Impact of Heterotropic Allosteric Modulation on the Time-Dependent Inhibition of Cytochrome P450 3A4.

The current study was designed to investigate the influence of allosteric effectors on the metabolism of the prototypical cytochrome P450 (CYP) 3A4 substrate midazolam (MDZ), and on the determination in vitro time-dependent inhibition (TDI) of CYP3A4 using human liver microsomes (HLM). As the concentration of midazolam increased to 250 µM in HLMs, homotropic cooperativity resulted in a decrease in the 1'-hydroxymidazolam to 4-hydroxymidazolam ratio to a maximum of 1.1. The presence of varying concentrations of testosterone, progesterone (PGS), or carbamazepine (CBZ) in HLMs with MDZ could recapitulate the effect of homotropic cooperativity such that the formation rates of the 1'hydroxymidazolam and 4-hydroxymidazolam were equal even at low concentrations of MDZ. The presence of PGS (10 or 100 µM) and CBZ (100 or 1000 µM) in in vitro TDI determination of four known CYP3A4 time-dependent inactivators (clarithromycin, troleandomycin, mibefradil, raloxifene) simultaneously decreased potency and inactivation rate constant, resulting in fold changes in inactivation efficiency on average of 1.6-fold and 13-fold for the low and high concentrations of allosteric modulator tested, respectively. The formation of a metabolic-intermediate complex (MIC) for clarithromycin and troleandomycin decreased in the presence of the allosteric modulators in a concentration-dependent manner, reaching a new steady state formation that could not be overcome with increased incubation time. Maximum reduction of the MIC formed by clarithromycin was up to ∼91%, while troleandomycin MIC decreased up to ∼31%. These findings suggest that the absence of endogenous allosteric modulators may contribute to the poor translation of HLM-based drug-drug interaction predictions. SIGNIFICANCE STATEMENT: The reported overprediction of in vitro human liver microsome time-dependent inhibition of CYP3A4 and observed drug interactions in vivo remains an issue in drug development. We provide characterization of allosteric modulators on the CYP3A4 metabolism of the prototypical substrate midazolam, demonstrating the ability of the modulators to recapitulate the homotropic cooperativity of midazolam. Furthermore, we demonstrate that allosteric heterotropic cooperativity of CYP3A4 can impact the time-dependent inhibition kinetics of known mechanisms-based inhibitors, providing a potential mechanism to explain the overprediction.

Numerical analysis of time-dependent inhibition kinetics: comparison between rat liver microsomes and rat hepatocyte data for mechanistic model fitting.

Time-dependent inhibition (TDI) may confound drug interaction predictions. Recently, models were generated for an array of TDI kinetic schemes using numerical analysis of microsomal assays. Additionally, a distinct terminal inactivation step was identified for certain mechanism based inhibitors (MBI) following reversible metabolite intermediate complex (MIC) formation. Longer hepatocyte incubations potentially allow analysis of slow TDI and terminal inactivation. In the experiments presented here, we compared the quality of TDI parameterization by numerical analysis between hepatocyte and microsomal data. Rat liver microsomes (RLM), suspended rat hepatocytes (SRH) and sandwich-cultured rat hepatocytes (SCRH) were incubated with the prototypical CYP3A MBI troleandomycin and the substrate midazolam. Data from RLM provided a better model fit as compared to SRH. Increased CYP3A expression after dexamethasone (DEX) induction improved the fit for RLM and SRH. A novel sequential kinetic scheme, defining inhibitor metabolite production prior to MIC formation, improved the fit compared to direct MIC formation. Furthermore, terminal inactivation rate constants were parameterized for RLM and SRH samples with DEX-induced CYP3A. The low expression of CYP3A and experimental error in SCRH resulted in poor data for model fitting. Overall, RLM generated data better suited for elucidation of TDI mechanisms by numerical analysis.

Evidence for the bioactivation of zomepirac and tolmetin by an oxidative pathway: identification of glutathione adducts in vitro in human liver microsomes and in vivo in rats.

