Pharmacology of Chenodeoxycholic Acid

73:310-313, 1977 Copyright © 1977 by the American Gastroenterological Association

GASTROENTEROLOGY

Vol. 73 , No.2

Printed in U.S A.

LACK OF TOXICITY OF CHENODEOXYCHOLIC ACID IN THE SQUIRREL MONKEY HIROSHI SuzuKI, TosHIO YosHIDA, HisAo MIKI, TAKURO SAno, PH.D., AND KEISUKE HASHIMOTO, M.D.

Central R esearch Laboratories , Yamanouchi Pharmaceutical Company, Ltd., and Department of Pathology, School of Medicine, Juntendo University , Tokyo, Japan

The toxicity of chenodeoxycholic acid was studied in squirrel monkeys of both sexes. The drug was orally administered to five groups of 26 animals each at a daily dose of 0, 10, 20, 40, and 80 mg per kg, respectively, for a maximum of 52 weeks. No clinical symptoms that could suggest drug toxicity were observed. All laboratory studies, including liver function tests, were within normal limits. The proportion of lithocholic acid in biliary bile acids remained unchanged, whereas that of chenodeoxycholic acid was dose-dependently increased. No histopathological changes considered to be attributable to drug administration were observed. It is now well known that the oral administration of chenodeoxycholic acid (CDCA) improves cholesterol solubility in bile 1 and induces dissolution of cholesterol gallstones in man.2- 5 Because CDCA is transformed to lithocholic acid (LCA), which is known to be hepatotoxic, by intestinal bacteria, hepatic dysfunction is thought to be one of the potential complications of CDCA medication. Studies in man, however, have demonstrated no apparent hepatotoxic effect; 2• 3 • 5• 6 only mild elevations of serum transaminase have been observed transiently during the prolonged intake of CDCA. 4 • 7 On the other hand, CDCA has been shown to cause proliferation of bile ducts in experimental animals such as rats, 8 rabbits, 9 rhesus monkeys, 10• 11 and baboons. 12 Thus, there seemed to exist species differences in susceptibility to CDCA between some animals and man. According to Osuga et al., 13 • 14 the squirrel monkey, whose bile acid composition in bile is known to be similar to that of man, developed pure cholesterol gallstones under some experimental conditions. This paper reports the toxicity study of long term administration of CDCA in the squirrel monkey.

Methods Materials and experimental design. CDCA was synthesized at Yamanouchi Pharmaceutical Co., Ltd., Tokyo, Japan. The lot used for the present studies contained more than 99% of CDCA; LCA was detected less than 0.1% by thin layer chromatography. One hundred and thirty squirrel monkeys (Saimiri sciureus) of both sexes obtained from Primate Import, Inc. (Port Washington, N.Y.) were used. The animals were housed as a group of 3 to 5 in metal cages and fed ad libitum a commercial diet (Oriental Kobo Kogyo Co., Ltd., Tokyo, Japan) and about 50 g per day of banana throughout the experimental period.

Received December 9, 1976. Accepted February 22, 1977. Address requests for reprints to: Hiroshi Suzuki, Yamanouchi Pharmaceutical Company, Ltd., Central Research Laboratories, 1-8, Azusawa-1-chome, Itabashi-ku, Tokyo, Japan.

The mean body weight of males was 650 g and that of females was 640 g at the beginning of the experiments. The animals were allocated to five study groups, each comprising 26 animals with 13 males and 13 females . Four groups were treated with CDCA, at a daily dose of 10, 20 , 40, and 80 mg per kg, respectively. CDCA was orally administered by tube as a suspension in 0.5 % carboxymethyl cellulose solution at a constant volume dose of 5 ml per kg, once a da y, 7 days a week . One group received the vehicle alone at the same volume dose and served as a control group. At the end of 13 and 26 weeks M dosing, 4 (2 males and 2 females) and 8 animals (4 males a nd 4 females), respectively, from each group, were killed. Remaining animals were killed after 52 weeks of CDCA administration, with the exception of 2 males and 2 females from each group, which were subjected to liver biopsies at the same time and were killed 10 weeks after discontinuation of CDC A. Hematology . Before dosing started and at intervals during the dosing period, blood samples were taken from saphenous vein of each monkey for the measurement of the following characteristics: erythrocyte count, packed cell volume, hemoglobin, and total and differential leukocyte count. Platelets were also counted on termination of the dosing period. S erum biochemistry . GPT and alkaline phosphatase (AP) were determined before dosing started and at intervals during the dosing period. Other determinations made on serum samples taken at the time of sacrifice included: GOT, isocitrate dehydrogenase, leucine aminopeptidase, y-glutamyl transpeptidase, glucose, urea nitrogen, total protein, albumin, bilirubin, electrolytes, triglycerides, cholesterol, phospholipids, and bromosulfophthalein (BSP) clearance rate. BSP clearance rate was estimated by determination of residual BSP in the plasma 20 min after intravenous injection of BSP at 15 mg per kg of body weight. Urinalysis. On termination of the dosing period, urinalysis was carried out by test strips (Ames Division, Miles-Sankyo Co., Ltd., Tokyo, Japan) for pH, protein, glucose, ketones , bilirubin, and urobilinogen. Determination of biliary bile acid composition. Bil'.l was obtained by needle aspiration from the gallbladder of the animals killed after 52 weeks of CDCA administration and those killed 10 weeks after discontinuation of CDCA. An ali310

