Pharmacological profile of BR-931, a new hypolipidemic agent that increases high-density lipoproteins

45

Atherosclerosis, 30 (19’78) 45-56 @ Elsevier/North-Holland Scientific Publishing, Ltd.

PHARMACOLOGICAL PROFILE OF BR-931, A NEW HYPOLIPIDEMIC AGENT THAT INCREASES HIGH-DENSITY LIPOPROTEINS

CESARE R. SIRTORI ‘, PIER0 GOMARASCA 2, GAETANO D’ATRI 3, SILVANO CERUTTI 4, GIANNI TRONCONI 5 and CARLO SCOLASTICO 6

’ Center E. Grossi Paoletti, University of Milan, 2~3~4~5LPB, Cinisello 6 Institute of Organic Chemistry, University of Milan, Milan (Italy)

Balsamo,

Milan, and

(Received 2 November, 1977) (Revised, received 19 December, 1977) (Accepted 19 December, 1977)

Summary [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio-(N-~-hydroxyethyl)BR-931 acetamide], a new hypolipidemic agent of low toxicity, was evaluated in several tests of lipolysis and hyperlipidemia in rats, and in the cholesterol-induced atherosclerosis in rabbits. Significant hypolipidemic activity was observed in rats with doses of the agent at 12.5-50 mg/kg. In the Triton-induced hyperlipidemia, 50 mg BR-931 per kg was equieffective as 200 mg of clofibrate (CPIB) per kg. In contrast with CPIB, BR-931 exerted a powerful antilipolytic activity against epinephrine, ACTH, nicotine and cold exposure. BR-931 was particularly effective in diet-induced hyperlipidemias. Ethanol lipemia was totally prevented by the agent at 100 mg/kg. With Nath’s diet, doses as low as 25 mg/kg significantly reduced hypercholesterolemia and hypertriglyceridemia. In these last two tests, the distribution of lipoprotein cholesterol was also determined. CPIB did not affect HDL cholesterol levels that had been decreased by the diets; in contrast, BR-931, already at doses of 50 mg/kg, brought the HDL/total cholesterol ratio back toward normal. A significant HDL cholesterol increase, together with some reduction of atheromatosis, was also observed in cholesterol-fed rabbits. BR-931, a potent inducer of liver peroxisomes and of mitochondrial carnitine acetyltransferase, appears to be a hypolipidemic agent of high efficacy and low toxicity for the clinical treatment of hyperlipidemias and atherosclerosis. Presented in part at the International Conference on Atherosclerosis, Milan, 9-11 November, 1977. Reprint requests to: Dr. Cesare R. Sirtori. Center E. Grossi Paoletti. Via A. de1 Sarto 21, I-29129 Milan, Italy.

46 Key words:

BR-931 - Clofibrate - Ethanol hyperlipidemia - High-density lipoproteins Hypolipidemic agents - Lipolysis - Nath’s diet - Procetofene - Rabbit atherosclerosis

