Serum LH correlates highly with intratesticular steroid levels in normal men.

Sex steroids are essential for spermatogenesis; however, normal intratesticular concentrations of these hormones in man have not been extensively studied. To improve our understanding of intratesticular hormone concentrations, we performed bilateral testicular aspirations in a group of normal men, determined sex steroid concentrations within each testis, and compared these levels to serum hormone concentrations. Ten healthy human subjects aged 20-49 underwent bilateral testicular aspirations. Intratesticular hormone concentrations of testosterone, dihydrotestosterone (DHT), and estradiol were measured using liquid chromatography-tandem mass spectrometry. Intratesticular testosterone concentrations ranged from 119 to 1251 ng/mL, with a mean of 635 +/- 368 ng/mL. Intratesticular estradiol ranged from 0.41 to 3.9 ng/mL, with a mean of 2.4 +/- 1.3 ng/mL. Intratesticular DHT ranged from 1.1 to 7.9 ng/mL, with a mean of 3.5 +/- 3.2 ng/mL. Intratesticular testosterone and estradiol concentrations correlated highly with serum luteinizing hormone (LH; r = 0.87 and r = 0.70 respectively, P < .01). Intratesticular testosterone correlated highly with serum testosterone. Moreover, a significant correlation between the right and left testes was observed for testosterone (r = 0.82, P = .003), but not for estradiol or DHT. Intratesticular hormone concentrations can be safely assessed by testicular aspiration. Intratesticular testosterone and estradiol correlate highly with serum LH concentrations, and variation in serum LH accounts for most of the variation in intratesticular testosterone among men. In addition, intratesticular testosterone is highly correlated between testes in a given individual. Direct measurement of intratesticular testosterone will improve our understanding of the relationship between intratesticular sex steroids and spermatogenesis, and may have implications for the development of male hormonal contraception.

Carbamazepine and oxcarbazepine decrease phenytoin metabolism through inhibition of CYP2C19.

Multiple studies suggest that phenytoin concentrations increase with CBZ co-medication. This study evaluated the hypothesis that CBZ and/or its major metabolite (CBZE) inhibit CYP2C19-mediated phenytoin metabolism using human liver microsomes and cDNA-expressed CYP2C19. Oxcarbazepine (OXC), and its 10-monohydroxy metabolite (MHD) were also evaluated. CBZ and MHD inhibited CYP2C19-mediated phenytoin metabolism at therapeutic concentrations. Thus, administration of CBZ and OXC with CYP2C19 substrates with narrow therapeutic ranges should be done cautiously.

Changes in free and esterified cholesterol: hallmarks of acute renal tubular injury and acquired cytoresistance.

Acute tubular cell injury is accompanied by plasma membrane phospholipid breakdown. Although cholesterol is a dominant membrane lipid which interdigitates with, and impacts, phospholipid homeostasis, its fate during the induction and recovery phases of acute renal failure (ARF) has remained ill defined. The present study was performed to ascertain whether altered cholesterol expression is a hallmark of evolving tubular damage. Using gas chromatographic analysis, free cholesterol (FC) and esterified cholesterol (CE) were quantified in: 1) isolated mouse proximal tubule segments (PTS) after 30 minutes of hypoxic or oxidant (ferrous ammonium sulfate) injury; 2) cultured proximal tubule (HK-2) cells after 4 or 18 hours of either ATP depletion/Ca(2+) ionophore- or ferrous ammonium sulfate-mediated injury; and 3) in renal cortex 18 hours after induction of glycerol-induced myoglobinuric ARF, a time corresponding to the so-called "acquired cytoresistance" state (ie, resistance to further renal damage). Hypoxic and oxidant injury each induced approximately 33% decrements in CE (but not FC) levels in PTS, corresponding with lethal cell injury ( approximately 50 to 60% LDH release). When comparable CE declines were induced in normal PTS by exogenous cholesterol esterase treatment, proportionate lethal cell injury resulted. During models of slowly evolving HK-2 cell injury, progressive CE increments occurred: these were first noted at 4 hours, and reached approximately 600% by 18 hours. In vivo myoglobinuric ARF produced comparable renal cortical CE (and to a lesser extent FC) increments. Renal CE accumulation strikingly correlated with the severity of ARF (eg, blood urea nitrogen versus CE; r, 0.84). Mevastatin blocked cholesterol accumulation in injured HK-2 cells, indicating de novo synthesis was responsible. Acute tubule injury first lowers, then raises, tubule cholesterol content. Based on previous observations that cholesterol has cytoprotectant properties, the present findings have potential relevance for both the induction and maintenance phases of ARF.

