Preincubation Of Human Liver Microsomes Research Articles

Interaction of a caffeine overdose with clinical doses of contraceptive ethinyl estradiol in a young woman.

The overdose of caffeine, a cytochrome P450 1A2 probe, in young women has become a problem. The aim of this study was to evaluate possible drug interactions between intentionally overdosed caffeine (12 g) and oral contraceptive doses of ethinyl estradiol prescribed to a young woman in a suicide attempt. The serum concentrations of caffeine in the patient and the time-dependent ethinyl estradiol inhibition of caffeine oxidation invitro were evaluated. The serum concentration of caffeine 4 h after overdose was 136 μg/mL; from the data obtained between 4 and 28 h after overdose, the half-life was estimated to be 33 h, which is many times larger than the normal value. Prescribed ethinyl estradiol prolonged caffeine elimination invivo and inhibited paraxanthine formation, as evidenced by the low serum concentrations. In human liver microsomes, ethinyl estradiol (50 nM) inhibited half of caffeine N 3 -demethylation. Pre-incubation of human liver microsomes with ethinyl estradiol resulted in a powerful time-dependent inhibitory effect on caffeine N 3 -demethylation in human liver microsomes. These results suggest that a prescription history of contraceptives at clinical doses may have a strong effect on the pharmacokinetics of overdosed caffeine in young women, resulting in dangerous drug interactions.

Lack of Correlation between In Vitro and In Vivo Studies on the Inhibitory Effects of (‒)-Sophoranone on CYP2C9 is Attributable to Low Oral Absorption and Extensive Plasma Protein Binding of (‒)-Sophoranone.

(‒)-Sophoranone (SPN) is a bioactive component of Sophora tonkinensis with various pharmacological activities. This study aims to evaluate its in vitro and in vivo inhibitory potential against the nine major CYP enzymes. Of the nine tested CYPs, it exerted the strongest inhibitory effect on CYP2C9-mediated tolbutamide 4-hydroxylation with the lowest IC50 (Ki) value of 0.966 ± 0.149 μM (0.503 ± 0.0383 μM), in a competitive manner. Additionally, it strongly inhibited other CYP2C9-catalyzed diclofenac 4′-hydroxylation and losartan oxidation activities. Upon 30 min pre-incubation of human liver microsomes with SPN in the presence of NADPH, no obvious shift in IC50 was observed, suggesting that SPN is not a time-dependent inactivator of the nine CYPs. However, oral co-administration of SPN had no significant effect on the pharmacokinetics of diclofenac and 4′-hydroxydiclofenac in rats. Overall, SPN is a potent inhibitor of CYP2C9 in vitro but not in vivo. The very low permeability of SPN in Caco-2 cells (Papp value of 0.115 × 10−6 cm/s), which suggests poor absorption in vivo, and its high degree of plasma protein binding (>99.9%) may lead to the lack of in vitro–in vivo correlation. These findings will be helpful for the safe and effective clinical use of SPN.

Guanfu base A, an antiarrhythmic alkaloid of Aconitum coreanum, Is a CYP2D6 inhibitor of human, monkey, and dog isoforms.

Guanfu base A (GFA) is a novel heterocyclic antiarrhythmic drug isolated from Aconitum coreanum (Lèvl.) rapaics and is currently in a phase IV clinical trial in China. However, no study has investigated the influence of GFA on cytochrome P450 (P450) drug metabolism. We characterized the potency and specificity of GFA CYP2D inhibition based on dextromethorphan O-demethylation, a CYP2D6 probe substrate of activity in human, mouse, rat, dog, and monkey liver microsomes. In addition, (+)-bufuralol 1'-hydroxylation was used as a CYP2D6 probe for the recombinant form (rCYP2D6), 2D1 (rCYP2D1), and 2D2 (rCYP2D2) activities. Results show that GFA is a potent noncompetitive inhibitor of CYP2D6, with inhibition constant Ki = 1.20 ± 0.33 μM in human liver microsomes (HLMs) and Ki = 0.37 ± 0.16 μM for the human recombinant form (rCYP2D6). GFA is also a potent competitive inhibitor of CYP2D in monkey (Ki = 0.38 ± 0.12 μM) and dog (Ki = 2.4 ± 1.3 μM) microsomes. However, GFA has no inhibitory activity on mouse or rat CYP2Ds. GFA did not exhibit any inhibition activity on human recombinant CYP1A2, 2A6, 2C8, 2C19, 3A4, or 3A5, but showed slight inhibition of 2B6 and 2E1. Preincubation of HLMs and rCYP2D6 resulted in the inactivation of the enzyme, which was attenuated by GFA or quinidine. Beagle dogs treated intravenously with dextromethorphan (2 mg/ml) after pretreatment with GFA injection showed reduced CYP2D metabolic activity, with the Cmax of dextrorphan being one-third that of the saline-treated group and area under the plasma concentration-time curve half that of the saline-treated group. This study suggests that GFA is a specific CYP2D6 inhibitor that might play a role in CYP2D6 medicated drug-drug interaction.

