The lipid-lowering profile in rodents AZ-1355, A New Dibenzoxazepine Derivative

263

40 (1981) 263-271 Elsevier/North-Holland Scientific Publishers, Ltd.

Atherosclerosis,

THE LIPID-LOWERINGPROFILE IN RODENTS AZ-1355, A New Dibenzoxazepine Derivative SAKAE WADA, MASUO KOIZUMI, KAZUO SASAHARA, TOMOHIRO NEICHI, HIROSHI NAKAKIMURA, FUSAYO ONODA and SHUN-ICHI HATA

Research Labomtories,

Chugai Pharmaceutical Co., Ltd., Toshima-ku, Tokyo 171 (Japan)

(Received 19 January, 1981) (Revised, received 31 March, 1981) (Accepted 31 March, 1981)

Summary The lipid-lowering profile of ethyl lO,ll-dihydro-4-methoxydibenz[b,f](1,4)oxazepine-&carboxylate (AZ-1355) has been evaluatedusing clofibrate as a reference compound. This compound is structurallyunrelated to any other hypolipidemic agent. AZ-1355 was selected not only for its effect in reducing serum lipids, but also because it inhibits platelet aggregationin vivo and elevates the prostaglandinI,/thromboxane Az ratio in vitro. It lowers serumtotal cholesterol in T&on-treated hyperlipidemic mice, and also lowers serum total cholesterol and triglyceride in dietary hyperlipidemic rats. In golden hamsters chosen for further evaluation, AZ-1355 reduced serum, liver and cardiac lipids, improved the p/o-lipoprotein ratio and increased the HDL cholesterol. Thus, it is apparentthat the lipid-loweringprofile of AZ-1355 differs from that of clofibrate. Key words:

AZ-l 355 - Clofibrate -High density lipoprotein cholesterol - @or-lipoprotein ratio -Serum cholesterol -Serum triglyceride

Introduction Atherosclerosis is a multifactorial disease which is associatedwith aging [ 11. A disorder in lipid metabolism has long been regarded as the major risltfactor in atherosclerosis[ 21. However, recent studieshave shown that disorders occurring in e.g. carbohydrate metabolism, endocrine system, blood coagulability, blood pressure, platelet aggregation and haemodynamics participate in the atherosclerotic process. Biochemical studies have also indicated that an ~~21-g16~/81/000~000/$~2.6~

0 1981 Elsevier/North-Holland

Scientific Publishers, Ltd.

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Fig. 1. The structure of AZ-1366.

increased level of lipid peroxide [3], and alterationsin cyclic-3’,5’-adenosine monophosphate [4] and the prostaglandinI,/th.romboxane Az ratio are related to atherosclerosis[ 51. In our search for new lipid-lowering drugs, we attempted to select a compound which not only lowers serum lipids but also affects platelet aggregation and the prostaglandin IJthromboxane A, ratio. Consequently, ethyl lO,lldihydro-4-methoxydibenz[ b,f] (1,4)oxazepine&carboxylate (AZ-1355) was selected for a clinical study. In this paper we compare the lipid-lowering property of AZ-1355 with the reference compound clofibrate in mice, rats and golden hamsters. Materialsand Methods Experimental animals Male mice, strain ddY and male rats, strain Sprague-Dawley, both 6 weeks

old, were purchasedfrom the Shizuoka AgriculturalAssociation for Laboratory Animals. Male golden hamsters,weighing 100-130 g, were obtained from the Nippon Institute for Biological Science.

Diets

The mice and the hamsterswere fed standardlaboratory diet pellets (Nippon CLEA, CE-2). The rats were fed either CE-2 or a high fat diet [6], composed of powdered CE-2 53%, butter 40%, cholesterol 5% and sodium cholate 2%. The animals were allowed free access to the diets and tap-water unless otherwise specified. Chemicals

The chemicals were obtained from the following sources: Triton WR-1399 from Ruger Chemicals (New York), clofibrate from Sowa Chemicals (Tokyo), cholesterol from Wako Pure Chemicals (Osaka) and sodium cholate from Nakarai Chemicals(Kyoto). AZ-1355 was synthesizedin our laboratories. Triton hyperlipidemia in mice

The mice were fasted overnight and then Triton WR-1399 (500 mg/kg) was intravenously injected. Immediately and 8 h after the injection, AZ-1366 or clofibrate (both 150 mg/kg, each) were given to the mice orally. The control mice received the vehicle, 1% aqueous methycellulose. The mice were maintained for 24 h on drinkingwater only and then the blood was withdrawnfrom the heart.

