Affinity of Drugs for Cytochrome P-450 Determined by Inhibition of p-Nitrophenetole O-Deethylation by Rat Liver Microsomes

Abstract: The rate of conversion of p-nitrophenetole to p-nitrophenol by rat liver microsomes was studied. Inhibition of the reaction by CO and by SKF 525A and the absolute dependence on NADPH and oxygen indicate that cytochrome P-450 catalyzes the reaction. The apparent K_m for oxygen was 0.07 μM. Furthermore, cytochrome b₅ seemed to be involved in the formation of p-nitrophenol. The effect on p-nitrophenol formation of drugs known to be involved in drug interaction in clinical practice was studied. There was a competitive inhibition by phenytoin (inhibitor constant, K_i, 30 μM), disulfiram (K_i, 2 μM) and chloramphenicol (K_i, 20 μM), whereas a mixed-type inhibition by isoniazid was observed (K_is, 1,3 mM and K_ii, 10,6 mM).     

Simulataneous and independent versus antagonistic inhibition of muscle carbonic anhydrase (CA III) by acetazolamide and cyanate

The inhibition by cyanate and acetazolamide of pig muscle carbonic anhydrase III (CA III) CO₂ hydratase activity was studied in order to explore mechanistic features possibly unique to the muscle isoenzyme. The turnover number for CO₂ hydration was found to be 6000 sec^?1 with a K_m of 83 mM for CO₂. Cyanate inhibition (K_i, 3 μM) and acetazolamide inhibition (K_i, 44 μM) were both found to be noncompetitive with respect to CO₂. Significantly, acetazolamide and cyanate displayed non-exclusive binding to pig muscle carbonic anhydrase. The similarity of mode and degree of inhibition of muscle carbonic anhydrase by cyanate as compared with the inhibition of the erythrocyte isoenzymes suggests the existence of a similar metal environment. However, the observation that cyanate and acetazolamide bind simultaneously to CA III and the comparatively large K_i for acetazolamide per se appear to be more compatible with a different mode of coordination of the zinc with the sulfonamide, thus supporting a five-coordinant zinc in the catalytic mechanism of CO₂ hydration for CA III.     

Calcium binding properties of rat heart plasma membrane and inhibition by structural analogues of dl-propranolol

The isolation and characterization of a plasma membrane preparation from rat heart is described. Enzymatic, chemical, and electron microscopic analysis revealed a relative lack of contamination with nuclear, mitochondrial, ribosomal, and sarcoplasmic reticulum membrane. One calcium binding site (K_d) = 265 μM, B_max = 65 $\text{nmoles}\text{mg}$ protein) was detected by equilibrium dialysis. Monovalent metal ions exhibited 10–100-fold less inhibition potency than divalent metal ions when analyzed by competitive inhibition of calcium binding. The range of K_i values found for divalent metal ions was similar to the K_d value for calcium. La⁺³ produced a potent non-competitive inhibition. A large variety of structural analogues of d,l-propranolol, many of which have been shown to lack β-adrenergic blocking activity, were competitive inhibitors of the calcium binding activity, with K_i values ranging from 40–900 μM. Electrophilic, hydrophobic, and diamino substituents greatly increased the inhibitory activity. There was no significant difference between related tertiary and quaternary amines. The experimental antiarrhythmic agent UM 272 had the least ability to inhibit calcium binding to the cardiac plasma membrane preparation (K_i = 795 μM). However, UM 424, another experimental antiarrhythmic agent, had an inhibitory activity similar to dl-propranolol (k_i = 115 μM and 108 μM, respectively).     

Synthesis of flurbiprofen thiadiazole urea derivatives and assessment of biological activities and molecular docking studies

Totally 15 novel flurbiprofen urea derivatives were synthesized bearing the thiadiazole ring. Their inhibition effects on tyrosinase were determined. 3c was found to be the strongest inhibitor with the IC₅₀ value of 68.0 μM against tyrosinase. The enzyme inhibition types of the synthesized compounds were determined by examining the kinetic parameters. The inhibition type of 3c was determined as uncompetitive and the K_i value was calculated as 36.3 μM. Moreover, their cytotoxic effects on hepatocellular carcinoma (HepG2), colorectal carcinoma (HT-29), and melanoma (B16F10) cell lines were evaluated. According to the cytotoxicity results, 3l (IC₅₀ = 14.11 μM) showed the highest cytotoxicity on the HT-29 cells, while 3o (IC₅₀ = 4.22 μM) exhibited the strongest cytotoxic effect on HepG2 cell lines. Also, 3j (IC₅₀ = 7.55 μM strongly affected B16F10. The effects of synthesized compounds on the healthy cell line were evaluated on the CCD-986Sk cell line. Molecular modelling studies have indicated the potential binding interactions of the uncompetitive inhibitor 3c with the enzyme-substrate complex.     

