Arch Toxicol (1983) 54:247-255 Archlves

TOXICOLOGY �9 Springer-Verlag 1983

The Acute Toxicity of Oxamniquine* in Rats; a Sex-Dependent Hepatotoxicity

Malcolm Gregory, Alastair Monro, Michael Quinton, and Norman Woolhouse

Pfizer Central Research, Sandwich, Kent, England

Abstract. Toxicity studies with oxamniquine in several laboratory animal species revealed an idiosyncratic sensitivity of rats, females being much more sensitive than males. After single p.o. doses of oxamniquine, rats died up to 14 days after the dose from hepatic failure. At doses near the LD-50, serum transaminases were high and proteins low from 24 h after the dose in females and from 48 h in males; serum and liver triglycerides showed no clear changes. Histologically the livers were characterised by cytoplasmic inclusion bodies, parenchymal necrosis, and bile duct proliferation. Metabolism and pharmacokinetic data were inadequate to explain the sex-dependency of this toxicity, but tissue distribution studies with carbon-14 labelled oxamniquine showed that 72 h after a given dose livers of female rats retained more label than males, and that little of this was due to unchanged drug.

Key words: Oxamniquine - Rats - Hepatotoxicity - Sex dependency

Introduction

Oxamniquine (6-hydroxymethyl-2-isopropylaminomethyl-7-nitro-l,2,3,4-tetra- hydroisoquinoline) is an agent widely used against schistosomiasis (Oxamni- quine Symposium 1980). Pre-clinical studies, reported briefly by Foster (1973), showed no significant toxicity after repeated doses to mice at 300 mg/kg p.o. or 750 mg/kg i.m., or to rabbits at 100 mg/kg i.m., while higher doses induced some mild hepatic changes. In dogs motor disturbance was seen at 50 mg/kg/day, but after 30 days' treatment there were no morphological changes. The LD-50 in mice was > 1,000 mg/kg (p.o. and i.m.) and in rabbits ca. 800 mg/kg p.o. and

Send offprint requests to: A. M. Monro, Centre de Recherche, Laboratoires Pfizer, B.P. 109, F-37401 Amboise Cedex, France * Mansil, Vansil (Pfizer)

248 M. Gregory et al.

> 1 ,000mg/kg (i .m.) . Several years ' widespread clinical use has shown o x a m n i q u i n e to be a wel l - tolerated drug; in part icular , there has b e e n no suggest ion of hepatotoxici ty , and indeed it has b e e n suggested to be the drug of choice in cases where hepat ic damage is a l ready presen t (Abaza et al. 1978; Clarke 1977; D o m i n g u e s and Cou th ino 1975).

By contrast , oxamniqu ine is fairly toxic to rats and is much more acutely lethal to female rats than to males. This paper summarises the ma in features of this toxicity and the subsequen t studies which were a imed at character is ing and unde r s t and ing the early hepat ic changes which appeared to be responsible for the lethal i ty after single p.o. doses of oxamniqu ine to male and female rats.

Methods

Rats were Charles River (CD) and were in the weight range 150-220 g (males) or 120-190 g (females), i.e., about 6-8 weeks of age, according to standard growth curves. They were housed singly and had free access to tap water. They were killed by exsanguination under ether anaesthesia. Clinical chemistry measurements (GPT, GOT, proteins, and triglycerides) were carried out by standard methods on liver and on blood taken from the vena cava at necropsy. Liver was processed by standard methods for light microscopy.

The drug, of pharmaceutical quality, was administered as a single p.o. gavage in a suspension in 10% Cremophor EL in saline, 10 ml/kg bodyweight. 14C-Oxamniquine was labelled in the methylene group of the side-chain (Kaye and Woolhouse 1976). Autoradiography followed the classical Ullman technique. Carbon-14 content of liver was determined by combustion of freeze-dried liver in a Packard apparatus and the unchanged drug in liver by reverse isotope dilution. Serum concentrations of oxamniquine were measured by a GLC method (Woolhouse and Wood 1977).