August 1977

311

TOXICITY OF CHENODEOXYCHOLIC ACID

quot of bile from each sample was hydrolyzed with cholyglycine hydrolase by the method of Klaassen et al. 15 The bile acid mixture was then methylated and acetylated as described by Okubo, 16 and individual bile acids were quantitatively analyzed by gas-liquid chromatography. The concentration of sulfated bile acids in bile was also determined in 4 animals of the control group and 7 animals of the group of 80 mg per kg per day from which sufficient volume of bile could be obtained. To determine this, an aliquot of bile was solvolyzed before alkaline saponification, as described by Palmer and Bolt. 17 Pathological studies. All killed animals were subjected to gross examination and the following organs were weighed: brain, heart, liver, kidneys, spleen, pancreas, gonads, pituitary, thyroids, thymus, adrenals , and submaxillary glands. Histopathological examination was carried out on these organs and on lungs, gallbladder, lymph nodes, gastrointestinal tract, urinary bladder, skeletal muscle , uterus, prostate, bone marrow, peripheral nerve, and aorta. Biopsy specimens of liver tissue were examined with the electron microscope (T. Kanetaka et al., unpublished data) and also with the light microscope. Statistical analysis . Statistical analysis was performed according to Student's t-test.

Results Mortality and clinical signs. No deaths were identified as being attributable to CDCA administration. Although 1 female of the control group, 1 female given 10 mg per kg per day, 2 males and 1 female given 40 mg per kg per day, and 1 female given 80 mg per kg per day died during the dosing period, their deaths seemed to be due to infectious diseases. All other monkeys were apparently in good health and no abnormal behavior was observed. Liquid or soft stools were occasionally found in the excreta at the bottom of each cage in which animals given CDCA were housed. The incidence, however, was considered to be comparable to that of the control group, in which such stools were also found. Body weight. The body weight of treated animals was not significantly affected by CDCA administration, although in the early stages the body weight of females given CDCA tended to be slightly diminished. Laboratory studies. All hematological parameters were within normal limits. Both serum GPT and AP remained unchanged during the administration of CDCA in comparison with control and/or pretreatment values. There were no intergroup differences in other biochemical findings, either. The urinalysis for pH, protein, glucose, ketones, bilirubin, and urobilinogen did not reveal any abnormality. Biliary bile acid composition. Biliary bile acid composition of the animals killed after 52 weeks of CDCA administration is shown in figure 1. The proportion of LCA remained unchanged. The proportion of CDCA increased dose-dependently and CDCA became the major component of biliary bile acids. Inversely, the proportion of cholic acid and deoxycholic acid was markedly decreased. These changes were almost completely reversed 10 weeks after discontinuation of CDCA (table 1). There were negligible differences between bile acid concentrations determined with solvolysis and those without solvolysis (table 2). Pathological studies. Gross examination revealed no

100

1-

z u a:

I I 0~

LCA DCA COCA CA

10

101

101

a. 80

a:

m

m

c(

..J

0 :1 40

20

j

j

_fi1i

0 10 (CON11IOL)

20

40

I l 80

DOSE (MG/KG/DA Y)

FIG. 1. Biliary bile acid composition of squirrel monkeys killed after 52 weeks of CDCA administration. Data are shown as means (±SE) for analyses in 6 control animals, 10 on 10 mg per kg per day, 7 on 20 mg per kg per day , 6 on 40 mg per kg per day, and 9 on 80 mg per kg per day. LCA , lithocholic acid; DCA , deoxycholic acid; CDCA, chenodeoxycholic acid; CA, cholic acid.