Potent in vitro inhibitors of tyrosinase: Metal-binding fractions of tobacco smoke condensate

A fraction of tobacco smoke condensate (TSC) containing copper-binding agents was subjected to a classical functional group extraxtion, yielding basic, acidic, phenolic and neutral subfractions. Two of these fractions (the) acidic and the phenolic) contain potent in vitro inhibitors of mushroom tyrosinase. The mechanism of inhibition was studied in comparison with known copper-binding agents. The concentration of active inhibitors in the TSC fractions has been estimated using the Easson-Stedman method and is of the same order of magnitude as their titratable acidity. This procedure is therefore applicable in the determination of concentrations of enzyme inhibitors in unidentified mixtures of compounds. The TSC fractions exhibited noncompetitive inhibition with K_i values of 1.3 × 10^?7m (acidic fraction) and 9.3 × 10^?8m (phenolic fraction), similar in magnitude to K_i values found for the most potent of the inhibitors studied.     

Phosphodiesterase in the liver fluke, Fasciola hepatica.

Phosphodiesterase was found in homogenates of the liver fluke, Fasciola hepatica, and was distributed between a supernatant and particulate fraction after centrifugation at 2000 g. Mg²⁺ was necessary for enzyme activity; Ca²⁺ in the presence of Mg²⁺ did not affect enzyme activity. Enzyme kinetics followed the Michaelis-Menten model with a K_m of 8 μM for cAMP and 300 μM for cGMP as the substrate. The most potent inhibitor tested was 1-ethyl-4-(isopropylidenehydrazino)-1 H-pyrazolo- (3,4-b)-pyridine-5-carboxylic acid, ethyl ester, HC1 (SQ 20009) which had a K_i of 26 μM. The K_i for isobutyl methyl xanthine (IBMX) was 45 μM; for 6,7 dimethyl-4 ethylquinazoline (Quazodine) 75 μM; papaverine. 100 μM; theophylline, 550 μM; and for caffeine or D-lysergic acid diethylamide (LSD), 800 μM. The effects on fluke motility of these phosphodiesterase inhibitors were tested. All phosphodiesterase inhibitors except caffeine stimulated the rhythmical movement of the flukes. None of the inhibitors tested significantly increased the endogenous cAMP concentrations of fluke heads. IBMX potentiated the rise in endogenous cAMP caused by 5-hydroxytryptamine (5-HT) but SQ 20009, LSD, and papaverine prevented it. The latter results could not be explained on the basis of phosphodies-terase inhibition, but might be attributed to interference with the stimulation of adenylate cyclase by 5-HT.     

Transport and interaction of drugs in leukocytes

The accumulation of selected CNS drugs by rat leukocytes was previously reported. This paper presents evidence for the transport into leukocytes of additional drugs. Also studied was the inhibition of the latter processes by various structurally related compounds. The markedly rapid and sodium-independent uptakes into rat leukocytes of amphetamine, codeine, methadone and naloxone fulfilled the basic criteria for active transport. The uptake of morphine was apparently accomplished by more than one process. The affinities of the high capacity transport systems (approximate V_max: 100 nmoles/g cells/5sec) varied considerably as reflected by the two extreme K_m values obtained for methadone (20 μM) and morphine (1.8 mM). A variety of amines inhibited the cellular transport of the drugs. Most potent inhibitors were quinacrine (K_i: 0.5 to 3 μM), desipramine (K_i: 6–20 μM) and methadone (K_i: 18–25 μM). Morphine and tryptamine exhibited inhibition constants higher than 1 mM. The cellular transport processes newly described in rat leukocytes apparently represent a novel addition to the heterogenous biological transport of basic amines. The structural specificity of amine transport in various tissues is discussed.     

Inhibition of cyclic AMP phosphodiesterase by 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-[2-(N-benzyl-N-methylamino)] ethyl ester 5-methyl ester hydrochloride (YC-93), a potent vasodilator.