Protocols

A. Groups of four to 10 rats/sex were used to characterise the symptomatology and time scale of mortality. The LD-50 was calculated by the probit method of Finney (1947). B. Groups of five female rats received 50 or 150 mg oxamniquine/kg and groups of five male rats 50 or 300 mg/kg. A group from each dose level was killed at 24, 48, and 72 h after the dose. Food was withheld for 4 h prior to the dose, but was continuously available thereafter. C. Bile flow was measured over 6 h periods by use of indwelling bile-duct cannulae in male and female rats anaesthetised with urethane (1-1.5 g/kg s.c.). Groups of rats (4/sex) received single doses of oxamniqnine (25 mg/kg) and bile was collected 2-8, 24-30, or 72-78 h after the dose. D. Groups of six rats/sex/time received 50 or 300 mg oxamniquine/kg and were used to provide blood samples for determination of serum concentrations of drug. E. Whole body autoradiography was carried out on pairs (1M, IF) of rats killed 6, 24, and 72 h after a p.o. dose of carbon-14 labelled oxamniquine (25 mg/kg).

Equivalent groups of control animals were used in all studies and these received vehicle only.

Results

Toxicology (Study A)

This s tudy yielded LD-50 values of ca. 3 0 0 - 5 0 0 mg/kg in male rats and 3 0 - 6 0 mg/kg in females (Fig. 1). For several days after receiving a dose nea r or

Oxamniquine in Rats 249

Males LDso: ~ 7 mg/kg IOOF / ~ (7001

801- / ? 0 6 0 0 / 6 5 0

- 500 / 550 60 L - 5 4001/,50 40

�9 . 3 0 0 / 3 5 0 20

r

I

m 0 1 2 3 l, 5 7 " 0

"6 0 g

e -

100

80 I

601

40

201

I 14

Fig. 1.

Females LDso: 57 90

i 60 /)71 ;,~ '

0 1 2 3 4 5 7 d a y s 14 Cumulative deaths after a single p.o. dose of oxamniquire

above the respective LD-50, rats displayed malaise, inanition and weight loss, chromodaccryohoea, hypomotility and orange coloration of the urine. Survivors tended to start gaining weight again at 3 -7 days, but some deaths were delayed till 7-14 days after dosage.

Pathology (Study B)

All male rats survived for 3 days after the dose but nine of 15 females died after 50 mg/kg and 12 of 15 after 150 mg/kg. Most of the animals in the low dose female group showed, from 24 h onwards, hepatic cytoplasmic inclusion bodies, focal necrosis, and bile-duct proliferation. Those which were moribund or died (most of the high dose group) showed more general parenchymal necrosis and extensive bile duct proliferation. In males these lesions were less marked, even at the high dose level while there was, in addition, some Kupffer cell hypertrophy present.

Biochemistry (Study B)

Transaminase values (Table 1) in females reflected the histological findings: very high and dose-related values were observed at 24 h, and these were higher still in the two of five survivors at 48 h. Only one of five survived to 72 h after the lower dose and none to 48 h after the high dose. In males transaminases were raised only at 48 h after the high dose, but not after the low dose. Serum protein levels were depressed to a similar degree in females at 50 mg/kg at 24 and 48 h and in males at 300 mg/kg at 48 and 72 h. Serum triglyceride concentrations rose

250 M. Gregory et al.

E 0./,

0

a

0.2

0 . 6 -

i

1 [ I ,, i 1 I /, 6 8 "" 73 75 77

Time hours af ter dose )

Fig. 2. Effect of a single dose of oxamniquine on biliary flow in the rat. ( 0 ) Control female; ( �9 Control male; (A) 25 mg/kg - female; (A) 25 mg/kg - male; (Z) Standard error

slightly in females at 24 h (after 50 mg/kg) but showed no clear trend in males. Liver triglyceride levels did not change in any obvious manner.

Bile Flow (Study C)

Oxamniquine-treated males and females exhibited normal bile flow at 2 - 8 h and 2 4 - 3 0 h after the dose. At 72 -78 h, however, bile flow in males was slightly but significantly increased whereas that of females was decreased (Fig. 2). In three of seven females the flow was virtually non-existent and in another female the bile was colourless.