Selective Inhibition of Cytochrome P450 2D6 by Sarpogrelate and Its Active Metabolite, M-1, in Human Liver Microsomes

The present study was performed to evaluate the in vitro inhibitory potential of sarpogrelate and its active metabolite, M-1, on the activities of nine human cytochrome (CYP) isoforms. Using a cocktail assay, the effects of sarpogrelate on nine CYP isoforms and M-1 were measured by specific marker reactions in human liver microsomes. Sarpogrelate potently and selectively inhibited CYP2D6-mediated dextromethorphan O-demethylation with an IC50 (Ki) value of 3.05 μM (1.24 μM), in a competitive manner. M-1 also markedly inhibited CYP2D6 activity; its inhibitory effect with an IC50 (Ki) value of 0.201 μM (0.120 μM) was more potent than that of sarpogrelate, and was similarly potent as quinidine (Ki, 0.129 μM), a well-known typical CYP2D6 inhibitor. In addition, sarpogrelate and M-1 strongly inhibited both CYP2D6-catalyzed bufuralol 1'-hydroxylation and metoprolol α-hydroxylation activities. However, sarpogrelate and M-1 showed no apparent inhibition of the other following eight CYPs: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, or CYP3A4/5. Upon 30-minute preincubation of human liver microsomes with sarpogrelate or M-1 in the presence of NADPH, no obvious shift in IC50 was observed in terms of inhibition of the nine CYP activities, suggesting that sarpogrelate and M-1 are not time-dependent inactivators. Sarpogrelate strongly inhibited the activity of CYP2D6 at clinically relevant concentrations in human liver microsomes. These observations suggest that sarpogrelate could have an effect on the metabolic clearance of drugs possessing CYP2D6-catalyzed metabolism as a major clearance pathway, thereby eliciting pharmacokinetic drug-drug interactions.

Potent inhibition of cytochrome P450 2B6 by sibutramine in human liver microsomes

The present study was performed to evaluate the potency and specificity of sibutramine as an inhibitor of the activities of nine human CYP isoforms in liver microsomes. Using a cocktail assay, the effects of sibutramine on specific marker reactions of the nine CYP isoforms were measured in human liver microsomes. Sibutramine showed potent inhibition of CYP2B6-mediated bupropion 6-hydroxylation with an IC50 value of 1.61μM and Ki value of 0.466μM in a competitive manner at microsomal protein concentrations of 0.25mg/ml; this was 3.49-fold more potent than the typical CYP2B6 inhibitor thio-TEPA (Ki=1.59μM). In addition, sibutramine slightly inhibited CYP2C19 activity (Ki=16.6μM, noncompetitive inhibition) and CYP2D6 activity (Ki=15.7μM, noncompetitive inhibition). These observations indicated 35.6- and 33.7-fold decreases in inhibition potency, respectively, compared with that of CYP2B6 by sibutramine. However, no inhibition of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, or CYP2E1 activities was observed. In addition, the CYP2B6 inhibitory potential of sibutramine was enhanced at a lower microsomal protein concentration of 0.05mg/ml. After 30min preincubation of human liver microsomes with sibutramine in the presence of NADPH, no shift in IC50 was observed in terms of inhibition of the activities of the nine CYPs, suggesting that sibutramine is not a time-dependent inactivator. These observations suggest that sibutramine is a selective and potent inhibitor of CYP2B6 in vitro, whereas inhibition of other CYPs is substantially lower. These in vitro data support the use of sibutramine as a well-known inhibitor of CYP2B6 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source.