Although zomepirac (ZP) and tolmetin (TM) induce anaphylactic reactions and form reactive acyl glucuronides, a direct link between the two events remains obscure. We report herein that, in addition to acyl glucuronidation, both drugs are subject to oxidative bioactivation. Following incubations of ZP with human liver microsomes fortified with NADPH and glutathione (GSH), a metabolite with an MH+ ion at m/z 597 was detected by LC/MS/MS. On the basis of collision-induced dissociation and NMR evidence, the structure of this metabolite was determined to be 5-[4'-chlorobenzoyl]-1,4-dimethyl-3-glutathionylpyrrole-2-acetic acid (ZP-SG), suggesting that the pyrrole moiety of ZP had undergone oxidation to an epoxide intermediate, followed by addition of GSH and loss of the elements of H2O to yield the observed conjugate. The oxidative bioactivation of ZP most likely is catalyzed by cytochrome P450 (P450) 3A4, since the formation of ZP-SG was reduced to approximately 10% of control values following pretreatment of human liver microsomes with ketoconazole or with an inhibitory anti-P450 3A4 IgG. A similar GSH adduct, namely 5-[4'-methylbenzoyl]-1-methyl-3-glutathionylpyrrole-2-acetic acid (TM-SG), was identified when TM was incubated with human liver microsomal preparations. The relevance of these in vitro findings to the in vivo situation was established through the detection of the same thiol adducts in rats treated with ZP and TM, respectively. Taken together, these data suggest that, in addition to the formation of acyl glucuronides, oxidative metabolism of ZP and TM affords reactive species that may haptenize proteins and thereby contribute to the drug-mediated anaphylactic reactions.

Metabolic activation of a pentafluorophenylethylamine derivative: formation of glutathione conjugates in vitro in the rat.

The aim was to investigate the metabolic activation potential of a pentafluorophenylethylamine derivative (compound I) in vitro in the rat and to identify the cytochrome P450 (CYP) enzymes that catalyse these metabolic activation processes. Reduced glutathione (GSH) was fortified in rat hepatocytes and liver microsomes to trap possible reactive intermediates. Four glutathione conjugates (M1-4) were identified by LC-MS(n) following incubation of compound I in GSH-enriched rat hepatocytes and liver microsomes. Three of these conjugates (M2-4) have not been reported previously for pentafluorophenyl derivatives. Elemental composition analysis of these conjugates was obtained using high-resolution quadrupole time-of-flight mass spectrometry. The formation of GSH conjugate M1 was rationalized as a direct nucleophilic replacement of fluoride by glutathione, whereas the formation of the GSH conjugates M2-4 was proposed to occur by NADPH-dependent metabolic activation of the pentafluorophenyl ring via arene oxide, quinone and/or quinoneimine reactive intermediates. Formation of these conjugates was enhanced three- to five-fold in liver microsomes obtained from phenobarbital- and dexamethasone-treated rats. In incubations with pooled rat liver microsomes and recombinant rat CYP3A1 and CYP3A2, troleandomycin (TAO) reduced the formation of GSH conjugates M2-4 by 80-90%, but it had no effect on the formation of M1. Incubation of compound I with rat supersomes indicated that only CYP3A1 and CYP3A2 were capable of mediating these metabolic activation processes.

Biochemical background of toxic interaction between tiamulin and monensin.

Tiamulin, a diterpene antibiotic, is used for treatment of pulmonary and gastrointestinal infections in swine and poultry. Combined administration of tiamulin and ionophores (e.g. monensin) to farm animals may lead to intoxication manifested in severe clinical symptoms. Tiamulin metabolite complex with cytochrome P450 has been suggested to be the basis of drug-interactions. However, the formation of metabolic intermediate complex is questionable. The effect of tiamulin-treatment on cytochrome P450 activities was investigated in rats. Ethylmorphine and aminopyrine N-demethylation activities as well as monensin metabolism (O-demethylation) increased in liver microsomes of tiamulin-treated (200 mg/kg) animals. CYP3A1 induction caused by tiamulin was confirmed by the results of Western blot analysis. To test metabolic intermediate complex formation as a result of tiamulin treatment, cytochrome P450 activities were also determined in the presence of potassium ferricyanide. The findings together with those of in vitro complex formation suggested that formation of metabolic intermediate complexes of tiamulin with cytochrome P450 could be excluded. On the other hand, the results of inhibition studies showed significant decrease of ethylmorphine or aminopyrine as well as monensin demethylation in the presence of tiamulin. Our results proved that tiamulin has dual effect on cytochromes P450. It is able to induce and directly inhibit CYP3A enzymes, which are predominantly responsible for monensin O-demethylation. The direct effect of tiamulin as an inhibitor might play a more important role in toxicity than its putative effect as a chemical inducer of CYP3A enzymes.

