| Cyclophosphamide (CPA), a common antineoplastic agent, is often used with dexamethasone (DEX) for the combined chemotherapy of various tumors, the imunosuppressive blockage of allograft rejection and treatment of autoimmune disease. Some clinical case reports have indicated that the drug interaction between CPA and DEX had developed after dosing them simultaneously, resulting in the enhancement of CPA toxicity. But the mechanism underlying is not entirely clear.Previous studies have shown that CPA is metabolized by CYP3A4 (N-dechloroethylation), a main cytochrome P450 (CYP450) in mammals, and CYP2B1/2 or CYP2C (4-hydroxylation). DEX is a typical inducer of CYP3A4, by a pregnane X receptor (PXR) mediated mechanism. In this study, we first hypothesized that DEX potentiates CPA toxicity by inducing CYP3A4 and therefor changing its metabolic fate and pharmacokinetics. It was scrutinized then the profiles and mechanisms of DEX induction, and the changes of CPA toxic manifestations after DEX induction. Finally, an in vitro cell-based cotransfection assay was established to assess CYP3A4 induction potential of drug candidates, based on these studies. The main results are as follows: 1. The profile of liver CYP450 induction by DEX in male ratAfter 4 daily i.p. administering of 0,25, 50 and lOOmg/kg/d DEX in male Wistar rat, it was determined the total CYP450 content, the mRNA and protein expression levels of CYP3A1, CYP3A2 and CYP2B1/2, and the ERD (CYP3A catalytic activity), PROD (CYP2B catalytic activity) and BROD (total CYP450 catalytic activity) in livers. The results showed that total CYP450 content and BROD, ERD or PROD was increased after multiple induction with DEX. The increases in CYP3A1 mRNA, protein and catalytic activity were both significant and dose-dependent. CYP3A2 protein also increased, but the level of mRNA did not increased significantly. It seems that CYP3A1 and CYP3A2 induction follow different mechanisms. The PROD, mRNA of CYP2B1/2 and total CYP450 content reached the maximum induction activity at the dose of 50mg/kg/d. Beyond this dose, there were an inhibitory effects, which may be attributedto toxicity of DEX itself to liver.2. The effects of DEX induction on CPA toxicity in male ratsAfter 4 daily i.p. Administering of 0 or 50 mg/kg/d DEX to male Wistar rats for CYP450 induction, animals were i.p. dosed then with 0,150 or 200 mg/kg CPA at the fifth day. After euthanatized 36h after last dosing, all animals were examined with the manifestations of liver, kidney, bone marrow and bladder toxicity of CPA. The results showed that DEX induction itself exhibited some general and liver toxicity to male rats, evidenced by decreased body weights of all DEX dosed animals, increase of plasma ALT, and mild bleeding and focal vacuoles in liver pathological examinations. Dosing CPA single time can bring a slight increase of G2/M myelocytes and a significant increase of urine protein and occult blood. Furthermore, DEX induction potentiates the hepatotoxicity, nephrotoxicity, myelotoxicity and bladder toxicity of CPA. The potentiation of CPA hepatotoxicity was evident by elevation of plasma ALT, decrease of plasma ALP, the decline of total and nonprtotein sulfhydryl groups, and narrowing of liver sinusoid and vacuolar degeneration of lobules. For CPA nephrotoxicity, this potentiation was evident by the increase of plasma BUN and Crea, elevation of urine protein and occult blood, and renal proximal tubule degeneration and bleeding in medulla, as compared with respective non-inductive animals. As for bone marrow toxicity of CPA, rats treated with DEX exhibited an increased myelocytes ratio of Gj/G, to S, after dosed with 200mg/kg CPA. The pathological examination showed that bladder inflammation and bleeding happened in CPA dosed rats after induced with 50mg/kg DEX.3. The mechanism of CYP3A induction by DEXAfter induction with 25, 50 and lOOmg/kg/d DEX, the mRNA and protein expression level of PXR in liver was all up regulated, which suggested that inductive effect of DEX to CYP3A was mediated... |
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