Contribution of CYP2E1 and CYP3A to acetaminophen reactive metabolite formation.

BACKGROUND: CYP2E1, 1A2, and 3A4 have all been implicated in the formation of N-acetyl-p-benzoquinone imine (NAPQI), the reactive intermediate of acetaminophen (INN, paracetamol), in studies in human liver microsomes and complementary deoxyribonucleic acid-expressed enzymes. However, recent pharmacokinetic evidence in humans has shown that the involvement of CYP1A2 is negligible in vivo. The purpose of this study was to evaluate the respective roles of CYP2E1 and 3A4 in vivo. METHODS: The involvement of CYP2E1 was assessed through pretreatment of adult human volunteers with disulfiram to inhibit the enzyme and the role of CYP3A4 through its induction in a second cohort of adults with rifampin (INN, rifampicin). Each of the respective studies was an open-label, balanced-randomized crossover design. Blood samples were obtained serially for 12 hours and urine was collected for 24 hours after acetaminophen administration. Acetaminophen was assayed in plasma, and acetaminophen and metabolites were assayed in urine. RESULTS: The recovery of the thiol metabolites formed by conjugation of NAPQI with glutathione was decreased by 69%, and the formation clearance of NAPQI was decreased by 74% (both P < .01) by pretreatment with disulfiram. Rifampin pretreatment had no effect on the formation of NAPQI or the recovery of thiol metabolites formed by conjugation of NAPQI with glutathione. CONCLUSIONS: CYP2E1 accounts for the formation of NAPQI in intact humans; the contribution of other isozymes of cytochrome P450 appears to be negligible. Under some conditions, disulfiram may be useful in diminishing the formation of NAPQI after acetaminophen overdose.

Analysis of cyclophosphamide and five metabolites from human plasma using liquid chromatography-mass spectrometry and gas chromatography-nitrogen-phosphorus detection.

An assay method for the quantification of cyclophosphamide (CY) and five metabolites from human plasma is presented. The procedure is adapted to the chemical properties of the compounds of interest: non-polar compounds are extracted into methylene chloride, concentrated and analyzed by GC-NPD after derivatization, and the remaining aqueous fraction is deproteinated with acetonitrile-methanol prior to separation via reversed-phase HPLC and detection using atmospheric pressure ionization (API)-MS. Standard curves are linear over the required range and reproducible over five months. Plasma concentration-time profiles of CY and metabolites from a patient receiving CY by intravenous infusion (60 mg/kg, once a day for two days) are presented.

Inhibition of human aldehyde dehydrogenase 1 by the 4-hydroxycyclophosphamide degradation product acrolein.

In a previous study, we observed that the elimination clearance of 4-hydroxycyclophosphamide (HCY) in patients receiving cyclophosphamide (CY) 60 mg/kg/day by 1-h i.v. infusion for 2 consecutive days decreased from day 1 to day 2 due to an apparent decrease in human aldehyde dehydrogenase 1 (ALDH1) activity. Here, the mechanism for the decrease in ALDH1 activity after CY administration was investigated. In human liver cytosol incubations, HCY inhibited ALDH activity mainly through its degradation product acrolein, whereas carboxyethylphosphoramide mustard inhibited ALDH activity only at supraclinical concentrations. Other CY metabolites evaluated, phosphoramide mustard and chloroacetaldehyde, did not inhibit ALDH. The inhibition of ALDH1 activity by acrolein in incubations with human erythrocyte ALDH1 was competitive with a Ki of 0.646 microM. The inhibition was independent of preincubation time and reversible by dialysis. The percentage of inhibition of ALDH1 activity in vivo by acrolein in patients receiving CY was calculated based on the in vitro Ki of acrolein, the in vitro Km of HCY, and the in vivo peak blood concentrations of HCY and acrolein. The calculations indicated that the activity of ALDH1 was inhibited by 85, 88, and 91% on days 1, 2, and 3 (24 h after the dose on day 2) of CY administration, respectively. The increase in ALDH1 inhibition with time is consistent with the decrease in HCY elimination clearance and the increase in HCY area under the plasma concentration time curve with time.