Pharmacokinetics (Study D)

Serum concentrations (Table 2) of oxamniquine were similar in males and females at 4 - 8 h after the dose, although at the lower dose (50 mg/kg) the serum

Oxamniquine in Rats

Table 1. Clinical chemistry mean values in serum (n = 4 or 5/group)

251

Parameter Sex Dose Hours post-dose (mg/kg)

24 48 72

SGOT (IU/L) M

F

SGPT (IU/L) M

F

Protein (g/100 ml) M

F

Triglycerides (mg %) M

0 113 50 86

300 102

0 71 50 1,462

150 4,033 c

0 32 50 13

300 38

0 9 50 1,847

150 4,433 c

0 6.3 50 5.7

300 6.0

0 6.3 50 5.6

150 5.5

0 59 50 56

300 58

0 52 50 122

150 43

146 109 600

97 5,625 a n . s .

12 12

391

18 1,900 a n . s .

6.4 6.4 5.4

6.7 5.2 a

n . s .

112 75

118

52 88 a

n . s .

176 180 238

232 1,040 b n . s .

25 10 25

10 31 b

n .S .

5.9 6.0 5.2

6.3 5.7 b

n , s .

136 132 72

54 n . s .

n . s .

n.s. = No sample due to unscheduled a Only two animals b Only one animal c Only three animals

death of animals

Table 2. Serum concentrations (~tg/ml) of oxamniquine (means of six different rats at each time)

Time post-dose Males Females (h)

50 mg/kg 300 mg/kg 50 mg/kg 300 mg/kg

4 1.17 27.7 1.29 17.1 5 0.66 13.5 0.95 29.4 6 0.49 18.0 0.55 26.9 7 0.44 23.1 0.52 18.2 8 n.s. 13.3 0.45 13.3

Approx. tl/2 (h) 1.9 Not calculable 2.5 3.2

n.s. = No sample

252 M. Gregory et al.

Table 3. Liver uptake of labelled oxamniquine after single p.o. doses (25 mg/kg)

Hours after dose

Males Females

n Total Oxamniquine n Total Oxamniquine radioactivity a (~tg/g) radioactivity" (~tg/g)

2 4 24.7 0.4 5 18.3" 0.2 6 3 19.6 0.2 5 15.4 0.3

24 5 3.8 0.03 5 9.5** 0.03

" Calculated as oxamniquine equivalents Significant difference between males and females *p < 0.05, **p < 0.01

elimination rate was slightly slower in females. Noteworthy was the finding that raising the dose by a factor of 6 increased serum levels by 20-40-fold in both sexes. In other experiments at 300mg/kg serum levels showed a large inter-animal variability. Serum concentrations of drug did not correlate on an animal-by-animal basis with the severity of liver damage detected microscop- ically.

Tissue Distribution (Study E)

Whole body autoradiography (WBAR) at 6 h post-dose showed a similar pattern in the male and female rats - a high level of activity in the intestine, stomach, liver, kidney, and salivary gland. At 24 and 72 h the only activity remaining in the male animals was in the lower intestine and a trace in the liver. By contrast, the female at 24 h appeared quite similar to that at 6 h, while at 72 h there was still considerable label in the liver and the lower gut.

Quantitation of the radioactivity in livers of male and female rats (Table 3) confirmed the pattern seen by WBAR. The females initially took up slightly less radioactivity than males, but by 24 h they retained considerably more. The proportion of the radioactive material corresponding to unchanged oxamniquine was minuscule in each sample.

Discuss ion

In seeking an explanation for the sensitivity to oxamniquine of the rat, and especially the female rat, one looks initially for possible interspecies differences in metabolism of the drug. While such differences can be quantitatively dramatic and thus explain species differences in response, differences in drug metabolism between males and females are generally modest (Kato 1974) and thus unlikely to explain the marked sex-related differences in acute toxicity observed in this study.

