Determination of l-α-acetylmethadol, l-α-noracetylmethadol and l-α-dinoracetylmethadol in plasma by gas chromatography—mass spectrometry

A method is described for the simultaneous determination of l-α-acetylmethadol (LAAM) and its N-demethylated metabolites, l-α-noracetylmethadol (norLAAM) and l-α-dinoracetylmethadol (dinorLAAM), in plasma by gas chromatography—chemical ionization mass spectrometry. Deuterated internal standards for each analyte serve as carriers and control for recovery during sample purification on a solid-phase extraction column (C₁₈), and subsequent separation and analysis on a DB-17 capillary column. With this method, we have determined levels of LAAM, norLAAM, and dinorLAAM in small volumes of plasma (100 μl). The limit of quantitation for all analytes was approximately 1.0 ng/g plasma and the limit of detection was approximately 0.5 ng/g plasma. An experimental application is also described where these analytes are quantitated in plasma obtained from rats before, during, and after chronic administration of LAAM-HCl. Since this technique affords a selective and sensitive means of detection of LAAM and its active, N-demethylated metabolites in small samples of blood, it may enable patient compliance to be more easily assessed by allowing samples to be collected by a simple finger-prick technique.     

Stereoselective metabolism of methadone N-demethylation by cytochrome P4502B6 and 2C19

Methadone is a clinically used opioid agonist that is oxidatively metabolized by cytochrome P450 (CYP) isoforms to a stable metabolite, EDDP. Methadone is a chiral drug administered as the racemic mixture of (R)-(-)- and (S)-(+)-methadone, but (R)-methadone is the active isomer. The cytochrome P450 (CYP) isoform involved in methadone's metabolism is thought to be CYP3A4, but human drug-drug interaction studies are not consistent with this. The ability of the common human drug-metabolizing CYPs (obtained from baculovirus-infected insect cell supersomes) to generate 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrilidine (EDDP) from racemic methadone was examined and then determined if the CYP isoforms metabolized methadone stereoselectively. Only CYP2B6, 2C19, and 3A4 generated measurable EDDP from 1 microg/ml of racemic methadone. The hierarchy of EDDP generation was CYP2B6 > CYP2C19 >/= CYP3A4. At 10 microg/ml of methadone, CYP2C9 and CYP2D6 also generated EDDP, but in at least 10-fold lower quantities than CYP2B6. Michaelis-Menten kinetic data demonstrated that CYP2B6 had the highest V(max) (44 ng/min/10pmol) and the lowest K(m) (12.6 microg/ml) for EDDP formation of all the CYP isoforms. In human liver microsomes with high and low CYP2B6 expression but equivalent CYP3A4 expression, high CYP2B6 expression microsomes generated twice the amount of EDDP from 10 microg/ml of methadone than low CYP2B6 expression microsomes. When stereoselective metabolism of racemic methadone by CYP2B6, 2C19, and 3A4 was examined using an enantiospecific methadone assay, CYP2B6 preferentially metabolized (S)-methadone, CYP2C19 preferentially metabolized (R)-methadone, and CYP3A4 showed no preference. These data suggest that multiple CYPs metabolized methadone but CYP2B6 had the highest V(max)/K(m). In addition, only CYP2B6 and 2C19 showed stereoselective metabolism. Our data could explain why the plasma concentration ratio of R/S methadone is variable and why drugs that induce CYP2B6 such as nevirapine and efavirenz also induce methadone metabolism, while the CYP3A4 inducer rifabutin has no effect on methadone pharmacokinetics.     

Preferential induction of the rat hepatic P450 I proteins by the food carcinogen 2-amino-3-methyl-imidazo[4,5-f]quinoline