Pharmacokinetics of cyclophosphamide and its metabolites in bone marrow transplantation patients.

OBJECTIVES: To characterize the pharmacokinetics of cyclophosphamide and 5 of its metabolites in bone marrow transplant patients and to identify the mechanism of the increase in 4-hydroxycyclophosphamide area under the plasma concentration-time curve (AUC) from day 1 to day 2 of cyclophosphamide administration. METHODS: Cyclophosphamide was administered by intravenous infusion (60 mg/kg over 1 hour, once a day) for 2 consecutive days to 18 patients. Cyclophosphamide and 4-hydroxycyclophosphamide concentration time data on day 1 and day 2 were fitted to a model to estimate 4-hydroxycyclophosphamide formation (CLf) and elimination (CLm) clearances. Erythrocyte aldehyde dehydrogenase-1 activity was measured ex vivo just before the first cyclophosphamide infusion was started (0 hours) and 24 hours after the second cyclophosphamide infusion (48 hours). RESULTS: From day 1 to day 2, the AUC of cyclophosphamide, deschloroethyl cyclophosphamide and phosphoramide mustard decreased 24.8%, 51%, and 29.4% (P < .02), the AUC of 4-hydroxycyclophosphamide and carboxyethylphosphoramide mustard increased 54.7% and 25% (P < .01), whereas the AUC of phosphoramide mustard was not significantly changed (P > .3). The CLf of 4-hydroxycyclophosphamide increased 60% (P < .001), its CLm decreased 27.7% (P < .001), and the fraction of cyclophosphamide dose converted to 4-hydroxycyclophosphamide increased 16% (P < .001) from day 1 to day 2. The activity of patient erythrocyte aldehyde dehydrogenase-1 decreased 23.3% (P < .02) from 0 hours to 48 hours. CONCLUSIONS: The AUC of 4-hydroxycyclophosphamide increased from day 1 to day 2 as a result of increased formation and decreased elimination clearances of 4-hydroxycyclophosphamide. Aldehyde dehydrogenase-1 activity appears to decline as a consequence of cyclophosphamide administration.

Oxidation of cyclophosphamide to 4-hydroxycyclophosphamide and deschloroethylcyclophosphamide in human liver microsomes.

We have investigated the formation of 4-hydroxycyclophosphamide (HCY) and deschloroethylcyclophosphamide (DCCY) from cyclophosphamide (CY) in human liver microsomes. For HCY, the estimated values (mean +/- SD; n = 3) of Km1 and Km2 were 0.095 +/- 0.072 and 5.09 +/- 4.30 mM, and the estimated values of Vmax1 and Vmax2 were 0.138 +/- 0.070 and 1.55 +/- 0.50 nmol/min/mg protein. For DCCY, Km1 and Km2 were 0.046 +/- 0.017 and 8.58 +/- 5.84 mM, and Vmax1 and Vmax2 were 0.006 +/- 0.003 and 0.274 +/- 0.214 nmol/min/mg protein. At CY concentrations of 0.1, 0.7, and 5 mM, HCY respectively accounted for 95.7 +/- 1.3, 95.1 +/- 2.4, and 90.7 +/- 2.7% of the total products of CY (HCY + DCCY; n = 6). In a separate experiment, 98.7 +/- 11.9% (n = 3) of CY loss could be accounted for by the formation of HCY at 0.1 mM CY. On the basis of cytochrome P450 (CYP) isoform-specific chemical inhibitor and cDNA-expressed human P450 isozyme studies, CYP2C9 and CYP3A4/5 seemed to be the major P450 isoforms responsible for HCY formation at low (0.1 mM) and high (0.7 and 5 mM) concentrations of CY, respectively. Although orphenadrine inhibition was observed in human liver microsomes (which has been taken to indicate CYP2B6 catalysis), orphenadrine inhibited cDNA-expressed CYP3A4 formation of HCY to the same extent observed in human liver microsomes, and the addition of orphenadrine to incubations containing sulfaphenazole (a specific inhibitor of CYP2C9) or troleandomycin (a specific CYP3A inhibitor) did not increase inhibition beyond that observed with sulfaphenazole or troleandomycin alone. Similar studies indicated that CYP3A4/5 was the major P450 isoform responsible for DCCY formation at high (0.7 and 5 mM) concentrations of CY. The P450 isoform responsible for DCCY formation at 0.1 mM CY could not be identified due to its very low formation rate.