A new, potent vasodilator (YC-93), 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-[2-(N-benzyl-N-methyl amino)] ethyl ester 5-methyl ester hydrochloride, competitively inhibited cyclic adenosine 3′,5′-monophosphate (cyclic AMP) phosphodiesterase in the 105,000 g supernatant solutions from canine basilar, carotid, coronary and femoral arteries. The inhibition constant (K_i) of YC-93 for these enzyme preparations was in the range of 2.0–4.3 μM at substrate concentrations near the low K_m (about 1 μM for each enzyme preparation), and was 4.0–12 μM at substrate concentrations near the high K_m (50–70 μM). The potency of YC-93 for inhibition of coronary phosphodiesterase at 1 μM cyclic AMP and 50 μM cyclic AMP was much greater than that of papaverine and 3-isobutyl-l-methyl xanthine (IBMX). Commercially available cyclic AMP phosphodiesterase purified from beef heart was also strongly inhibited by YC-93 in a competitive manner and its K_i value was 2.0 μM in the wide range of substrate concentrations tested. Studies on the structure-activity relationship using low K_m phosphodiesterase from canine coronary artery and high K_m phosphodiesterase from beef heart, demonstrated that 3,5-diethoxycarbonyl-1,4-dihydro-2, 4,6-collidine, the simplest 1,4-dihydropyridine derivative (tested in the present studies) resulted in slightly less inhibition than papaverine, and the inhibitory potency of the former compound was greatly increased mainly by two structural modifications. Firstly, addition of a nitrophenyl group at position 4 of the 1,4-dihydropyridine ring, secondly, the replacement of ethylester at position 3 of the 1,4-dihydro-pyridine ring by N-benzyl-N-methylaminoethyl ester. A few dihydropyridine derivatives together with YC-93 were the most potent inhibitors of cyclic AMP phosphodiesterase among the compounds tested. The finding that the level of cyclic AMP in canine arterial strips was increased by 64 per cent (P < 0.01) even after 1 min exposure to 1 μM YC-93 supports the possibility of at least a partial involvement of phosphodiesterase inhibition in vasodilation by the drug.     

Stereospecificity at carbon 6 of fomyltetrahydrofolate as a competitive inhibitor of transport and cytotoxicity of methotrexate in vitro

The unnatural diastereoisomer of l-5-formyltetrahydrofolate was 20-fold less effective as a competitive inhibitor of [³H] methotrexate influx than the natural diastereoisomer during carrier-mediated membrane transport in L1210, S180 and Ehrlich cells. Values derived for K_i, were 1.84 to 2.29 μM for the natural derivative and 35.2 to 53.8 μM for the unnatural derivative. Values for K_i derived with a chemically synthesized mixture containing equal amounts of both natural and unnatural diastereoisomers were 2-fold greater than values obtained for the natural diastereoisomer. The unnatural diastereoisomer was 100-fold less effective and the chemically synthesized mixture was 2-fold less effective than the natural diastereoisomer in preventing inhibition by methotrexate of L1210 cell growth in culture. These results indicate that the unnatural diastereoisomer competes relatively ineffectively with the natural diastereoisomer or methotrexate for transport in these murine tumor cells.     

Inhibition of gastric K+ ATPase by phenylbutazone and indomethacin.

Inhibition of gastric H⁺ secretion by phenylbutazone and indomethacin was investigated by examining the effects of these agents on a putative H⁺ transport enzyme, a K⁺ stimulated ATPase. unique to gastric mucosa. Phenylbutazone and indomethacin were found to inhibit both the K⁺ ATPase and the K⁺ pNPPase. K_is were 430 μM and 710 μM for the K⁺ ATPase and 330 μM and 670 μM for the K⁺ pNPPase for phenylbutazone and indomethacin respectively. Inhibition was not reversed by Mg²⁺, ATP, pNPP, or KCl and obeyed non-competitive kinetics. Inhibition of the pNPPase suggested that the mechanism of inhibition involved the K⁺ sensitive dephosphorylation of the phosphoenzyme. In the presence of 500 μM phenylbutazone dephosphorylation was significantly less at 3, 5, 7.5, 10 and 15 sec following KCl addition. These studies provide an alternate mechanism for inhibition of gastric H⁺ secretion by phenylbutazone and indomethacin.     