Introduction A large number of drugs are now available to the clinician for the treatment of hyperlipidemias [ 11. Hypolipidemic agents, generally classified into drugs affecting lipoprotein production and drugs affecting lipoprotein catabolism [ 21, significantly decrease plasma cholesterol and/or triglyceride levels, particularly when these are elevated. Treatment with hypolipidemic agents follows the rationale that lowering of lipids may be beneficial for the prevention of atherosclerosis and particularly of ischemic heart disease (IHD). Results of recent long-term studies, particularly of secondary prevention of IHD, however, have cast some doubts on the real efficacy of these drugs in the prevention of clinical manifestations of atherosclerosis [ 31. To explain this suggested clinical inadequacy, alterations of lipoprotein cholesterol distribution induced by hypolipidemic agents, have been given particular attention. It has been noted by several authors [4,5] that, in subjects with hypertriglyceridemia, increased catabolism of very low density lipoproteins (VLDL) may lead to increased concentrations of low density lipoproteins (LDL), a larger fraction of plasma cholesterol being thus transported in the most atherogenic lipoproteins [6]. An increase of LDL cholesterol may be seen both with carbohydrate-restricted diets and with drugs of the clofibrate or nicotinic acid types, the addition of an anion-exchange resin to the regimen becoming necessary in some patients [ 71. Recent epidemiological studies have also pointed out that high-density lipoprotein (HDL) cholesterol levels are inversely correlated with the incidence of atherosclerosis in selected populations [ 8,9]. Data on available hypolipidemic drugs show that HDL cholesterol may be raised somewhat in patients by clofibrate or other agents, but the effect is not striking [lo]. The most recent studies on new drugs affecting lipid and lipoprotein levels have therefore been aimed at the development of agents that decrease atherogenie lipoproteins (particularly LDL) [ 111, or increase high-density lipoproteins [ 121. Whereas the first effort failed to develop clinically useful agents [ 111, procetofene, a clofibrate derivative, was recently shown significantly to increase HDL levels in rats after hyperlipidemic diets [ 131 and to exert similar effects in hyperlipidemic patients [14]. Recent clinical data also suggest that two other clofibrate derivatives, gemfibrozil [15] and bezafibrate [16], may raise HDL cholesterol levels in man. The pharmacological activities of BR-931 [4-chloro-6-(2,3-xylidino)2-pyrimidinyl thio-(N-/3-hydroxyethyl)-acetamide] are described in this report. BR931 (Fig. 1) is the ethanolamine derivative of Wy-14,643, a previously described hypolipidemic agent [ 171, which has not entered clinical testing. BR931 retains most of the activities of Wy-14,643, and has a markedly decreased toxicity. Moreover, the observation of HDL cholesterol increases in both rats

47

NH CH3

CH3

CH3

CH3

BR 931

Wy 14.643 Fig. 1. Structures of WY 14,643

and BR-931.

and rabbits treated with high lipid regimens, makes BR-931 of potential est for the clinical treatment of hyperlipidemias and atherosclerosis.

inter-

Materials and Methods Animals and drugs Mule rats of the Sprague-Dawley

strain, weighing between 200 and 300 g, were used in all experiments. They were kept at constant humidity in air-conditioned rooms, and fed standard Purina chow, with free access to water, unless otherwise specified. BR-931 was administered by gastric tube, 10% gum arabic as vehicle. Control rats received the same vehicle by gastric tube. Clofibrate (courtesy of I.C.I., Macclesfield, U.K.) used as a reference compound, was also administered dispersed in the same vehicle. Rabbits, in the experiment of cholesterol-induced hyperlipidemia and atheromatosis, were males of the New Zealand strain, weighing from 2.8 to 3.0 kg. In this experiment, BR-931 was mixed with the diet, and food consumption was monitored to ascertain intake of the selected daily dose.

Pharmacological

evaluation

BR-931 underwent oral acute and sub-acute tests in rats and mice, and chronic toxicity tests in rats and dogs. Hypolipidemic activity, in rats on a normal diet, was determined according to Buchanan [18]. Treated rats received daily doses of 12.5-100 mg of BR931 per kg orally for 4 days. Plasma cholesterol and triglycerides were determined in the control and treated groups. Z’riton hyperlipidemia was achieved by i.p. administration of Triton WR1339 (400 mg/kg) [19]. BR-931 was given in 3 doses (50,100 and 150 mg/kg), clofibrate (200, 400 and 600 mg/kg) being the reference compound. Rats were killed 18 h after the administration of Triton and of drugs and vehicle. Plasma cholesterol and triglyceride levels were measured in all groups. Dietary hyperlipidemias were induced by ethanol and by Nath’s diet. Ethanol hyperlipidemia followed the 4-day protocol described by Puglisi et al. [ 201. This required ad libitum administration of 10% ethanol, from the 2nd to the