Characterization of carbamazepine metabolism in a mouse model of carbamazepine teratogenicity.

The disposition of carbamazepine (CBZ) was investigated in the SWV mouse. A 14C-CBZ dose was administered to CBZ pretreated mice, and the distribution of radiolabeled material was determined. Twenty-four hours after the 14C-CBZ dose, 92.5% of the dose was accounted for in urine (56%), in the visera and carcass (22%), in feces (11%), and expired as 14CO2 (2%). CBZ metabolites present in hydrolyzed urine were also identified using a combination of spectroscopic techniques. CBZ, CBZ-10,11-epoxide (CBZE), 2- and 3-hydroxy-CBZ, methylsulfonyl-CBZ, and glucuronides of CBZ and CBZE accounted for 64% of total urinary radioactivity (0-24 hr) in CBZ pretreated mice. Minor metabolites of CBZ included novel cysteine and N-acetylcysteine conjugates of CBZ, as well as a methylsulfonyl conjugate of CBZE not previously reported. The urinary excretion of these thioether conjugates was increased in CBZ/phenobarbital pretreated mice and decreased in CBZ/stiripentol pretreated mice in comparison with CBZ-only treated mice. Preliminary studies of the effects of phenobarbital and stiripentol on the urinary abundance of these metabolites are consistent with the modulation of teratogenicity in the SWV mouse by the same pretreatments. These data suggest the formation of thioether metabolites of CBZ may be related to CBZ teratogenicity in the SWV mouse.

Effects of probenecid on brain-cerebrospinal fluid-blood distribution kinetics of E-Delta 2-valproic acid in rabbits.

E-Delta 2-valproic acid (E-Delta 2-VPA), a major active metabolite of VPA, has been proposed as an alternative to VPA because it is less hepatotoxic and is nonteratogenic. In rodents, VPA and E-Delta 2-VPA have a brain tissue/free plasma concentration ratio less than unity, which suggests rapid removal of the alkanoate anticonvulsants from the central nervous system. This study in rabbits employed a simultaneous iv infusion-ventriculocisternal (VC) perfusion technique to investigate the steady-state kinetics of E-Delta 2-VPA transport at the blood-brain barrier, the blood-cerebrospinal fluid (CSF) barrier, and the neural cell membrane. Probenecid (PBD) was coadministered to probe the mediation of transport by organic anion transporter(s). Rabbits in the control group (N = 6) received an iv infusion of E-Delta 2-VPA to achieve a steady-state plasma concentration of 50 to 60 microg/ml. Blood and cisternal outflow of mock CSF perfusate were continuously sampled. Midway through the experiment, the VC perfusate was switched to one containing [3H]E-Delta 2-VPA. At 225 min, the rabbits were sacrificed, and each brain was removed and dissected into ten regions. Rabbits in the PBD group (N = 9) received an iv infusion and VC perfusion as in the control group as well as concomitant iv infusion of the inhibitor. The mean steady-state VC extraction ratio for [3H]E-Delta 2-VPA did not differ between the control and PBD groups (63.7 +/- 8.3% vs. 60. 6 +/- 9.6%), indicating the lack of a significant PBD-sensitive transport at the choroidal epithelium. Coadministration of PBD elevated brain concentration of cold E-Delta 2-VPA in the absence of a significant change in total or free steady-state plasma concentration. Mean E-Delta 2-VPA brain tissue/free plasma concentration ratios in the various brain regions were 3.5- to 5.2-fold higher in PBD-treated animals than in the controls. Significant increases (3.0- to 4.5-fold) in the mean brain tissue/cisternal perfusate concentration ratios were also observed. Compartmental modeling of the steady-state distribution data suggested that clearance of E-Delta 2-VPA from the brain parenchyma is governed jointly by efflux transporters at the neural cell membrane and brain capillary endothelium. Moreover, PBD-induced elevation of E-Delta 2-VPA tissue concentrations is attributed primarily to inhibition of E-Delta 2-VPA efflux transport at the neural cell membrane, resulting in both intracellular trapping and greater tissue retention of E-Delta 2-VPA.

