Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi-shi, Tokyo 186-8650, Japan
Received June 26, 2001; accepted September 10, 2001
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ABSTRACT |
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Key Words: rats; ACAT inhibitor; toxicity; sex differences; hepatic CYP enzymes; CYP3A2.
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INTRODUCTION |
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YIC-C8-434, chemically designated as N-(3,5-dimethoxy-4-n-octyloxycinnamoyl)-N`-(3,4-dimethylphenyl)piperazine, is a potent ACAT inhibitor that acts in rat liver microsomes, in hepatocyte-like HPG2 cells, and in intestinal enterocyte-like Caco-2 cells (Ohishi et al., 2001). Furthermore, YIC-C8-434 showed anti-hypercholesterolemic activity in cholesterol-fed rats at 2.4 mg/kg/day (Ohishi et al., 2001
). Considering the predominant expression of ACAT-1 and ACAT-2 in HPG2 cells and Caco-2 cells, respectively (Chang et al., 2000
), it is assumed that YIC-C8-434 might have sufficient ability to inhibit both ACAT-1 and ACAT-2. However, we are investigating the possibility that YIC-C8-434 strongly inhibits ACAT-2 rather than ACAT-1, since YIC-C8-434 did not inhibit ACAT in THP-1 macrophages (unpublished communication) in which ACAT-1 is expressed.
We demonstrated that the toxicity of YIC-C8-434 was restricted to female rats. To date, there have been no reports on sex-related differences in the toxicity of ACAT inhibitors. The purpose of this study is to show that the sex-related toxicity is caused not by the inhibition of ACAT in steroidogenic tissues, especially in ovaries, but by sex-related differences in YIC-C8-434 metabolism in rats.
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MATERIALS AND METHODS |
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Twenty-eight-day repeated oral toxicity study.
YIC-C8-434 was suspended in a 0.5% carboxymethylcellulose solution at concentrations of 0.4, 2, 10, and 50 mg/ml. The dosing volume was 10 ml/kg. Each group consisted of 10 rats, which were administered the drug by gavage once daily for 28 days. Control rats were given the same volume of 0.5% carboxymethylcellulose as received by drug-treated rats. Body weight was measured weekly during the test period. Food weight was measured twice a week, and we calculated food consumption per day. After the final administration of YIC-C8-434, rats were fasted overnight. The next day, they were sacrificed under pentobarbital anesthesia. Blood was collected from the abdominal vena cava. Blood for hematological analysis was collected in tubes containing potassium EDTA. Hematological determinations, including erythrocyte, leukocyte, platelet, hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and leukocyte differential (lymphocyte, neutrophil, eosinophil, basophil, monocyte) were performed using an automated cell counter (Bayer H 1E, Bayer Co., Tarrytown, NY). Blood was collected in tubes with heparin for plasma chemistry and without anticoagulant for serum protein analyses. In blood chemical examinations, we analyzed the following items; transaminase (GOT, GPT), alkaline phosphatase, glucose, total cholesterol, triglyceride, phospholipid, total bilirubin, urea nitrogen, creatinine, calcium, sodium, potassium, chloride, and total protein using an Automatic Clinical Analyzer (7170, HITACHI Ltd., Tokyo, Japan). The weights of organs (brain, pituitary, thymus, heart, liver, spleen, kidney, adrenal, testis, prostate, uterus, and ovary) were determined at necropsy of the animals. Tissues were fixed in 10% neutral buffered formalin and processed for paraffin sectioning followed by hematoxylin and eosin (H&E) staining for microscopic observation.
The following tissues from the 500-mg/kg dose group and control group were examined microscopically: brain, pituitary, thymus, heart, liver, spleen, kidney, adrenal, femur with marrow, skin, mammary gland, harderian gland, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), pancreas, mesenteric lymph nodes, thyroid gland, parathyroid gland, muscle, testis, epididymis, seminal vesicle, prostate, uterus, and ovary. Adrenal, ovary and bone marrow were examined in the 100-mg/kg dose group, for female rats.
In vivo pharmacokinetic study.
Three rats of each sex received a single oral administration of YIC-C8-434 at 500 mg/kg. Blood was collected from the cervical vein at 1, 4, 10, 24, and 48 h after the administration. Plasma was obtained after immediate centrifugation and the concentration of YIC-C8-434 in plasma was assayed by HPLC. The area under the blood drug concentration-time curve (AUC) was calculated from the drug-concentration for 48 h after a single administration by the linear trapezoidal method.
Absorption and elimination kinetics was investigated by a residual method. It is assumed that YIC-C8-434 is absorbed into the body and is eliminated from the systemic circulation by a first-order process. Based on this assumption, kinetics of absorption and elimination were compared between male and female rats. Utilizing the data from a single administration test, separate linear regression analyses were performed with plots for 448 h (elimination term) and residual plot-time profiles based on the slope of the residual plot (14 h for absorption term) were constructed. The constants of absorption rate (Ka) and elimination rate (Ke) were calculated from the slope.
