Studies on the Mechanism of Action of Halofenate I LEWIS R. MANDEL, Merck Institute for Therapeutic Research, Rahway, New Jersey 07065 ABSTRACT
INTRODUCTION
This paper reviews most of the preclinical studies on the mode of action of halofenate, an established hypolipidemichypouricemic agent in man. In yeast cultures and in isolated rat adipocytes, halofenate was found to inhibit the conversion of pyruvate to acetyl CoA. While pyruvate dehydrogenase was inhibited in vitro, halofenate also inhibited the activity of various other isolated enzymes. In rats maintained on halofenate in the diet (0.02-0.10%) for 2-14 days, there were 20-40% decreases in plasma cholesterol, t r i g l y c e r i d e s , phospholipids, and free fatty acids. Inhibition of liver HMG-CoA reductase does not appear to account for the hypocholesterolemic effect, and activation of mitochondrial t~-glycerophosphate dehydrogenase does not explain the hypotriglyceridemic action. Kinetic measurements of the serum appearance a n d disappearance of triglycerides in drug-treated rats suggest that the hypotriglyceridemic activity is due to a net inhibition of hepatic triglyceride synthesis. Reduction of very low density lipoprotein (VLDL) and high density lipoprotein (HDL) levels in rats with sucrose-induced hyperlipidemia and normalization of the altered apolipoprotein profiles are in accord with the effects of halofenate on plasma triglyceride and cholesterol levels. T h e reduced insulin-to-glucagon ratio observed in Zucker obese hyperlipemic rats is also consistent with halofenate's hypotriglyceridemic activity. Preliminary experiments in rats on the mechanism of its hypoglycemic activity, observed in some diabetic hyperlipidemic patients, indicate that halofenate acts differently t h a n conventional oral hypoglycemic agents. Some, but not all, of the effects of halofenate were observed with clofibrate at two to ten times higher levels.
The question of whether plasma lipid lowering agents exert their action by affecting a s i n g l e e n z y m e or by influencing several enzymes or biochemical processes has been investigated for many years in a number of laboratories. In the case of clofibrate, for example, there are a variety of mechanisms which could account for its hypotriglyceridemic and hypocholesterolemic activity (1-6), How these different mechanisms relate to one another and to the clinical effects of clofibrate is not yet established. The intent of this paper is to outline the highlights of several preclinical investigations on the mode of action of the hypolipidemic agent halofenate [ 2-acetamidoethyl(p-chlorophenyl) (m-trifluoromethylphenoxy) acetate]. Some of these studies were carried out by members of the Department of Biochemistry at the Merck Sharp & Dohme Research Laboratories while others were undertaken by collaborating investigators. Data sources include unpublished studies and personal communications as well as published reports on halofenate. Figure 1 shows the structure and nomenclature for halofenate (MK-185). Like clofibrate, halofenate produces reductions primarily in serum triglycerides but less frequently in serum cholesterol. Like probenecid, halofenate produces consistent reductions in uric acid. The changes in metabolic parameters at the end of 48 wk of treatment with 1 g of halofenate in a single daily oral dose are shown in Table I. These data indicate the order of the hypolipemic activity of halofenate and are representative of the results obtained in various trials during 5 y r of clinical evaluation. A few selected papers on the clinical effects of halofenate are given in the reference section (7-13). RESULTS AND DISCUSSION Isolated Systems
1The list of literature references on halofenate cited in this paper is not a complete one. Since 1970, there have been over I00 publications on halofenate of which 20 relate to mechanism of action studies in laboratorY animals or man. Fifty are primarily concerned with clinical hypolipemic and hypouricemic activity and the balance on other aspects of its properties. 34
In the initial mechanism studies, the effect of halofenate on lipid synthesis in cultures of S a c c h a r o m y c e s cerevisiae was investigated. Since the enzymatic steps of lipid biosynthesis are similar in yeast and mammalian cells, it was reasoned that the simpler system might identify a primary site of action more readily (14). The results of this line of investigation are summarized in Table II. The free acid of halofenate
MECHANISM OF ACTION OF HALOFENATE ( H F A ) , t h e active m o i e t y ( 1 5 ) , was f o u n d to i n h i b i t yeast g r o w t h reversibly; at 0.34 mM, 50% i n h i b i t i o n o f g r o w t h was achieved. T h e i n h i b i t o r y effect o f H F A was p r e v e n t e d by a d d i n g 0.35 mM oleic or 3.7 mM acetic acids to t h e c u l t u r e m e d i u m . Pyruvic acid at m u c h higher c o n c e n t r a t i o n s (to 72 m M ) c o u l d n o t by-pass t h e block. H F A i n h i b i t e d the convers i o n of 14C-glucose a n d 1 4 C - p y r u v a t e t o labelled f a t t y acids a n d sterols, b u t c o n v e r s i o n of 14C-acetate to lipids was n o t affected. These findings t a k e n w i t h t h e i n h i b i t i o n o f t h e decarboxylation o f p y r u v a t e - l - 1 4 C t o 14CO2 strongly suggest t h a t in t h e y e a s t system, g r o w t h and lipogenesis are i n h i b i t e d by a b l o c k at t h e p y r u v a t e d e h y d r o g e n a s e c o m p l e x . In e x t e n d i n g these o b s e r v a t i o n s to a n i m a l tissues, e x p e r i m e n t s were carried o u t w i t h i s o l a t e d a d i p o c y t e s p r e p a r e d f r o m rat fat pads (16). White H F A i n h i b i t e d t h e u p t a k e of 14C-glucose a n d 1 4 C - p y r u v a t e t o t h e same e x t e n t as it b l o c k e d t h e i r i n c o r p o r a t i o n i n t o f a t t y acids, it did n o t i n h i b i t t h e u p t a k e of l e u c i n e , p a l m i t a t e , or 2 - d e o x y g t u c o s e , suggesting t h a t it was n o t acting as a n o n s p e c i f i c t r a n s p o r t i n h i b i t o r . T h a t t h e drug i n h i b i t e d lipogenesis was d e m o n s t r a t e d in e x p e r i m e n t s w h e r e i n H F A i n h i b i t e d c o n v e r s i o n of p y r u v a t e 2-14C to labelled f a t t y acids t o t h e same e x t e n t as it b l o c k e d t h e d e c a r b o x y l a t i o n of p y r u v a t e 1-14C to 14CO2 ( T a b l e III). A c o m p a r i s o n of t h e activity of H F A w i t h t h r e e e s t a b l i s h e d
35
C1
0
0
O--CH-- C - O - C H 2 CH2-NH--C-- CH 3
CF3 Halofenate
(MK-185)
2-Acetarnidoethyl (p_-chlorophenyl ~ (m-trifluoromethylphenoxy}acetate
FIG. 1. Structure and nomenclature of halofenate. lipogenesis i n h i b i t o r s h y d r o x y c i t r a t e , k y n u r e hate a n d c e r u l e n i n - s h o w s t h a t at levels w h i c h i n h i b i t f a t t y acid s y n t h e s i s 50%, i n h i b i t i o n of p y r u v a t e d e c a r b o x y l a t i o n decreased w i t h t h e p r o x i m i t y of c y t o p l a s m i c e n z y m e to p y r u v a t e d e h y d r o g e n a s e (16). These findings f u r t h e r suggest t h a t i n h i b i t i o n of e n z y m e s involved in f a t t y acid b i o s y n t h e s i s in the c y t o p l a s m results in p r o d u c t i n h i b i t i o n o f m i t o c h o n d r i a l p y r u v a t e d e h y d r o g e n a s e . While t h e exact site of a c t i o n of h a l o f e n a t e o n lipogenesis in a d i p o c y t e s c a n n o t be determined u n e q u i v o c a l l y f r o m these studies, t h e p a t t e r n of data suggests t h a t H F A acts prior t o t h e c o n v e r s i o n o f citrate to acetyl CoA, n a r r o w i n g t h e site t o p y r u v a t e d e h y d r o genase or citrate s y n t h e t a s e . In c o n t r a s t to t h e results o b t a i n e d in yeast and adipocytes, halofenate-induced inhibition of lipid s y n t h e s i s in isolated liver systems was
TABLE I Changes in Metabolic Parameters at the End of 48 Weeks of Halofenate Treatment Measurement a
Pretreatment mean
Body weight (lbs) Fasting blood sugar (mg %) Serum triglycerides (mg %) Serum cholesterol (mg %) Serum uric acid (mg %)
17"7.4 116.4 399.4 293.3 6.87
Change from pretreatment mean
% Change
+1.2 -8.0 -93.4 -15.3 -2.32
+0.7 -6.9 -23.4 -5.2 -33.8