1. Administration of the food carcinogen, 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) to rats gave rise to significant dose-dependent increases in the microsomal O-deethylations of ethoxycoumarin and ethoxyresorufin but had no effect on the O-dealkylation of pentoxyresorufin and the NADPH-dependent reduction of cytochrome c, and decreased the N-demethylation of dimethylnitrosamine. Microsomal cytochrome b5 and total cytochrome P-450 levels decreased following the administration of the carcinogen. 2. Hepatic microsomal preparations from IQ-treated animals were much more efficient than control in activating the premutagen 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole to mutagenic intermediates in the Ames test. 3. Immunoquantification of two of the major families of cytochrome P-450, namely P450 I and P450 II B, using ELISA techniques showed that treatment with IQ induced the apoprotein levels of the P450 I family but not of P450 II B. 4. Immunoblot analysis employing polyclonal antibodies against P450 I revealed that IQ induced both isoenzymes of this family, namely P450 I A1 and A2. 5. It is concluded that IQ is an inducer of the rat hepatic monooxygenases, selectively inducing the P450 I family as predicted by a computer-graphic analysis of its dimensions which showed that it is a large, essentially planar, molecule.     

Catalyzation of cocaine N-demethylation by cytochromes P4502B,P4503A,and P4502D in fish liver

Cocaine N-demethylation by microsomal cytochrome P450s is the principal pathway in cocaine bioactivation and hepatotoxicity. P450 isozymes involved in N-demethylation of cocaine have not been elucidated yet and they differ from species to species. In humans and mice, P4503A contributes to cocaine N-demethylase activity, whereas in rats, both P4503A and P4502B participate. In the present study, contribution of different P450 isozymes to cocaine N-demethylase activity was studied in vitro with fish liver microsomes. The specific cocaine N-demethylase activity was found to be 0.672 +/- 0.22 nmol formaldehyde formed/min/mg protein (mean +/- SD, n = 6). Cocaine N-demethylase exhibited biphasic kinetics, and from the Lineweaver-Burk plot, two K(m) values were calculated as 0.085 and 0.205 mM for the high- and low-affinity enzyme. These results indicate that N-demethylation of cocaine in mullet liver microsomes is catalyzed by at least two cytochrome P450 isozymes. Inhibitory effects of cytochrome P450 isozyme-selective chemical inhibitors, ketoconazole, cimetidine, SKF-525A, and quinidine, on cocaine N-demethylase activity were studied at 50, 100, and 500 micro M concentrations of these inhibitors. At 100 micro M final concentrations, ketoconazole (P4503A inhibitor), SKF-525A (inhibitor of both P4502B and P4503A), and cimetidine (P4503A inhibitor) inhibited N-demethylation activity by 73, 69, and 63%, respectively. Quinidine, P4502D-specific inhibitor, at 100 micro M final concentration, reduced N-demethylation activity down to 64%. Aniline, a model substrate for P4502E1, did not alter N-demethylase activity in the final concentration of 100 micro M. IC(50) values were calculated to be 20 micro M for ketoconazole, 48 micro M for cimetidine (both specific P4503A inhibitors), 164 micro M for quinidine (P4502D inhibitor), and 59 micro M for SKF-525A (inhibitor of both P4503A and P4502B). The contribution of P4502B to cocaine N-demethylase activity in mullet liver microsomes was further explored by the use of purified mullet cytochrome P4502B in the reconstituted system containing purified mullet P450 reductase and lipid. The turnover number was calculated as 4.2 nmol HCOH/(min nmol P450). Overall, these results show that P4503A and P4502B are the major P450s responsible for N-demethylation of cocaine, whereas contribution of P4502D is a minor one, and P4502E1 is not involved in the N-demethylation of cocaine in mullet liver microsomes.     

Isolation and characterization of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats

Chromatography on 1.8-diaminooctyl-Sepharose and DEAE-Sephacel resulted in 4 fractions of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats. All the four fractions differed in terms of their absorption maxima in the CO-reduced state, Mr and catalytic activity. Only one cytochrome fraction (cytochrome P-450 C) possessed a high activity upon benz(a)pyrene hydroxylation. All cytochrome P-450 forms were characterized by a low rate of aminopyrine N-demethylation. Antibodies against cytochrome P-450 C (P-448) (anti-P-448) were raised. Cytochromes of fractions A, B1 and B2 in the Ouchterlony reaction of double immunodiffusion did not give precipitation bands with anti-P-448. Neither of the four cytochrome P-450 forms interacted with the antibodies raised against cytochrome P-450 isolated from liver microsomes of rats induced with phenobarbital. The procedure developed is applicable to the isolation of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced rats. Using rocket immunoelectrophoresis, cytochrome P-450 C possessing a high (as compared to benz(a)pyrene metabolism) activity (18 nmol/min/nmol cytochrome) and a high (60-70%) content in 3-methylcholanthrene-induced rat liver microsomes was shown to give a relatively high yield.     