A Comparison of 2-Phenyl-2-(1-piperidinyl)propane (PPP), 1,1′,1″-Phosphinothioylidynetrisaziridine (ThioTEPA), Clopidogrel, and Ticlopidine as Selective Inactivators of Human Cytochrome P450 2B6

The use of selective chemical inhibitors of human cytochrome P450 (P450) enzymes represents a powerful method by which the relative contributions of various human P450 enzymes to the metabolism of drugs can be determined. However, the identification of CYP2B6 in the metabolism of drugs has been more challenging because of the lack of a well established inhibitor of this enzyme. In this report, we describe the selectivity of 2-phenyl-2-(1-piperidinyl)propane (PPP) as an inactivator of CYP2B6 and compare this selectivity versus other CYP2B6 inactivators: 1,1',1''-phosphinothioylidynetrisaziridine (thioTEPA), clopidogrel, and ticlopidine. Values of K(I) and k(inact) for PPP were 5.6 microM and 0.13/min for bupropion hydroxylase catalyzed by pooled human liver microsomes, and values for thioTEPA were similar (4.8 microM and 0.20/min, respectively). Intrinsic inactivation capability was considerably greater for clopidogrel because of a greater k(inact) value (1.9/min). Ticlopidine was potent with K(I) and k(inact) values of 0.32 microM and 0.43/min, respectively. The selectivity of these four agents for CYP2B6 was determined by testing their effects on other human P450 enzyme activities using conditions that yield approximately 90% inactivation of CYP2B6 activity. The results showed that preincubation of human liver microsomes with PPP at 30 microM for 30 min provided more selective inhibition for CYP2B6 than thioTEPA, clopidogrel, and ticlopidine. Furthermore, the use of clopidogrel is complicated by the observation that this agent is not stable in the presence of human liver microsomes, even without addition of NADPH. Therefore, PPP can serve as a selective chemical inactivator of CYP2B6 and be used to define the role of CYP2B6 in the metabolism of drugs.

In Vitro Activation of Valproate Glucuronidation by Carbapenem Antibiotics

The serum concentration of valproic acid (VPA) in epilepsy patients decreased by the administration of carbapenem antibiotics, such as meropenem, panipenem or imipenem, to a sub-therapeutic level. Studies to explain the decrease were carried out using almost rats by the following steps: absorption of VPA in the intestine, glucuronidation in the liver, disposition in blood and renal excretion. It is difficult to consider the inhibition of intestinal absorption, because carbapenem antibiotics are intravenously administered and do not reach the intestine at an effective concentration. The liver is the key organ for the decrease of VPA concentration by carbapenem antibiotics, because it has been reported that no decrease of the VPA level by carbapenem was found in hepatectomized rats. The most likely mechanism in liver is the activation of UDP-glucuronosyltransferase by carbapenem antibiotics. We found a 35% increase of VPA-glucuronidation activity by the pre-incubation of human liver microsomes with meropenem. We estimated that this increase fully compensates for the decrease of serum VPA level by carbapenem antibiotics in patients. Meanwhile, we could not find the in vitro activation of CYP2A6, CYP3A4, P-glycoprotein (P-gp, MDR1) and Multidrug resistance-associated protein 2 by pre-incubation with carbapenem antibiotics. Thus, we considered that the activation of VPA glucuronidation by carbapenem antibiotics is a key point in the decrease of plasma VPA level.