265

Hypolipidemic property in rats A total of 56 rats were randomly assignedto 2 equal groups. One group was

fed the CE-2 diet and the other the high fat diet. Each group was further subdivided into 4 equal groups (n = 7). The rats in the first subgroup received the vehicle, 5% aqueous gum arabic solution, orally (1 ml/100 g body weight). The second sub-group received clofibrate (100 mg/kg daily), and the third and the fourth received AZ-1355 in daily doses of 50 and 100 mg/kg, respectively.The drug was administeredonce a day for 4 consecutive weeks. Body weightswere monitored daily. There was no difference in weight gains between the treated and corresponding control groups. Effects of AZ-1 355 on golden hamsters

Two setsof experimentswere carried out with the hamsters.During the treatment the animals were weighed daily. There was no difference in the weight gains among the groups. In experiment 1,18 hamsterswere randomly assigned to 3 equal groups (n = 6). The first group was given AZ-1355 (daily 100 mg/kg), the second group clofibrate (daily 100 mg/kg) and the third (control) group received the vehicle orally. The treatment was administeredonce a day for 4 consecutive weeks. In experiment 2, the dose--response relationship of AZ-1355 was examined using 30 hamsters. They were randomly assignedto 5 equal groups (n = 6). The first (control) group was given the vehicle, and the other four groups received AZ-1355 orally at daily doses of 12.5, 25, 50 and 100 mg/kg, respectively. The treatmentwas administeredonce a day for 4 consecutive weeks. The serum and organs

The rats and the hamsterswere sacrificed 24 h after the final administration. Before being killed, the animalswere fasted for 5 h. Blood was withdrawnfrom the abdominal vein. After exsanguination, the liver and heart were quickly removed, rinsed with saline, blotted with filter paper and weighed. Extraction of the organ lipids

The liver and heart (200 mg, each) were homogenized in a chloroformmethanol mixture (2 : 1) using a Polytron homogenizer (Kinematica GmbH, Switzerland). The homogenates were kept at room temperatureovernight and then filtered. The lipids were determined as follows: total cholesterol by Zurkowski’s method [ 71, triglyceride by van Handel’s method [ 81 and phospholipid by Hoeflmeyr’s method [ 91. Serum chemistry

The serum free fatty acids (FFA) were determined by the method of Itaya [lo], lipid peroxide by the method of Yagi [ll], high density lipoprotein cholesterol by the method of Gordon [ 121, P/a-lipoprotein ratio by disc electrophoresis using a QDL Kit (Ames Co., Indianapolis) [13], bilirubin by the method of Malloy [14] and creatine phosphokinaseby the method of Rosalki [15].

Statistics

The results are represented as the mean ? SEM. Statisticalsignificancewas calculated using Student’s t-test.

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Results The efficacy of AZ-1355 was compared with clofibrate using Triton hyperlipidemic mice. The experiments were repeated 10 times and the results were averaged(Table 1). AZ-1355 reproducibly lowered serum total cholesterol (TC) and the magnitude of reduction was similar to that of clofibrate; the former reduced it by 13.8% and the latter by 15.5%. Both agents were given orally for 4 weeks to rats fed either CE-2 or the high fat diet. The results are presented in Tables 2 and 3. When the rats were fed CE-2, clofibrate reduced the serum TC, triglyceride (TG) and phospholipid (PL) but AZ-1355 did not. But with the high fat diet, AZ-1356 was more effective than clofibrate. AZ-1355 (50 mg/kg) significantlyreduced serum TG and the 100 mg/kg dose resulted in serum TC and TG reduction. Clofibrate reduced only serum TG in these groups. Clofibrate reduced hepatic TC on a mg/g tissue basis, but this reduction was masked on the total liver basis due to enlargementof the liver (Table 3). On the other hand, AZ-1355 (100 mg/kg) reduced total liver TC in rats fed CE-2, and