Identification of human liver cytochromes P450 by using MALDI-TOF mass spectrometry

Proteomic approaches have been used for detection and identification of cytochromes P450 forms from highly purified membrane preparations of human liver. These included the protein separation by 2D-and/or 1D-electrophoresis and molecular scanning of a SDS-PAGE gel fragment in a range 45–66 kDa (this area corresponds molecular weights of cytochromes P450). The analysis of protein content was statistically evaluated by means of an original 1D-ZOOMER software package which allowed to carry out the processing of mass spectra mixture instead of individual mass spectra used by standard techniques. In the range 45–66 kDa we identified 13 microsomal membrane proteins including such cytochrome P450 forms as CYPs 1A2, 1B1, 2A6, 2E1, 2C8, 2C9, 2C10, 2D6, 3A4, 4A11, 4F2. Study of enzymatic activities of human liver microsomal cytochrome P450 isoforms CYP 1A, 2B, 3A, and 2E revealed the decrease in the rates of O-dealkylation and N-demethylation catalyzed by CYP 450 1A1/1A2 and 3A4 under pathological conditions, whereas 7-benzyloxyresorufin-O-debenzylase activity (which characterizes the total activity of CYP 2B and CYP 2C), the activities of CYP 2E1 (methanol oxidation), 7-pentoxyresorufin-O-dealkylation (CYP 2B), 7-ethoxy-and 7-methoxycoumarin-O-dealkylases (CYP 2B1) remained basically unchanged.     

A liquid chromatographic-electrospray ionization-tandem mass spectrometric method for determination of buprenorphine,its metabolite,norbuprenorphine, and a coformulant,naloxone, that is suitable for in vivo and in vitro metabolism studies

A liquid chromatographic-electrospray ionization-tandem mass spectrometric method has been developed and validated for determination of the antiabuse medication, buprenorphine, its primary metabolite, norbuprenorphine, and a proposed coformulant, naloxone. The method uses deuterated internal standards and a simple liquid-liquid extraction. Mass spectrometry employed selected reaction monitoring of the transitions of m/z 468 to 396 for buprenorphine, 472 to 400 for [2H4]buprenorphine, 414 to 101 for norbuprenorphine, 423 to 110 for [2H9]norbuprenorphine, 328 to 310 for naloxone, and 345 to 327 for its internal standard, [2H3]naltrexone. The method was accurate and precise across the dynamic range of 0.1 to 10 ng/ml. All analytes were stable in human plasma stored at room temperature for up to 24 h and after three freeze-thaw cycles. Reconstituted extracts were stable at -20 degrees C for up to 3 days. In human subjects receiving a sublingual tablet of 8 mg buprenorphine and 2 mg naloxone, buprenorphine and norbuprenorphine were detected for up to 24 h with respective maximum concentrations at 1 and 1.5 h. Maximal concentrations ranged from 2.2 to 2.8 and 1.5 to 2.4 ng/ml for buprenorphine and norbuprenorphine, respectively (i.e., approximately 6 nM). The method detected norbuprenorphine formation in human liver microsomes incubated with 5-82 nM buprenorphine, which encompasses the therapeutic plasma concentration range. When cDNA-expressed P450s were incubated with 21 nM buprenorphine, norbuprenorphine formation was detected for P450s 3A4, as previously described, but also for 3A5, 3A7, and 2C8. Buprenorphine utilization generally exceeded norbuprenorphine formation, suggesting that P450s 2C18, 2C19, 2D6, and 2E1 may also be involved in buprenorphine metabolism to other products. These results suggest this method is suitable for both in vivo and in vitro studies of buprenorphine metabolism to norbuprenorphine.     