a328 patients, primarily type IV. TABLE II Effect of Halofenate Free Acid (HFA) on Growth and Lipid Synthesis in S. c e r e v i s i a e a 1. HFA inhibits yeast growth 50% at 0.34 raM. 2. Inhibitory effect prevented by added oleate or acetate but not pyruvate. 3. HFA inhibits conversion of glucose-U-14C or pyruvate-2-14C, but not acetate-2-14C, into labelled fatty acids and sterols. 4. HFA inhibits conversion of pyruvate-l-14C to 14CO2. 5. In vitro yeast PDH is inhibited 50% by 0.4 mM HF#~ aAdapted from the report of Greenspan and Germershausen (14). LIP1DS, VOL. 12, NO. 1
L.R. MANDEL
36
TABLE III Effect of HFA on Lipid Synthesis in Isolated Rat Adipocytesa mitochondria glu--§ p y r - ~ acetylCoA-----~ c i t r a t e ~ HFA
Compound 1.5 mM 10.0 mM 10.0 mM 0.12 mM
HFA Hydroxy citrate Kynurenate Cerulenin
c i t r a t e - ~ a c e t y l C o A - - ~ malonylCoA-!l~ fattyacids hydroxy kynurenate cer enin citrate % Inhibition pyr-2-14C to pyr-l-14C to Site fatty acids 14CO2
[PDH] ATP-citrate lyase Acetyl CoA carboxylase Fatty acid synthetase
50 50 50 50
50 40 27 16
aAdapted from the report of Greenspan et al. (16). TABLE IV Effect of Halofenate (free acid) on the Activity of Various Enzymes in vitro a
En zy me
Source
Conc. (mM) of HFA producing 50% inhibition
Pyruvat e dehydrogenase
Yeast Pig heart
0.4-3.0 2.0
Acetyl CoA carboxylase
Chicken liver Yeast
Citrate cleavage enzyme
Rat liver
2.0
HMG CoA reductase
Yeast Rat liver
0.1 >1.0
1.5 0.2
Fatty acid synthetase
Rat liver
0.3
a-Glycerophosphate dehydrogenase
Rat liver
0.3
Glucose 6-phosphate dehydrogenase Lactic acid dehydrogenase Prostaglandin synthetase Insulinase
Yeast Rabbit muscle Sheep seminal vescicle Rat liver
5.0 9.2 0.2 1.0
aTaken from Vol. I of the preclinical evaluation of halofenate, Merck Sharp & Dohme Research Laboratories, March 22, 1974. associated with an i n h i b i t i o n o f p r o t e i n synthesis. Thus, in liver slices f r o m n o r m a l adult rats, Morris 7777 h e p a t o m a and fetal rat liver, or in p r i m a r y rat fetal h e p a t o c y t e s g r o w n in culture, neither halofenate nor clofibrate i n h i b i t e d lipid s y n t h e s i s out o f p r o p o r t i o n to the inhibition o f t o t a l p r o t e i n synthesis ( G r e e n s p a n ; Weinstein and Steinberg, u n p u b lished results). With isolated h e p a t o c y t e s f r o m adult rats, i n h i b i t i o n o f lipogenesis was also associated w i t h i n h i b i t i o n o f p r o t e i n synthesis ( 1 7 ; G o u l d and S h r e w s b u r y , u n p u b l i s h e d results). In h e p a t o c y t e s , h a l o f e n a t e was also s h o w n t o stimulate f a t t y acid m e t a b o l i s m , suggesting the drug causes t h e i r d i s p l a c e m e n t f r o m a l b u m i n (17). While liver m i t o c h o n d r i a l preparations f r o m rats m a i n t a i n e d on h a l o f e n a t e or LIPIDS, VOL. 12, NO. 1
clofibrate s h o w e d a s t i m u l a t i o n o f c h o l e s t e r o l 26-14C o x i d a t i o n to 14CO2, an e f f e c t apparently related t o an increase in liver size comm o n to several p h e n o x y a c e t i c acid s t r u c t u r e s (18-19), the significance o f this activity in t e r m s o f u n d e r s t a n d i n g its h y p o l i p e m i c activity is n o t clearly e s t a b l i s h e d (1). Another approach undertaken to determine the m o d e o f action o f plasma h y p o l i p e m i c drugs has b e e n t o evaluate t h e c o m p o u n d s in vitro as i n h i b i t o r s o f isolated e n z y m e s involved in the b i o s y n t h e s i s o f triglycerides and sterols. As in the case o f clofibrate, this does n o t seem to r e p r e s e n t a fruitful line o f e n d e a v o r for halofenate. Table IV s h o w s t h a t H F A inhibits the activity of e n z y m e s involved in a variety o f b i o c h e m i c a l reactions. Moreover, e n z y m e s n o t
MECHANISM OF ACTION OF HALOFENATE
37
TABLE V Effect of Halofenate on Plasma Lipid Parameters in Rats a % of Halofenate in diet (9 days) 0 (Control) 0.00625 0.0t25 0.025 0.05
% Decrease from control b
Body weight gain (gm)
Chol.
TG
PL
FFA
48 46 50 50 49
12 24 31 38
12 18 29 43
10 18 24 31
0 23 38 34
aAdapted from the report of Gilfillan et al. (24). bChol. = cholesterol, TG = triglycerides, PL = phospholipid, FFA = free fatty acids. TABLE VI Effect of Dietary (0.05%) Halofenate on Rat Liver HMG-CoA Reductase Activity and Sterol Synthesis in Vitro
Exp. no.
Animal group
Serum cholesterol
1 2 3 4
midnight rats- 7 days midnight rats - 7 days midnight rats - 2 days basal rats - 2 days
-50 -26 -36 -27
directly i n v o l v e d in lipogenesis are also i n h i b ited b y m i l l i m o l a r levels of H F A . C l o f i b r a t e free acid (CPIB) p r o d u c e d similar results at f o u r t o t e n t i m e s h i g h e r levels ( d a t a n o t s h o w n ) a n d t h e p u b l i s h e d l i t e r a t u r e i n d i c a t e s t h a t CPIB inhibits many enzymes and biochemical processes in v i t r o (3-6,20-23). Studies in Rats
The in vivo h y p o l i p e m i c a c t i v i t y of halofen a t e was first d e m o n s t r a t e d in rats given t h e d r u g orally (24). As s h o w n in T a b l e V, h a l o f e n a t e t r e a t m e n t ( 0 . 0 5 % in t h e diet for 9 days) p r o d u c e d ca. 3 5 - 4 0 % r e d u c t i o n in cholesterol, triglycerides, p h o s p h o l i p i d s , a n d free f a t t y acids in male H o l t z m a n rats (165 g) m a i n t a i n e d o n a 72% sucrose diet. A small (12%) b u t statistically significant r e d u c t i o n in c h o l e s t e r o l a n d triglycerides was f o u n d w i t h h a l o f e n a t e at 0 . 0 0 6 2 5 % in t h e diet, In this a n i m a l assay, h a l o f e n a t e was a b o u t six t i m e s m o r e p o t e n t t h a n c l o f i b r a t e as a h y p o c h o l e s t e r o l e m i c a g e n t a n d n e a r l y t w i c e as p o t e n t as a plasma h y p o t r i glyceridemic agent. To d e t e r m i n e w h e t h e r h a l o f e n a t e i n h i b i t e d p y r u v a t e m e t a b o l i s m , rats m a i n t a i n e d o n 0 . 0 5 % dietary h a l o f e n a t e for 10 days were given p y r u v a t e - 2 -14C b y i.p. i n j e c t i o n . One h o u r later t h e y were sacrificed a n d t h e livers r e m o v e d for t h e i s o l a t i o n of glycogen. T h e labelled g l y c o g e n
Percent change in Incorporation of 14C into digitonide precipitable sterols by liver homogenates from HM G-CoA 14C.acetat e 14C.mevalona t e reductase -13 +2 +30 -26
-8
+52
was h y d r o l y z e d t o glucose, w h i c h was d e g r a d e d (25). T h e r e was n o d i f f e r e n c e in t h e distribut i o n o f r a d i o a c t i v i t y in t h e c a r b o n a t o m s f r o m 14C-glucose derived f r o m t h e g l y c o g e n of control rats c o m p a r e d t o t h a t of t h e d r u g - t r e a t e d animals ( L a n d a u , u n p u b l i s h e d results). Had c o n v e r s i o n o f p y r u v a t e - 2 -14C t o labelled acetyl C o A b e e n i n h i b i t e d , t h e n t h e p e r c e n t of 14 C in c a r b o n a t o m s 3 a n d 4 w o u l d have b e e n expected t o b e l o w e r in t h e rats given h a l o f e n a t e . While this result, t a k e n w i t h t h e failure o f 4-10% d i e t a r y t r i a c e t i n or s o d i u m a c e t a t e t o reverse t h e h y p o l i p e m i c effect o f h a l o f e n a t e , does n o t c o n f i r m t h a t p y r u v a t e d e h y d r o g e n a s e is t h e site o f a c t i o n for t h e drug, a d d i t i o n a l in vivo e x p e r i m e n t a t i o n is r e q u i r e d t o validate or i n v a l i d a t e t h e p y r u v a t e d e h y d r o genase m e c h a n i s m . In view o f the i m p o r t a n t role of H M G - C o A r e d u c t a s e in c o n t r o l l i n g c h o l e s t e r o l b i o s y n t h e s i s (26), t h e effect of h a l o f e n a t e o n its activity was investigated. M a l e Sprague D a w l e y rats, 2 0 0 - 2 5 0 g, were given t h e drug at 0.05 a n d 0.15% in t h e diet f o r 2 a n d 7 days; e n z y m e activity was m e a s u r e d in liver e x t r a c t s at t h e t i m e o f its p e a k a c t i v i t y ( m i d n i g h t ) a n d at n o o n , t h e t i m e of its basal activity (27). Incorp o r a t i o n of 2-14C a c e t a t e a n d 2-14C-mevalo n a t e i n t o sterols was also m e a s u r e d in o n e o f t h e e x p e r i m e n t s (28). T h e d a t a in T a b l e VI LIPIDS, VOL. 12, NO. 1
38
L.R. MANDEL TABLE VII Comparison of the Effect of Halofenate and Clofibrate on Rat Liver c~-Glycerophosphate Dehydrogenase Drug
% Decrease in plasma triglycerides
0.05% Halofenate 0.1% Halofenate
42 51
77 (45% i") 92 ( 7 4 % t )
0.1% Clofibrate 0.2% Clofibrate
23 28
156 (195%i") 229 ( 3 3 2 % t )
None
Enzyme activity a 53
a~1/O2/50 mg tissue/hr.