TABLE 1 EFFECT OF CLOFIBRATE LIPIDEMIC MICE

AND AZ-1366

ON SERUM TOTAL CHOLESTEROL

IN TRITON-HYPER-

Results are presented as the mean f SEM. Groups

Serum total cholesterol

n

5%Change

(mg/dl)

-

Control

64

608i

Clofibrate AZ-1366

64 67

119*14*** 624 f 11 ***

***

P < 0.001,

4

-15.1 -13.8

ekgnifioance of difference from control.

TABLE 2 SERUM LIPID LEVELS

OF RATS TREATED

WITH CLOFIBRATE

AND AZ-1366

Resulta plasented ea mean f SEM (n = 7) Groups

Dose

Diets

(me/kg)

Total cholesterol

Triglyceride

Phospholipid

(mg/dl)

(mg/dl)

(mg/dl) CE-2

49*

3

Cloflbrate AZ-1366 AZ-1366

100 60 100

CE-2 CE-2 CE-2

as* 47* 61t

3* 2 2

Control Clofibrate AZ-1366 AZ-1366

100 60 100

IiFP HFe HFe HF*

Control

219 214 180 163

i f f f

21 11 13 12 *

172 88 184 148

f f f f

16 13 ** 22 13

231 f 34 s* 149f 143 f 6 * 107 f 4*

* P < 0.06 end ** P < 0.01, eigniflcance of difference from the corresponding controls. * High fat diet.

3 7 * 7 6

148 130 149 160

f + i f

169 182 166 162

f 11 f 10 f 8 f 10

CE-2 CE-2 CE-2 CE-2

-

100 50 100

100 50 100

Control Clofibrate AZ-1355 AZ-1355

Control Clofibrate AZ-1355 AZ-1355

22.6 f 0.9 19.3 f 0.9 * 17.3 *ox *** 19.3 k1.5

1.83 f 0.19 1.06 ?z0.12 ** 1.41 f 0.21 1.23 k 0.12

(mrlr)

Totalcholesterol

of difference *P< 0.05.** P< O.Oland ***P-C O.OOl,significance a The high fat diet.

HF* HFa HFa HFa

Diets

Dose (mslkg)

Groups

ResuItspresentedasmean+SEM(n=7)

HEPATICLIPIDLEVELSOFRATSTREATEDWITHCLOFIBRATEANDAZ-1355

TABL83

+21 k32 +19* 534

f 2.6 f 2.0 f 3.2 f 1.3 * 104 61 80 83

9.68 8.32 8.23 7.55

(me/e) 0.87 0.41 0.63 0.11

+8 +5*** *4* *7

f + f f

from the correspondingcontrols.

390 368 304 312

23.6 16.8 17.7 15.1

(mrlliver)

Triglyceride

1790 1160 1410 1340

11 6 10 8+ 5160 +llO +130 * 170

124+ 131+ 102* 93k

(mg/Uver)

20.8 21.8 18.7 21.4

22.2 22.7 22.2 21.5

+ 1.1 f 1.0 k 0.5 f 0.1

f 1.0 f 0.8 + 0.8 * 0.6

(mrlg)

Phospholipid

358 +22 411525 328 514 338 k23

287 214 361i23 * 273 +16 265 t10

(mg/liver)

268 TABLE 4 SERUM BIOCHEMISTRY

OF GOLDEN

HAMSTERS

TREATED

WITH CLOFIBRATE

AND AZ-1366

Results presented as mean f SEM (n = 6) Groups

Control

Total cholesterol (mg/dl) Triglyceride (mg/dl) Phospholipid (mg/dl) Free fatty acid &Eafl) s/orlipoprotein ratio Lipid peroxide @mole/ml) Bilirubin (mg/dl) Creatine phosphokinase (W/l)

187 766 210 1024 1.288 5.6 1.34 170

f 21 f 107 f 24 f 185 f 0.220 f 1.0 f 0.46 * 67

Clofibrate

AZ-1355

(lOOmg/h?)