4th days of experimentation: in addition, in the evening of the 3rd and the morning of the 4th days a supplement of ethanol (0.5 g/rat) was given by gastric tube. Drugs were administered every morning from day 1 to day 4, and rats were killed 4 h after the last administration of ethanol and 2 h after the last dose of drug or vehicle. BR-931 was given in doses of 50, 100 and 150 mg/kg per day, and clofibrate at 200 mg/kg. Plasma triglycerides were determined in all groups. In a second experiment, ethanol was administered in the same way to rats treated with BR-931 (100 mg/kg). At the end of the experiment the distribution of lipoprotein cholesterol was assayed in control rats, and in rats treated with ethanol and ethanol + BR-931. Separation of heparin-precipitable and non-precipitable lipoproteins [ 211 was carried out as described below. Dietary induction of hyperlipidemia was also achieved with a slightly modi24% hydrogenated cocofied Nut/z’s diet [22], of the following composition: nut oil, 1% cholesterol, 1% cholic acid, 1% casein with vitamins, 4% mineral salts, 1% corn oil and 49% sucrose. Groups of 10 rats were treated for 12 days with the Nath’s regimen with or without the addition of BR-931 (12.5, 25 and 50 mg/kg), clofibrate (200 mg/kg) being the reference compound. After 12 days of treatment, controls, dietary controls and diet + drug-treated animals were killed, and plasma cholesterol and triglyceride levels were estimated, as well as cholesterol distribution in heparin-precipitable and non-precipitable lipoproteins. Anti-lipolytic activity of BR-931 was tested in acute experiments against: cold exposure (8 h at 8°C); noradrenaline (0.1 mg/kg i.p., the animals being killed 40 min after treatment); ACTH (Synacthen 200 III/kg s.c., 5 h before killing); and nicotine tartrate [23] (6 mg/kg i.p., the animals being killed 120 min after treatment). BR-931 was given in all of these experiments at doses of 25, 50 and 100 mg/kg, with the exception of the cold exposure test, where only the 100-mg/kg dose was tested. BR-931 was administered immediately before injection of the lipolytic agents or before the rats were exposed to cold. Clofibrate (200 mg/kg) was given as the reference compound in the noradrenaline and ACTH experiments. Hypercholesterolemia in rabbits was induced by feeding cholesterol in the diet (2%) for 8 weeks [ 241. Controls, cholesterol-fed rabbits and rabbits treated with cholesterol + BR-931 (50 mg/kg) were bled from an ear vein after 4 and 8 weeks, for the determination of plasma cholesterol, triglycerides and heparinprecipitable lipoproteins. After 8 weeks, animals were killed and the extent of atherosclerosis of the arch and thoracic aorta was assessed by a scoring method v51. Biochemical methods Plasma cholesterol and triglycerides were estimated by calorimetric [ 261 and enzymatic methods [27]. Free fatty acids (FFA) in the lipolysis experiments were determined according to Trout et al. [ 281. Cholesterol precursors [ 291 in plasma after sub-acute and chronic toxicity were analyzed by gas-liquid chromatography [ 301. Lipoprotein cholesterol distribution was determined by the heparin-manganese chloride procedure [ 211. The validity of this method in both rats and rab-

