METABOLITE COMPLEX-FORMATION OF ORPHENADRINE WITH CYTOCHROME-P450 - INVOLVEMENT OF CYP2C11 AND CYP3A ISOZYMES

Expression and inhibition of cytochrome P450 (CYP) isozymes capable of forming an orphenadrine metabolite complex were studied in microsomes of untreated and inducer-treated male and female rats. High levels of complex-forming isozymes were found in microsomes of untreated male a s compared to female rats. Treatment of male rats with several P450 in ducers did not considerably increase the extent of in vitro complex fo rmation. In female rats, however, phenobarbital or dexamethasone treat ments led to pronounced induction. The isozyme specifity of complex fo rmation was investigated by several approaches including: 1. inhibitio n by orphenadrine of isozyme-specific P450 activities, such as hydroxy lation of testosterone, O-dealkylation of pentoxy- and ethoxyresorufin and complex formation with triacetyloleandomycin (TAO), 2. inhibition of orphenadrine complex formation by metyrapone, TAO, and cimetidine, and 3. correlation of complex levels with immunochemically, enzymatic ally, or spectroscopically determined amounts of P450 isozymes. Our da ta suggest that CYP2C11, a CYP3A isozyme and an unidentified P450 spec ies are involved in complex formation with orphenadrine, but exclude t he involvement of CYP1A1/2 and CYP2B1/2. The capability of CYP2C11 to form a metabolite complex with orphenadrine is strongly suggested for the following reasons: 1. Efficient inhibition of testosterone 2 alpha - and 16 alpha-hydroxylation by complex formation with orphenadrine in microsomes of untreated male rats, 2. high expression of orphenadrine -complexing isozymes in untreated male compared to female rats, 3. spe cific inhibition of in vitro complex formation by cimetidine, 4. suppr ession of complex-forming isozymes by 3-methylcholanthrene and beta-na phthoflavone, and 5. concomitant induction of complex-forming isozymes , immunodetectable CYP2C11, and testosterone 2 alpha-hydroxylase by st anozolol. That at least one, but not all, CYP3A isozymes is involved i n complex formation is concluded from inhibition experiments with TAO that show that orphenadrine complexation can be significantly inhibite d in microsomes of dexamethasone-treated, but not in microsomes of unt reated rats. Furthermore, complex formation with TAO is not inhibited by orphenadrine in microsomes of phenobarbital (PB)-treated rats. In P B-treated female rats, a further unidentified complex-forming isozyme can be detected that is not inhibited by complex formation with TAO.