abnormalities. No intergroup differences in organ weights existed. In the histological specimens there were no significant findings attributed to CDCA administration in the hepatic parenchyma or in the periportal areas. Histopathological findings of the liver obtained at 52 weeks are given in table 3. There were no case of bile ductular proliferation. No histopathological abnormalities resulting from CDCA administration were encountered in other tissues examined. Discussion The administration of CDCA for 52 weeks was not associated with any functional or histological liver abnormalities in squirrel monkeys. No abnormalities were also encountered in the ultrastructure of the liver ( unpublished data). In previous studies in man and animals, special attention has been focused on hepatotoxicity as one of the potential side effects of CDCA. In our preliminary studies, therefore, susceptibility of the squirrel monkey to hepatic injury was examined; it was shown that the ligation of common bile duct or short term administration of a-naphthylisothiocyanate caused marked elevations of serum GOT, GPT, AP, and bilirubin, and also resulted in hepatic damage including bile duct proliferation. According to Hunt, 18 feeding of LCA also led to bile duct proliferation in this species. It was, therefore, assumed that the squirrel monkey was not unsuitable for examining the bile acid-induced injuries in hepatobiliary systems, and it was concluded that CDCA was not toxic in this monkey, at least at a maximum daily dose of 80 mg per kg. With regard to the effect on bowel function, however, present studies failed to reveal whether CDCA caused diarrhea.

312

SUZUKI ET AL .

TABLE

Vol . 73, No.2

1. Biliary bile acid composition of squirrel monkeys 10 weeks after discontinuation ofCDCA administration which lasted for 52 weeks"· b Molar %

No. of animals

Group

LCA

DCA mean±

Control CDCA (80 mg/kg/day) withdrawn for 10 weeks

4 4

0.5 ± 0.2 0.9 ± 0.6

6.4 ± 1.4 5.4 ± 0.7

CDCA

CA

50.3 ± 2.8 45 .5 ± 3.1

42.8 ± 3.5 48.2 ± 2.7

SE

: Abb~evi.ations a~e: CDCA, chenodeoxycholic acid; LCA, lithocholic acid; DCA, deoxycholic acid; CA, cholic acid. No s1gmficant differences were found between control and withdrawn animals. 2. Sulfated bile acids: biliary bile acid concentration of squirrel monkeys killed after 52 weeks ofCDCA administration"·b JLmoles/ml of bile Group No. of animals LCA DCA CDCA CA

TABLE

mean±

SE

Without solvolysis Control 80 mg/kg/day

4 7

0.4 ± 0.2 0.2 ± 0.1 NS

6.4 ± 2.6 0.1 ± 0.1 p < 0.01

31.8 ± 5.2 96.5 ± 9.3 p < 0.01

26.9 ± 4.1 9.7 ± 5.1 p < 0.01

With solvolysis Control 80 mg/kg/day

4 7

0.7 ± 0.3 0.4 ± 0.1 NS

4.7 ± 1.7 0.1 ± 0.1 p < 0.01

37.3 ± 12.2 96.4 ± 10.3 p < 0.01

26.1 ± 8.7 8.2 ± 4.1 p < 0.01

Abbreviations are: CDCA, chenodeoxycholic acid; LCA, lithocholic acid; DCA, deoxycholic acid; CA, cholic acid; NS, not sig;;ificant. Significant differences between groups indicated by P values; no significant differences were found between without solvolysis and with solvolysis. a b

TABLE

Group

Control

3. Summary of histological findings of liver of squirrel monkeys given chenodeoxycholic acid (CDCAJ for 52 weeks" Parenchyma Portal triad

No.