49

bits was confirmed by parallel ultracentrifugal separation of lipoproteins [ 311, which showed that in both species the heparin non-precipitable fraction corresponded to d > 1.065 lipoproteins, i.e. heparin-precipitable lipoproteins (HPL) were equivalent to VLDL + LDL in both species of animal. Statistical evaluation of the results was carried out by one-way analysis of variance, followed by inspection of all differences between pairs of means; in specific cases, i.e. repeated estimations in the same animal, a t-test of the differences was used [32]. Results Toxicity of BR-931 The oral LD 50 of BR-931 in mice and in rats could not be determined, being in excess of 5,000 mg/kg (the oral LD 50 of the parent compound Wy14,643 was, respectively, 1,600 and 4,150 mg/kg). In sub-acute toxicity tests, daily doses up to 400 mg/kg did not produce significant toxicity; however, liver enlargement was noted with doses above 50 mg/kg [33]. Chronic toxicity tests in both rats (6 months with 25, 50 and 100 mg/kg) and beagle dogs (18 months with 25, 50 and 75 mg/kg) failed to elicit significant changes. Gasliquid chromatographic analysis of plasma sterols also did not indicate any increase of cholesterol precursors in treated animals. Effect on basal plasma lipids and in Triton hyperlipidemia Administration of BR-931 (50 mg/kg) to normal rats in 4 different experiments, produced a fall of total cholesterol and triglycerides between 21 and 41.3%; a slightly higher effect was noted with 100 mg/kg. After these preliminary tests, doses of 12.5-150 mg/kg were selected for further pharmacological testing. In Triton-induced hyperlipidemia, doses of 50-150 mg of BR-931 proved about equivalent in hypocholesterolemic and hypotriglyceridemic activity to 200-600 mg/kg doses of clofibrate; 150 mg BR-931 per kg proved significantly more effective in lowering triglycerides than 600 mg clofibrate per kg (Fig. 2).

C

C

TRITON

CP10rn

200

Fig. 10

2.

400

50

BR 9310 Comparative

animals.

X

f SEM).

effects *

200

600

100 of

TRITON

BR-931

P < 0.05;

**

and

400

50

150 clofibrate

P < 0.01

(CPIB)

“S controls

(C);

100

in Triton ??P <

600 m@Kg 150 mglKg

hyperlipidemia

0.05;

**P

< 0.01

in rats “s CPIB.

(groups

of

50

s ol

E

200

SI :: 6 ::$:

:::::

E ..:.:. :::::: :::::

ii

0

::::::

f15 Y ;

::::::

:#

:::::: .:::::

100

E

:::::: 3 g$ :::::. :::::: :q .:3:.

i0 100 200

c Fig.

3.

(groups

EtOH

BR 931

Comparative of

10

200

effects

animals,

mg/Kg

CPIB of

BR-931 *

N + SEM).

and

P < 0.05;

clofibrate **

(CPIB)

in

ethanol

P < 0.01 “S controls

(C);

hypertriglyceridemia ??P

< 0.05;

in rats

P < 0.01

??*

“S

CPIB.

Diet-induced hyperlipidemias In Ethanol hypertriglyceridemia was evaluated in two different experiments. the first (Fig. 3), doses of BR-931 (50-200 mg/kg) were remarkably effective, as compared with clofibrate (200 mg/kg), a significant hypotriglyceridemic effect being evident with 50 mg/kg. In a successive experiment, the distribution of lipoprotein cholesterol was analyzed in control rats and in rats treated with ethanol and ethanol + BR-931 (100 mg/kg). Whereas ethanol significantly increased HPL, as compared with controls, the distribution of HPL and HDL in ethanol + BR-931-treated rats was similar to that of normal rats (Table 1). BR931-treated rats also had total cholesterol levels significantly lower than those of control rats. Hyperlipidemia induced with N&h’s diet is characterized by both hypercholesterolemia and hypertriglyceridemia. BR-931 at 25 mg/kg already exerted a significant effect on both cholesterol and triglycerides, a 50 mg/kg dose being significantly more effective than 200 mg clofibrate per kg (Table 2). In this experiment, HPL cholesterol levels were also determined. Nath’s diet inverted the normal HPL/HDL cholesterol ratio, and this change was not modified by clofibrate. In contrast, BR-931 more than doubled the percentage of HDL cholesterol, as compared with dietary controls, thus bringing the HPL/HDL cholesterol ratio towards normal (Fig. 4).