show that 0.05% halofenate lowers plasma cholesterol but does not markedly reduce HMG-CoA reductase activity or the incorporation of labelled acetate or mevalonate into digitonide precipitable sterols (Erickson and Gould, unpublished results). At a level of 0.15% halofenate in 2-day experiments, there was a 40% decrease in food intake and the drop in plasma cholesterol was accompanied by a reduction in HMG-CoA reductase activity and incorporation of both 1-14C-acetate and 2 -14C mevalonate into sterols. In LM-cell culture, n e i t h e r h a l o f e n a t e n o r c l o f i b r a t e (at 12.5/ag/ml) inhibited desmosterol synthesis in assays which detected the HMG-CoA reductase inhibitory activity of 25-hydroxy or 7-keto cholesterol (29; 30 and Mandel, unpublished results), These results suggest that inhibition of mevalonate formation does not account for the hypocholesterolemic activity of halofenate in the rat, in contrast to results on clofibrate in which a marked and specific inhibition in cholesterol synthesis from 1-14C acetate but n o t from 2-14C mevalonate was reported ( 3 1 , 3 2 ) . However, preliminary data from another study led to the opposite conclusion. White had reported that clofibrate regulates hepatic cholesterol biosynthesis by inhibiting the microsomal reduction of HMG-CoA to mevalonate (33). When halofenate was given to 170 g male Sprague-Dawley rats at 0.05% in the diet for 7 days, the reduction of l 4C.HMG_Co A to labelled mevalonate was inhibited 52% in assays employing a liver microsomal system ( W h i t e , u n p u b l i s h e d results). Twenty-nine m/amoles of 14C-HMG-CoA was incorporated into mevalonate with preparations from control animals compared to 14.4 m/lmoles for the drug-treated rats. Clearly, more experimentation is required to understand the involvement of this key enzyme in the hypocholesterolemic action of halofenate. W i t h regard to the hypotriglyceridemic LIPIDS, VOL. 12, NO. 1
effect of halofenate, activation of mitochondrial c~-glycerophosphate dehydrogenase was considered because this should decrease the amount of a-glycerophosphate available for triglyceride synthesis. One effect of clofibrate is to induce the synthesis of liver mitochondrial proteins such as the dehydrogenase, and the hypolipemic activity of this drug has been attributed to this (34-36). Rats (Holtzman, 150 g) were maintained on dietary halofenate or clofibrate at least 7 days prior to enzyme assay. Liver mitochondrial enzymes were assayed by the method of Ruegamer et al. (34). As shown in Table VII under conditions where halofenate was more than four times as potent as clofibrate in lowering triglycerides, clofibrate was about seven times more effective than halofenate in stimulating the mitochondrial c~-glycerophosphate dehydrogenase (Huff, unpublished results). It would appear that if the mechanism of the hypotriglyceridemic action of these two drugs is similar, then their effect is probably not mediated by activation of the dehydrogenase. A l t e r n a t i v e l y , if clofibrate does influence plasma triglycerides because it activates this enzyme, then the mode of action of halofenate should be different. In another study designed to establish the mode of the hypotriglyceridemic action of halofenate, male Wistar rats (200 g) were fed a fat-free diet containing no drug, 0.02% or 0.10% halofenate for 14 days. They were then given equivalent amounts of either 2-3H - or l(3)-3H-glycerol i.v., and blood samples were collected from 10 to 1 5 0 r a i n later for the analysis of labelled triglycerides (37). Kinetic analysis of the serum appearance and elimination curves of 3H-triglyceride permitted an estimation of total serum 3H-triglyceride formation and fractional turnover rates. The experimental design was similar to that described by Nikkila and Kekki for measuring plasma triglyceride kinetics in man (38). It was found that the total amount of 3H-triglyceride formed
39
MECHANISM OF ACTION OF HALOFENATE
VLDL Plasma m ( j % of Lipoprolein Constituents
TG
Chol
,if
250
200
150
6
Ioo [I
12
24
N
N S S+H
t
TG ~1.9
T(; 80.3
TG "/7.,9 ~q
6.I
14...99 17.5
Pr 14.2
~
~176 1
TG
80
i
6O
i
'i
50
~"~ S+H
Protein
18
IOO
O
PL
TG / Pr Ratio
% Composition
o
N S S+H
6O
4O
40
ZO
PL
01 V//A N S S+H
o
2O
'~
N
PL II,O S
PL
12.l
S+H
0
N
S
;+H
FIG. 2. The abbreviations used are: VLDL = very low density lipoprotein; LDL = low density lipoprotein; HDL = high density lipoprotein; TG = triglyceride; Chol = cholesterol; PL = phospholipid, Pr = protein; N = normal diet (for rats maintained on a normal chow diet); S = sucrose fortified semi-purified diet (for rats maintained on the 60% sucrose diet); S+H = sucrose plus halofenate diet (for rats given 0.05% halofenate in the 60% sucrose diet). from the 2-3H - or l(3)-3H-glycerol in control rats was similar. Over 95% of the 3H serum triglyceride formed from either substrate circulated in a rapidly turning-over tirglyceride pool (t89 = 8 min). Treatment of the rats with 0.10% halofenate decreased by about 75% the labelling of serum triglycerides without increasing the fractional turnover rates. The drug was further found to decrease the incorporation of U-14C-glycerol into hepatic but not intestinal t r i g l y c e r i d e s without significantly altering incorporation of 14C into total phospholipids of either tissue. From these results, it appears that halofenate reduces serum trigiyceride levels by inhibiting hepatic synthesis. This inhibition could occur at a site subsequent to the formation of diacylgiycerophosphate from either a-glycerophosphate or dihydroxyacetone phosphate. Clofibrate at 0.25% in the diet produced a similar pattern of data in all the experiments with the 2-3H, 1(3)-3H, and UA4C-glycerot (39). In related studies, it was established that these drugs do not decrease net triglyceride synthesis by stimulating hepatic lipase activities but reduce the availability of fatty acids for de novo triglyceride synthesis (40). Studies on the effect of halofenate on lipoand apolipoprotein patterns were carried out on hyperlipidemic rats (Sprague-Dawley, 4 0 0 g
males) in which the hyperlipidemia was induced by feeding a semipufified diet containing 60% sucrose and 5% lard for 3 wk. This regimen produces about a threefold increase in the VLDL fraction, a 50% increase in the HDL fraction and a 40% decrease in LDL (41). After this period, the animals were fed the same diet containing 0.05% halofenate for 10 more days. Serum lipids, lipoproteins and apolipoproteins of the drug-treated rats were compared with patterns obtained from non-drug treated hyperlipidemic animals and with normolipidemic rats kept on a regular chow diet. U n d e r these test conditions, halofenate produced about a 60% reduction in total serum triglycerides and a 30% reduction in cholesterol levels. Figure 2 shows the concentration of VLDL constituents expressed on a plasma mg/dl basis. The dietary regimen induced a marked increase in VLDL triglyceride, cholesterol, phospholipid, and protein, and halofenate produced reductions in all four components. While the values for cholesterol and protein were normalized by the drug, triglycerides were not, and thus the composition analysis shows the triglyceride:protein ratio remained high at 12.5. It is possible that if drug treatment were continued beyond 10 days the triglyceride values would also return to baseline. FigL I P I D S , V O L . 12, N O . 1
L.R. MANDEL
40
LDL Plasma rag% of Lipoprotein Constituents
TG
Chol
PL
Protein
m
I00
Pr ~.4 8.0
4.0
4,0
8.0
8C
6.0
3.0
3.0
6.0
6O
4.0
2.0
2.0
4.0
2.0
1.0
1.0
Chol / Pr Rotio
% Composition
Pr ~.8
IOO Pr 24.7
.T4 Pr
.8l
Pr
5t!
Pr
i92
80
TG BA
TG ~A
TG 37.1
60
m
4O
25.C
~4.1
17,(]
PL
~4.fl
PL
!43
-PL ~0.5
N
S
S+H
20
2.0
.69
;m 16.4
~et ~8
20 I
0
N S S+H
0
N S S+H
0
N S S+H
0
N S S+H
o
O
t
N
S
S§
FIG. 3. The abbreviations used are: VLDL = very low density lipoprotein; LDL = low density lipoprotein; HDL = high density lipoprotein; TG = triglyceride; Chol = cholesterol; PL = phospholipid, Pr = protein; N = normal diet (for rats maintained on a normal chow diet); S = sucrose fortified semi-purified diet (for rats maintained on the 60% sucrose diet); S+H = sucrose plus halofenate diet (for rats given 0.05% halofenate in the 60% sucrose diet). ure 3 shows the data for the LDL analysis, where only small changes in the components are seen due to sucrose feeding, with and without halofenate. As only a small percent of the total plasma lipoprotein of rat serum is LDL, the changes in amounts and composition (e.g., drop in LDL protein due to sucrose which shows a tendency to normalize with halofenate, and an elevation in LDL triglyceride due to halofenate) may not be critical to the mode of action of halofenate. Figure 4 shows the pattern for t h e HDL analysis. Sucrose feeding induces a rise in all four components and halofenate treatment prevents this increase. The reduction in HDL cholesterol is expected as this is the major cholesterol carrying lipoprotein in the rat. The apoprotein profiles were determined by quantitative immunoelectrophoresis described by Laurell (42). While the total serum concentration of apoprotein B is unchanged in the sucrose fed rats, its distribution between the VLDL and LDL fraction is altered (Table VIII). In the normal rat, 22% of apoprotein B is present in the VLDL fraction compared to 76% in the serum of the sucrose hyperlipidemic rat. Halofenate tends to normalize the distribution of this apoprotein, but not to baseline values, again possibly because drug treatment for 10 LIP1DS, VOL. 12, NO. 1