Comparison of substrate metabolism by cytochromes P450 2B1, 2B4, and 2B6: relationship of heme spin state,catalysis, and the effects of cytochrome b5

The metabolism of selected substrates by cytochromes P450 (P450) 2B1, 2B4, and 2B6 was compared, and the effects of cytochrome b(5) (b(5)) on these reactions were assessed. There did not appear to be any trends regarding the effects of b(5) when the metabolism of a given substrate by the different P450 enzymes was compared. The changes in spin states of the P450 enzymes as a result of interactions with substrates and cytochrome b(5) were also determined. Only P450 2B4 demonstrated a relationship between spin state, reaction coupling and b(5) effects. The rates of benzphetamine and 7-ethoxy-4-trifluoromethylcoumarin metabolism by the three enzymes could be correlated with the proportions of high spin heme. Similarly, the proportion of reaction coupling during the metabolism of selected substrates was approximately equal to the proportion of high spin P450. The data are interpreted to indicate that a P450 conformational equilibrium coordinately regulates catalysis and spin state changes.     

Dose-dependent, mechanism-based inactivation of cytochrome P450 monooxygenases in vivo by 1-aminobenzotriazole in liver, lung, and kidney of untreated, phenobarbital-treated, and beta-naphthoflavone-treated guinea pigs.

The mechanism-based inactivation of the cytochrome P450 (P450) dependent monooxygenase system was studied in vivo in liver, lung, and kidney of untreated, phenobarbital-treated, and beta-naphthoflavone-treated guinea pigs 24 h after administration of 1-aminobenzotriazole (1-100 mg/kg, i.p.). Microsomal isozyme-selective or -specific monooxygenase activities were inhibited in a dose-dependent manner in all three organs. In the liver of untreated and phenobarbital-treated animals, 7-pentoxyresorufin O-depentylation (catalyzed primarily by P450 2Bx, an orthologue of rabbit P450 2B4/rat 2B1) was inhibited more than 7-ethoxyresorufin O-deethylation (P450 1A1), 4-aminobiphenyl N-hydroxylation (P450 1A2), erythromycin N-demethylation (P450 3A), or benzphetamine N-demethylation; in beta-naphthoflavone-treated animals, 4-amino-biphenyl N-hydroxylation activity was preferentially inhibited. In lung, the order of inactivation of monooxygenase activities was 4-aminobiphenyl N-hydroxylation (4Bx, the orthologue of rabbit 4B1) > 7-pentoxyresorufin O-depentylation activity (2Bx) > 7-ethoxyresorufin O-deethylation (1A1; for example 72, 53, and 29% inactivation, respectively, in phenobarbital-treated animals at 100 mg/kg). In all three tissues the loss in spectrally assayed P450 content corresponds quite well to the inhibition of monooxygenase activities. Thus, these studies show that 1-aminobenzotriazole is an effective inactivator of the pulmonary, hepatic, and renal P450 systems in guinea pigs following i.p. administration, and that P450 1A2 (liver) and P450 4Bx (lung), isozymes efficient for the oxidation of primary aromatic amines, are preferentially inactivated.     

N-terminal truncated cytochrome P450 2B4: catalytic activities and reduction with alternative electron sources.

It was shown that riboflavin binds to the truncated cytochrome P450 2B4 and forms a complex with the K(d) = 26 microM. Noncovalent complex of truncated (Delta2-27) cytochrome P450 2B4 with riboflavin was essential for electron transfer realization and catalyzed the NADH-dependent and hydrogen peroxide-supported monooxygenase reactions of aminopyrine N-demethylation and aniline p-hydroxylation. Flavocytochrome molecular maquette was capable of supporting photoactivatable electron transfer and could be photoreduced and electroreduced quantitatively in the absence of pyridine nucleotides.     

The cytochrome P450 2B4-NADPH cytochrome P450 reductase electron transfer complex is not formed by charge-pairing.