TABLE

1

TOTAL (x

AND

f SEM

HDL

FOR

CHOLESTEROL

GROUPS

OF

10

LEVELS

IN

RATS

TREATED

WITH

ETHANOL

AND

ANIMALS)

Total

cholesterol

(mg/dl)

%inHDL

Controls

83.1

r 3.0

Ethanol

99.6

t 3.0

a

69.1

? 3.8

a

59.3

?: 1.9

b.d

76.2

_+ 2.1

c

d P

< 0.01

Ethanol

a P

+ BR-931

< 0.05:

b P

(100

< 0.01

me/kg)

vs controls;

c P

< 0.05;

71.7

vs ethanol

group.

f 1.4

BR-931

2

LIPID

CHANGES

3

64.2 f 2.4

8 weeks

27.2 ? 3.6

8 weeks

0.25 ? 0.16

Thor&c

e P < 0.05: b P < 0.01 as compared

0.36 i 0.17

Arch

scores [251

33.2 f 2.1

4 weeks

atherosclerosis

34.5 f 1.6

base

1.9

1.9

1.42 ? 0.34

1.87 + 0.23

75.4 I 12.4

77.3 r 19.3

32.9 *

(mg/dl)

2415.1 r 182.4

1125.8 ? 123.4

68.5 +

Diet

diet group.

cholesterol

64.7 f 3.3

4 weeks

lipoprotein

69.8 ? 2.4

levels (mg/dl)

base

C. Aortic

242.4 ? 14.4 461.2 ? 35.4

64.5 ? 1.5

Diet

CLOFIBRATE

107.4 f 3.2

AND

mg/kg/day) IN CHOLESTEROL-FED

Controls

OF BR-931(50

B. High-density

BR-931

Controls

AFTER

2.1

1.0

1.12 i 0.23

1.75 * 0.16 b

115.2 * 26.7 a

133.9 f 22.0 a

35.5 *

2184.2 t 184.1

996.7 fz 107.8

68.5 ?

Diet + BR-931

lo.9 b

DIET

166.5 f 2.0

66.6 ? 2.3

67.1 t 2.3

A. Plasma triglyceride

Controls

12 DAYS

GROUP)

166.5 ? 5.8

124.4 _+6.7

levels (mg/dI) 69.4 ? 1.9

Diet

lo.9 c-d

OF

111.0 + 9.3 b

88.0 !z 6.1 e

68.8 r 2.2

Diet + BR-931

195.8 ? 15.6 b

200.2 + 13.5 e

200 mg/kg

+ CPIB,

(X ? SEM FOR GROUPS

135.0 + 11.0 c.d

111.2 +

50 mglkg

+ BR-931,

FOR

PER EXPERIMENTAL

242.1 ? 27.9 b

(% f SEM OF 8 ANIMALS

365.3 t 20.1 =

189.0 ?

25 mglkg

218.5 f 13.7

+ BR-931,

NATH’S

12.5 mglkg

FED A MODIFIED

+ BR-931,

IN RATS

RABBITS

P < 0.01; c P < 0.001 vs. diet; d P < 0.01 vs. CPIB.

A. Plasma cholesterol

EFFECTS

TABLE

e P < o.05;b

Total cholesterol (mg,d,) Triglycerides (mg/dl)

10 ANIMALS)

PLASMA

TABLE

52 a-LIPOPROTEIN ??

CHOL.

B-LIPOPROTEIN

CHOL.

Isi! 100

in z

6O

:

0 a

60

8 3 2

40

E .-' 20

0 NATH’S DIET

STD DIET

25

12.5

NATH’S

DIET

after

DIET

CLOFtBRATE

t3R’931 Fig. 4. Lipoprotein cholesterol distribution diet (groups of 10 animals, X ? SEM).

mglKg

200

50

NATH’S

BR-931

and clofibrate

in rats fed on a modified

N&h’s

An ti-lipoly tic activity BR-931 exerted a significant anti-lipolytic effect against all lipolytic agents tested (Fig. 5), the effect being most marked against ACTH and nicotine. In the cold-exposure test a significant antilipolytic effect of BR-931 was also observed (basal, 546 + 21 pEq/l; cold; 2365 ? 165 pEq/l; cold + 100 mg BR-931,1082 +

1800

NICOTINE

,

NORADRENALINE

ACTH

1600 1400 1200

25

C

NIC

+

50 loo BR 931

; I

25 C

NA

+

50 100 BR 931

1 I

25 C ACTH

+

50 loo BR 931

Fig. 5. Antilipolytic effects in different tests of BR-931 and clofibrate SEM). * P < 0.05: * * P < 0.01 vs controls (C); * P < 0.05; ** P < 0.01

200

mglKg’

CPM

(CPIB) (groups vs CPIB.

of 10 animals. X ?