Studies on the reaction catalyzed by transport (Na, K) adenosine triphosphatase. I. Effects of divalent metals

The effects of divalent metals (Cu²⁺, Zn²⁺, Fe²⁺ and Pb²⁺) on a microsomal preparation of NaK-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from beef cerebral cortex were studied. These metals are all potent inhibitors of the enzyme with I₅₀ values of 1 μM for Cu²⁺ and Zn²⁺, 3 μM for Fe²⁺ and 20 μM for Pb²⁺. Kinetic studies examining the effect of low concentrations of divalent metals on K_m and V for MgATP are reported. The results indicate that Fe²⁺ and Pb²⁺ are competitive inhibitors of NaK-ATPase with K_i values of 1.60 μM and 1.90 μM respectively. Cu²⁺ and Zn²⁺ are noncompetitive inhibitors of NaK-ATPase with K_i values of 1.18 μM and 3.48 μM respectively.     

Aspartate carbamoyltrasferase activity,drug concentrations,and pyrimidine nucleotides in tissue from patients treated with N-(phosphonacetyl)-L-aspartate

Biopsy specimens were obtained from patients treated with $\text{N-(}\text{phosphonacetyl)-}\text{L}\text{-aspartate}$ (PALA) in a phase I clinical trial. Activities of aspartate carbamoyltransferase (ACTase), the target enzyme, in ten specimens before treatment varied from 0.4 to 1.7 units/mg. PALA was measured in protein-free extracts of thirteen specimens by inhibition of rat ACTase. At 1.5 to 145 hr after doses of 1 to 6 g/m², PALA concentrations were 0.9 to 89 μg/g; at 4 hr or later the tissue concentrations were similar to those in plasma (five samples). The observed inhibition of ACTase (17–87%) correlated with the PALA concentrations. Pyrimidine nucleotides were decreased (relative to purine nucleotides) in nine of ten specimens, by 16–72%. ACTase partially purified from human spleen had a K_m for carbamoyl phosphate of 20.6 μM and the K_i for PALA was 0.011 μM. The results suggest that inhibition of ACTase by PALA affects the concentration of pyrimidine nucleotides in human tumors in a dose-dependent manner.     

Identification of efflux systems for large anions and anionic conjugates as the mediators of methotrexate efflux in L1210 cells

Two ATP-dependent efflux systems for methotrexate have been identified in inside-out vesicles from an L1210 mouse cell variant with a defective influx carrier for methotrexate. Transport at 40 μM [³H]methotrexate was separated by inhibitors into two components comprising 62 and 38% of total transport activity. The predominant route was inhibited by low concentrations of indoprofen (K_i = 2.5 μM), 4-biphenylacetic acid (K_i = 5.3 μM), and flurbiprofen (K_i = 5.2 μM), whereas the second component showed a high sensitivity to the glutathione conjugates of bromosulfophthalein (K_i = 0.08 μM), ethacrynic acid (K_i = 0.52 μM), and 1-chloro-2,4-dinitrobenzene (K_i = 0.77 μM). Bilirubin ditaurate was a potent inhibitor of both transport components (K_i = 1.5 and 0.17 μM, respectively). Separation of transport activities without interference from the other route was achieved by adding an excess (100 μM) of either the glutathione conjugate of ethacrynic acid or biphenylacetic acid. Double-reciprocal plots of transport at various substrate concentrations gave K_m values of 170 and 250 μM for methotrexate transport via the anion-sensitive and conjugate-sensitive routes, respectively. A comparison of inhibitor specificities indicated that the anion-sensitive transport activity in vesicles represents efflux system II for methotrexate in intact cells and is the same system identified previously in vesicles as an anion/anion conjugate pump. The conjugate-sensitive activity corresponds to efflux system I for methotrexate in intact cells and is the same system identified in vesicles as the high-affinity glutathione conjugate pump.     

In vitro and in vivo effects of alpha-acetylenic DOPA and alpha-vinyl DOPA on aromatic L-amino acid decarboxylase.

In vitro α-acetylenic DOPA (RMI 71.858) is a potent inhibitor of aromatic l-amino acid decar?ylase (AADC). Inhibition appears to consist of both a competitive, with a K_i of 0.3 μM, and an irreversible component, the enzyme losing 50 per cent of its activity in 20 min at an inhibitor concentration of 100 μM. After inhibition, the activity can only be partially restored by dialysis. α-Vinyl DOPA (RMI 71.816) is a less potent inhibitor of the enzyme, with a K_i of 39 μM. No transformation of the inhibitors by AADC can be detected in the incubation medium. Ex viro, both compounds (100–500 mg/kg, i.p. (reduce the activity of AADC in different organs, with a more pronounced effect in peripheral tissues than in brain. In vivo, α-acetylenic DOPA (10–500 mg/kg, i.p.) inhibits the peripheral decar?ylation of [³H]l-DOPA and 5-hydroxytryptophan with a consequent short-lasting elevation of brain catecholamines and serotonin.     