(100mg/W

148 337 235 166 0.709 2.6 0.31 363

143 326 245 627 0.494 3.4 0.20 147

* 5 f 31** f 9 f 46 f 0.050 * f 0.2 * * 0.09 * * 104

f 4 +22 ** * 6 f 13 * + 0.088 ** + 0.3 ?: 0.06 * * 32

* P < 0.06 and ** P < 0.01. significance of difference from control.

liver

lipids

(mg/g)

heart

lipids

I 18

I.8

16

I.6

14

1.4

12

1.2

10

1.0

8

0.8

6

3.6

I

cl.4

2

d.2

0

0

(mg/g) I

Fig. 2. The liver and heart levels of lipids in golden hamsters treated with clofibrate and AZ-1356. a) P < 0.06,and b) P < 0.01, significance of difference from the control. The black bars are the control group, the white bars the clofibrate group and the striped bars AZ-1366. Each bar represents the mean f SEM (n = 6). 400

I

10

3 300. ?

E $ 200, ._

t :: %I ‘; 100.

oFig. 3. The do-erponse relationship of the seam lipids in golden hamsters treated with AZ-1366. a) P < 0.06 and c) P < 0.001. significance of difference from the control. Each bar represents the mean f SEM. The white bars are the control, the black bars AZ-1356. 12.6 ml/kg. the stziped bars AZ-1356.26 mg/kg, the dotted bars AZ-1356.50 ml/kg and the slanted bars AZ-1366,100 mg/lrg.

269 IO

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0 Fig. 4. The doseieeponee relation&p of the liver lipids in golden hamsters treated with AZ-1366. a) P < 0.05, c) P < 0.01, eigniflcence of difference from the control. Each bar represents the mean f SEM (n = 6). The white bars a the controls, the black bars AZ-1366,lZ.C~ mg/kg. the striped ban AZ-1355.28 the dotted bars AZ-1365.60 me/kg end the slanted bars AZ-1356,100 mg/kg.

mglkg.

the 50 mg/kg dose reduced hepatic TC in rats fed the high fat diet on both bases. Total hepatic TG of the CE-2 fed rats was also reduced by AZ-1355 (100 mg/kg). In the high fat diet groups, both agentsreduced hepatic TG on a mg/g basis. However, neither treatmentproduced any difference in the lipid levels of the heart (data not presented). In subsequent experiments, golden hamsterswere used because of their similarity to man with regard to lipid metabolism. In experiment 1, the efficacy of AZ-1355 was again compared with clofibrate. The resultsare shown in Table 4 and Fig. 2. Clofibrate and AZ-1355 reduced serum TC by 21% and 24%, but the reduction was not significantdue to the largevariancein the control group. Both agentsshowed a marked hypotriglyceridemiceffect. In addition, AZ-1355 reduced serum FFA, the /3/e-lipoprotein ratio and bilirubin. Clofibrate also reduced the serum p/o-lipoprotein ratio and lipid peroxide but doubled serum creatine phosphokinase. The hepatic and cardiac lipid levels were significantlyaffected by the AZ1356 treatment, which resulted in decreases of hepatic TC and PL, and of car: disc TC and TG (Fig. 2). On the other hand, clofibrate failed to affect these lipid levels. The dose-response relationship of AZ-1355 was examined using hamsters. Such treatment reduced serum TC by l,O-15%, but the difference was not significant (Fig. 3). Serum TG was reduced by the higherdoses (above 25 mg/kg). In addition, the highest dose (100 mg/kg) elevated HDL cholesterol by 20%. The hepatic levels of TC and TG were againreduced, indicatingthat the reductions are reproducible (Fig. 4). However, the efficacy was not always doserelated. Discussion AZ-1355 is novel and structurally unrelated to any hypolipidemic agent. The primary screen for our system was Triton hyperlipidemia [16]. Among