Kaye and Woolhouse (1976) studied the metabolism of oxamniquine (I) in dog, rabbit, hamster, mouse, rhesus monkey and rat. The major metabolite in

Oxamniquine in Rats

Fig. 3. Structure of oxamniquine N02 R2 and metabolites H

253

R1 R2 I CH2OH CH2NHPr i

1I CO2H CH2NHPr i

11I CH2OH CO2H

1V CH 3 CH2NHPr i

V CH 3 CH2NH 2

37[ CH 3 CO2H

all species was the 6-carboxylic acid (II), while the lesser metabolite, the 2-carboxylic acid (III), was barely detectable in the rat and monkey (Fig. 3). There was no evidence to suggest that the rat produces a metabolite not produced by other species. That the rat was also deficient at oxidation at the 2-position of the desoxy analogue (IV) of oxamniquine (Kaye and Woolhouse 1972). Interestingly, however, administration of the primary amine (V) did result in deamination to the acid (VI), and this occurred in the liver mitochondrial fraction and not in the microsomes (Woolhouse et al. 1979). Liver mitochondria from mice had a similar activity.

In summary, an inter-species difference in metabolism of oxamniquine does not at present provide an immediate explanation of its toxicity in the rat. Nevertheless, the greater persistence of radioactivity (little of which corre- sponded to unchanged drug) in the liver of female than of male rats after a dose of 14C- oxamniquine suggests the possibility of a causative role for a minor, as yet unidentified, metabolite. This greater persistence could, of course, be a result of and not the cause of the liver toxicity.

Functional criteria were not clearly indicative of mechanism. Although the mitochondrion may be the site of species differences in metabolism of oxamniquine, oxidative phosphorylation was unaffected in vitro after incubation of mitochondria isolated from livers of male and female rats with oxamniquine at concentrations up to 150 ~tg/ml (J. Nachbaur, personal communication). It seems unlikely, therefore, that the first point of attack is the mitochon- drion.

The disproportionate rise in serum concentrations of drug when the dose level was increased from 50 to 300 mg/kg might be due to an impaired clearance, but the data did not permit calculation of the kinetics of elimination. Alternatively the volume of distribution might have been greatly reduced, perhaps as a result of changes in the binding capacity of serum proteins. Preliminary estimates (Quinton, unpublished) of liver microsomal enzyme activity showed that O- and N-demethylase activities were unaffected 24 h after oxamniquine doses of 300 mg/kg to male or female rats. This suggests that the smooth endoplasmic reticulum is not an early target of oxamniquine.

The sex-dependent changes in bile flow seen in females at 72 h post-dose may simply be the expected consequences of the observed early morphological changes to the bile-ducts, although the enhancement of flow in males appears enigmatic.

254 M. Gregory et al.

The overall pattern of histological changes does not seem to resemble that of any other known hepatotoxic agent. The marked sex difference in response leads one to consider similarities in mechanism with those of other agents which are more hepatotoxic to female rats than to males. The best known of these are probably ethionine (Farber 1967; Yokota et al. 1982), cycloheximide (Bar-on et al. 1972), orotic acid (Negishi and Aizawa 1975) and D-galactosamine (Katterman and Sirowej 1979). All these agents, however, produce essentially fatty liver which was not seen to any obvious extent in our studies. Similarly, although the extensive bile duct proliferation observed in our studies, especially in females from 24 h onwards, is reminiscent of the effects of a-naphthyliso- thiocyanate (ANIT) in rats, this latter also produces its effects in mice and guinea-pigs (Capizzo and Roberts 1971). Furthermore, the pattern and chronology of events after ANIT - bile stasis and early inhibition of microsomal enzyme activity (Drew and Priestley 1976) is rather different from that of oxamniquine. Again one is forced to conclude that a mechanistic parallel does not seem to be valid.

In conclusion, there is at present no satisfactory explanation for the species-specific and sex-dependent hepatotoxicity of oxamniquine. The species and sex differences in metabolism may contribute to the explanation, although one cannot exclude the possibility that they are merely a consequence of the pathology.

Acknowledgements. We wish to thank Mr M. H. Tarbit for the autoradiographs, Mr R. G. Lawrence for the bile-flow data, and Melle M Cochard for preparation of the manuscript.

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Oxamniquine in Rats 255

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Received May 20, 1983