Interaction Between Valproic Acid and Carbapenem Antibiotics

The serum concentration of valproic acid (VPA) in epilepsy patients decreased by the administration of carbapenem antibiotics, such as meropenem, panipenem or imipenem, to a sub-therapeutic level. This review summarized several case reports of this interaction between VPA (1–4 g dose) and carbapenem antibiotics to elucidate the possible mechanisms decreasing VPA concentration by carbapenem antibiotics. Studies to explain the decrease were carried out using rats by the following sites: absorption of VPA in the intestine, glucuronidation in the liver, disposition in blood and renal excretion. In the intestinal absorption site, there are two possible mechanisms: inhibition of the intestinal transporter for VPA absorption by carbapenem antibiotics, and the decrease of β-glucuronidase supplied from enteric bacteria, which were killed by antibiotics. This is consistent with a view that the decrease of VPA originated from VPA-Glu, relating to entero-hepatic circulation. The second key site is in the liver, because of no decreased in VPA level by carbapenem antibiotics in hepatectomized rats. There are three possible mechanisms in the liver to explain the decreased phenomenon: first, decrease of the UDPGA level by carbapenem antibiotics. UDPGA is a co-factor for UDP-glucuronosyltransferase (UGT)-mediated glucuronidation of VPA. Second, the direct activation of UGT by carbapenem antibiotics. This activation was observed after pre-incubation of human liver microsomes with carbapenem antibiotics. Third, the inhibition of β-glucuronidase in liver by carbapenem antibiotics and the decreased VPA amount liberated from VPA-Glu. The third site is the distribution of VPA in blood (erythrocytes and plasma). Plasma VPA distributed to erythrocytes by the inhibition of transporters (Mrp4), which efflux VPA from erythrocytes to plasma, by carbapenem antibiotics. The increase of renal excretion of VPA as VPA-Glu depends on the increase of VPA-Glu level by UGT. One or a combination of some factors in these mechanisms might relate to the carbapenem-mediated decrease of the plasma VPA level.

Inhibition of the Human Liver Microsomal and Human Cytochrome P450 1A2 and 3A4 Metabolism of Estradiol by Deployment-Related and Other Chemicals

Cytochromes P450 (P450s) are major catalysts in the metabolism of xenobiotics and endogenous substrates such as estradiol (E2). It has previously been shown that E2 is predominantly metabolized in humans by CYP1A2 and CYP3A4 with 2-hydroxyestradiol (2-OHE2) the major metabolite. This study examines effects of deployment-related and other chemicals on E2 metabolism by human liver microsomes (HLM) and individual P450 isoforms. Kinetic studies using HLM, CYP3A4, and CYP1A2 showed similar affinities (Km) for E2 with respect to 2-OHE2 production. Vmax and CLint values for HLM are 0.32 nmol/min/mg protein and 7.5 microl/min/mg protein; those for CYP3A4 are 6.9 nmol/min/nmol P450 and 291 microl/min/nmol P450; and those for CYP1A2 are 17.4 nmol/min/nmol P450 and 633 microl/min/nmol P450. Phenotyped HLM use showed that individuals with high levels of CYP1A2 and CYP3A4 have the greatest potential to metabolize E2. Preincubation of HLM with a variety of chemicals, including those used in military deployments, resulted in varying levels of inhibition of E2 metabolism. The greatest inhibition was observed with organophosphorus compounds, including chlorpyrifos and fonofos, with up to 80% inhibition for 2-OHE2 production. Carbaryl, a carbamate pesticide, and naphthalene, a jet fuel component, inhibited ca. 40% of E2 metabolism. Preincubation of CYP1A2 with chlorpyrifos, fonofos, carbaryl, or naphthalene resulted in 96, 59, 84, and 87% inhibition of E2 metabolism, respectively. Preincubation of CYP3A4 with chlorpyrifos, fonofos, deltamethrin, or permethrin resulted in 94, 87, 58, and 37% inhibition of E2 metabolism. Chlorpyrifos inhibition of E2 metabolism is shown to be irreversible.