Attempts to covalently link NADPH-cytochrome P450 reductase to cytochrome P450 2B4 using a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylisopropyl)carbodiimide, were unsuccessful, despite the fact that under the same conditions about 30% of P450 2B4 could be covalently linked with cytochrome b5 in a functionally active complex (Tamburini, P. P., and Schenkman, J. B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 11-15). This suggested that the functional electron transfer complex between P450 2B4 and reductase is not stabilized by electrostatic forces. Raising the ionic strength of the medium is disruptive to salt bridges and was used to further test whether P450 2B4 and the reductase form charge-pairing complexes. Instead of inhibiting electron transfer, high ionic strength increased the apparent fast phase rate constant and the fraction of P450 2B4 reduced in the fast phase. The possibility that electron transfer between NADPH-cytochrome P450 reductase and P450 2B4 is diminished by charge repulsion was examined. Consistent with this hypothesis, the Km of P450 2B4 for reductase was decreased 26-fold by increasing the ionic strength from 10 to 100 mM sodium phosphate without affecting the Vmax. The rate of benzphetamine N-demethylation also was increased by elevation of the ionic strength. Electron transfer from the reductase to other charged redox acceptors, e.g. cytochrome c and ferricyanide, was also stimulated by increased ionic strength. However, no similar stimulation was observed with the uncharged acceptor 1,4-benzoquinone. Polylysine, a polypeptide that binds to anionic sites, enhanced electron transfer from NADPH to ferricyanide and the apparent fast phase of reduction of cytochrome P450. The results are consistent with the hypothesis that charges on NADPH-cytochrome P450 reductase and cytochrome P450 decrease the stability of the electron transfer complex.     

Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4

The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b(5) (cyt b(5)) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b(5) molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 +/- 2 and 29 +/- 4.5 s(-1), respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of benzphetamine congruent with100-fold (k = 0.15 +/- 0.05 s(-1)) and cyclohexane congruent with10-fold (k = 2.5 +/- 0.35 s(-1)) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b(5), and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b(5) and CPR. Increasing the concentration of cyt b(5) enhanced the amount of product formed by cyt b(5) while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b(5):cyt P450 molar ratios examined, cyt b(5) inhibits the rate of NADPH consumption. Nevertheless, at low cyt b(5):cyt P450 molar ratios 相似文献   

Isosafrole-induced cytochrome P2-450 in DBA/2N mouse liver. Characterization and genetic control of induction

Mouse "cytochrome P2-450" is defined as that form of isosafrole-induced P-450 in DBA/2N liver most specifically correlated with isosafrole metabolism. Isosafrole pretreatment does not induce aryl hydrocarbon hydroxylase activity ("cytochrome P1-450") in C57BL/6N or DBA/2N mice, induces acetanilide 4-hydroxylase activity ("cytochrome P3-450") more than 3-fold in C57BL/6N but not in DBA/2N mice, and induces isosafrole metabolite formation more than 3-fold in both C57BL/6N and DBA/2N mice. P2-450 was, therefore, purified from isosafrole-treated DBA/2N liver microsomes having negligible amounts of contaminating P1-450 and P3-450. The apparent molecular weight of P2-450 is 55,000, and the protein appears homogeneous on sodium dodecyl sulfate-polyacrylamide gels. The Soret peak of the reduced purified cytochrome X CO complex is 448 nm. Purified P2-450, reconstituted in vitro, metabolizes acetanilide poorly and benzo[a]pyrene hardly at all. Anti-(P2-450) inhibits (90 to 100%) liver microsomal isosafrole metabolite formation, yet has no effect on aryl hydrocarbon hydroxylase, acetanilide 4-hydroxylase, biphenyl 2- or 4-hydroxylase, or 7-ethoxycoumarin O-de-ethylase activities. 3-Methylcholanthrene induces anti-(P2-450)-precipitable protein about 12-fold in C57BL/6N and 2-fold in DBA/2N liver; 2,3,7,8-tetrachlorodibenzo-p-dioxin (10 micrograms/kg), about 12-fold in both C57BL/6N and DBA/2N liver; isosafrole, more than 3-fold in both C57BL/6N and DBA/2N. Benzo[a]anthracene at maximal doses induces anti-(P2-450)-precipitable protein in C57BL/6N liver no more than 2-fold, yet is known to be a highly potent inducer of P1-450 mRNA in C57BL/6N liver. The sensitivity of the P2-450 induction process to isosafrole is inherited as an autosomal additive trait; studies of offspring from the C57BL/6N(DBA/N)F1 X DBA/2N backcross confirm involvement of the Ah locus or s closely segregating gene. In contrast, among crosses between C57BL/6N and DBA/2N, sensitivity of the P1-450 and P3-450 induction process to 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin is inherited as an autosomal dominant trait. These data suggest that, although P1-450, P2-450, and P3-450 proteins are controlled by the Ah locus, either a P-450 protein polymorphism exists between C57BL/6N and DBA/2N mice or subtle differences may exist in the interaction of various inducers with Ah receptor.     