270

those which passed the screen, AZ-1355 was finally selected because it inhibited platelet aggregation, protecting rabbits from arachidonic acid shock and elevatedprostaglandinIJthromboxane A. ratio [ 171. Rats have been used as a standardmodel for evaluatingthe efficacy of hypolipidemic agents. However, the present study indicates that the Triton hyperlipidemic mouse is a useful tool and the resultsprovide some information about the mechanism. Although the real mechanism of the hyperlipidemiais still obscure, Friedman has shown that Triton WR-1399 increaseshepatic lipogenesis and delays lipoprotein uptake in peripheraltissuesfrom blood [ 181. Therefore, at least two possibilitieswere considered for AZ-1355 reduction of serumTC in hyperlipidemic mice. One was that AZ-1355 inhibits hepatic cholesterogenesis. Clofibrate inhibits hepatic cholesterogenesis [19] and this mechanism causes cholesterol-loweringin hyperlipidemicmice. However, AZ-1355 did not inhibit incorporation of acetate or mevalonate into cholesterol (unpublished observation), so this possibility was unlikely. Rats are relatively resistant to an atherogenic regimen, but a high-fat diet, supplemented with bile acids, is able to induce hyperlipidemiain rats 1201. The present study shows that the lipid-lowering profile of AZ-1355 differs from that of clofibrate in rats: the former was more effective in the hyperlipidemic rats whereas the latter reduced the serum lipids in normolipidemic rats. Clofibrate induces enlargementof the liver, while the AZ-1355 reduces the hepatic levels of lipids in rats without causing hepatomegaly, and the reduction was seen regardlessof the kind of diet. Golden hamstershave rarely been used for evaluatinghypolipidemic agents [21]. However, as shown in the present study, hamstershave advantagesover other rodents, because the serum lipid levels are higherand, as Ohharapointed out, the bile acid composition, hepatic cholesterol turnoverand intestinalflora are similar to those in man [ 221. Beher et al. [ 231 have also shown that hepatic and serum levels of cholesterol are higher in hamstersthan in other rodents due to the slower turnoverrate. Hamstersresponded well to the lipid-loweringaction of AZ-1355, although the dose--response relationship was not always clear. As expected, treatment did not cause hepatomegaly, nor did it induce CPK elevation as did clofibrate. AZ-1355 markedly reduced the serum /3/4ipoprotein ratio. This reduction may be effected by a combination of two mechanisms.The first is the hypotriglyceridemic effect, suggestingthat the agent selectively lowers serum p-lipoproteins. The other is through an increasein serum HDL cholesterol, indicating an increase in serum a-lipoproteins. These combined effects would cause a much sharper fall in the p/e-ratio. Hepatic and cardiac TC levels were also reduced by AZ-1365, suggestingthat it acceleratesthe excretion of sterols in the feces. This idea is supported by increased HDL cholesterol in the treated hamsters. Thus, the lipid-loweringprofile of AZ-1355 differs from that of clofibrate. AZ-1355 is more effective when administeredto animalswith a high /3-lipoprotein level; improving the serum P/o-lipoprotein ratio through a decrease of &lipoproteins and an increaseof cy-lipoproteins.