THE EFFECT OF CIMETIDINE ON DEXTROMETHORPHAN O-DEMETHYLASE ACTIVITY OF HUMAN LIVER MICROSOMES AND RECOMBINANT CYP2D6

Clinically, cimetidine therapy impairs the clearance of various drugs metabolized by CYP2D6, such as desipramine and sparteine. Cimetidine is known to reversibly inhibit CYP2D6 in vitro; however, Ki values are greater than plasma concentrations observed in vivo. There is evidence suggesting that this drug may act as an inactivator of cytochrome P450 (P450) enzymes after metabolic activation. Therefore, the purpose of this study was to determine whether cimetidine acts as a mechanism-based inactivator of CYP2D6. Dextromethorphan O-demethylation was used as a probe of CYP2D6 activity. The Vmax and Km of this reaction were 0.82 +/- 0.06 nmol/min/nmol of P450 and 4.1 +/- 0.1 microM, respectively, in pooled human liver microsomes; and 15.9 +/- 0.8 nmol/min/nmol P450 and 1.4 +/- 0.6 microM, respectively, with recombinant CYP2D6. With human liver microsomes, cimetidine competitively inhibited CYP2D6 (Ki = 38 +/- 5 microM) and was a mixed inhibitor of recombinant CYP2D6 (Ki = 103 +/- 17 microM). Preincubation of human liver microsomes with cimetidine and NADPH did not increase the inhibitory potency of cimetidine; however, preincubation with recombinant CYP2D6 resulted in enzyme inactivation that could be attenuated by the CYP2D6 inhibitor quinidine. The KI and kinact were estimated to be 77 microM and 0.03 min-1, respectively, and the half-life of inactivation was 25 min. Therefore, cimetidine may represent a class of compounds capable of inactivating specific cytochromes P450 in vivo, but for which conditions may not be achievable in vitro using human liver microsomes.

In vitro metabolism of carbaryl by human cytochrome P450 and its inhibition by chlorpyrifos

Carbaryl is a widely used anticholinesterase carbamate insecticide. Although previous studies have demonstrated that carbaryl can be metabolized by cytochrome P450 (CYP), the identification and characterization of CYP isoforms involved in metabolism have not been described either in humans or in experimental animals. The in vitro metabolic activities of human liver microsomes (HLM) and human cytochrome P450 (CYP) isoforms toward carbaryl were investigated in this study. The three major metabolites, i.e. 5-hydroxycarbaryl, 4-hydroxycarbaryl and carbaryl methylol, were identified after incubation of carbaryl with HLM or individual CYP isoforms and analysis by HPLC. Most of the 16 human CYP isoforms studied showed some metabolic activity toward carbaryl. CYP1A1 and 1A2 had the greatest ability to form 5-hydroxycarbaryl, while CYP3A4 and CYP1A1 were the most active in generation of 4-hydroxycarbaryl. The production of carbaryl methylol was primarily the result of metabolism by CYP2B6. Differential activities toward carbaryl were observed among five selected individual HLM samples with the largest difference occurring in the production of carbaryl methylol. Co-incubations of carbaryl and chlorpyrifos in HLM greatly inhibited carbaryl metabolism. The ability of HLM to metabolize carbaryl was also reduced by pre-incubation of HLM with chlorpyrifos. Chlorpyrifos inhibited the generation of carbaryl methylol, catalyzed predominately by CYP2B6, more than other pathways, correlating with an earlier observation that chlorpyrifos is metabolized to its oxon primarily by CYP2B6. Therefore, carbaryl metabolism in humans and its interaction with other chemicals is reflected by the concentration of CYP isoforms in HLM and their activities in the metabolic pathways for carbaryl. (Supported by NCDA Environmental Trust Fund)

Diclofenac-induced inactivation of CYP3A4 and its stimulation by quinidine.

Incubation of human liver microsomes with diclofenac in the presence of NADPH resulted in a decrease in testosterone 6 beta-hydroxylation activity. The decrease in the activity followed time- and concentration-dependent kinetics, required oxidative metabolism, and was resistant to reduced glutathione, suggesting that diclofenac causes a mechanism-based inactivation of cytochrome p450 (p450) 3A4 (CYP3A4). The inactivation was reproduced by using microsomes from B-lymphoblastoid cell lines expressing CYP3A4 instead of human liver microsomes. No other monooxygenase activities measured as indexes of p450 enzymes; CYP2C8, CYP2C9, or CYP2C19 was inactivated by the same incubation procedure. Quinidine, a stimulant of CYP3A4-mediated diclofenac 5-hydroxylation, did not affect the inactivation of CYP3A4 assessed by testosterone 6 beta-hydroxylation activity but accelerated the inactivation assessed by diazepam 3-hydroxylation activity. These results supported the idea that diclofenac 5-hydroxylation is involved in the inactivation of CYP3A4 and described for the first time a stimulation of mechanism-based inactivation attributable to CYP3A4 heterotropic cooperativity. Preincubation of human liver microsomes with 5-hydroxydiclofenac instead of diclofenac did not cause the inactivation of CYP3A4, suggesting that 5-hydroxydiclofenac is not a precursor of a postulated reactive metabolite that inactivates CYP3A4, and thus 5-hydroxylation step is critical to inactivation of CYP3A4.