Formation in vitro of an inhibitory cytochrome P450 x Fe2+-metabolite complex with roxithromycin and its decladinosyl, O-dealkyl and N-demethyl metabolites in rat liver microsomes.

1. Roxithromycin and its major metabolites found in rat and human urine, namely the decladinosyl derivative (M1), O-dealkyl derivative (M2) and N-demethyl derivative (M3), were incubated with rat liver microsomes and formation of an inhibitory cytochrome P450 (CYP)-metabolite complex and of formaldehyde (measurement of N-demethylation) were determined in vitro. Troleandomycin and erythromycin were also used for comparison. 2. Dexamethasone very significantly induced the microsomal N-demethylations of these macrolide antibiotics. The order of magnitude for the Vmax/Km ratio of N-demethylations by liver microsomes from dexamethasone-treated rats was troleandomycin > erythromycin = M2 > roxithromycin > M3, M1. 3. Formation of an inhibitory P450 x Fe2+-metabolite complex was detected on incubation of these macrolide antibiotics with rat liver microsomes in the presence of an NADPH-generating system and the order of maximum complex formation was troleandomycin > erythromycin > M2 > roxithromycin > M3 > M1. 4. Troleandomycin, erythromycin and M2 inhibited CYP3A-dependent testosterone 6beta-hydroxylation catalysed by liver microsomes from the dexamethasone-treated rat by 54, 33 and 23%, respectively, but roxithromycin, M3 and M1 were very weak by comparison. In the untreated rat, only testosterone 6beta-hydroxylation, but not testosterone 16alpha- and 2alpha-hydroxylation and androstenedione formation, activities were inhibited, indicating that inhibitory actions of these antibiotics are specific for CYP3A enzymes in liver microsomes. 5. These results support the view that formation of an inhibitory P450-metabolite complex is prerequisite for the inhibition of CYP3A-dependent substrate oxidations by rat liver microsomes and that M2 (and M3, to a lesser extent) may be the active metabolite that can form an inhibitory P450-metabolite complex by CYP3A enzyme(s).

Characterization of the effects of tebufelone on hepatic cytochromes P450 in the beagle dog.

Tebufelone (1-[3,5-bis(1,1-dimethylethyl)-4-hydroxy-phenyl]-hex-5-yne-1-one) is an investigational ditertiary butylphenol nonsteroidal anti-inflammatory drug. The purpose of the present study was to assess the effects of tebufelone on hepatocyte ultrastructure and hepatic cytochromes p450 (P450s) in the beagle dog after 2 weeks of oral administration at dose levels of 0, 5, 15, 50, and 100 mg/kg/day (N = 1/sex/dose level). Hepatic tissue was obtained at necropsy for histologic, ultrastructural, and biochemical evaluation. Hepatocellular hypertrophy was observed in only a single tebufelone-treated dog (50 mg/kg). Electron microscopic evaluation, however, revealed marked dose-dependent increases in smooth endoplasmic reticulum in all of the tebufelone treatment groups. Biochemical indicators suggested that tebufelone produced mixed effects on hepatic P450s. p-Nitroanisole O-demethylase and, to a greater extent, ethoxyresorufin O-deethylase activities were decreased with increasing tebufelone dose. The precise mechanism by which tebufelone decreased ethoxyresorufin O-deethylase activity in dogs in unknown, but it was not by competitive inhibition, P450 inactivation, or reduced CYP1A expression. Tebufelone treatment increased NADPH-dependent cytochrome c reductase, total P450, and indicators of CYP2B11 (chloramphenicol covalent binding and immunochemically determined 2B11) and CYP3A12 (erythromycin N-demethylase, triacetyloleandomycin spectral complex formation, testosterone 6 beta-hydroxylase, and immunochemically determined 3A12). The largest increase in the 2B11 and 3A12 markers occurred in the 50 or 100 mg/kg treatment groups. The greatest increase in CYP2B11 markers produced by tebufelone treatment ranged from 2- to 3-fold, whereas the increase in CYP3A12 markers ranged from 5- to 10-fold. The changes in hepatic ultrastructure and increases in CYP2B11 and CYP3A12 markers produced by tebufelone in dogs are similar to that reported for phenobarbital.