271

Acknowledgements We are grateful to Dr. S. Tomioka, the Director of our laboratories, for his interest in and encouragement of .the present work. We are also grateful to the members of our pharmacological department for their discussion and assistance. References 1 Hollander, W., Suppression of atheromatous fibrous plaque formation by antiprolferative and antiinBammatory drugs, Circ. Res.. SuppI. I, 34 and 36 (1074) 131. 2 Day, Al.. Dynamics of iipoproteins and Iipids in the arterisd waB. In: G. Sehettler, Y. Goto, Y. Hata and G. Gloss (Ed%). AtheroscIerosIs-IV, Springer-VerIag. New York. 1077, p. 46. 3 Tsai. A.C., Thie. GM. and Lin. CR., Effect of cholesterol feeding on tissue lipid peroxidation, gtutathione peroxidase activity and Iiver microsomal functions in rats and guinea pigs. J. Nutr.. 107 (1077) 310. 4 Shhnamoto. T.. Hyperreactive arterial endotheliaI ceBs in atherosclerosis and cyclic AMP phosphodie&erase inhibitor in prevention and treatment of atherosclerotic disorders, Jap. Heart J., 16 (1076) 76. 6 HoIIis. TM., Rabbit aortic histamine synthesis following short-term cholesterol feeding, Atherosclerosis. 21 (1076) 126. 6 Hartroft, W.S. and Thomus, W.A.. Discussion of some of our own observations of atheroscierotic lesions in rats associated with thrombogenic diets. In: hf. Sadler and G.H. Boume (Ed%), Atherosclerosis and its Origin, Academic Press, New York, 1063. p. 447. 7 Zurkowski. P., A rapid method for cholesterol determination with a singIe reagent, Chn. Chem., 10 (1064) 461. 8 Van Handel. E.. EiIversmit, D.B. and Bowman, K., Micromethod for the direct determination of serum triglyceride. J. Lab. Clin. Med., 60 (1067) 162. 0 Hoefhneyr. J. and Fried, It.. Eine Methode aur routine-miissigen Besthnmung des Lipidphosphorus und der Phosphatide, Med. Emfihr., 7 (1066) 0. 10 Itaya. K. and Ui. M., Calorimetric determhration of free fatty acids in biological fluids, J. Lipid Res., 6 (1066) 16. 11 Yagi, K., Nishigaki. I. and Ohara. H., Messurement of serum TBA value, Vitamins (Tokyo). 37 (1068) 106. 12 Gordon, T.. Predicting coronary heart disease in middle-aged and older persons, J. Amer. Med. Ass., 238 (1077) 407. 13 Dangerfield. W.G. and Pratt, J.J., An investigation of plasma liDOPrOthI8 by polyacryiamide ale&ophoresis. Clin. Chim. Acta, 30 (1070) 273. 14 Mailoy. HT. and Evelyn, K.A.. The determination of biIirubin with the photoelectric calorimeter, J. Biol. Chem.. 110 (1037) 481. 16 NuttaB. F.Q. and Wedin. D.S., A simple rapid calorimetric method for determination of creatine kinase activity, J. Lab. Cl&. Med., 681 (1066) 324. 16 Schurr. P.E., Schultz. J.R. and Rarkinson. T.M., Triton induced hypedipidemia in rats as an animal model for screening hypoBpidemic drugs, Lipids, 7 (1072) 68. 17 Neichi. T., Yamasaki. T., Onoda. F., Koizumi, M., Hata. S. and Wada, S., Stimulation of prostagiandin synthesis and prevention of arachidonic acid shock by A&1366, a new lipid-lowering agent, RostagIandins. In press. 18 Friedman, M. and Byers. S.C.. Mechanism underlying hypercholesterolemia induced by Mton WR1300, Amer. J. Physiol.. 100 (1067) 430. 10 Steinberg. D.. Drugs inhibiting cholesterol biosynthesis, with speciaI reference to cioflbrate. In: R.J. Jones (Ed.). Atherosclerosis (Proceedings of the 2nd Int. Symposium). SpringerVerIag. New York. 1070. p. 600. 20 Page. I.H. and Brown, H.B., Mechedem of iodide action on cholesterol action, Circulation. 6 (1062) 881. 21 SchoenfieId. L.J., Bonorris. G.G. and Ganz. P., Induced aRerations in the rate-Ihniting enzymes of hepatic choleeterol and bile acid synthesis in the hamsters, J. Lab. CBn. Med., 82 (1073) 868. 22 Ohhara. H.. Shiojiri. H. and Ono. H.. Time course of bile acid composition of normal and experimental gaBstony hamater. Clin. Report (Tokyo). 12 (1078) 2463. 23 Beher. W.T.. FiIus. A.M., Rae, B. and Beher. ME.. A comparative study of bile acid metaboBsm in the rat. mouse, hamster and gerbile. Rot. Sot. EXP. BioI. Med., 180 (1069) 1067.