Mechanism-based inactivation of cytochrome P450s 1A2 and 3A4 by dihydralazine in human liver microsomes.

Dihydralazine is known to induce immunoallergic hepatitis. Since anti-liver microsome (anti-LM) autoantibodies found in the serum of the patients react with P450 1A2, it is suggested that dihydralazine is biotransformed into a reactive metabolite, which covalently binds to cytochrome P450 1A2 and triggers an immunological response as a neoantigen. We investigated inactivation of P450 enzymes, including P450 1A2, during the metabolism of dihydralazine to evaluate the selectivity of P450 1A2 as a catalyst and a target of dihydralazine. Human liver microsomes or microsomes from lymphoblastoid cells expressing P450 enzymes were preincubated with dihydralazine in the presence of NADPH, followed by an assay of several monooxygenase activities. Preincubation of human liver microsomes with dihydralazine in the presence of NADPH resulted in decreases in phenacetin O-deethylase activity (an indicator of P450 1A2 activity) and testosterone 6beta-hydroxylase activity (P450 3A4), but not in diclofenac 4'-hydroxylase activity (P450 2C9), an indication of inactivation of P450s 1A2 and 3A4 during the dihydralazine metabolism. The inactivation of both of the P450s followed pseudo-first-order kinetics and was saturable with increasing dihydralazine concentrations. Similar time-dependent decreases in the activities were obtained in the case for use in microsomes expressing P450 1A2 and P450 3A4 instead of the human liver microsomes. The data presented here demonstrated that dihydralazine was metabolically activated not only by P450 1A2 but also by P450 3A4, and the chemically reactive metabolite bound to and inactivated the enzyme themselves, suggesting that dihydralazine is a mechanism-based inactivator of P450s 1A2 and 3A4. The data support the postulated covalent binding of a reactive metabolite of dihydralazine to P450 1A2 as a step in the formation of anti-LM antibodies in dihydralazine hepatitis, but it is not the unique factor for determining the specificity of the autoantibodies.

Inhibition of Coumarin 7-Hydroxylase Activity in Human Liver Microsomes

Nine organic solvents and 47 commonly used P450 substrates and inhibitors were examined for their effects on coumarin 7-hydroxylase (CYP2A6) activity in human liver microsomes. Of the nine organic solvents examined (final concentration 1%, v/v), only methanol did not inhibit the 7-hydroxylation of coumarin (0.5 to 50 μm) by human liver microsomes. Dioxane and tetrahydrofuran, which are structurally related to coumarin, were the most inhibitory solvents examined. Although the rates of coumarin 7-hydroxylation varied enormously among nine samples of human liver microsomes and cDNA-expressed CYP2A6 (Vmax= 179 to 2470 pmol/mg protein/min), theKmfor coumarin 7-hydroxylation was fairly constant (ranging from 0.50 to 0.70 μm). The following chemicals caused little or no inhibition of CYP2A6 as defined by aKi> 200 μm: caffeine, chlorzoxazone, cimetidine, dextromethorphan, diazepam, diclofenac, erythromycin, ethinylestradiol, ethynyltestosterone, fluconazole, furafylline, furfural, hexobarbital, itraconazole, mephenytoin, methimazole, metronidazole, naringenin, naringin, nifedipine, norfloxacin, norgestrel, orphenadrine, quinidine, papaverine, phenacetin, pyrimethamine, ranitidine, spironolactone, sulfaphenazole, sulfinpyrazone, testosterone, tolbutamide, troleandomycin, and warfarin. In other words, these chemicals, at a final concentration of 100 μm, failed to inhibit CYP2A6 when the concentration of coumarin was equal toKm(0.50 μm). The following chemicals were classified as strong inhibitors of CYP2A6 (defined byKi< 200 μm): clotrimazole, diethyldithiocarbamate, ellipticine, ketoconazole, 8-methoxypsoralen, 4-methylpyrazole, metyrapone, miconazole, α-naphthoflavone, nicotine,p-nitrophenol, and tranylcypromine. The potency with which each chemical inhibited the 7-hydroxylation of coumarin was independent of which sample of human liver microsomes was studied. One of the most potent inhibitors of coumarin 7-hydroxylase was 8-methoxypsoralen (methoxsalen), which was determined to be a mechanism-based inhibitor (suicide substrate) of CYP2A6 (kinactivation0.5 min−1). With the exception of 8-methoxypsoralen, preincubation of human liver microsomes and NADPH with the aforementioned inhibitors did not increase their ability to inhibit CYP2A6. The most potent competitive inhibitor of CYP2A6 was tranylcypromine (Ki= 0.04 μm). Several of the chemicals that strongly inhibited CYP2A6, such as ketoconazole and tranylcypromine, are often used with the intention of selectively inhibiting human P450 enzymes other than CYP2A6. The results of this study underscore the need for a systematic evaluation of the specificity of commonly used P450 inhibitors.