Formation of ligand and metabolite complexes as a means for selective quantitation of cytochrome P450 isozymes.

The suitability of triacetyloleandomycin (TAO) metabolite complex formation and metyrapone binding to reduced cytochrome P450 as a means for selective isozyme quantitation has been studied. Although isozymes of both subfamilies bind metyrapone in the reduced state, selective quantitation of 2B isozymes through the metyrapone complex is possible after complex formation of P450 3A with a TAO metabolite. Thus, consecutive application of both reactions allows the spectroscopic quantitation of P450 3A and 2B isozymes. Complete conversion of P450 3A into the complex, a precondition for P450 3A quantitation, requires NADH in addition to NADPH. A precise collective quantitation of 3A + 2B isozymes as metyrapone complexes alone is not possible because the corresponding complexes possess different molar extinction coefficients, i.e 71.5 and 52 mM-1 cm-1 at 446-490 nm, respectively. The formation of the TAO complex appears to be quite specific, since it correlates well with 3A-specific enzymatic activities, i.e. TAO N-demethylation and formation of 2 beta-hydroxy-, 15 beta-hydroxy- and 6-dehydrotestosterone. P450 3A levels in liver microsomes of male rats either untreated or treated with TAO, dexamethasone (DEX), phenobarbital or hexachlorobenzene amount to 13%, 78%, 66%, 24% and 11% of total P450, respectively. Good correlation between these values and P450 3A-specific enzymatic activities is obtained. By the spectroscopic method, P450 2B isozymes could not be detected in microsomes of untreated rats. With TAO, DEX and hexachlorobenzene the microsomal 2B level is elevated to about 20% of total P450, i.e. to 0.8, 0.4 and 0.4 nmol P450/mg protein, respectively. 2B levels of about 60% of total P450 (0.75 nmol P450/mg protein) are obtained by phenobarbital treatment. Immunoblotting with anti-P450 2B shows that the ratio of expressed 2B1 and 2B2 differs depending on the type of inducer. DEX predominantly leads to induction of 2B2, which may explain the low pentoxyresorufin O-depentylase activity in these microsomes.

Cytochrome P-450 complex formation by dirithromycin and other macrolides in rat and human livers.

Some macrolide antibiotics cause clinical drug interactions, resulting in altered metabolism of concomitantly administered drugs, via formation of an inactive cytochrome P-450 complex. In the present study, the formation of a cytochrome P-450 type I binding spectrum and a metabolic intermediate complex by troleandomycin and dirithromycin was assessed in liver microsomes obtained from untreated rats and phenobarbital- or dexamethasone-pretreated rats. Troleandomycin produced a type I binding spectrum and metabolic intermediate complex in microsomes from dexamethasone- and phenobarbital-pretreated rats. Dirithromycin did not produce a detectable type I binding spectrum but formed a small cytochrome P-450 metabolic intermediate complex (6% of that formed by troleandomycin) in microsomes from dexamethasone-pretreated rats only. The formation of a cytochrome P-450 type I binding spectrum and a metabolic intermediate complex by troleandomycin, erythromycin, dirithromycin, and erythromycylamine was also assessed in microsomes prepared from human livers. Troleandomycin and erythromycin formed a type I binding spectrum and a metabolic intermediate complex which were larger in microsomes from subjects on barbiturate therapy than in microsomes from subjects with no recent barbiturate exposure. Erythromycylamine did not form a detectable type I binding spectrum with any of the human microsomal samples, but a small metabolic intermediate complex was formed with microsomes from a subject on phenobarbital, phenytoin, and propranolol therapy. Dirithromycin did not form a detectable type I binding spectrum or a metabolic intermediate complex in any human liver sample. Preclinical quantitation of the human metabolic intermediate complex may be helpful in predicting the possibility of clinical drug interactions of new drug candidates.

Identification of a constitutive form of cytochrome P-450 with high activity towards macrolide antibiotics in Syrian golden hamster liver.

The presence of cytochromes P450 belonging to the P450IIIA family was investigated in the liver of untreated Syrian golden hamsters and compared to those of untreated, phenobarbital- and dexamethasone-treated rats. The microsomal preparations from untreated Syrian golden hamster livers exhibited higher activities of N-demethylation towards the macrolide antibiotics, erythromycin and troleandomycin, than those from untreated and phenobarbital-treated rats. Western blots analysis revealed larger amounts of proteins immunorelated to the rat cytochrome P450IIIA1 isozyme in Syrian golden hamster liver than in untreated and phenobarbital-treated rats. The N-demethylation of erythromycin and troleandomycin were strongly inhibited after preincubation of the microsomes from Syrian golden hamsters with anti-P450IIIA1. We conclude that a cytochrome P450 belonging to the P450IIIA family is expressed constitutively in the liver from Syrian golden hamsters, whereas it is not in rat liver.