53

17 pEq/l, P < 0.001). Against noradrenaline, only the higher doses of the drug were effective. Clofibrate did not show a significant activity in the ACTH (Fig. 5) or noradrenaline (data not shown) experiments. Rabbit hypercholesterolemia and atherosclerosis Cholesterol administration to rabbits induced, both in control and BR-931treated animals, a marked hypercholesterolemia. The difference of plasma total cholesterol levels between the two groups did not reach statistical significance, whereas triglycerides were significantly reduced by the drug treatment after 2, 4 and 8 weeks of cholesterol feeding (Table 3A). Analysis of the lipoprotein cholesterol distribution indicates that the percentage of HDL cholesterol was almost doubled after 4 weeks in diet + BR-931treated rabbits, and it was still significantly increased after 8 weeks of treatment (Table 3B). The extent of atherosclerosis, estimated after the rabbits were killed [24], was marked in both treatment groups. However, atherosclerosis of the arch was significantly decreased in BR-931-treated rabbits, the same not being true for atherosclerosis of the thoracic aorta (Table 3C). Discussion BR-931 is a component of a new series of hypolipidemic compounds structurally unrelated to clofibrate. Available data on the most potent member of this series of 2-pyrimidinylthio-acetic acid derivatives, Wy-14,643, indicate both hypolipidemic properties [15] and a significant protective effect in cholesterol atherosclerosis in rabbits [ 341. BR-931, the ethanolamine derivative of Wy-14,643, shows a markedly different toxicity from the parent compound, suggesting that the addition of the ethanolamine group, already described as being hypolipidemic per se [ 351, somewhat changes the toxicological, although probably not the pharmacological, properties of BR-931. In particular, evaluation of the acute toxicity tests suggests that BR-931 is not broken down to Wy-14,643. During determination of the LD 50, rats and mice are, in practice, followed for one week after drug administration; during this period there would be ample chance for BR-931 to break down to Wy-14,643 and ethanolamine, thus giving an LD 50 identical with that of Wy-14,643. The same would obviously be true for the sub-acute and chronic toxicity tests. More direct evidence is provided by pharmacokinetic studies now in progress [36], demonstrating a very short plasma half-life of BR-931, without formation of specific metabolites. Detailed studies on the pharmacological effects of BR-931 point out interesting differences between this agent and clofibrate. BR-931 is 4-8 times more potent than clofibrate in standard tests of hypolipidemic activity (ad libitum diet, Triton hyperlipidemia, diet-induced hyperlipidemias); it also displays a significant anti-lipolytic activity, particularly against ACTH, cold exposure and nicotine. In the two tests of lipolysis where clofibrate was tested as the reference compound, it failed to show a significant effectiveness. Reduction of lipolysis probably plays an important role in the over-all hypolipidemic activity of BR-931. Major hypolipidemic drugs now available have