days was not adequate. Nevertheless, the trend of the data suggests that hatofenate may correct a defect in VLDL catabolism and thus increase its rate of removal. The sucrose-induced increases in the apolipoproteins A-I, A-IV, and C-III and in the arginine-rich peptide were also prevented by halofenate; these observations are in accord with the changes seen in the serum lipoproteins. The details of the studies on the effect of halofenate on lipoprotein metabolism will be reported separately (Roheim, to be published). It might be added that, while clofibrate produced a similar pattern of data at a five times higher level (0.25% in the diet), it had a much more rapid onset of action, showing hypolipemic activity after only one day of administration, and halofenate clearly did not. The influence of halofenate therapy on i n s u l i n a n d glucagon secretion was also examined. For these studies, the Zucker obese rat with genetic endogenous hyperlipemia was used. Coincident with the plasma lipid lowering effects of halofenate, the insulin to glucagon molar ratio decreased from 2.72 in the control animals to 0.96 in rats treated with the drug for 10 days (43). Following arginine stimulation, the ratio remained reduced (0.87), in contrast to the greater ratio (2.5) seen in the control animals. The halofenate induced reduction in
MECHANISM OF ACTION OF HALOFENATE
41
HDL Chol / Pr Ratio
% Composition
Plasma m g % of Lipoprotein Constituents Protein PL
TG
Chol
60
r
50
60
--
I00
50
Pr
14.1 4.0
8O
3.0
60
Pr 15.(
I00
Pr l/l)
.41
.40
Pr N.I
71+4
70J
N
S
;+H
Pr
.41 h
80
60
[] 2.0
1.0
II
O ' +']"
N S S+H
O '' N
40
20
S+H
O ~ ...._ '_ S S+H
N S S+H
0
192
40
20
~L6
PL 91.1
51.4
N
S
;+H
0
FIG. 4. The abbreviations used are: VLDL = very low density lipoprotein; LDL = low density lipoprotein; HDL = high density lipoprotein; TG = triglyceride; Chol = cholesterol; PL = phospholipid, Pr = protein; N = normal diet (for rats maintained on a normal chow diet); S = sucrose fortified semi-purified diet (for rats maintained on the 60% sucrose diet); S+H = sucrose plus halofenate diet (for rats given 0.05% halofenate in the 60% sucrose diet). t h e insulin t o glucagon r a t i o was associated with hypolipemia, postarginine hyperglycemia, and hyperketonemia-metabolic parameters characteristic of glucagon excess relative t o insulin. These results are similar to t h o s e seen in rats given c l o f i b r a t e (44) a n d s h o w t h a t t h e h y p o l i p i d e m i c a c t i o n of h a l o f e n a t e is associated w i t h a r e d u c t i o n in t h e i n s u l i n to glucagon m o l a r r a t i o b o t h in t h e basal state a n d a f t e r arginine challenge. While a r e d u c t i o n in arginine-stimulated insulin secretion with potentiat i o n of glucagon release has also b e e n r e p o r t e d t o o c c u r w i t h c l o f i b r a t e in m a n (45), it w o u l d be at p r e s e n t i n a p p r o p r i a t e t o e x t r a p o l a t e t h e data w i t h h a l o f e n a t e t o m a n . These findings relate t o t h e r e c e n t l y o b s e r v e d b l o o d glucose l o w e r i n g effect o f h a l o f e n a t e in T y p e I V h y p e r l i p o p r o t e i n e m i c p a t i e n t s w h o s e p r e t r e a t m e n t fasting b l o o d sugar values were n e a r 2 0 0 m g %. In this s t u d y w i t h d i a b e t i c p a t i e n t s , m e a n b l o o d sugar values for the group ( 1 9 p a t i e n t s ) were decreased 25-50 mg % a f t e r 12, 24, 36, a n d 4 8 wk of halofenate t r e a t m e n t . A b o u t 60% of t h e p a t i e n t s h a d decreases o f 20 m g % or greater in t h e i r i n d i v i d u a l m e a n fasting b l o o d sugar values, a n d t h e m e a n decrease was a b o u t 80 m g % for this s u b s e t o f p a t i e n t s . T h e r e was also a n i m p r o v e m e n t in t h e glucose t o l e r a n c e t e s t (46;
TABLE VIII Distribution of Apoprotein B between VLDL and LDL Fractions Distribution (%) Animal group
VLDL
LDL
Normal Sucrose hyperlipidemic Sucrose hyperlipidemic plus halofenate (0.05% in the diet for 10 days)
22 76
78 24
39
61
aVLDL = very low density lipoprotein; LDL = low density lipoprotein. White, u n p u b l i s h e d results). In t h e case of clofibrate, t h e c h a n g e s were less c o n s i s t e n t , w i t h n o significant d r o p in t h e m e a n fasting b l o o d sugar value or i m p r o v e m e n t in glucose tolerance. H y p o g l y c e m i c a c t i v i t y was n o t o b s e r v e d in h a l o f e n a t e - t r e a t e d p a t i e n t s w h e r e t h e p r e t r e a t m e n t m e a n value was less t h a n 1 4 0 m g %. P r e l i m i n a r y e x p e r i m e n t s designed t o s t u d y the m e c h a n i s m b y w h i c h h a l o f e n a t e r e d u c e s b l o o d sugar have s h o w n t h a t this a g e n t does n o t l o w e r b l o o d glucose in e i t h e r t h e s t r e p t o z o t o c i n d i a b e t i c or a d r e n a l e c t o m i z e d rat ( T a b l e IX). T h e s e a n i m a l m o d e l s d e t e c t e d t h e h y p o LIPIDS, VOL. 12, NO. 1
42
L.R. MANDEL TABLE IX Comparison of Halofenate with Other Hypoglycemic Agents % Reduction in blood sugar Compound
Streptozotocin diabetic rat
Adrenalectomized rat
Halofenate Clofibrate Phenformin Tolb utamide Insulin
8% (300 mg/kg p.o.) 10% (300 mg/kg p.o.) 55% (200 mg/kg p.o.)
12% (100 mg/kg p.o.) 0% (100 mg/kg p.o.) 57% ( 25 mg/kg p.o.)
Insulinase in vitro; % inhibition at 1 mM 47 a 20 a 3 8
67% ( 1 unit/rat s.c.)
aFree acid.
glycemic activity of phenformin and insulin, and tolbutamide, respectively. Moreover, when normal and streptozotocin diabetic rats were treated with halofenate at 0.05% in the diet for 21 days, there was no reduction in blood glucose (Glitzer, unpublished results). The ability of halofenate to inhibit insulinase activity in vitro (47) is only of marginal interest as it has been shown that the drug inhibits several other enzymes in vitro (see Table IV). (Mandel, unpublished results). In summary, it is evident from this review that it is not possible to pinpoint a single enzyme or major site of action to account for t h e p l a s m a h y p o l i p i d e m i c a c t i v i t y of halofenate. However, several possibilities have been explored both with isolated intact systems and in short- and long-term studies in rats. The c o n t i n u i n g studies on hepatic triglyceride synthesis and on lipo- and apolipoprotein metabolism will undoubtedly shed more light on the mode of activity of this compound. Exploration of the effects of halofenate on plasma insulin levels both in rats and in man may offer an explanation for the hypoglycemic activity observed clinically. It should also be ponted out that there are many sites of action reported on in the published literature to explain the biological activity of clofibrate, and, though this drug has been known for a much longer period of time, the precise mechanism for its hypolipidemic effects still remains to be established unequivocally. ACKNOWLEDGMENT The author has been involved with the work described as a coordinator and reviewer. The contributions of Drs. W.A. Bolhofer, J.W. Huff, M.S. Glitzer, M.G. Greenspan, S.F. Kulaga, J.S. Walker and their associates of the Merck Sharp & Dohme Research Laboratories are gratefully acknowledged. I wish to thank our collaborating investigators, Drs. D. Weinstein and D. Steinberg (University of California), S.K. Erickson, M.A. Shrewsbury and R.G. Gould (Stanford University), S.A. Tepper and D. Kritchevsky (Wistar Institute), LW. White and B.R. Landau (Case Western LIPIDS, VOL. 12, NO. 1
Reserve U n i v e r s i t y ) , R.J. Cenedella and W.G. Crouthamel (West Virginia University), P.S. Roheim (Yeshiva University), R.P. Eaton (University of New Mexico), and their associates for their contributions and assistance.
REFERENCES 1. Steinberg, D., Drugs Inhibiting Cholesterol Biosynthesis with Special Reference to Clofibrate in "Atherosclerosis: Proc. of the 2nd Int'l. Symposium," Edited by R.J. Jones, Springer-Verlag, New York, NY, 1970, p. 500. 2. Levy, R.I., D.S. Fredrickson, R. Shulman, D.W. Bilheimer, J . L Breslow, N.J. Stone, S.E. Lux, H.R. Sloan, R.M. Krauss, and P.N. Herbert, Ann. Int. Med. 77:267 (1972). 3. Havel, R.J., and J.P. Kane, Ann. Rev. Pharmacology 13:287 (1973). 4. Sirtori, C.R., D. Torreggiani, and R. Fomagalli, Mechanism of Action of Hypolipidemic Drugs in "Lipids, Lipoproteins and Drugs," Edited by D. Kritchevsky, R. Paoletti, and W.L. Holmes, Plenum Press, New York, NY, 1975, p. 123. 5. Gamble, W., J. Theor. Biol. 54:181 (1975). 6. Yeshurun, D., and A.M. Gotto, Jr., Am. J. Med. 60:379 (1976). 7. J a i n , A., J.R. Ryan, D. Hague, and F.G. M c M a h o n , Clin. Pharmacol. Therap. 11:551 (t970). 8. Morgan, J.P., J.R. Bianchini, T-H Hsu, and S. Margolis, Ibid. 12:517 (1971). 9. Sirtori, C., A. Hurwitz, K. Sabih, and D.L. Azarnoff, Lipids 7:96 (1972). 10. Aronow, W.S., M.D. Vicario, IC Moorthy, J. King, M. Vawter, and N.P. Papageorge, Current Therapeutic Res. 18:855 (1975). 11. Bluestone, R., D. Campion, and J.R. Klinenberg, Arthritis Rheumatism 18:859 (1975). 12. Lisch, H.-J., J. Patsch, S. Sailer, and H. Braunsteiner, Atherosclerosis 21:391 (1975). 13. Dujovne, C.A., D.L. Azarnoff, D.H. Huffman, P. Pentikainen, A. Hurwitz, and D.W. Shoeman, Clin. Pharmacol. Therap. 19:352 (1976). 14. Greenspan, M.D., and J.I. Germershausen, J. Bacteriol. 113:847 (1973). 15. Hucker, H.B., L T . Grady, B.M. Michniewicz, S.C. Stauffer, S.E. White, G.E. Maha, and F.G. McMahon, J. Pharm. Exptl. Therap. 179:359 (1971). 16. Greenspan, M.D., J.I. Germershausen, and R. M a c k o w , B i o c h i m . Biophys. Acta 380:190 (1975). 17. Homcy, C.J., and S. Margolis, Atherosclerosis 19:381 (1974).