Effect of glutathione depletion on the in vivo inhibition of drug metabolism by agents forming an inactive cytochrome P-450 Fe(II):metabolite complex. Studies with amiodarone and troleandomycin.

The relative contribution of competitive inhibition versus formation of a P-450:metabolite complex to the in vivo inhibition of drug metabolism for several agents is unclear. The present investigation examined the contribution of these two mechanisms to the in vivo inhibition of drug metabolism by amiodarone through manipulation of glutathione turnover. In vivo P-450-dependent metabolism in rats was assessed by determining antipyrine clearance. Pretreatment with amiodarone (50 mg/kg, iv) decreased antipyrine clearance with or without prior glutathione depletion. Depletion of glutathione by buthionine sulfoximine (1.6 g/kg, ip) did not enhance the magnitude of inhibition of antipyrine clearance by amiodarone. Moreover, administration of a normally subinhibitory dose of amiodarone after buthionine sulfoximine pretreatment did not influence antipyrine clearance. Similarly, depletion of glutathione via buthionine sulfoximine or diethylmaleate (1 mL/kg, po) did not influence the magnitude of inhibition caused by a single po dose of troleandomycin (500 or 350 mg/kg, respectively). These data indicate that glutathione content may not be a critical determinant for the in vivo inhibition of drug metabolism by agents which form a P-450:metabolite complex.

Studies on the role of liver cytochrome P-450 and oestradiol metabolism in the effects of nutrition and phenobarbital on ovulation rate in the ewe.

Coopworth ewes were differentially fed to produce 60 heavy (62 kg) and 80 light (45 kg) ewes. They were then fed a low protein (100 g protein kg-1 dry matter) pelleted ration. On Day 7 of the oestrous cycle after synchronization the following treatments were commenced in groups of 10 ewes: 4 low liveweight groups received low protein (LP), high protein (HP; 230 g protein kg-1 dry matter), LP + phenobarbital (PB; 1 g per os per day in gelatin capsules for 10 days) and LP + triacetyloleandomycin (TAO, 0.5 g day-1 in capsule for 10 days); while 3 high liveweight groups received LP, HP and HP + carbon tetrachloride (CCl4, 0.1 mL kg-1 bodyweight as a single dose). The experiment was repeated using another 7 groups of 10 ewes at an interval of 3 weeks. PB, TAO, high liveweight, and protein diet increased the ovulation rate whereas treatment with CCl4 reduced the ovulation rate. Because of the small number of ewes in some treatment protocols, only changes due to liveweight and protein diet were statistically significant. Liver weight and microsomal protein were increased by all treatments except CCl4 which caused a decrease. PB and TAO increased cytochrome P-450 and associated enzyme activities, in particular those related to cytochrome P-450p or P-450NF (including oestradiol 2-hydroxylation) in the human liver. In vitro, TAO binding indicated that the specific cytochrome was induced by PB and TAO but there were no direct effects of protein diet and liveweight. Most of the data support the theory that nutritionally induced increases in ovulation rate in ewes could result from changes in oestradiol metabolism, but the lack of induction of the specific cytochrome by protein diet and high liveweight suggests that increased ovulation caused by these factors may be the physiological response to several metabolic changes.

Metabolism of dihydroergotamine by a cytochrome P-450 similar to that involved in the metabolism of macrolide antibiotics.

1. Previous studies have shown that the macrolide antibiotics, such as oleandomycin and erythromycin, enhance their own transformation into a stable metabolite-cytochrome P-450 complex, thus impairing monooxygenase activity. This cytochrome P-450 induced by macrolides is similar to the major form induced in rats by pregnenolone-16 alpha-carbonitrile (PCN) (III A1 isozyme). 2. The cytochrome P-450 isozyme induced in rats by PCN or macrolide antibiotics bound dihydroergotamine (DHE) with high affinity and was also capable of metabolizing the drug. However, phenobarbital administration enhanced the metabolism of DHE to a greater extent than would be expected from the levels of the PB-PCNE isoenzyme, indicating that other cytochrome P-450 proteins may also be involved in DHE metabolism. 3. DHE metabolism was inhibited by macrolide antibiotics both ex vivo and in vitro. The metabolite-cytochrome P-450 complex formed by the antibiotics impairs the metabolism of DHE, so that when the complex is dissociated the metabolic activity is restored. These findings explain the observed clinical interactions between macrolides and other drugs, and such an approach may prove useful in their prediction.

