Absence of enantioselectivity in the pharmacodynamics of P450 2B induction by 5-ethyl-5-phenylhydantoin in the male rat liver or in cultured rat hepatocytes.

To explore the enantioselectivity of ligand interaction with the putative phenobarbital receptor, the pharmacodynamics of cytochrome P450 2B (CYP2B) induction by racemic 5-ethyl-5-phenylhydantoin and its two enantiomers were investigated in the male F344/NCr rat and in cultured adult male rat hepatocytes. Steady-state serum drug concentrations, measured following 14 days of administration of the compounds in the diet (0-1320 ppm, n = 3 rats per group), were used as an approximation of intrahepatocellular drug concentration. The serum xenobiotic concentrations associated with half-maximal hepatic CYP2B induction were 5-10 microM, based on measurement of pentoxy- or benzyloxyresorufin O-dealkylation activities, or immunoreactive CYP2B1 protein. The corresponding potency values in the hepatocyte culture experiments were 8-12 microM, based on measurement of total cellular RNA coding for CYP2B1. In both the in vivo and the hepatocyte culture experiments, the potencies for CYP2B induction were essentially equivalent for the racemate and the individual enantiomers of 5-ethyl-5-phenylhydantoin. In the case of this compound, there would appear to be no enantioselectivity for CYP2B induction. This finding may be interpreted as evidence against receptor mediation in the induction of CYP2B activity, although it is also possible that a receptor is involved that does not exhibit enantioselectivity.

Hepatic cytochrome P450 2B induction by ethyl/phenyl-substituted congeners of phenobarbital in the B6C3F1 mouse.

The abilities of structural congeners of phenobarbital to induce immunoreactive hepatic cytochrome P450 2B (CYP2B) protein and associated catalytic activity (benzyloxyresorufin O-dealkylation) in the male B6C3F1 mouse were examined. Interspecies differences in inducing ability were examined through comparison of the results with induction data obtained previously with the male F344/NCr rat. The congeners were administered in the diet for 2 weeks at concentrations equimolar to 500 ppm of the prototype CYP2B inducer, phenobarbital. Of the series of compounds tested, phenobarbital was the most effective inducer of benzyloxyresorufin O-dealkylation and immunoreactive CYP2B protein, with 2-ethyl-2-phenylsuccinimide, 5-ethyl-5-phenylhydantoin, primidone, and glutethimide being only 19-42% as effective. 5-Ethyl-5-phenyloxazolidinedione and the ring-opened and decarboxylated congeners, N-(2-ethyl-2-phenylacetyl)urea and 2-ethyl-2-phenylmalonamide, displayed minimal induction of these catalytic activities. Dose-response experiments performed with 5-ethyl-5-phenylhydantoin indicated that the intrinsic CYP2B-inducing activity of this congener was as great as that of phenobarbital in the mouse, although a fourfold greater dietary concentration of this hydantoin (2000 ppm) was required to elicit a response equivalent to that caused by 500 ppm phenobarbital. When extent of induction was related to serum total xenobiotic concentration rather than to administered dietary concentration, the potencies of the two congeners were determined to be more similar (58 vs. > or = 78 microM for phenobarbital and 5-ethyl-5-phenylhydantoin, respectively).

Comparative pharmacodynamics of hepatic cytochrome P450 2B induction by 5,5-diphenyl- and 5,5-diethyl-substituted barbiturates and hydantoins in the male F344/NCr rat.

To explore further the structural requirements for ligand interaction with the putative phenobarbital receptor, the pharmacodynamics of CYP2B induction by 5,5-diphenylbarbituric acid, phenytoin (5,5-diphenylhydantoin), barbital (5,5-diethylbarbituric acid) and 5,5-diethylhydantoin were investigated in the male F344/NCr rat. Steady-state total (free plus protein-bound) serum drug concentration, measured after 14 days of administration of the compounds in the diet, was used as an approximation of intrahepatocellular drug concentration. The serum concentrations associated with half-maximal hepatic CYP2B induction (EC50 values) were 6 to 11 microM and 15 to 18 microM for the diphenyl-substituted barbiturate and hydantoin, respectively, based on measurement of pentoxy- or benzyloxyresorufin O-dealkylation activities, or immunoreactive CYP2B1 protein. The corresponding potency values for the diethyl-substituted barbiturate and hydantoin were 16 to 20 microM and > or = 500 microM, respectively. The magnitudes of the maximal CYP2B induction responses elicited by the diphenyl-substituted congeners and by barbital were 94 to 122% of the responses resulting from phenobarbital itself. In contrast, the maximum induction responses elicited by 5,5-diethylhydantoin were only 24% as great as those elicited by phenobarbital. The finding of a CYP2B inducer with a potency value 2 to 3 orders of magnitude lower than those for certain other prototype CYP2B inducers is suggestive but not proof of receptor mediation in the induction process.