54

different effects on lipolysis in adipose tissue. Experiments with clofibrate, in vivo and in vitro, both in man and animals [37-391, have given irregular results, some suggesting a reduced lipolysis, some no effect of. More recent studies on isolated adipose tissue in vitro [ 401, however, have shown a significant decrease of cyclic AMP, concomitant with a reduction of basal glycerol release, after the addition of clofibrate to the system. These results have suggested that clofibrate may shift the balance between the adipose-tissue hormone-sensitive lipase and lipoprotein lipase [41], significantly increasing the latter enzyme. This hypothesis is supported by recent findings in treated patients, indeed showing an increase of adipose tissue lipoprotein lipase [ 421. Although similar data are not available for BR-931, the powerful effect on Triton hyperlipidemia is consistent with some activity of the drug on the lipase system. BR-931 seems, on the other hand, more similar to nicotinic acid, which has a marked anti-lipolytic activity in vivo. Nicotinic acid does not affect basal cyclic AMP levels in adipose tissue, but significantly reduces them after stimula[ 431. In the case of nicotinic acid, a temtion by noradrenaline or theophylline poral relationship may be seen between decreased lipolysis and fall of plasma triglycerides, which is later followed by total and LDL cholesterol decreases [44]. The existence of a similar temporal relationship for BR-931 is currently under study. The most interesting finding with BR-931 remains, however, that of increased HDL cholesterol levels in animals treated with atherogenic diets. Increased HDL levels were accompanied, in cholesterol-fed rabbits, by significantly reduced atheromatosis of the aortic arch, confirming a previous similar observation by Kritchevsky et al. [34]. It is obviously difficult to reconcile these morphological findings with the increase of HDL cholesterol, because the other authors did not meausre lipoprotein distribution in their animals. The potential protective effect against atherosclerosis of increased HDL is, however, suggested by so many clinical [8,9] and experimental [45] observations that a correlation between the two groups of observations should not be ruled out. Increased HDL levels in BR-931 rats were associated with marked hepatomegaly. A preliminary report by Reddy [33] shows that BR-931 increases liver peroxisomes as well as the activities of hepatic catalase and camitine acetyltransferase. Liver peroxisome proliferation appears to be closely associated with the hypolipidemic activity of several compounds [46]. BR-931 is similar in activity to Wy-14,643 in inducing peroxisome proliferation in liver cells, i.e. these compounds are 2-4 times more potent than clofibrate. Detailed studies on the comparative effects of BR-931 and Wy-14,643 on hepatic peroxisomes and peroxisome-associated enzymes will be reported elsewhere [ 471. Drugs that decrease heparin-precipitable lipoproteins were the object of a large screening effort by Day et al. [ll]. These authors described several compounds that decreased HPL in rats, the most potent of which, U-14,792, an adamantyloxyphenylpiperidine, however, showed unacceptable liver toxicity in rats. Procetofene, a clofibrate derivative, increases HDL levels in rats on a fatty diet [ 131. The same finding has been reported for hyperlipidemic, particularly type IV, patients [14], who also showed a less significant LDL cholesterol increase than noted with clofibrate. Although a comparison of the procetofene and BR-931 findings in rats is not possible, since the dietary treatment schedule