Characterization of a phenobarbital-inducible dog liver cytochrome P450 structurally related to rat and human enzymes of the P450IIIA (steroid-inducible) gene subfamily.

A cytochrome P450 called PBD-1 isolated from liver microsomes of an adult male Beagle dog treated with phenobarbital (PB) is structurally and functionally similar to members of the P450IIIA gene subfamily in rat and human liver microsomes. The sequence of the first 28 amino-terminal residues of PBD-1 is identical in 15 and 20 positions, respectively, to the P450IIIA forms P450p from rat and P450NF (and HLp) from human. Upon immunoblot analysis, anti-PBD-1 IgG recognizes PCNa (P450p) and PCNb (PB/PCN-E) from rat, P450NF from human, and two proteins in liver microsomes from both untreated and PB-treated dogs. Similarly, anti-PCNb IgG cross-reacts with PBD-1 and with at least one protein in microsomes from untreated dogs and two proteins in microsomes from PB-treated dogs. P450IIIA-form marker steroid 6 beta-hydroxylase activities increase 2.5-fold upon PB-treatment of dogs and are selectively inhibited by anti-PBD-1 IgG. NADPH-dependent triacetyloleandomycin (TAO) complex formation and erythromycin demethylase, also marker activities for P450IIIA forms from rats and humans, increase 4- and 5-fold in dog liver microsomes upon PB treatment, whereas immunochemically reactive PBD-1 is induced 3-fold. In microsomes from PB-treated dogs, 5 mg anti-PBD-1 IgG/nmol P450 inhibits greater than 75 and 50% of TAO complex formation and erythromycin demethylase activity, respectively. TAO complex formation is not inhibited by chloramphenicol, a selective inhibitor of the major PB-inducible dog liver cytochrome P450, PBD-2. These data suggest that PBD-1 or another immunochemically related form is responsible for a major portion of macrolide antibiotic metabolism by microsomes from PB-treated dogs and for steroid 6 beta-hydroxylation by microsomes from both untreated and PB-treated dogs. Major species differences were noted, however, in the apparent Km for 6 beta-hydroxylation of androstenedione by liver microsomes from untreated rats (24 microM), humans (380 microM), and untreated dogs (4700 microM).

Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6 beta-hydroxylase cytochrome P-450 enzyme.

Cytochrome P-450-dependent steroid hormone metabolism was studied in isolated human liver microsomal fractions. 6 beta hydroxylation was shown to be the major route of NADPH-dependent oxidative metabolism (greater than or equal to 75% of total hydroxylated metabolites) with each of three steroid substrates, testosterone, androstenedione, and progesterone. With testosterone, 2 beta and 15 beta hydroxylation also occurred, proceeding at approximately 10% and 3-4% the rate of microsomal 6 beta hydroxylation, respectively, in each of the liver samples examined. Rates for the three steroid 6 beta-hydroxylase activities were highly correlated with each other (r = 0.95-0.97 for 25 individual microsomal preparations), suggesting that a single human liver P-450 enzyme is the principal microsomal 6 beta-hydroxylase catalyst with all three steroid substrates. Steroid 6 beta-hydroxylase rates correlated well with the specific content of human P-450NF (r = 0.69-0.83) and with its associated nifedipine oxidase activity (r = 0.80), but not with the rates for debrisoquine 4-hydroxylase, phenacetin O-deethylase, or S-mephenytoin 4-hydroxylase activities or the specific contents of their respective associated P-450 forms in these same liver microsomes (r less than 0.2). These correlative observations were supported by the selective inhibition of human liver microsomal 6 beta hydroxylation by antibody raised to either human P-450NF or a rat homolog, P-450 PB-2a. Anti-P-450NF also inhibited human microsomal testosterone 2 beta and 15 beta hydroxylation in parallel to the 6 beta-hydroxylation reaction. This antibody also inhibited rat P-450 2a-dependent steroid hormone 6 beta hydroxylation in uninduced adult male rat liver microsomes but not the steroid 2 alpha, 16 alpha, or 7 alpha hydroxylation reactions catalyzed by other rat P-450 forms. Finally, steroid 6 beta hydroxylation catalyzed by either human or rat liver microsomes was selectively inhibited by NADPH-dependent complexation of the macrolide antibiotic triacetyloleandomycin, a reaction that is characteristic of members of the P-450NF gene subfamily (P-450 IIIA subfamily). These observations establish that P-450NF or a closely related enzyme is the major catalyst of steroid hormone 6 beta hydroxylation in human liver microsomes, and furthermore suggest that steroid 6 beta hydroxylation may provide a useful, noninvasive monitor for the monooxygenase activity of this hepatic P-450 form.

