10 in single or divided dosages. The exact dose of any component of the combination to be administered is determined by the attending clinician and is dependent on the potency of the compound administered, the age, weight, condition and response of the patient. Generally, to reduce the plasma cholesterol levels in 15 mammals needing such treatment, the compounds in the combination may be administered to patients in dosage ranges as follows: for HMG CoA reductase inhibitors, about 10 to about 40 mg per dose is given 1 to 2 times a day, giving a total daily dose of about 10 to 80 mg per day, and for {he other cholesterol biosynthesis inhibitors, about 1 20 to 1000 mg per dose is given 1 to 2 times a day, giving a total daily dose of about 1 mg to about 2 g per day. About 1 to about 1000 mg per dose of the p-lactam cholesterol absorption Inhibitor is given 1 to 4 times a day. Where the components of a combination are administered separately, the number of doses of each component given per day may not necessarily 25 be the same, e.g. where one component may have a greater duration of activity, and will therefore need to be administered less frequently. A method for the reduction of plasma cholesterol levels by treatment with a combination of active ingredients wherein said active ingredients may be administered separately is described herein. 30 Accordingly, also described herein is the combining of separate pharmaceutical compositions in kit form. That is, a kit is contemplated wherein two separate units are combined: a cholesterol biosynthesis inhibitor pharmaceutical composition and a (J-lactam cholesterol absorption inhibitor pharmaceutical composition. The kit will preferably 35 include directions for the administration of the separate components. The kit form is particularly advantageous when thn Ropm-nto rr>mpg|-|pntc be administered in different dosage forms (e.g administered at different dosage intervals. oral and parefifffiffi or 10 APR 1997 are RECEIVED 299704 10 -15- WHAT WE CLAIM IS:

1. The use of a (J-lactam cholesterol absorption inhibitor for the manufacture of a medicament for the combined use with a cholesterol biosynthesis inhibitor in the treatment or prevention of athersclerosis, or for the reduction of plasma cholesterol levels.