Functional Interactions between Cytochromes P450 1A2 and 2B4 Require Both Enzymes to Reside in the Same Phospholipid Vesicle: EVIDENCE FOR PHYSICAL COMPLEX FORMATION*

Previous studies have shown that the combined presence of two cytochrome P450 enzymes (P450s) can affect the function of both enzymes, results that are consistent with the formation of heteromeric P450·P450 complexes. The goal of this study was to provide direct evidence for a physical interaction between P450 1A2 (CYP1A2) and P450 2B4 (CYP2B4), by determining if the interactions required both enzymes to reside in the same lipid vesicles. When NADPH-cytochrome P450 reductase (CPR) and a single P450 were incorporated into separate vesicles, extremely slow reduction rates were observed, demonstrating that the enzymes were anchored in the vesicles. Next, several reconstituted systems were prepared: 1) CPR·CYP1A2, 2) CPR·CYP2B4, 3) a mixture of CPR·CYP1A2 vesicles with CPR·CYP2B4 vesicles, and 4) CPR·CYP1A2·CYP2B4 in the same vesicles (ternary system). When in the ternary system, CYP2B4-mediated metabolism was significantly inhibited, and CYP1A2 activities were stimulated by the presence of the alternate P450. In contrast, P450s in separate vesicles were unable to interact. These data demonstrate that P450s must be in the same vesicles to alter metabolism. Additional evidence for a physical interaction among CPR, CYP1A2, and CYP2B4 was provided by cross-linking with bis(sulfosuccinimidyl) suberate. The results showed that after cross-linking, antibody to CYP1A2 was able to co-immunoprecipitate CYP2B4 but only when both proteins were in the same phospholipid vesicles. These results clearly demonstrate that the alterations in P450 function require both P450s to be present in the same vesicles and support a mechanism whereby P450s form a physical complex in the membrane.     

Diclofenac and its derivatives as tools for studying human cytochromes P450 active sites: particular efficiency and regioselectivity of P450 2Cs

A comparison of the oxidations of diclofenac with microsomes of yeasts expressing various human liver cytochromes P450 showed that P450 2C9 regioselectively led to 4'-hydroxy diclofenac (4'-OHD) whereas P450 3A4 only led to 5-hydroxy diclofenac (5-OHD). P450 2C19, 2C18, and 2C8 led to the simultaneous formation of 4'-OHD and 5-OHD (respective molar ratios of 1.3, 0.37, and 0.17), and P450 1A1, 1A2, 2D6, and 2E1 failed to give any detectable hydroxylated metabolite under identical conditions. P450 2C9 was found to be much more efficient for diclofenac hydroxylation than all the other P450s tested (k(cat)/K(M) of 1.6 min(-1) microM(-1) instead of 0.025 for the second more active P450), mainly because of markedly lower K(M) values (15 +/- 8 instead of values between 170 and 630 microM). Oxidation of diclofenac with chemical model systems of cytochrome P450 based on iron porphyrin catalysts exclusively led to the quinone imine derived from two-electron oxidation of 5-OHD, in an almost quantitative yield. Two derivatives of diclofenac lacking its COO(-) function were then synthesized; their oxidation by recombinant human P450 2Cs always led to a major product coming from their 5-hydroxylation. Substrate 2, which derives from reduction of the COO(-) function of diclofenac to the CH(2)OH function, was studied in more detail. All the P450s tested (1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6, and 3A4) almost exclusively led to its 5-hydroxylation. P450s of the 2C subfamily were found to be the most efficient catalysts for this reaction, with k(cat)/K(M) values between 0.2 and 1.6 min(-1) microM(-1). Oxidation of 2 with an iron porphyrin-based chemical model of cytochrome P450 also led to a product derived from the oxidation of 2 at position 5. These results show that oxidation of diclofenac and its derivative 2, either with chemical model systems of cytochrome P450 or with recombinant human P450s, generally occurs at position 5. This position, para to the NH group on the more electron-rich aromatic ring of diclofenac derivatives, is thus, as expected, the privileged site of reaction of electrophilic, oxidant species. The most spectacular exception to this chemoselective 5-oxidation of diclofenac derivatives was found for oxidation of diclofenac itself with P450 2C9 (and P450 2C19 and 2C18 to a lesser extent), which only led to 4'-OHD. A likely explanation for this result is a strict positioning of diclofenac in the P450 2C9 active site, via its COO(-) function, to completely orientate its hydroxylation toward position 4', which is not chemically preferred. P450 2C19, 2C18, and 2C8 would not lead to such a strict positioning as they give mixtures of 4'-OHD and 5-OHD. The above results show that diclofenac derivatives are interesting tools to compare the active site topologies of human P450 2Cs.     