Inhibition of cyclosporine and tetrahydrocannabinol metabolism by cannabidiol in mouse and human microsomes.

1. The in vitro and in vivo effects of cannabidiol on mouse and human liver microsomal metabolism of the immunosuppressive drug cyclosporine and the psychoactive compound tetrahydrocannabinol have been examined. 2. Preincubation of mouse or human liver microsomes with cannabidiol decreased the formation of all detectable cyclosporine metabolites by 73-89%. 3. In vivo cannabidiol treatment of mouse similarly decreased the formation of all detectable cyclosporine metabolites by 60-86%. 4. Preincubation of human liver microsomes with cannabidiol selectively decreased the formation of tetrahydrocannabinol metabolites catalyzed by cytochrome P4503A by 60% but had no effect on P4502C9-catalyzed metabolites. 5. Cannabidiol has the potential to clinically affect cyclosporine metabolism which may result in increased cyclosporine blood levels and an increase in its toxic side effects, and likewise may also affect tetrahydrocannabinol and its metabolite levels in man.

Nature of cytochromes P450 involved in the 2-/4-hydroxylations of estradiol in human liver microsomes

Kinetics of the 2- and 4-hydroxylations of estradiol (E2) by human liver microsomal samples were studied to determine the major P450 isoform involved in these endogenous reactions. Thirty human liver microsomal samples were analysed. Metabolism of 25 μM [ 14C]E2 produced 2-hydroxy and 4-hydroxy derivatives with a ratio of 3.2 ± 1.5 and a great inter-individual variation. Kinetic analysis of the 2- and 4-hydroxylations of E2 exhibited a curvilinear double reciprocal plot with an apparent K m of 15 μM. Further experiments demonstrated that α-naphthoflavone, testosterone and progesterone increased the 2-hydroxylation activity, suggesting the involvement of a substrate activation mechanism. These two hydroxylations of E2 were shown to be catalysed by cytochrome P450 with an apparent dissociation constant K s of 0.8 μM. These 2- and 4-hydroxylations inter-correlated significantly ( r = 0.93; N = 30). The 2-hydroxylation of E2 correlated with four monooxygenase activities known to be supported by P450 3A4/3A5, namely nifedipine oxidation ( r = 0.78; N = 29); erythromycin N-demethylation ( r = 0.69; N = 27), testosterone 6β-hydroxylation ( r = 0.66; N = 25) and tamoxifen N-demethylation ( r = 0.64; N = 29). On the other hand, E2-hydroxylations did not correlate with activities supported by P450 1A2 and P450 2E1. Furthermore, drugs as cyclosporin, diltiazem. triacetyl-oleandomycin and 17α-ethynylestradiol inhibited more than 90% of the E2-hydroxylations at concentrations < 250 μM, while weak inhibition was shown with 500 μM cimetidine and no significant inhibition with caffeine, phenacetin and omeprazole. Finally, 2- and 4-hydroxylations of E2 correlated significantly with the content of P450 3A4/3A5 immunodetected by a monoclonal antibody anti-human P450-nifedipine ( r = 0.84; N = 28). E2-hydroxylation activities were inhibited by more than 80% with polyclonal anti-human anti-P450-nifedipine. Preincubation of human liver microsomes with 100 μM gestodene (a suicide substrate of P450 3A4) inactivated this P450 isoform and accordingly allowed evaluation of the contribution of other P450 isoforms to the E2 metabolism to about 21% (± 17%. N = 29). All these results taken together suggest that P450 3A4 3A5 are the major forms involved in the formation of catecholestrogens in the human liver microsomes.