43
MECHANISM O F ACTION OF H A L O F E N A T E 18. Kritchevsky, D., S.A. Tepper, P. Sallata, J.R. Kabakjian, and V.J. Cristofalo, Proc. Soc. Exp. Biol. Med. 132:76 (1969). 19. Kritehevsky, D., and S.A. Tepper, Ibid 139:1284 (1972). 20. Carter, E.A., and K.J. Isselbacher, Life Sci. 13:907 (1973). 21. Capuzzi, D.M., R.D. Lackman, M.O. Uberti, and M.A. Reed, Biochem. Biophys. Res. Comm. 60:1499 (1974). 22. D'Costa, M.A., and A. Angel, J. Clin. Invest. 55:138 (1975). 23. Landriscina, C., G.V. Gnoni, and E. Quagliariello, Biochem. Med. 12:356 (1975). 24. Gilfiilan, J.L., V.M. Hunt, and J.W. Huff, Proc. Soc. Exp. Biol. Med. 136:1274 (1971). 25. Bernstein, I.A., and H.G. Wood, "Determination of Isotopic Carbon Patterns in Carbohydrate by Bacterial F e r m e n t a t i o n " in Methods in Enzymology, Vol. 4, Edited by S.P. Colowick and N.O. Kaplan, Academic Press, New York, NY, 1957, p. 561. 26. Rodwell, V.W., D.J. McNamara, and D.J. Shapiro, Adv. Enzymol. 38:373 (1973). 27. Heller, R.A., and R.G. Gould, Biochem. Biophys. Res. C o m m . 50:859 (1973). 28. Gould, R.G., and E.A. Swyryd, J. Lipid Res. 7:698 (1966). 29. Brown, M.S., and LL. Goldstein, J. Biol. Chem. 2 4 9 : 7 3 0 6 (1974). 30. Kandutsch, A.A., and H.W. Chen, J. Cell Physiol. 85:415 (1975). 31. Avoy, D.R., E.A. Swyryd, and R.G. Gould, J. Lipid Res. 6:369 (1965). 32. Gould, R.G., E.A. Swyryd, B.J. Coan, and D.R.
Avoy, J. Athero. Res. 6:555 (1966). 33. W h i t e , L . W . , J. Pharm. Exp. Therapeutics 178:361 (1971). 34. Ruegamer, W.R., W.W. Westerfield, and D.A. Richert, Endocrinology 75:908 (1964). 35. Hess, R., W. Staubli, and W. Riess, Nature 2 0 8 : 8 5 6 (1965). 36. Pereira, J.N., and G.F. Holland, Studies on the Mechanism of Action of p-Chloro-phenoxy isobutyrate (CPIB), in "Atherosclerosis: Proc. of t h e 2nd Int'L S y m p o s i u m , " Edited by R.J. Jones, Springer-Verlag, New York, NY, 1970, p. 549. 37. Cenedella, R.J., and W.G. Crouthamel, Fed. Proc. 34:Abstr No. 3226 (1975). 38. Nikkila, E.A., and M. Kekki, Acta Med. Scand. 190:49 (1971). 39. Cenedella, R.J., and W.G. Crouthamel, J. Lipid l~es. 17:156 (1976), 40. Cenedella, R.J,, Biochem. Pharmacol. (1976) (In press). 41. Segal, P., P.S. Roheim, and H.A. Eder, J. Clin. Invest. 51:1632 (1972). 42. Laurell, C.-B., Anal. Biochem. 15:45 (1966). 43. Eaton, R.P., R. Oase, and D.S. Schade, Metablism 25:245 (1976). 44. Eaton, R.P., Ibid. 22:763 (1973). 45. Eaton, R.P., and D.S. Schade, Ibid. 23:445 (1974). 46. Feldman, E.B., F. Gluck, and A.C. Carter, Ciin. Res. 2 3 ( 1 ) : 4 3 A (1975). 47. Mirsky, I.A., G. Perisutti, and F.J. Dixon, J. Biol. Chem. 2 1 4 : 3 9 7 (1955).
[ Received August 13, 1976]
LIPIDS, VOL. 12, NO. 1
INTRODUCTION
This paper reviews most of the preclinical studies on the mode of action of halofenate, an established hypolipidemichypouricemic agent in man. In yeast cultures and in isolated rat adipocytes, halofenate was found to inhibit the conversion of pyruvate to acetyl CoA. While pyruvate dehydrogenase was inhibited in vitro, halofenate also inhibited the activity of various other isolated enzymes. In rats maintained on halofenate in the diet (0.02-0.10%) for 2-14 days, there were 20-40% decreases in plasma cholesterol, t r i g l y c e r i d e s , phospholipids, and free fatty acids. Inhibition of liver HMG-CoA reductase does not appear to account for the hypocholesterolemic effect, and activation of mitochondrial t~-glycerophosphate dehydrogenase does not explain the hypotriglyceridemic action. Kinetic measurements of the serum appearance a n d disappearance of triglycerides in drug-treated rats suggest that the hypotriglyceridemic activity is due to a net inhibition of hepatic triglyceride synthesis. Reduction of very low density lipoprotein (VLDL) and high density lipoprotein (HDL) levels in rats with sucrose-induced hyperlipidemia and normalization of the altered apolipoprotein profiles are in accord with the effects of halofenate on plasma triglyceride and cholesterol levels. T h e reduced insulin-to-glucagon ratio observed in Zucker obese hyperlipemic rats is also consistent with halofenate's hypotriglyceridemic activity. Preliminary experiments in rats on the mechanism of its hypoglycemic activity, observed in some diabetic hyperlipidemic patients, indicate that halofenate acts differently t h a n conventional oral hypoglycemic agents. Some, but not all, of the effects of halofenate were observed with clofibrate at two to ten times higher levels.
The question of whether plasma lipid lowering agents exert their action by affecting a s i n g l e e n z y m e or by influencing several enzymes or biochemical processes has been investigated for many years in a number of laboratories. In the case of clofibrate, for example, there are a variety of mechanisms which could account for its hypotriglyceridemic and hypocholesterolemic activity (1-6), How these different mechanisms relate to one another and to the clinical effects of clofibrate is not yet established. The intent of this paper is to outline the highlights of several preclinical investigations on the mode of action of the hypolipidemic agent halofenate [ 2-acetamidoethyl(p-chlorophenyl) (m-trifluoromethylphenoxy) acetate]. Some of these studies were carried out by members of the Department of Biochemistry at the Merck Sharp & Dohme Research Laboratories while others were undertaken by collaborating investigators. Data sources include unpublished studies and personal communications as well as published reports on halofenate. Figure 1 shows the structure and nomenclature for halofenate (MK-185). Like clofibrate, halofenate produces reductions primarily in serum triglycerides but less frequently in serum cholesterol. Like probenecid, halofenate produces consistent reductions in uric acid. The changes in metabolic parameters at the end of 48 wk of treatment with 1 g of halofenate in a single daily oral dose are shown in Table I. These data indicate the order of the hypolipemic activity of halofenate and are representative of the results obtained in various trials during 5 y r of clinical evaluation. A few selected papers on the clinical effects of halofenate are given in the reference section (7-13). RESULTS AND DISCUSSION Isolated Systems
1The list of literature references on halofenate cited in this paper is not a complete one. Since 1970, there have been over I00 publications on halofenate of which 20 relate to mechanism of action studies in laboratorY animals or man. Fifty are primarily concerned with clinical hypolipemic and hypouricemic activity and the balance on other aspects of its properties. 34
In the initial mechanism studies, the effect of halofenate on lipid synthesis in cultures of S a c c h a r o m y c e s cerevisiae was investigated. Since the enzymatic steps of lipid biosynthesis are similar in yeast and mammalian cells, it was reasoned that the simpler system might identify a primary site of action more readily (14). The results of this line of investigation are summarized in Table II. The free acid of halofenate
MECHANISM OF ACTION OF HALOFENATE ( H F A ) , t h e active m o i e t y ( 1 5 ) , was f o u n d to i n h i b i t yeast g r o w t h reversibly; at 0.34 mM, 50% i n h i b i t i o n o f g r o w t h was achieved. T h e i n h i b i t o r y effect o f H F A was p r e v e n t e d by a d d i n g 0.35 mM oleic or 3.7 mM acetic acids to t h e c u l t u r e m e d i u m . Pyruvic acid at m u c h higher c o n c e n t r a t i o n s (to 72 m M ) c o u l d n o t by-pass t h e block. H F A i n h i b i t e d the convers i o n of 14C-glucose a n d 1 4 C - p y r u v a t e t o labelled f a t t y acids a n d sterols, b u t c o n v e r s i o n of 14C-acetate to lipids was n o t affected. These findings t a k e n w i t h t h e i n h i b i t i o n o f t h e decarboxylation o f p y r u v a t e - l - 1 4 C t o 14CO2 strongly suggest t h a t in t h e y e a s t system, g r o w t h and lipogenesis are i n h i b i t e d by a b l o c k at t h e p y r u v a t e d e h y d r o g e n a s e c o m p l e x . In e x t e n d i n g these o b s e r v a t i o n s to a n i m a l tissues, e x p e r i m e n t s were carried o u t w i t h i s o l a t e d a d i p o c y t e s p r e p a r e d f r o m rat fat pads (16). White H F A i n h i b i t e d t h e u p t a k e of 14C-glucose a n d 1 4 C - p y r u v a t e t o t h e same e x t e n t as it b l o c k e d t h e i r i n c o r p o r a t i o n i n t o f a t t y acids, it did n o t i n h i b i t t h e u p t a k e of l e u c i n e , p a l m i t a t e , or 2 - d e o x y g t u c o s e , suggesting t h a t it was n o t acting as a n o n s p e c i f i c t r a n s p o r t i n h i b i t o r . T h a t t h e drug i n h i b i t e d lipogenesis was d e m o n s t r a t e d in e x p e r i m e n t s w h e r e i n H F A i n h i b i t e d c o n v e r s i o n of p y r u v a t e 2-14C to labelled f a t t y acids t o t h e same e x t e n t as it b l o c k e d t h e d e c a r b o x y l a t i o n of p y r u v a t e 1-14C to 14CO2 ( T a b l e III). A c o m p a r i s o n of t h e activity of H F A w i t h t h r e e e s t a b l i s h e d
35
C1
0
0
O--CH-- C - O - C H 2 CH2-NH--C-- CH 3
CF3 Halofenate
(MK-185)
2-Acetarnidoethyl (p_-chlorophenyl ~ (m-trifluoromethylphenoxy}acetate