-

+

++

+++

-

M

7 6 7 6 7 7 5 6 7 6

0 0 0 0 0 0 2 1 0 0

0 1 1 2 1 0 0 0 0 0

1 2 1 1 0 0 0 3 0 2

6 3 5 3 6 7 3 2 7 4

6 5 6 6 5 5 3 5 5 5

M

F 20 mg/kg/day

M

F 40 mg/kg/day

M

F 80 mg/kg/day

Hydropic degeneration

Sex

F 10 mg/kg/day

Glycogen contents

Animals

M

F

+

1 0 2 2 2 1 2 I

Fatty degeneration

++

-

+

++

0 0 0 0 0 0 0 0 0 0

3 6 4 6 5 6 4 6 3 6

4 0 3 0 2 1 1 0 4 0

0 0 0 0 0 0 0 0 0 0

lnflamatory cell infiltration -

+

6 1 3 2 5 1 5 2 5 4 2 1 3 2 5 2 3

Mobilization of Kupffer cell

lnflamatory cell infiltration

Proliferation of bile duct

++

-

+

++

-

+

++

-

+

++

0 2 0 0 0 2 2 2 0 1

7 6 7 5 6 6 5 5 6 5

0 0 0

0 0 0 0 0 0 0 0 0 0

3 2 5 4 6 3 2 1 4 4

4 4 2 1 1 2 2 4 3 2

0 0 0 1 0 2 1 1 0 0

7 6 7 6 7 7 5 6 7 6

0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0

1 1 0

o· 0 0 0

a-, none;+, slight;++, moderate;+++, marked . Values given are number of animals.

The changes in biliary bile acid composition caused by the administration of CDCA in squirrely monkeys- a marked increase in the proportion of CDCA associated with diminished cholic acid and deoxycholic acidseemed to be common to other animals9--12 • 19 and man. 1• 20• 21 The proportion of LCA in bile, however, did not increase in our animals, unlike rabbits, 9 rhesus monkeys, 10 • 19 and baboons. 12 In rhesus monkeys and baboons fed CDCA, the increase in the proportion of LCA in bile and 'hepatic changes such as bile duct proliferation were observed under conditions similar to ours. 1(}-- 12 Salen et al. 19 commented in his report that CDCA produced hepatotoxicity presumably through bacterial metabolism and the formation of LCA in rhesus monkeys. The effects of CDCA treatment on the proportion ofLCA in bile have also been studied in man. In most of the studies, to our knowledge, the patients

with cholelithiasis showed no apparent increase in the proportion of LCA in bile after the prolonged intake of CDCA, 1• 21• 22 although Coyne et al.2 3 reported that the patients on CDCA developed a significant increase in the proportion of LCA in bile. In any case, no apparent hepatic damage has been demonstrated in man given CDCA. From these findings in man and animals, it is suggested that species differences in susceptibility to CDCA correlate with the differences in the increases of the biliary LCA proportion. Our results in squirrel monkeys seemed to be consistent with this interpretation. Sulfate conjugation is known to be a main metabolic pathway of LCA in man. 17• 24 In fact, marked increases in biliary sulfated LCA in the patients with cholelithiasis after CDCA medication have been found .20• 25 Thus, it was hypothesized that low susceptibility of man

TOXICITY OF CHENODEOXYCHOLIC ACID

August 1977

to the hepatotoxic effects of CDCA was due to enhanced excretion of LCA through this sulfation, which plays an important role in detoxyfying LCA.26 • 27 LCA accumulation and hepatotoxicity observed when CDCA was ingested by the rhesus monkey was explained by deficiency of the detoxifying mechanism in this species.28 Our studies in squirrel monkeys demonstrated neither accumulation of LCA nor appreciable changes in sulfated LCA contents. The absence of hepatotoxicity of CDCA in squirrel monkeys may be due to poor bacterial formation of LCA from exogenous CDCA, poor intestinal absorption of LCA, or its accelerated urinary excretion. In this connection, Osuga et al. 14 reported that DCA and LCA predominated in the large intestine of nontreated squirrel monkeys. There have been, however, no data on the metabolism of exogenous CDCA in this species. The urinary and fecal excretions of bile acids including sulfated LCA in the squirrel monkey fed CDCA are now under investigation. REFERENCES 1. Thistle JL, Schoenfield LJ: Induced alterations in composition of bile of persons having cholelithiasis . Gastroenterology 61:488496, 1971 2. Bell GD, Whitney B, Dowling RH: Gallstone dissolution in man using chenodeoxycholic acid. Lancet 2:1213-1216, 1972 3. Dam:inger RG, Hofmann AF, Schoenfield LJ, et al: Dissolution of cholesterol gallstones by chenodeoxycholic acid. N Eng! J Med 286:1-8, 1972 4. Thistle JL, Hofmann AF: Efficacy and specificity of chenodeoxycholic acid therapy for dissolving gallstones. N Eng! J Med 289:655-659, 1973 5. Iser JH, Dowling RH, Mok HYI, et al: Chenodeoxycholic acid treatment of gallstones. A follow-up report and analysis of factors influencing response to therapy. N Eng! J Med 293:378-383 , 1975 6. Bell GD, Mok HYI, Thwe M, et al: Liver structure and function in cholelithiasis. Effect of chenodeoxycholic acid . Gut 15:165172, 1974 7. Mok HYI,. Bell GD, Dowling RH: Effect of different doses of chenodeoxycholic acid on bile· lipid composition and on frequency of side effects in patients with gallstones. Lancet 2:253-257, 1974 8. Tsujii T, Tamura M, Matsuoka Y, et al: Hepatotoxicity of bile acids. Effects of long term administration of lithocholate or chenodeoxycholate. Acta Hepatol Jpn 16:293, 1975 9. Fischer CD, Cooper NS, Rothschild MA, et al: Effect of dietary chenodeoxycholic acid and lithocholic acid in the rabbit. Am J Dig Dis 19:877-886, 1974 10. Webster KH, Lancaster MC, Hofmann AF, et al: Influence of primary bile acid feeding on cholesterol metabolism and hepatic