Cytochrome P‐450 Enzymes Contributing to Demethylation of Maprotiline in Man

Abstract: From case reports of patients treated with the tetracyclic antidepressant drug maprotiline, it appears that this drug is subject to polymorphic metabolism. Thus, we studied formation of the major maprotiline metabolite desmethylmaprotiline to identify the human cytochrome P‐450 enzymes (CYP) involved. In incubations with human liver microsomes from two different donors, the substrate maprotiline was used at five different concentrations (5 to 500 μM). For selective inhibition of CYPs, quinidine (0.5–50 μM; CYP2D6), furafylline (0.3–30 μM; CYP1A2), ketoconazole (0.2–20 μM; CYP3A4), mephenytoin (20–200 μM; CYP2C19), chlorzoxazone (1–100 μM; CYP2E1), sulphaphenazole (0.2–100 μM; CYP2C9) and coumarin (0.2–100 μM; CYP2A6) were used. Desmethylmaprotiline concentrations were measured by HPLC, and enzyme kinetic parameters were estimated using extended Michaelis‐Menten equations with non‐linear regression. Relevant inhibition of the desmethylmaprotiline formation rate was observed in incubations with quinidine, furafylline and ketoconazole only. Formation rates of desmethylmaprotiline were consistent with a two enzyme model with a high (K_M=71 and 84 μM) and a low (K_M=531 and 426 μM) affinity site for maprotiline in the two samples, respectively. The high affinity site was competitively inhibited by quinidine (K_i,_c 0.13 and 0.61 μM), the low‐affinity site was non‐competitively inhibited by furafylline (K_i,_nc 0.11 and 1.3 μM). Thus it appears that CYP2D6 and CYP1A2 contribute to maprotiline demethylation. Based on the parameters obtained, for plasma concentrations of 1 μM 83% (mean) of desmethylmaprotiline formation in vivo is expected to be mediated by CYP2D6 while 17% only may be attributed to CYP1A2 activity.     

Inhibition of pseudorenin by pepstatin

The pentapeptide pepstatin was found to inhibit the ability of rat spleen pseudorenin to form angiotensin I from tetradecapeptide renin substrate. Dixon and Webb plots showed that this inhibition was noncompetitive in nature. Lineweaver-Burk analysis also showed noncompetitive inhibition. K_i values determined by the three graphical methods ranged from 1.8 to 3.8 × 10^?10M. The K_m for pseudorenin was determined to be between 0.82 and 1.23 μM. The concentration of enzyme used was estimated to be 3.1 × 10^?10M. Pepstatin should prove useful in the future for the analysis and purification of pseudorenin.     

Biochemical pharmacology of acivicin in rat hepatoma cells

The antiglutamine agent acivicin, l-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, inhibited the growth of hepatoma 3924A cells in culture. After 7 days of incubation with the drug, an lc₅₀ of 1.4 μM was observed by determination of colony forming ability. A combination of cytidine (1 mM), deoxycytidine (10 μM) and guanosine (10 μM) completely protected the hepatoma cells against the cytotoxic action of acivicin, but each nucleoside by itself had no effect. Acivicin (0.1 mM) inhibited the incorporation of uridine and thymidine into macromolecules, but not that of leucine. Acivicin depressed the pools of CTP, GTP, dCTP, dGTP and dTTP to 46, 62, 40, 64 and 53%, respectively, but it increased UTP level to 152% of the values of untreated cancer cells. The activity of a highly purified CTP synthetase (EC 6.3.4.2) from rat liver and hepatoma 3924A was inhibited by acivicin. The inhibition was competitive with respect to l-glutamine, and the K_i values with liver and hepatoma enzymes, determined by Dixon and reciprocal plots, were 1.1 and 3.6 μM respectively. The hydroxy analog of acivicin was also a competitive inhibitor, but it was less effective than acivicin, with a K_i value of 1.8 mM for the hepatoma enzyme. Our observations on the impact of acivicin on the behavior of pools of ribonucleotides and deoxyribonucleotides and the competitive inhibition of purified CTP synthetase from hepatoma cells suggest that a major mechanism of action for this drug is the inhibition of CTP synthetase and GMP synthetase (EC 6.3.5.2).     