FIG. 1. Structure and nomenclature of halofenate. lipogenesis i n h i b i t o r s h y d r o x y c i t r a t e , k y n u r e hate a n d c e r u l e n i n - s h o w s t h a t at levels w h i c h i n h i b i t f a t t y acid s y n t h e s i s 50%, i n h i b i t i o n of p y r u v a t e d e c a r b o x y l a t i o n decreased w i t h t h e p r o x i m i t y of c y t o p l a s m i c e n z y m e to p y r u v a t e d e h y d r o g e n a s e (16). These findings f u r t h e r suggest t h a t i n h i b i t i o n of e n z y m e s involved in f a t t y acid b i o s y n t h e s i s in the c y t o p l a s m results in p r o d u c t i n h i b i t i o n o f m i t o c h o n d r i a l p y r u v a t e d e h y d r o g e n a s e . While t h e exact site of a c t i o n of h a l o f e n a t e o n lipogenesis in a d i p o c y t e s c a n n o t be determined u n e q u i v o c a l l y f r o m these studies, t h e p a t t e r n of data suggests t h a t H F A acts prior t o t h e c o n v e r s i o n o f citrate to acetyl CoA, n a r r o w i n g t h e site t o p y r u v a t e d e h y d r o genase or citrate s y n t h e t a s e . In c o n t r a s t to t h e results o b t a i n e d in yeast and adipocytes, halofenate-induced inhibition of lipid s y n t h e s i s in isolated liver systems was
TABLE I Changes in Metabolic Parameters at the End of 48 Weeks of Halofenate Treatment Measurement a
Pretreatment mean
Body weight (lbs) Fasting blood sugar (mg %) Serum triglycerides (mg %) Serum cholesterol (mg %) Serum uric acid (mg %)
17"7.4 116.4 399.4 293.3 6.87
Change from pretreatment mean
% Change
+1.2 -8.0 -93.4 -15.3 -2.32
+0.7 -6.9 -23.4 -5.2 -33.8
a328 patients, primarily type IV. TABLE II Effect of Halofenate Free Acid (HFA) on Growth and Lipid Synthesis in S. c e r e v i s i a e a 1. HFA inhibits yeast growth 50% at 0.34 raM. 2. Inhibitory effect prevented by added oleate or acetate but not pyruvate. 3. HFA inhibits conversion of glucose-U-14C or pyruvate-2-14C, but not acetate-2-14C, into labelled fatty acids and sterols. 4. HFA inhibits conversion of pyruvate-l-14C to 14CO2. 5. In vitro yeast PDH is inhibited 50% by 0.4 mM HF#~ aAdapted from the report of Greenspan and Germershausen (14). LIP1DS, VOL. 12, NO. 1
L.R. MANDEL
36
TABLE III Effect of HFA on Lipid Synthesis in Isolated Rat Adipocytesa mitochondria glu--§ p y r - ~ acetylCoA-----~ c i t r a t e ~ HFA
Compound 1.5 mM 10.0 mM 10.0 mM 0.12 mM
HFA Hydroxy citrate Kynurenate Cerulenin
c i t r a t e - ~ a c e t y l C o A - - ~ malonylCoA-!l~ fattyacids hydroxy kynurenate cer enin citrate % Inhibition pyr-2-14C to pyr-l-14C to Site fatty acids 14CO2
[PDH] ATP-citrate lyase Acetyl CoA carboxylase Fatty acid synthetase
50 50 50 50
50 40 27 16
aAdapted from the report of Greenspan et al. (16). TABLE IV Effect of Halofenate (free acid) on the Activity of Various Enzymes in vitro a
En zy me
Source
Conc. (mM) of HFA producing 50% inhibition
Pyruvat e dehydrogenase
Yeast Pig heart
0.4-3.0 2.0
Acetyl CoA carboxylase
Chicken liver Yeast
Citrate cleavage enzyme
Rat liver
2.0
HMG CoA reductase
Yeast Rat liver
0.1 >1.0
1.5 0.2
Fatty acid synthetase
Rat liver
0.3
a-Glycerophosphate dehydrogenase
Rat liver
0.3
Glucose 6-phosphate dehydrogenase Lactic acid dehydrogenase Prostaglandin synthetase Insulinase
Yeast Rabbit muscle Sheep seminal vescicle Rat liver
5.0 9.2 0.2 1.0
aTaken from Vol. I of the preclinical evaluation of halofenate, Merck Sharp & Dohme Research Laboratories, March 22, 1974. associated with an i n h i b i t i o n o f p r o t e i n synthesis. Thus, in liver slices f r o m n o r m a l adult rats, Morris 7777 h e p a t o m a and fetal rat liver, or in p r i m a r y rat fetal h e p a t o c y t e s g r o w n in culture, neither halofenate nor clofibrate i n h i b i t e d lipid s y n t h e s i s out o f p r o p o r t i o n to the inhibition o f t o t a l p r o t e i n synthesis ( G r e e n s p a n ; Weinstein and Steinberg, u n p u b lished results). With isolated h e p a t o c y t e s f r o m adult rats, i n h i b i t i o n o f lipogenesis was also associated w i t h i n h i b i t i o n o f p r o t e i n synthesis ( 1 7 ; G o u l d and S h r e w s b u r y , u n p u b l i s h e d results). In h e p a t o c y t e s , h a l o f e n a t e was also s h o w n t o stimulate f a t t y acid m e t a b o l i s m , suggesting the drug causes t h e i r d i s p l a c e m e n t f r o m a l b u m i n (17). While liver m i t o c h o n d r i a l preparations f r o m rats m a i n t a i n e d on h a l o f e n a t e or LIPIDS, VOL. 12, NO. 1
clofibrate s h o w e d a s t i m u l a t i o n o f c h o l e s t e r o l 26-14C o x i d a t i o n to 14CO2, an e f f e c t apparently related t o an increase in liver size comm o n to several p h e n o x y a c e t i c acid s t r u c t u r e s (18-19), the significance o f this activity in t e r m s o f u n d e r s t a n d i n g its h y p o l i p e m i c activity is n o t clearly e s t a b l i s h e d (1). Another approach undertaken to determine the m o d e o f action o f plasma h y p o l i p e m i c drugs has b e e n t o evaluate t h e c o m p o u n d s in vitro as i n h i b i t o r s o f isolated e n z y m e s involved in the b i o s y n t h e s i s o f triglycerides and sterols. As in the case o f clofibrate, this does n o t seem to r e p r e s e n t a fruitful line o f e n d e a v o r for halofenate. Table IV s h o w s t h a t H F A inhibits the activity of e n z y m e s involved in a variety o f b i o c h e m i c a l reactions. Moreover, e n z y m e s n o t
MECHANISM OF ACTION OF HALOFENATE
37
TABLE V Effect of Halofenate on Plasma Lipid Parameters in Rats a % of Halofenate in diet (9 days) 0 (Control) 0.00625 0.0t25 0.025 0.05
% Decrease from control b
Body weight gain (gm)
Chol.
TG
PL
FFA
48 46 50 50 49
12 24 31 38
12 18 29 43
10 18 24 31
0 23 38 34
aAdapted from the report of Gilfillan et al. (24). bChol. = cholesterol, TG = triglycerides, PL = phospholipid, FFA = free fatty acids. TABLE VI Effect of Dietary (0.05%) Halofenate on Rat Liver HMG-CoA Reductase Activity and Sterol Synthesis in Vitro
Exp. no.
Animal group
Serum cholesterol
1 2 3 4
midnight rats- 7 days midnight rats - 7 days midnight rats - 2 days basal rats - 2 days
-50 -26 -36 -27
directly i n v o l v e d in lipogenesis are also i n h i b ited b y m i l l i m o l a r levels of H F A . C l o f i b r a t e free acid (CPIB) p r o d u c e d similar results at f o u r t o t e n t i m e s h i g h e r levels ( d a t a n o t s h o w n ) a n d t h e p u b l i s h e d l i t e r a t u r e i n d i c a t e s t h a t CPIB inhibits many enzymes and biochemical processes in v i t r o (3-6,20-23). Studies in Rats
The in vivo h y p o l i p e m i c a c t i v i t y of halofen a t e was first d e m o n s t r a t e d in rats given t h e d r u g orally (24). As s h o w n in T a b l e V, h a l o f e n a t e t r e a t m e n t ( 0 . 0 5 % in t h e diet for 9 days) p r o d u c e d ca. 3 5 - 4 0 % r e d u c t i o n in cholesterol, triglycerides, p h o s p h o l i p i d s , a n d free f a t t y acids in male H o l t z m a n rats (165 g) m a i n t a i n e d o n a 72% sucrose diet. A small (12%) b u t statistically significant r e d u c t i o n in c h o l e s t e r o l a n d triglycerides was f o u n d w i t h h a l o f e n a t e at 0 . 0 0 6 2 5 % in t h e diet, In this a n i m a l assay, h a l o f e n a t e was a b o u t six t i m e s m o r e p o t e n t t h a n c l o f i b r a t e as a h y p o c h o l e s t e r o l e m i c a g e n t a n d n e a r l y t w i c e as p o t e n t as a plasma h y p o t r i glyceridemic agent. To d e t e r m i n e w h e t h e r h a l o f e n a t e i n h i b i t e d p y r u v a t e m e t a b o l i s m , rats m a i n t a i n e d o n 0 . 0 5 % dietary h a l o f e n a t e for 10 days were given p y r u v a t e - 2 -14C b y i.p. i n j e c t i o n . One h o u r later t h e y were sacrificed a n d t h e livers r e m o v e d for t h e i s o l a t i o n of glycogen. T h e labelled g l y c o g e n
Percent change in Incorporation of 14C into digitonide precipitable sterols by liver homogenates from HM G-CoA 14C.acetat e 14C.mevalona t e reductase -13 +2 +30 -26
-8
+52
was h y d r o l y z e d t o glucose, w h i c h was d e g r a d e d (25). T h e r e was n o d i f f e r e n c e in t h e distribut i o n o f r a d i o a c t i v i t y in t h e c a r b o n a t o m s f r o m 14C-glucose derived f r o m t h e g l y c o g e n of control rats c o m p a r e d t o t h a t of t h e d r u g - t r e a t e d animals ( L a n d a u , u n p u b l i s h e d results). Had c o n v e r s i o n o f p y r u v a t e - 2 -14C t o labelled acetyl C o A b e e n i n h i b i t e d , t h e n t h e p e r c e n t of 14 C in c a r b o n a t o m s 3 a n d 4 w o u l d have b e e n expected t o b e l o w e r in t h e rats given h a l o f e n a t e . While this result, t a k e n w i t h t h e failure o f 4-10% d i e t a r y t r i a c e t i n or s o d i u m a c e t a t e t o reverse t h e h y p o l i p e m i c effect o f h a l o f e n a t e , does n o t c o n f i r m t h a t p y r u v a t e d e h y d r o g e n a s e is t h e site o f a c t i o n for t h e drug, a d d i t i o n a l in vivo e x p e r i m e n t a t i o n is r e q u i r e d t o validate or i n v a l i d a t e t h e p y r u v a t e d e h y d r o genase m e c h a n i s m . In view o f the i m p o r t a n t role of H M G - C o A r e d u c t a s e in c o n t r o l l i n g c h o l e s t e r o l b i o s y n t h e s i s (26), t h e effect of h a l o f e n a t e o n its activity was investigated. M a l e Sprague D a w l e y rats, 2 0 0 - 2 5 0 g, were given t h e drug at 0.05 a n d 0.15% in t h e diet f o r 2 a n d 7 days; e n z y m e activity was m e a s u r e d in liver e x t r a c t s at t h e t i m e o f its p e a k a c t i v i t y ( m i d n i g h t ) a n d at n o o n , t h e t i m e of its basal activity (27). Incorp o r a t i o n of 2-14C a c e t a t e a n d 2-14C-mevalo n a t e i n t o sterols was also m e a s u r e d in o n e o f t h e e x p e r i m e n t s (28). T h e d a t a in T a b l e VI LIPIDS, VOL. 12, NO. 1
38
L.R. MANDEL TABLE VII Comparison of the Effect of Halofenate and Clofibrate on Rat Liver c~-Glycerophosphate Dehydrogenase Drug
% Decrease in plasma triglycerides
0.05% Halofenate 0.1% Halofenate
42 51
77 (45% i") 92 ( 7 4 % t )
0.1% Clofibrate 0.2% Clofibrate
23 28
156 (195%i") 229 ( 3 3 2 % t )
None
Enzyme activity a 53
a~1/O2/50 mg tissue/hr.