313

function in the rhesus monkey. Mayo Clin Proc 50:134-138, 1975 11. Dyrszka H, Salen G, Zaki FG, et al: Hepatic toxicity in the rhesus monkey treated with chenodeoxycholic acid for 6 months: biochemical and ultrastructural studies. Gastroenterology 70:93-104, 1976 12. Morrissey KP, McSherry CK, Swarm RL, et a!: Toxicity of chenodeoxycholic acid in the non-human primate. Surgery 77:851-860, 1975 13. Osuga T, Portman OW: Experimental formation of gallstones in the squirrel monkey. Proc Soc Exp Biol Med 136:722-726, 1971 14. Osuga T, Portman OW: Relationship between bile composition and gallstone formation in squirrel monkey. Gastroenterology 63:122-133 , 1972 15. Klaassen CD: Gas-liquid-chromatographic determination of bile acids in bile. Clin Chim Acta 35:225-229, 1971 16. Okubo H: Clinical studies on bile acids in bile by gas-liquid chromatography . J Jpn Surg Soc 69:865-885, 1968 17. Palmer RH, Bolt MG: Bile acid sulfates . I. Synthesis of lithocholic acid sulfates and their identification in human bile. J Lipid Res 12:671-679, 1971 18. Hunt RD: Proliferation of bile ductules (the ductular cell reaction) induced by lithocholic acid (abstr.). Fed Proc 24:431, 1965 19. Salen G, Dyrszka H, Zacki G, et a!: Hepatic metabolism of chenodeoxycholic acid (CDCA) in the rhesus monkey (abstr). Gastroenterology 69:802, 1975 20 . Danzinger RG, Hofmann AF, Thistle JL, et al: Effect of oral chenodeoxycholic acid on bile acid kinetics and biliary lipid composition in women with cholelithiasis. J Clin Invest 52:28092821, 1973 21. Salen G, Tint GS, Eliav B, et a!: Increased formation of ursodeoxycholic acid in patients treated with chenodeoxycholic acid. J Clin Invest 53:612-621, 1974 22. Bremmelgaard A, Pedersen L: Bile acids in bile during longterm chenodeoxycholic acid treatment. Scand J Gastroenterol 11:161-165, 1976 23. Coyne MJ, Bonorris GG, Chung A, et al: Treatment of gallstones with chenodeoxycholic acid. and phenobarbital. N Eng! J Med 292:604-607 , 1975 24. Cowen AE, Korman MG, Hofmann AF, et al: Metabolism of lithocholate in healthy man. I. Biotransformation and biliary excretion of intravenously administered lithocholate, lithocholylglycine and their sulfates . Gastroenterology 69:59-66, 1975 25 . Stiehl A, Raedsch R: Increasing sulphation of lithocholate in patients during chenodeoxycholate therapy (abstr) . Digestion 10:323, 1974 26 . Palmer RH: Bile acids, liver injury, and liver disease. Arch Intern Med 130:606-617, 1972 27. Cowen AE, Korman MG, Hofmann AF, et al: Metabolism of lithocholate in healthy man. II. Enterohepatic circulation. Gastorenterology 69:67-76, 1975 28. Gadacz TR, Allan RN, Mack E, et al: Impaired lithocholate sulfation in the rhesus monkey: a possible mechanism for chenodeoxycholate toxicity. Gastroenterology 70:1125-1129, 1976