Kinetic studies with 5-azacytidine-5'-triphosphate and DNA-dependent RNA polymerase.

In order to further understand the biochemical mode of action of 5-azacytidine, a potent antileukemic agent, kinetic studies were performed with 5-azacytidine-5'-triphosphate (5-aza-CTP) and purified DNA-dependent RNA polymerase from Escherichia coli and calf thymus. RNA polymerase could catalyze the incorporation of the fradulent nucleotide, 5-aza-CTP, into RNA. The apparent K_m value for 5-aza-CTP was estimated to be 350 and 390 for the E. coli and calf thymus enzymes respectively. The K_m value for 5-aza-CTP was about 18-fold greater than the K_m value for CTP (20 μM). The apparent V_max value for CTP was about 2-fold greater than the V_max value for 5-aza-CTP. 5-Aza-CTP was a weak competitive inhibitor with respect to CTP; the apparent K_i value for 5-aza-CTP was estimated to be 680 and 810 μM for the E. coli and calf thymus enzymes respectively. On the other hand, CTP was a potent competitive inhibitor with respect to 5-aza-CTP; the apparent K_i value of CTP was estimated to be 16 μM. 5-Aza-CTP did not appear to inhibit the incorporation of UTP into RNA in the reaction catalyzed by RNA polymerase. These data suggest that the inhibition of RNA synthesis in cells by 5-aza-cytidine is not produced by the inhibition of RNA polymerase by 5-aza-CTP.     

Anti-convulsants and brain aldehyde metabolism: Inhibitory characteristics of ox brain aldehyde reductase

The major isoenzyme of aldehyde reductase has been purified from ox brain by affinity chromatography. Carbamazepine (K_i = 7.3 × 10^?4 M) and phenacemide (K_i = 2.5 × 10^?4 M), in common with all other established anti-convulsant drugs tested, have been shown to inhibit the activity of this enzyme. A selection of structural analogues of the anti-convulsant sodium valproate were found to be potent inhibitors of the reductase (K_ivalues in the range 10^?3 M ?5 × 10^?5 M) and these analogues also showed anti-convulsant activity in the mouse maximal electroshock test. A third group of compounds, the flavonoids, constitute the most potent group of aldehyde reductase inhibitors yet reported. Quercetin and morin exhibited K_i values less than 1 μM. The possible relationship between aldehyde metabolism and anti-convulsant action is discussed and structural characteristics pre-disposing to potent inhibition of aldehyde reductase are described.     

Induction and inhibition of hepatic drug metabolizing enzymes by rifampin.

The effects of the antibiotic rifampin on microsomal drug metabolizing enzymes were investigated. On acute administration, rifampin (100 mg/kg i.p.) doubled hexobarbital sleeping time and zoxazolamine paralysis time in mice. The in vitro metabolism of zoxazolamine (0.25 mM) and 17β-estradiol (10 μM) were inhibited 50% by rifampin in concentrations of 0.4 and 0.3 mM, respectively. The inhibition of ethylmorphine N-demethylase was competitive with an apparent K_i, of 52 μM. Repeated administration of rifampin to mice (50 mg/kg i.p. daily for 6 days) increased liver weight by 20%a cytochrome P-450 (50%), NADPH cytochrome c reductase (43%,). ethylmorphine N-demethylase (85%), zoxazolamine hydroxylase (77%), benzpyrene hydroxylase (174%). and 17β-estradiol metabolism (89%). Microsomal protein (mg/g). aniline hydroxylase and p-nitrophenol glucuronyl transferase activities were unaffected.Rat microsomal drug metabolizing enzyme activity was also inhibited after acute administration of rifampin as exemplified by an increase in hexobarbital sleeping time of 44% and a competitive inhibition of ethylmorphine N-demethylase by rifampin. The K_i, (33 μM) was close to that obtained with the mouse enzyme. The similarity in the in vivo and in vitro inhibition suggests that rifampin binds to microsomes in a similar manner in both species. Chronic administration of rifampin to rats (50 mg/kg i.p.) twice daily for six days did not lead to induction, indicating a species difference with respect to rifampin's inducing ability. Rifampin did not modify microsomal induction in rats by phenobarbital when both drugs were administered concomitantly. The mechanism responsible for the species difference and the clinical relevance of these results are discussed.