Accumulation of mitochondrial P450MT2, NH(2)-terminal truncated cytochrome P4501A1 in rat brain during chronic treatment with beta-naphthoflavone. A role in the metabolism of neuroactive drugs

The biochemical and molecular characteristics of cytochrome P4501A1 targeted to rat brain mitochondria was studied to determine the generality of the targeting mechanism previously described for mitochondrial cytochrome P450MT2 (P450MT2) from rat liver. In rat brain and C6 glioma cells chronically exposed to beta-naphoflavone (BNF), P450MT2 content reached 50 and 95% of the total cellular pool, respectively. P450MT2 from 10 days of BNF-treated rat brain was purified to over 85% purity using hydrophobic chromatography followed by adrenodoxin affinity binding. Purified brain P450MT2 consisted of two distinct molecular species with NH(2) termini identical to liver mitochondrial forms. These results confirm the specificity of endoprotease-processing sites. The purified P450MT2 showed a preference for adrenodoxin + adrenodoxin reductase electron donor system and exhibited high erythromycin N-demethylation activity. Brain mitoplasts from 10-day BNF-treated rats and also purified P450MT2 exhibited high N-demethylation activities for a number of neuroactive drugs, including trycyclic anti-depressants, anti-convulsants, and opiates. At 10 days of BNF treatment, the mitochondrial metabolism of these neuroactive drugs represented about 85% of the total tissue activity. These results provide new insights on the role of P450MT2 in modulating the pharmacological potencies of different neuroactive drugs in chronically exposed individuals.     

Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli

Drug oxidation activities of 12 recombinant human cytochrome P450s (P450) coexpressed with human NADPH-P450 reductase (NPR) in bacterial membranes (P450/NPR membranes) were determined and compared with those of other recombinant systems and those of human liver microsomes. Addition of exogenous membrane-bound NPR to the P450/NPR membranes enhanced the catalytic activities of CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5. Enhancement of activities of CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, and CYP2E1 in membranes was not observed after the addition of NPR (4 molar excess to each P450). Exogenous purified human cytochrome b5 (b5) further enhanced catalytic activities of CYP2A6, CYP2B6, CYP2C8, CYP2E1, CYP3A4, and CYP3A5/NPR membranes. Catalytic activities of CYP2C9 and CYP2C19 were enhanced by addition of b5 in reconstituted systems but not in the P450/NPR membranes. Apo b5 (devoid of heme) enhanced catalytic activities when added to both membrane and reconstituted systems, except for CYP2E1/NPR membranes and the reconstituted system containing purified CYP2E1 and NPR. Catalytic activities in P450/NPR membranes fortified with b5 were roughly similar to those measured with microsomes of insect cells coexpressing P450 with NPR (and b5) and/or human liver microsomes, based on equivalent P450 contents. These results suggest that interactions of P450 and NPR coexpressed in membranes or mixed in reconstituted systems appear to be different in some human CYP2 family enzymes, possibly due to a conformational role of b5. P450/NPR membrane systems containing b5 are useful models for prediction of the rates for liver microsomal P450-dependent drug oxidations.     

Topological analysis of the active sites of cytochromes P450IIB4 (rabbit), P450IIB10 (mouse), and P450IIB11 (dog) by in situ rearrangement of phenyl-iron complexes.