Teratogenicity of carbamazepine-10, 11-epoxide and oxcarbazepine in the SWV mouse.

The mechanism of carbamazepine (CBZ)-related teratogenicity was investigated in the SWV mouse by contrasting the effects of CBZ-10, 11-epoxide (CBZE) and oxcarbazepine (OXC) treatments. Dietary CBZE administration was initiated 2 weeks before mating and continued through day 18 of gestation. OXC was administered to pregnant dams by gavage on day 6 of gestation and continued through day 18 of gestation. Maternal plasma concentrations of CBZE ranged from 1.4 to 17.7 micrograms/ml and OXC ranged from 6.1 to 15.9 micrograms/ml. In comparison, clinical plasma concentrations of CBZE ranged from 1 to 2 micrograms/ml and OXC plasma concentrations were 1 microgram/ml or less. The incidence of malformation were 14%, 27% and 26% after daily CBZE doses of 300, 600 and 1000 mg/kg, respectively, compared with a 6% incidence in no-drug control mice, P < .05. The incidence of malformation was 8% after exposure at the highest tolerable dose of OXC (1100 mg/kg/day), compared with a 5% incidence in no-drug controls, P > .05. Phenobarbital cotreatment (45 mg/kg/day) with OXC (1100 mg/kg/day) did not lead to changes in the incidence of malformation when compared with OXC (1100 mg/kg/day) dosed alone. These data are consistent with a teratogenic CBZ metabolite, possibly CBZE, or with oxidation of CBZE or CBZ at positions on the aromatic ring leading to the formation of reactive intermediates such as arene oxides or quinones.

Metabolism of lisofylline and pentoxifylline in human liver microsomes and cytosol.

The metabolism of lisofylline and pentoxifylline were examined in cytosol and microsomes prepared from four human livers to determine whether pentoxifylline is likely to serve as an efficient prodrug for the more active inhibitor of phosphatidic acid-dependent cell signaling, lisofylline, and to determine the extent to which lisofylline is converted to pentoxifylline, a hemorheologic agent used for the treatment of intermittent claudication. Pentoxifylline is exclusively reduced to the optical antipode of lisofylline (S M-1) in human liver cytosol, whereas the reduction in microsomes is 85% stereoselective in favor of S M-1 formation. The intrinsic clearance (Vmax/KM) of S M-1 formation in cytosol was 4 times that in microsomes. In human liver microsomes, S M-1 is exclusively converted to pentoxifylline, whereas approximately 45% of lisofylline oxidation is accounted for by the formation of pentoxifylline and the balance by aliphatic diols. It is concluded that pentoxifylline is an inefficient prodrug for delivery of lisofylline and that formation of pentoxifylline accounts for approximately 40% of the microsomal metabolites formed from lisofylline at substrate concentrations likely to be encountered in human therapeutic applications.

Conditioning regimen-dependent disposition of cyclophosphamide and hydroxycyclophosphamide in human marrow transplantation patients.

PURPOSE: The pharmacokinetics of cyclophosphamide (CY) and 4-hydroxycyclophosphamide (HCY) were studied in 14 patients being prepared for bone marrow transplantation with either busulfan (BU)/CY (n = 7) or CY/total-body irradiation (TBI) (n = 7) to determine whether exposure to CY and its proximate toxic metabolite HCY is modulated by other agents used in the preparative regimen. PATIENTS AND METHODS: HCY was assayed by a new method that stabilized the metabolite at bedside. In BU/CY patients (who also received phenytoin), CY clearance was 112% greater (P = .0014), half-life 54% less (P = .0027), peak HCY concentration in plasma/CY dose 113% greater (P = .0006), and the ratio of area under the plasma concentration-time curves (AUCs) of HCY to CY 166% greater (P = .0116) than in CY/TBI patients. The ratio of the AUC of HCY/CY dose was 48% greater in BU/CY patients than in CY/TBI patients when one CY/TBI patient with an apparent impaired ability to eliminate HCY was excluded from analysis. In CY/TBI patients, there was an inverse correlation between the AUC of HCY and that of CY (R2 = .740, P = .028). Also, the ratio of the AUC of HCY/CY dose was correlated with the average concentration of BU at steady-state (Css, Bu) (R2 = .646, P = 0.29). Variability in CY and HCY pharmacokinetics among the 14 patients overall was pronounced, with the highest variability (15-fold) observed in the ratio of the AUC of HCY to that of CY. CONCLUSION: Prior administration of BU and/or phenytoin significantly alters exposure to CY and HCY. Interpatient variability in HCY exposure at a given CY dose is substantial.