In vitro metabolism studies.
Hepatic microsomes were prepared freshly from livers of male and female rats. Microsomes were washed, and the pellets were resuspended in 0.1 M phosphate buffer (pH 7.4). The metabolism of YIC-C8-434 by hepatic microsomes was assayed in a reaction mixture consisting of an NADPH-generating system (20 mM glucose-6-phosphate, 1.2 IU glucose-6-phosphate dehydrogenase, 20 mM MgCl2), hepatic microsomes (0.5 mg protein/ml) and YIC-C8-434 (0.67, 1.33, 2.66, 6.67, 13.3, 26.6, or 53.3 µM). After pre-incubation for 5 min at 37°C, the reaction was initiated by adding NADPH (1 mM). The final volume of the reaction mixture was 1.5 ml. After incubation for 10 min, the reaction was terminated by the addition of 2 ml of ice-cold acetonitrile. Since metabolites of YIC-C8-434 were unknown, the metabolism was evaluated as the difference in the amount of residual YIC-C8-434 between the start (0 min) and the end of the reaction (10 min).
For inhibition by SKF525A, hepatic microsomes were mixed with various amounts of SKF525A (0, 2, 10, 50, or 250 µM) and pre-incubated at 37°C for 5 min. An aliquot of this microsome solution was transferred to a reaction mixture containing the NADPH-generating system and YIC-C8-434 (2.66 µM). After pre-incubation at 37°C for 5 min, the reaction was initiated by adding NADPH (1 mM) and terminated after a 10-min incubation (final volume of 1.5 ml).
Immunoinhibition of the YIC-C8-434 metabolism with microsomes prepared from the liver of male rats was performed in the presence of 50 µl of anti-P450 antisera (0.4 mg protein/ml) against CYP1A1, CYP1A2, CYP2B1, CYP2C11, CYP2E1, CYP3A2, or CYP4A1. Microsomes were premixed with each antibody for 30 min and then were added to a reaction mixture containing the NADPH-generating system and YIC-C8-434 (15 µM). After incubation for 10 min, the amount of residual YIC-C8-434 with/without anti-P450 antiserum was measured and the inhibition rates were calculated.
Measurement of YIC-C8-434 in plasma and the reaction mixture.
The concentration of YIC-C8-434 was measured by HPLC. Acetonitrile (2 ml) was added to 0.4 ml of plasma or 1.5 ml of reaction mixture. After shaking and ultrasonic treatment for 5 min, the sample was centrifuged (3000 rpm, 15 min). The upper layer was transferred to a clean glass tube, dried under N2 gas, and reconstituted with 100 µl of acetonitrile/water (9/1). Twenty µl of the solution was injected onto a Symmetry C18 analytical column (3.5 µm, 4.6 x 75 mm. Water Co., Milford, MA). The temperature of the column was adjusted to 40°C. The flow rate of the mobile phase, acetonitrile/water/acetic acid/triethylamine (500:475:25:1), was adjusted according to the retention time of YIC-C8-434 to be about 12 min. Column effluent was monitored by UV absorption at 310 nm, and the limit of detection was 15 ng/ml.
Statistical analysis.
Data were represented as the mean ± SD. The homogeneity of variance was tested by Bartlett's method. If homogeneous, the data were analyzed by one-way ANOVA, and if not, by the Kruskal Wallis test. In significant cases, Dunnett's test for multiple group comparisons was performed. Significance was accepted at p < 0.05.
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RESULTS |
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In Figure 2, the changes in body weight during the 28 days are shown. In male rats, the body weight gains were normal. In contrast, weight gain was markedly suppressed in the high-dose group (500 mg/kg) of female rats compared with the control and other groups. The mean body weight of this group was significantly lower than that of the control group during the test period.
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DISCUSSION |
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Studies have shown that the adrenal gland is very susceptible to the toxicity of ACAT inhibitors (Dominick et al., 1993a,b
; Matsuo et al., 1996
; Vernetti et al., 1993
; Wolfgang et al., 1995
). However, unlike other ACAT inhibitors, YIC-C8-434 had little effect on adrenal glands compared with thymus and ovaries. The inhibition of ACAT by YIC-C8-434 is potentially toxic to the ovary and thymus since the ACAT enzyme is expressed in these organs, as well as the adrenal gland (Matsuda et al., 1998
; Meiner et al., 1997
; Uelmen et al., 1995
). Thus, if toxic effects on the thymus and ovary resulted from inhibition of ACAT that was expressed in these organs, we may point out that the thymus and ovary are the target organs of toxicity induced by YIC-C8-434.