show that 0.05% halofenate lowers plasma cholesterol but does not markedly reduce HMG-CoA reductase activity or the incorporation of labelled acetate or mevalonate into digitonide precipitable sterols (Erickson and Gould, unpublished results). At a level of 0.15% halofenate in 2-day experiments, there was a 40% decrease in food intake and the drop in plasma cholesterol was accompanied by a reduction in HMG-CoA reductase activity and incorporation of both 1-14C-acetate and 2 -14C mevalonate into sterols. In LM-cell culture, n e i t h e r h a l o f e n a t e n o r c l o f i b r a t e (at 12.5/ag/ml) inhibited desmosterol synthesis in assays which detected the HMG-CoA reductase inhibitory activity of 25-hydroxy or 7-keto cholesterol (29; 30 and Mandel, unpublished results), These results suggest that inhibition of mevalonate formation does not account for the hypocholesterolemic activity of halofenate in the rat, in contrast to results on clofibrate in which a marked and specific inhibition in cholesterol synthesis from 1-14C acetate but n o t from 2-14C mevalonate was reported ( 3 1 , 3 2 ) . However, preliminary data from another study led to the opposite conclusion. White had reported that clofibrate regulates hepatic cholesterol biosynthesis by inhibiting the microsomal reduction of HMG-CoA to mevalonate (33). When halofenate was given to 170 g male Sprague-Dawley rats at 0.05% in the diet for 7 days, the reduction of l 4C.HMG_Co A to labelled mevalonate was inhibited 52% in assays employing a liver microsomal system ( W h i t e , u n p u b l i s h e d results). Twenty-nine m/amoles of 14C-HMG-CoA was incorporated into mevalonate with preparations from control animals compared to 14.4 m/lmoles for the drug-treated rats. Clearly, more experimentation is required to understand the involvement of this key enzyme in the hypocholesterolemic action of halofenate. W i t h regard to the hypotriglyceridemic LIPIDS, VOL. 12, NO. 1
effect of halofenate, activation of mitochondrial c~-glycerophosphate dehydrogenase was considered because this should decrease the amount of a-glycerophosphate available for triglyceride synthesis. One effect of clofibrate is to induce the synthesis of liver mitochondrial proteins such as the dehydrogenase, and the hypolipemic activity of this drug has been attributed to this (34-36). Rats (Holtzman, 150 g) were maintained on dietary halofenate or clofibrate at least 7 days prior to enzyme assay. Liver mitochondrial enzymes were assayed by the method of Ruegamer et al. (34). As shown in Table VII under conditions where halofenate was more than four times as potent as clofibrate in lowering triglycerides, clofibrate was about seven times more effective than halofenate in stimulating the mitochondrial c~-glycerophosphate dehydrogenase (Huff, unpublished results). It would appear that if the mechanism of the hypotriglyceridemic action of these two drugs is similar, then their effect is probably not mediated by activation of the dehydrogenase. A l t e r n a t i v e l y , if clofibrate does influence plasma triglycerides because it activates this enzyme, then the mode of action of halofenate should be different. In another study designed to establish the mode of the hypotriglyceridemic action of halofenate, male Wistar rats (200 g) were fed a fat-free diet containing no drug, 0.02% or 0.10% halofenate for 14 days. They were then given equivalent amounts of either 2-3H - or l(3)-3H-glycerol i.v., and blood samples were collected from 10 to 1 5 0 r a i n later for the analysis of labelled triglycerides (37). Kinetic analysis of the serum appearance and elimination curves of 3H-triglyceride permitted an estimation of total serum 3H-triglyceride formation and fractional turnover rates. The experimental design was similar to that described by Nikkila and Kekki for measuring plasma triglyceride kinetics in man (38). It was found that the total amount of 3H-triglyceride formed
39
MECHANISM OF ACTION OF HALOFENATE
VLDL Plasma m ( j % of Lipoprolein Constituents
TG
Chol
,if
250
200
150
6
Ioo [I
12
24
N
N S S+H
t
TG ~1.9
T(; 80.3
TG "/7.,9 ~q
6.I
14...99 17.5
Pr 14.2
~
~176 1
TG
80
i
6O
i
'i
50
~"~ S+H
Protein
18
IOO
O
PL
TG / Pr Ratio
% Composition
o
N S S+H
6O
4O
40
ZO
PL
01 V//A N S S+H
o
2O
'~
N
PL II,O S
PL
12.l
S+H
0
N
S
;+H
FIG. 2. The abbreviations used are: VLDL = very low density lipoprotein; LDL = low density lipoprotein; HDL = high density lipoprotein; TG = triglyceride; Chol = cholesterol; PL = phospholipid, Pr = protein; N = normal diet (for rats maintained on a normal chow diet); S = sucrose fortified semi-purified diet (for rats maintained on the 60% sucrose diet); S+H = sucrose plus halofenate diet (for rats given 0.05% halofenate in the 60% sucrose diet). from the 2-3H - or l(3)-3H-glycerol in control rats was similar. Over 95% of the 3H serum triglyceride formed from either substrate circulated in a rapidly turning-over tirglyceride pool (t89 = 8 min). Treatment of the rats with 0.10% halofenate decreased by about 75% the labelling of serum triglycerides without increasing the fractional turnover rates. The drug was further found to decrease the incorporation of U-14C-glycerol into hepatic but not intestinal t r i g l y c e r i d e s without significantly altering incorporation of 14C into total phospholipids of either tissue. From these results, it appears that halofenate reduces serum trigiyceride levels by inhibiting hepatic synthesis. This inhibition could occur at a site subsequent to the formation of diacylgiycerophosphate from either a-glycerophosphate or dihydroxyacetone phosphate. Clofibrate at 0.25% in the diet produced a similar pattern of data in all the experiments with the 2-3H, 1(3)-3H, and UA4C-glycerot (39). In related studies, it was established that these drugs do not decrease net triglyceride synthesis by stimulating hepatic lipase activities but reduce the availability of fatty acids for de novo triglyceride synthesis (40). Studies on the effect of halofenate on lipoand apolipoprotein patterns were carried out on hyperlipidemic rats (Sprague-Dawley, 4 0 0 g
males) in which the hyperlipidemia was induced by feeding a semipufified diet containing 60% sucrose and 5% lard for 3 wk. This regimen produces about a threefold increase in the VLDL fraction, a 50% increase in the HDL fraction and a 40% decrease in LDL (41). After this period, the animals were fed the same diet containing 0.05% halofenate for 10 more days. Serum lipids, lipoproteins and apolipoproteins of the drug-treated rats were compared with patterns obtained from non-drug treated hyperlipidemic animals and with normolipidemic rats kept on a regular chow diet. U n d e r these test conditions, halofenate produced about a 60% reduction in total serum triglycerides and a 30% reduction in cholesterol levels. Figure 2 shows the concentration of VLDL constituents expressed on a plasma mg/dl basis. The dietary regimen induced a marked increase in VLDL triglyceride, cholesterol, phospholipid, and protein, and halofenate produced reductions in all four components. While the values for cholesterol and protein were normalized by the drug, triglycerides were not, and thus the composition analysis shows the triglyceride:protein ratio remained high at 12.5. It is possible that if drug treatment were continued beyond 10 days the triglyceride values would also return to baseline. FigL I P I D S , V O L . 12, N O . 1
L.R. MANDEL
40
LDL Plasma rag% of Lipoprotein Constituents
TG
Chol
PL
Protein
m
I00
Pr ~.4 8.0
4.0
4,0
8.0
8C
6.0
3.0
3.0
6.0
6O
4.0
2.0
2.0
4.0
2.0
1.0
1.0
Chol / Pr Rotio
% Composition
Pr ~.8
IOO Pr 24.7
.T4 Pr
.8l
Pr
5t!