2. The use as claimed in claim 1, wherein the p-lactam cholesterol absorption inhibitor is represented by the structural formula R a d- O R< wherein A is -CH=CH-B; -C«C-B; 15 -(CH2)p-X-B, wherein p is 0,1 or 2 and X is a bond, -NH- or -S(0)o.2-; heteroaryl, benzofused heteroaryl, W-substituted heteroaryl or W-substituted benzofused heteroaryl, wherein heteroaryl is selected from the group consisting of pyrrolyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, 20 imidazolyl, thiazofyl, pyrazoiyl, thienyi, oxazolyl and furanyl, and for nitrogen-containing heteroaryls, the N-oxides thereof, and wherein W is 1-3 substituents on the iuig carbon atoms selected from the group consisting of lower alkyl, hydroxy lower alkyl, lower alkoxy, alkoxyalkyl, alkoxyalkoxy, alkoxycarbonytalkoxy, (lower alkoxy-imino)lower alkyl, lower alkanedioyl, 25 lower alkyl lower alkanedioyl, allyloxy, -CF3, -OCF3, benzyl, Ri4-benzyl, benzyloxy, Ru-benzyioxy, phenoxy, Ri4-phenoxy, dioxolanyl, NO2, -NR10R11, NRioRn(lower alkyl)-, NRioRn(lower alkoxy)-, OH, halogeno, -NHC(0)0Rs, -NHC(0)Rs, Re02SNH-, (R602S)2N-, -S(0)2NH2, -S(0)o-2Rio. tert-butyldimethylsilyloxymethyl, -C(0)Ri2and -CHa-N Rtt 30 w , and wherein the substituenti on the substituted heteroaryl ring nitrogen atoms, when present, are selected from the group consisting of lower alkyl, lower alkoxy, -C(0)ORs, -C(0)Rs, OH, NRioRn(lower alkyl)-, NR10R11 (lower alkoxy)-, -S(0)2NH2 and 2-(trimethylsilyl)ethoxymethyl; -16- -C(0)-B; or /—\ •(CHaK-N Ris N—' , wherein k is 1 or 2; 259704 D is B'-(CH2)mC(0)-, wherein m is 1,2,3,4 or 5; B'-(CH2)q-, wherein q is 2, 3,4, 5 or 6; 5 B'-(CH2)e-Z-(CH2)r. wherein Z is -O-, -C(O)-, phenylene, -NRe- or -8(0)0.2-, e is 0,1,2, 3, 4 or 5 and r is 1, 2, 3, 4 or 5, provided that the sum of e and r is 1,2,3,4,5 or 6; B'-(C2-C6 alkenylene)-; B'-fC^i-Ce alkadienylene)-; B'-(CH2)t-Z-(C2-C6 alkenylene)-. wherein Z is as defined above. 10 and wherein t is 0,1,2 or 3, provided that the sum of t and the number of carbon atoms in the alkenylene chain is 2,3,4. 5 or 6; S:-(CH2)f-V-(CH2)o-. wherein V is C3-C6 cycloalkyiene, f is 1,2, 3,4orf andgisO. 1.2.3,4 or 5,-provided that the sum off andgis 1.2. 3, 4, 5 cr 6; 15 B'-(CH2)t-V-(C2-C6 alkenylene)- or B'-(C2-Ce alkenylene)-V- (CH2)t-. wherein V and t are as defined above, provided that the sum of t and the number of carbon atoms in the alkenylene chain is 2,3,4, 5 or 6; B*-(CH2)a-Z-(CH2)b-V-(CH2)d-. wherein Z and V are as defined above and a ,b and d are independently 0,1,2,3.4. 5 or 6. provided that 20 thesumofa, banddisO. 1,2,3,4,5or6; T-(CH2)r. wherein T is cycloalkyl of 3-6 carbon atoms and s is 1,2,3,4,5 or 6; or naphthylmethyl, heteroarylmethyl, or W-substituted heteroarylmethyl, wherein heteroaryl and W are as defined above; 25 Bis Ri -Ra B' is naphthyl, heteroaryl or W-substituted heteroaryl, wherein heteroaryl is as defined above, or "V t 30 R is hydrogen, fluoro, C1-C15 alkyl, C1-C15 alkeriyl. or B-(CH2)h wherein B is as defined above and h is 0, 99 9 7 o ft - 17- Rl, R2 and R3 are independently selected from the group consisting of H and W (as defined above), provided that when W is halogeno, it is o-halogeno or m-haolgeno; or Ri is hydrogen and R2 and R3, together with adjacent carbon atoms to which they are attached, form a dioxolanyl ring; 5 Rv, R2' and R3' are independently selected from the group consisting of H and W; or Ri* is hydrogen and R2* and R3', together with adjacent carbon atoms to which they are attached, form a dioxolanyl ring; R4 Is W , wherein n is 0,1,2 or 3, fndanyi, benzofuranyl, benzodioxolyl, tetrahydronaphthyl, pyridyl, pyrazinyl. 10 pyrimldinyl or quinolyl; R5 is lower alkyl, phenyl, Ri4*phenyl, benzyl or R-u-benzyl; Re Is OH, lower alkyl, phenyl, benzyl, Ru-phenyl or R-u-benzyl; R7 is lower alkyl, lower alkoxy, OH, halogeno, -NR10R11, -NHC(0)0Rtf -NHC(0)R^ NO2. -CN. -N3. -SH, -S(0)o-2-(k>wer alkyO, 15 -COORo, -CONR10R11, -COR12, phenoxy, benzyloxy, -OCF3, or tert-butyldimethylsilyioxy, and where n is 2 or 3, the R7 groups can be the same or different; R8 is H, lower alkyl, phenyl lower alkyl, or -C(0)Rs; Rb is H, lower alkyl, phenyl or phenyl lower alkyl; 20 Rio and R11 are independently selected from H and lower alkyl; ■*W" R12 is H, OH, alkoxy, phenoxy, benzyloxy, x' , -NR10R11 (wherein r^q and are as defined above), lower alkyl, phenyl or Ri4-phenyl; R13 Is -O-, -CH2-. -NH- or -N(lower alkyl)-; and 25 R14 is 1-3 groups independently selected from the group consisting of lower alkyl, lower alkoxy, -COOH, NO2, -NRioRh, OH or halogeno; or a pharmaoeutically acceptable salt thereof.