In vivo and in vitro effects of a new macrolide antibiotic roxithromycin on rat liver cytochrome P-450: comparison with troleandomycin and erythromycin.

The effects of a new macrolide antibiotic (Roxithromycin) and one of its major metabolite (RU 39001) on rat hepatic drug metabolizing enzymes were compared to those of erythromycin, erythralosamine and troleandomycin (TAO) both in vitro and in vivo. In contrast to erythromycin, erythralosamine and TAO, roxithromycin and its metabolite RU 39001 exhibit: (i) a very poor affinity for rat liver cytochrome P-450, (ii) an unability to be metabolized into a stable inhibitory metabolite-cytochrome P-450 complex and (iii) a decreased ability to induce liver cytochrome P-450 PCNE, an isozyme implicated in drug associations involving some macrolide antibiotics.

Clotrimazole induction of cytochrome P-450: dose-differentiated isozyme induction.

Treatment of male rats for 3 days with the N-substituted imidazole, clotrimazole, produced up to a 4-fold induction of hepatic microsomal cytochrome P-450. The monooxygenase activities induced varied with the dose administered. At low doses (less than 25 mg/kg), p-nitroanisole demethylase and aniline hydroxylase activities were induced. Only at higher doses were other monooxygenase activities (erythromycin and ethylmorphine demethylases and cytochrome P-450 metabolic-intermediate complex formation from troleandomycin) induced. Microsomal UDP-glucuronosyltransferase activity toward morphine was induced at low doses in a manner similar to that of p-nitroanisole demethylase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of microsomes indicated that low doses of clotrimazole caused the intensification of a 48,000 molecular weight protein band, whereas at high doses, there was a marked intensification of an additional 50,500 molecular weight protein, the same molecular weight band as was intensified in phenobarbital- and dexamethasone-induced microsomes. The observations suggest a phenomenon of "dose-differentiated" isozyme induction for cytochrome P-450.

Drug interactions with macrolide antibiotics: specificity of pseudo-suicide inhibition and induction of cytochrome P-450.

Macrolide antibiotics like Erythromycin and Tri-acetyl oleandomycin (TAO) are metabolized to nitrosoderivatives which cause inactivation of Cytochrome P-450 by forming stable complex with the Iron of the hemoporphyrin. Several derivatives of erythromycin having lost their cladinose moiety are stronger inducer of liver cytochrome P-450 itself. The major form of cytochrome P-450 induced by all these macrolides in rat liver electrophoretically and immunologically indistinguishable from the major form induced by pregnenolone 16 alpha carbonitrile (PCN). This form is particularly able to metabolize macrolide and to lead to the corresponding 456 nm absorbing cytochrome P-450 complexes in vivo and in vitro.

Carbamazepine drug interactions.

Carbamazepine (CBZ) is commonly prescribed as an anticonvulsant or for the pain of trigeminal neuralgia. The potential for clinically important drug interactions exists because CBZ may induce the hepatic metabolism of other drugs or, conversely, other drugs may induce or inhibit the metabolism of CBZ. Studies and case reports demonstrate that CBZ may accelerate the metabolism of phenytoin, phenobarbital (PB), primidone, valproic acid, and warfarin. Likewise, phenytoin, PB, and primidone may increase the hepatic metabolism of CBZ. Inhibition of the metabolism of CBZ has been caused by triacetyloleandomycin, erythromycin, propoxyphene, isoniazid, and cimetidine. Future investigations will document the clinical significance of the CBZ interactions as well as reveal new interactions.