Pr
i92
80
TG BA
TG ~A
TG 37.1
60
m
4O
25.C
~4.1
17,(]
PL
~4.fl
PL
!43
-PL ~0.5
N
S
S+H
20
2.0
.69
;m 16.4
~et ~8
20 I
0
N S S+H
0
N S S+H
0
N S S+H
0
N S S+H
o
O
t
N
S
S§
FIG. 3. The abbreviations used are: VLDL = very low density lipoprotein; LDL = low density lipoprotein; HDL = high density lipoprotein; TG = triglyceride; Chol = cholesterol; PL = phospholipid, Pr = protein; N = normal diet (for rats maintained on a normal chow diet); S = sucrose fortified semi-purified diet (for rats maintained on the 60% sucrose diet); S+H = sucrose plus halofenate diet (for rats given 0.05% halofenate in the 60% sucrose diet). ure 3 shows the data for the LDL analysis, where only small changes in the components are seen due to sucrose feeding, with and without halofenate. As only a small percent of the total plasma lipoprotein of rat serum is LDL, the changes in amounts and composition (e.g., drop in LDL protein due to sucrose which shows a tendency to normalize with halofenate, and an elevation in LDL triglyceride due to halofenate) may not be critical to the mode of action of halofenate. Figure 4 shows the pattern for t h e HDL analysis. Sucrose feeding induces a rise in all four components and halofenate treatment prevents this increase. The reduction in HDL cholesterol is expected as this is the major cholesterol carrying lipoprotein in the rat. The apoprotein profiles were determined by quantitative immunoelectrophoresis described by Laurell (42). While the total serum concentration of apoprotein B is unchanged in the sucrose fed rats, its distribution between the VLDL and LDL fraction is altered (Table VIII). In the normal rat, 22% of apoprotein B is present in the VLDL fraction compared to 76% in the serum of the sucrose hyperlipidemic rat. Halofenate tends to normalize the distribution of this apoprotein, but not to baseline values, again possibly because drug treatment for 10 LIP1DS, VOL. 12, NO. 1
days was not adequate. Nevertheless, the trend of the data suggests that hatofenate may correct a defect in VLDL catabolism and thus increase its rate of removal. The sucrose-induced increases in the apolipoproteins A-I, A-IV, and C-III and in the arginine-rich peptide were also prevented by halofenate; these observations are in accord with the changes seen in the serum lipoproteins. The details of the studies on the effect of halofenate on lipoprotein metabolism will be reported separately (Roheim, to be published). It might be added that, while clofibrate produced a similar pattern of data at a five times higher level (0.25% in the diet), it had a much more rapid onset of action, showing hypolipemic activity after only one day of administration, and halofenate clearly did not. The influence of halofenate therapy on i n s u l i n a n d glucagon secretion was also examined. For these studies, the Zucker obese rat with genetic endogenous hyperlipemia was used. Coincident with the plasma lipid lowering effects of halofenate, the insulin to glucagon molar ratio decreased from 2.72 in the control animals to 0.96 in rats treated with the drug for 10 days (43). Following arginine stimulation, the ratio remained reduced (0.87), in contrast to the greater ratio (2.5) seen in the control animals. The halofenate induced reduction in
MECHANISM OF ACTION OF HALOFENATE
41
HDL Chol / Pr Ratio
% Composition
Plasma m g % of Lipoprotein Constituents Protein PL
TG
Chol
60
r
50
60
--
I00
50
Pr
14.1 4.0
8O
3.0
60
Pr 15.(
I00
Pr l/l)
.41
.40
Pr N.I
71+4
70J
N
S
;+H
Pr
.41 h
80
60
[] 2.0
1.0
II
O ' +']"
N S S+H
O '' N
40
20
S+H
O ~ ...._ '_ S S+H
N S S+H
0
192
40
20
~L6
PL 91.1
51.4
N
S
;+H
0
FIG. 4. The abbreviations used are: VLDL = very low density lipoprotein; LDL = low density lipoprotein; HDL = high density lipoprotein; TG = triglyceride; Chol = cholesterol; PL = phospholipid, Pr = protein; N = normal diet (for rats maintained on a normal chow diet); S = sucrose fortified semi-purified diet (for rats maintained on the 60% sucrose diet); S+H = sucrose plus halofenate diet (for rats given 0.05% halofenate in the 60% sucrose diet). t h e insulin t o glucagon r a t i o was associated with hypolipemia, postarginine hyperglycemia, and hyperketonemia-metabolic parameters characteristic of glucagon excess relative t o insulin. These results are similar to t h o s e seen in rats given c l o f i b r a t e (44) a n d s h o w t h a t t h e h y p o l i p i d e m i c a c t i o n of h a l o f e n a t e is associated w i t h a r e d u c t i o n in t h e i n s u l i n to glucagon m o l a r r a t i o b o t h in t h e basal state a n d a f t e r arginine challenge. While a r e d u c t i o n in arginine-stimulated insulin secretion with potentiat i o n of glucagon release has also b e e n r e p o r t e d t o o c c u r w i t h c l o f i b r a t e in m a n (45), it w o u l d be at p r e s e n t i n a p p r o p r i a t e t o e x t r a p o l a t e t h e data w i t h h a l o f e n a t e t o m a n . These findings relate t o t h e r e c e n t l y o b s e r v e d b l o o d glucose l o w e r i n g effect o f h a l o f e n a t e in T y p e I V h y p e r l i p o p r o t e i n e m i c p a t i e n t s w h o s e p r e t r e a t m e n t fasting b l o o d sugar values were n e a r 2 0 0 m g %. In this s t u d y w i t h d i a b e t i c p a t i e n t s , m e a n b l o o d sugar values for the group ( 1 9 p a t i e n t s ) were decreased 25-50 mg % a f t e r 12, 24, 36, a n d 4 8 wk of halofenate t r e a t m e n t . A b o u t 60% of t h e p a t i e n t s h a d decreases o f 20 m g % or greater in t h e i r i n d i v i d u a l m e a n fasting b l o o d sugar values, a n d t h e m e a n decrease was a b o u t 80 m g % for this s u b s e t o f p a t i e n t s . T h e r e was also a n i m p r o v e m e n t in t h e glucose t o l e r a n c e t e s t (46;
TABLE VIII Distribution of Apoprotein B between VLDL and LDL Fractions Distribution (%) Animal group
VLDL
LDL
Normal Sucrose hyperlipidemic Sucrose hyperlipidemic plus halofenate (0.05% in the diet for 10 days)
22 76
78 24
39
61
aVLDL = very low density lipoprotein; LDL = low density lipoprotein. White, u n p u b l i s h e d results). In t h e case of clofibrate, t h e c h a n g e s were less c o n s i s t e n t , w i t h n o significant d r o p in t h e m e a n fasting b l o o d sugar value or i m p r o v e m e n t in glucose tolerance. H y p o g l y c e m i c a c t i v i t y was n o t o b s e r v e d in h a l o f e n a t e - t r e a t e d p a t i e n t s w h e r e t h e p r e t r e a t m e n t m e a n value was less t h a n 1 4 0 m g %. P r e l i m i n a r y e x p e r i m e n t s designed t o s t u d y the m e c h a n i s m b y w h i c h h a l o f e n a t e r e d u c e s b l o o d sugar have s h o w n t h a t this a g e n t does n o t l o w e r b l o o d glucose in e i t h e r t h e s t r e p t o z o t o c i n d i a b e t i c or a d r e n a l e c t o m i z e d rat ( T a b l e IX). T h e s e a n i m a l m o d e l s d e t e c t e d t h e h y p o LIPIDS, VOL. 12, NO. 1
42
L.R. MANDEL TABLE IX Comparison of Halofenate with Other Hypoglycemic Agents % Reduction in blood sugar Compound
Streptozotocin diabetic rat
Adrenalectomized rat
Halofenate Clofibrate Phenformin Tolb utamide Insulin
8% (300 mg/kg p.o.) 10% (300 mg/kg p.o.) 55% (200 mg/kg p.o.)
12% (100 mg/kg p.o.) 0% (100 mg/kg p.o.) 57% ( 25 mg/kg p.o.)
Insulinase in vitro; % inhibition at 1 mM 47 a 20 a 3 8
67% ( 1 unit/rat s.c.)
aFree acid.
glycemic activity of phenformin and insulin, and tolbutamide, respectively. Moreover, when normal and streptozotocin diabetic rats were treated with halofenate at 0.05% in the diet for 21 days, there was no reduction in blood glucose (Glitzer, unpublished results). The ability of halofenate to inhibit insulinase activity in vitro (47) is only of marginal interest as it has been shown that the drug inhibits several other enzymes in vitro (see Table IV). (Mandel, unpublished results). In summary, it is evident from this review that it is not possible to pinpoint a single enzyme or major site of action to account for t h e p l a s m a h y p o l i p i d e m i c a c t i v i t y of halofenate. However, several possibilities have been explored both with isolated intact systems and in short- and long-term studies in rats. The c o n t i n u i n g studies on hepatic triglyceride synthesis and on lipo- and apolipoprotein metabolism will undoubtedly shed more light on the mode of activity of this compound. Exploration of the effects of halofenate on plasma insulin levels both in rats and in man may offer an explanation for the hypoglycemic activity observed clinically. It should also be ponted out that there are many sites of action reported on in the published literature to explain the biological activity of clofibrate, and, though this drug has been known for a much longer period of time, the precise mechanism for its hypolipidemic effects still remains to be established unequivocally. ACKNOWLEDGMENT The author has been involved with the work described as a coordinator and reviewer. The contributions of Drs. W.A. Bolhofer, J.W. Huff, M.S. Glitzer, M.G. Greenspan, S.F. Kulaga, J.S. Walker and their associates of the Merck Sharp & Dohme Research Laboratories are gratefully acknowledged. I wish to thank our collaborating investigators, Drs. D. Weinstein and D. Steinberg (University of California), S.K. Erickson, M.A. Shrewsbury and R.G. Gould (Stanford University), S.A. Tepper and D. Kritchevsky (Wistar Institute), LW. White and B.R. Landau (Case Western LIPIDS, VOL. 12, NO. 1
Reserve U n i v e r s i t y ) , R.J. Cenedella and W.G. Crouthamel (West Virginia University), P.S. Roheim (Yeshiva University), R.P. Eaton (University of New Mexico), and their associates for their contributions and assistance.
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43
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[ Received August 13, 1976]
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