55

for procetofene has not been made available, it may be noted that procetofene increases HDL levels in rats with doses above 100 mg/kg, i.e. far in excess of the therapeutic range, whereas a similar effect is achieved with lower doses of BR-931. In conclusion, BR-931 is a new hypolipidemic agent structurally unrelated to clofibrate, and markedly different from clofibrate in the pharmacological profile. Its effects, in particular the increase of high density lipoprotein cholesterol, are of potentially high interest, and encourage application of this new agent to the treatment of hyperlipidemias and to the prevention of IHD. References 1 Sirtori, C.R., Catapsno, A. and Paoletti. R.. Therapeutic significance of hypolipidemic and antiatherosclerotic drugs. In: Atherosclerosis Reviews, Vol. 2, Raven Press., New York, N.Y., 1977. p. 113. 2 Levy. R.I.. The effect of hypolipidemic drugs on plasma lipoproteins, Ann. Rev. Pharmacol. Toxic&. 17 (1977) 499. 3 The Coronary Drug Project: Clofibrate and niacin in coronary heart disease, J. Amer. Med. Ass., 231 (1975) 360. 4 Wilson, D.E. and Lees, R.S.. Metabolic relationships among the plasma lipoproteins, J. Clin. Invest., 51 (1972) 1051. 5 Carlson. L.A., Olsson, A.G.. Or6, L., Rtissner. S. and Walldius, G.. Effect of hypolipidemic regimens on serum lipoproteins. In: Atherosclerosis III, Springer Verlag. Berlin, 1974, p. 768. 6 Goldstein, J.L. and Brown. M.D., Lipoprotein receptors, cholesterol metabolism and atherosclerosis. Arch. Path., 99 (1975) 181. 7 Rose, H.G., Haft, G.K. and Juliano. J., Clofibrate-induced low density lipoprotein elevation - Therapeutic implications and treatment by colestipol resin, Atherosclerosis, 23 (1976) 413. 8 Miller. G.J. and Miller, N.E.. Plasma-high~ensity-lipoprotein concentration and development of ischaemit heart disease, Lancet. 1 (1975) 16. 9 Stanhope. J.M., Sampson. V.N. and Clarkson. P.M., High-density-lipoprotein cholesterol and other serum lipids in a New Zealand biracial adolescent sample, Lancet, 1 (1977) 968. 10 Carlson, L.A., Olsson. A.G. and Ballantyne, D.. On the rise in low density and high density llpoproteins in response to the treatment of hypertriglyceridemia in type IV and type V hyperlipoproteinemiss. Atherosclerosis, 26 (1977) 603. 11 Day, C.E., Schurr. P.E. and Heyd, W.E., Biological activity of a hypobetalipoproteinemic agent. Adv. EXP. Med. Biol., 67 (1976) 215. 12 Day, C.E.. Personal communication. 13 Eckhardt. B., Lipanthyl (W 13,653/LF178): Summary of pharmacological activity, Warner Lambert Res. Invest. R.R. 915-0163.1976. 14 Riissner, S. and Or6, L.. Effects on serum lipoproteins of procetofene (Lipanthyl) in hyperlipoproteinemia - Dose-response studies and a comparison with clofibrate. In: Proc. Int. Conference on Atherosclerosis, Milan, 9-11 Nov. 1977. 15 Howard, A.N.. Gemfibrozil -A review of current clinical pharmacology. In: Proc. Int. Conference on Atherosclerosis, Milan, 9-11 Nov.. 1977. 16 Olsson, A.G.. Carlson. L.A.. RBssner, S.. Walldius. G. and Lang. P.D.. Long term effects of Bezafibrate on serum lipoproteins in hyperlipoproteinemia (HLP). In: Proc. Int. Conference on Atherosclerosis. Milan, 9-11 Nov.. 1977. 17 Santilli, A., Scotese, A.C. and Tomarelli. R.M.. A potent antihypercholesterolemic agent: I-chlorod(2,3-xylidino)-2-pyrimidinylthio acetic acid (WY-14.643). Experientia. 30 (1974) 1110. 18 Buchanan, R.L., Sprancmanis, V. and Partyka. R.A.. Hypocholesterolemic 5substituted tetrazoles. J. Med. Chem.. 12 (1969) 1001. 19 Schurr, P.E.. Schultz, J.R. and Parkinson, T.M.. Triton-induced hyperlipidemia in rats as an animal model for screening hypolipidemic drugs, Lipids. 7 (1972) 68. R. and Sirtori, C.R.. Effect of hypolipidemic and hypo20 Puglisi, L.. Caruso, V., Conti. F.. Fumagalli. glycemic drugs on ethanol-induced hypertrlglyceridemia in rats, Pharmacol. Res. Comm., 9 (1977) 1. 21 Burstein. M., Scholnick. M.R. and Morfin, R.J., Rapid method for the isolation of lipids from human serum by precipitation with polyanions. J. Lipid Res. 11 (1970) 583. 22 Nath, N.. Wiener, R., Harper, A.E. and Elvehjem. C.A.. Diet and cholesterolemia, Part 1 (Development on a diet for the study of nutritional factors affecting cholesterolemia in rats), J. Nutr., 67 (1959)

289.

56

23 24

25 26 27 28 29 30 31 32 33 34 35

36 37 38 39 40 41

42 43 44 45 46 47

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