Biotransformation of lovastatin: IV. Identification of cytochrome P450 3A proteins as the major enzymes responsible for the oxidative metabolism of lovastatin in rat and human liver microsomes

Previous studies from our laboratories have shown that the metabolism of the cholesterol-lowering drug lovastatin by rat and human liver microsomes occurs primarily at the 6′-position, giving 6′β-hydroxy- and 6′-exomethylene-lovastatin and that these oxidations are catalyzed by cytochrome P450-dependent monooxygenases. In the present study, the specific cytochrome P450 form involved in lovastatin oxidation was identified through immunoinhibition studies. Among several antibodies prepared against various cytochrome P450s, only anti-rat P450 3A IgG inhibited lovastatin metabolism in liver microsomes from untreated, phenobarbital treated, and pregnenolone-16α-carbonitrile-treated rats. Lovastatin metabolism at the 6′-position was markedly inhibited (6′β-hydroxy, greater than 95%; 6′-exomethylene, 70–80%) by this antibody whereas the effect of anti-rat P450 3A on the 3″-hydroxylation was variable depending on the source of the microsomes. With human liver microsomes, both anti-rat P450 3A and anti-human P450 3A inhibited lovastatin metabolism. Correlation between lovastatin oxidation and the P450 3A content in human liver microsomes (measured by immunoblot analysis) was excellent ( r 2 = 0.97). In addition, preincubation of human liver microsomes with troleandomycin and NADPH inhibited metabolism by 60%. These results clearly indicate that cytochrome P450 3A enzymes are primarily responsible for the metabolism of lovastatin in rat and human liver microsomes.

3-hydroxy-3-methylglutaryl coenzyme A reductase in human liver microsomes: active and inactive forms and cross-reactivity with antibody against rat liver enzyme.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the enzyme catalyzing the rate-limiting step in cholesterol biosynthesis, exists in one active (dephosphorylated) and one inactive (phosphorylated) form in liver microsomes obtained from several animal species. The present study was undertaken in order to determine a) whether the human enzyme also exists in active and inactive readily interconvertible forms; b) whether the large inter-individual variation in HMG-CoA reductase activity observed in normal man can be explained by variations in the activation state of the enzyme; and c) to characterize the reactivity of antibodies raised against rat liver HMG-CoA reductase with the intact human microsomal enzyme. HMG-CoA reductase activity, assayed in microsomes prepared in the presence of 50 mM NaF, was only 17 +/- 3% of the activity observed in microsomes prepared from the same liver in the absence of fluoride. Preincubation of microsomes prepared in NaF with alkaline phosphatase resulted in a tenfold increase of enzyme activity, while the activity of microsomes prepared without fluoride was increased also (by about 45%) with this treatment. On the other hand, the activated enzyme could be inactivated by incubation of microsomes with Mg-ATP. In eleven normal weight, normolipidemic gallstone patients, the HMG-CoA reductase activity determined in microsomes prepared without NaF ("standard procedure") reflected well both the "expressed" activity (in microsomes prepared with NaF) and the "total" (fully activated) enzyme activity; correlation coefficients were +0.80 and +0.84, respectively. Preincubation of human liver microsomes with rabbit antiserum against partially purified HMG-CoA reductase from rat liver resulted in a 72 +/- 6% inhibition of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)