The reaction of phenyldiazene with purified, phenobarbital-inducible rabbit cytochrome P450IIB4, mouse cytochrome P450IIB10, and dog cytochrome P450IIB11 yields complexes with absorbance maxima at 480 nm. Treatment of the cytochrome P450 complexes with K3Fe(CN)6 results in disappearance of the 480-nm absorption. Extraction of the prosthetic group from the proteins after these reactions yields the two isomers of N-phenylprotoporphyrin IX with the N-phenyl group on pyrrole rings A and D as the major products and the regioisomer with the N-phenyl on pyrrole ring C as a minor product. The A:C:D arylated pyrrole ring ratio is 3:2:3 for rabbit P450IIB4, 3:1:3 for mouse P450IIB10, and 4:1:2 for dog P450IIB11. Formation of the A and D regioisomers is consistent with the results obtained previously for rat isozymes IA1, IIB1, IIB2, and IIE1, but the rabbit, mouse, and dog P450IIB enzymes differ from the four rat enzymes in that a substantial amount of the isomer with the N-phenyl on pyrrole ring C is also formed. The results indicate that the region over pyrrole ring B is masked by protein residues in all the active sites and suggest that the region over pyrrole ring C is more hindered by protein residues in the rat than in the rabbit, mouse, or dog enzymes so far examined.     

Electrospray ionization mass spectrometric analysis of intact cytochrome P450: identification of tienilic acid adducts to P450 2C9

A general scheme for the purification of baculovirus-expressed cytochrome P450s (P450s) from the crude insect cell pastes has been designed which renders the P450s suitable for analysis by high-performance liquid chromatography (HPLC) electrospray ionization mass spectrometry (ESI-MS). An HPLC/ESI-MS procedure has been developed to analyze small amounts of intact purified P450 (P450s cam-HT, 1A1, 1A2, 2A6, 2B1, 2C9, 2C9 C175R, 3A4, 3A4-HT) and rat NADPH cytochrome P450 reductase (P450 reductase). The experimentally determined and predicted (based on the amino acid sequences) molecular masses (MMs) of the various proteins had identical rank orders. For each individual protein, the difference between the experimentally determined (+/-SD, based on experiments performed on at least 3 different days) and predicted MMs ranged from 0.002 to 0.035%. Each experimentally determined MM had a standard deviation of less than 0.09% (based on the charge state distribution). Application of this HPLC/ESI-MS technique made the detection of the covalent modification to P450 2C9 following mechanism-based inactivation by tienilic acid possible. In the absence of glutathione, three P450 2C9 species were detected that produced ESI mass spectra corresponding to native P450 2C9 and both a monoadduct and a diadduct of tienilic acid to P450 2C9. In the presence of glutathione, only native P450 2C9 and the monoadduct were detected. Based on the observed mass shifts for the P450 2C9/tienilic acid adducts, a mechanism for the inactivation of P450 2C9 by tienilic acid is proposed.     

Comparative cytochrome P450 proteomics in the livers of immunodeficient mice using 18O stable isotope labeling

Quantitative changes in cytochrome P450 (CYP) proteins involved in drug metabolism as a consequence of drug treatment are important parameters in predicting the fates and pharmacological consequences of xenobiotics and drugs. In this study we undertook comparative P450 proteomics using liver from control and 1,4-bis-2-(3,5-dichloropyridyloxybenzene) (TCPOBOP)-dosed mice. The method involved separation of microsomal proteins by SDS-PAGE, trypsin digestion, and postdigest 18O/16O labeling followed by nano-LC-MS/MS for peptide identification and LC-MS for relative quantification. Seventeen P450 proteins were identified from mouse liver of which 16 yielded data sufficient for relative quantification. All the P450s detected were unambiguously identified except the highly homologous CYP2A4/2A5. With the exception of CYP2A12, -2D10, and -2F2, the levels of all the P450s quantified were affected by treatment with TCPOBOP (3 mg/kg). CYP1A2, -2A4/5, -2B10, -2B20, -2C29, -2C37, -2C38, -3A11, and -39A1 were up-regulated, and CYP2C40, -2E1, -3A41, and -27A1 down-regulated. The response of CYP2B20 to stimulation has not been distinguished previously from that of CYP2B10 because of the poor discrimination between these two proteins (they share 87% sequence identity). Differential response to chemical stimulation by closely related members of the CYP2C subfamily was also observed.