However, the increase in plasma bilirubin, and the atrophies of thymus, ovary, and fatty marrow are arguably not irrelevant to the decrease in food consumption. Our findings indicated that YIC-C8-434 induced anorexia in female rats. Food consumption was reduced to about 40% of control in female rats during the treatment period at the highest dose (500 mg/kg) and weight gain was suppressed. Generally, in food-restriction studies, decreases of tissue weight (thymus and ovary) and increase of fat in the marrow are exhibited in rats (Bronson, 1986; Levin et al., 1993
; Seki et al., 1997
). The restricted rats also exhibit a decrease of hematopoiesis and increase of total bilirubin (Levin et al., 1993
; Seki et al., 1997
). We observed similar changes caused by the toxicity of YIC-C8-434. It seems entirely possible that the loss of bone marrow is reflected in the appearance of fatty marrow and a decrease in the number of circulating leukocytes in rats. As for the suppression of food consumption occurring from the first week of treatment in female rats, it is conceivable that YIC-C8-434 has an ability to induce hypophagia.
Meiner et al. (1996) found that ACAT knockout mice had increased plasma cholesterol levels. Our finding of an increase in lipids among female rats is similar to this. Although strong inhibition of the ACAT enzyme could be a causal factor in plasma lipid disorders, the increase of plasma lipids might also be explained by anorexia. Stone (1994) reported that anorexia could be correlated with hyperlipidemia. However, we cannot explain why anorexia should be induced in female rats during treatment with YIC-C8-434. Further study into the influence of YIC-C8-434 on the hypophagic effect is required.
The major characteristic of the toxicity induced by YIC-C8-434 was that it was sex-related. We suggested that the finding was attributable to a higher concentration of YIC-C8-434 in the blood of female than of male rats. As shown in Table 4, greater values of the concentration of YIC-C8-434 in blood and AUC in female rats were interpreted to reflect a difference in the rate of metabolism or absorption of YIC-C8-434 from the intestinal tract in males and females. To investigate the mechanism behind the higher concentration of YIC-C8-434 in blood in female rats, in vivo and in vitro studies were performed. From the result of the in vivo study with a single oral administration, sex-related differences in the metabolism of YIC-C8-434 were predicted reasonably well. Kato and Kamataki (1982) have reported sex differences in the toxicological and pharmacological potency of certain drugs. There is evidence that sexual dimorphism in drug metabolism in rats results from a sex-related differential expression of hepatic P450s (Kobliakov et al., 1991
). Therefore, we performed additional experiments on the metabolism of YIC-C8-434 by hepatic microsomes of the rat. As shown in Figure 8
, these in vitro experiments indicated that the microsomal cytochrome P450 from male rats could metabolize YIC-C8-434. However, consistent with the observations in vivo, hepatic microsomes from female rats poorly metabolized YIC-C8-434. Furthermore, inhibition of the metabolism in microsomes from female rats was not observed with the P450 inhibitor, SKF525A. This result indicated that cytochrome P450 did not play a role in YIC-C8-434 metabolism in female rats. It is known that the sexual dimorphism of drug metabolism in the rat is due to the differential expression of certain cytochromes P450. For example, CYP2A2, CYP3A2, and CYP2C11 are male-predominant or expressed only in male rats, whereas CYP2A1, CYP2C7, and CYP2C12 are female-predominant or limited to female rats (Bandiera, 1990
; Kobliakov et al., 1991
; Legraverend et al., 1992
; Waxman et al., 1985
, 1990
). In the examination with anti-rat P450 (CYP) antisera, we tried to identify the CYP isoform for metabolism. Anti-rat CYP3A2 antiserum showed the most potent inhibition of YIC-C8-434 metabolism: 50%. Because the CYP3A2 isoform is responsible for the metabolism of YIC-C8-434 in rats, and because there is more CYP3A2 isoform in the liver of males than females, these results strongly suggest that the sexual dimorphism in the metabolism of YIC-C8-434 in rats results from the sex-related differential expression of the CYP3A2 isoform.
In summary, the results of the present study implicate the sex-related differential expression of CYP3A2 in the toxicological findings relevant to reduced metabolism and the accumulation of YIC-C8-434 in female rats. It would be reasonable to conclude that the metabolite of YIC-C8-434 had no effect on the toxicity, since toxic findings were not observed in male rats in which YIC-C8-434 was well metabolized. The existence of sex differences in drug metabolism is not unique to rats. Such differences have been shown in humans (Hunt et al., 1992). However, the magnitude of the sex differences in humans is invariably far subtler than that found in rats. Further study will be required to ascertain whether YIC-C8-434 shows a sex-related difference in clearance in humans.
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NOTES |
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