Dose-response relationships for the induction of P450 2B by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzne (TCPOBOP) in rat and cultured rat hepatocytes.

1. The dose-response relationships for hepatic CYP2B induction by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) were examined in the male F344/NCr rat. TCPOBOP, administered for 14 days at 0-1000 ppm in the diet, caused concentration-dependent induction of hepatic CYP2B1 protein and RNA, and of CYP2B-mediated catalytic activities (benzyloxy- and pentoxyresorufin O-dealkylation, and testosterone 16 beta-hydroxylation). ED50 values for CYP2B induction were > or = 300 ppm dietary TCPOBOP. The maximal inductions observed were 66-88% of those resulting from exposure of the rats to 500 ppm dietary phenobarbital. 2. The EC50 values for hepatic CYP2B induction were 1.5-3.0 microM (based on serum TCPOBOP) and 15-20 mumol/kg liver. 3. The maximal inductions of isozymes of the CYP3A subfamily, of microsomal epoxide hydrolase, and of glutathione S-transferases Ya/Yc and Yb1/Yb2 in rats exposed to TCPOBOP were 58-74% of those resulting from exposure of the rats to 500 ppm dietary phenobarbital. 4. The ED50 value for induction of benzyloxyresorufin O-dealkylation in cultured rat hepatocytes by TCPOBOP was determined to be 0.93 microM. The maximal induction of this activity caused by TCPOBOP was 87% of the maximum increases caused by phenobarbital. 5. The results indicate that TCPOBOP is a highly effective phenobarbital-type inducer in the rat when administered in the diet for 2 weeks at 1000 ppm. When extent of induction is related to serum total xenobiotic level, TCPOBOP would appear to be at least as potent as, if not more potent than, phenobarbital in the rat.

Pharmacodynamics of cytochrome P450 2B induction by phenobarbital, 5-ethyl-5-phenylhydantoin, and 5-ethyl-5-phenyloxazolidinedione in the male rat liver or in cultured rat hepatocytes.

The pharmacodynamics of rat hepatic cytochrome P450 2B (P450 2B) induction by phenobarbital (PB) and two structural congeners, dl-5-ethyl-5-phenylhydantoin (EPH) and dl-5-ethyl-5-phenyloxazolidinedione (EPO), were investigated. The in vivo induction of P450 2B was probed in F344/NCr rats by measuring immunoreactive hepatic P450 2B1 protein and by assaying the hepatic 16 beta-hydroxylation of testosterone and O-dealkylation of (benzyloxy)- and pentoxyresorufin. The induction of (benzyloxy)resorufin O-dealkylation activity was also measured in adult rat hepatocyte cultures exposed to the three xenobiotics. The concentration of xenobiotic at the putative active site in the in vivo studies was approximated by measuring serum total xenobiotic levels, while in the hepatocyte culture studies, the nominal xenobiotic concentration in the culture medium was used. Concentration-dependent induction of P450 2B activities was observed in the in vivo and hepatocyte culture studies. The in vivo ED50 values for P450 2B induction were approximately 110, approximately 100, and approximately 3000 dietary ppm (14 days administration) for PB, EPH, and EPO, respectively. The in vivo EC50 values for P450 2B induction were approximately 9, approximately 6, and approximately 130 microM (total serum) for PB, EPH, and EPO, respectively. In cultured rat hepatocytes, the ED50 values for induction of (benzyloxy)resorufin O-dealkylation activity were 14.5, 14.2, and 108 microM for PB, EPH, and EPO, respectively. These data indicate that pharmacodynamic results obtained with cultured hepatocytes represent a good qualitative and quantitative approximation of the in vivo hepatic responses in male rats caused by PB-type inducers.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative pharmacodynamics of CYP2B induction by phenobarbital in the male and female F344/NCr rat.

The phenobarbital dose-CYP2B induction response relationships and pharmacodynamics of CYP2B induction have been characterized in female and male F344/NCr rats. The ED50 and EC50 values for the induction, by phenobarbital, of hepatic CYP2B1 or 2B1/2B2 protein or associated catalytic activities (benzyloxy- or pentoxyresorufin O-dealkylation or testosterone 16 beta-hydroxylation) were 2- to 7-fold higher in the female than in the male rat, indicating a somewhat decreased potency for this effect in the female rat. In contrast, the maximal induction, expressed as the ratio of induced activity to control activity, was as great or greater in the female rat than in the male. Thus, any difference in the responsiveness of female rats to hepatic CYP2B induction by phenobarbital, compared to male rats, is reflected in potency but not degree of induction of catalytic activity or immunoreactive protein.