3. The use as claimed in claim 2, wherein the p-lactam 30 cholesterol absorption inhbitor is as defined in claim 2, wherein: RisH; D is B'-(CH2)q-, B,-(CH2)®-Z-(CH2)r. B'-(C2-Ce alkenylene)-, or B'-(CH2^V-(CH2)g-, wherein B', Z. V, q, e, r, f, and g are as defined in claim 2; * (wherein R$ is as defined above) -18- 99970 FU is phenyl, R7-substitiited phenyl or indanyl, wherein R7 is lower alkyl, lower alkoxy, halogeno, -OCF3, lower alkylthio. -NR10R11. -CN, OH or -CORi2, wherein R10. R11 and R12 are as defined in claim 2; and A is -(CH2 )p-X-B, wherein X, B and p are as defined in claim 2. 5

4. The use as claimed in claim 3, wherein the P-lactam cholesterol absorption inhibitor as defined in claim 2, wherein: D is B-(CH2)q-, wherein B' is phenyl and q is 3 or 4; B'-(CH2)e-Z-(CH2)r 1 wherein B' is p-fluoro-phenyl or p-mbthoxyphenyl, e is zero, Z is 10 -O-, and r is 2; B'-(C2-C6 alkenylene)- is 3-phenyM-propenyi; or B'-(CH2)f-V-(CH2)g-. wherein B' is phenyl, f is 1, V is cyclopropylene, and g is zero; A is -(CH2 )p-X-B wherein p is zero, X is a bond and B is as defined in claim 2; 15 Ri. R2 and R3 are selected from the group consisting of H, OH, •NO2. lower aftoxy, alkoxyalkoxy, lower alkyl lower alkandioyl, m-halogeno, NR10R11 (lower alkoxy)-, allyloxy, phenoxy, alkoxycarbonyl-alkoxy and -C(0)Ri2, wherein R10, R11 and R12 are as defined in claim 2; R4 is (R7)n-substituted phenyl, wherein n is 1 and R7 is lower alkyl, ?.0 lower alkoxy, halogeno, -OCF3, lower alkylthio. -NR10R11. -CN, OH or -COR12. wherein R10, R11 and R12 are as defined in claim 2.

5. The use as claimed in claim 4, wherein the p-lactam cholesterol absorption inhibitor is (3R-4S)-1,4-bis-(4-methoxy-25 phenyl)-3-(3-phenylpropyl)'2-azet!dinone.

6. Tha use of a cholesterol btosynthesis inhibitor for the manufacture of a medicament for the combined use with a p-lactam cholesterol absorption Inhibitor in the treatment or prevention of 30 athersclerosis, or for the reduction of plasma cholesterol levels.

7. The use as claimed in claim 6, wherein the cholesterol biosynthesis inhbitor is selected from the group consisting of HMG CoA reductase inhibitors, squalene synthesis inhibitors and squalene 35 epoxidase inhibitors.

8. The use as claimed in claim 7, wherein th biosynthesis inhibitor is selected from the group consi WO 94/14433 PCT/US93/12291 29 9 70 4 pravastatin, ftuvastatin, simvastatin. Ci-981, L-659,699, squalestatin 1 and NB-598.

9. The use as claimed in any of claims 1 to 5. wherein the 5 cholesterol biosynthesis inhibitor is as specified in claim 7 or claim 8.

10. The use as claimed in any one of claims 6 to 8 wherein the p-lactam cholesterol absorption inhibitor is as defined in any one of claims 2 to 5. 10 15 20 25 11 - The use as defined in claim 1 of a p-lactam cholesterol absorption inhibitor substantially as herein described with reference to any example thereof. ,12 • The use as defined in claim 6 of a cholesterol biosynthesis inhibitor substantially as herein described with reference to any example thereof. Sof-tr? m/r, uo/i aa• By the authorised agents A J PARK & SON ,/1 / 30 end of claims 35 10 APR <99? BECOVFP J </div>