Protein-reactive metabolites of carbamazepine in mouse liver microsomes.

The character of reactive metabolites formed from carbamazepine (CBZ) was sought in incubations of [14C]CBZ in hepatic microsomes prepared from adult female mice of a strain (SWV/Fnn) susceptible to CBZ-induced teratogenicity. The formation of radio-labeled protein adducts was used as an index of reactive metabolite exposure. A dependence on cytochrome P450 was shown by a requirement for NADPH and inhibition by carbon monoxide, 1-aminobenzotriazole, piperonyl butoxide, and stiripentol. The addition of ascorbic acid, caffeic acid, N-acetylcysteine, and glutathione decreased the rate of binding of the radiolabel from [14C]CBZ to microsomal protein by more than 50%. The addition of glutathione transferases diminished protein adduct formation beyond that seen with glutathione alone. Evidence for the formation of an arene oxide was sought through the use of inhibitors of epoxide hydrolases, including cyclohexene oxide, chalcone oxides (with the addition of cytosol as appropriate), and by the addition of recombinant human soluble and microsomal epoxide hydrolases and recombinant rat microsomal epoxide hydrolase. The microsomal epoxide hydrolases decreased the velocity of 14C-labeled protein adduct formation by approximately 23%, whereas inhibitors had no effect, most likely because of the low native activity of microsomal epoxide hydrolase in mice. Both DT-diaphorase and catechol-O-methyltransferase diminished 14C-labeled protein adduct formation by 54% and 45%, respectively. The data suggest that the major reactive metabolites formed from CBZ by adult female SWV/Fnn liver microsomes are quinones and arene oxides.

Inhibition of sulfamethoxazole hydroxylamine formation by fluconazole in human liver microsomes and healthy volunteers.

Sulfamethoxazole toxicity is putatively initiated by the formation of a hydroxylamine metabolite by cytochromes P450. If this reaction could be inhibited, toxicity may decrease. We have studied--in vitro and in vivo--fluconazole, ketoconazole, and cimetidine as potentially suitable clinical inhibitors of sulfamethoxazole hydroxylamine formation. Both fluconazole and ketoconazole in human liver microsomal incubations competitively inhibited sulfamethoxazole N-hydroxylation, with the inhibitory constant (Ki) values of 3.5 and 6 micromol/L, respectively. Cimetidine exhibited a mixed type of inhibition of sulfamethoxazole hydroxylamine formation in human liver microsomes, with IC 50 values (the concentration required to decrease hydroxylamine formation by 50%) of 80 and 800 micromol/L, the lower value being observed when cimetidine was preincubated with microsomes and reduced nicotinamide adenine dinucleotide phosphate. In an in vivo study in six healthy volunteers the inhibition of the cytochrome P450-mediated generation of the toxic metabolite in the presence of fluconazole was shown by a 94% decrease in the area under the plasma concentration-time curve of sulfamethoxazole hydroxylamine. In contrast, the recovery of hydroxylamine in urine decreased by only 60%. Total clearance of sulfamethoxazole was decreased by 26% by fluconazole, most likely because of the inhibition of unidentified P450 elimination pathways. There was close agreement between the predicted (87%) and observed inhibition (94%) of sulfamethoxazole hydroxylamine formation in vivo. Similarly, there was close agreement between in vivo and in vitro Ki values--1.6 and 3.5 micron/L, respectively.

Metabolism of dapsone to its hydroxylamine by CYP2E1 in vitro and in vivo.

Dapsone toxicity is putatively initiated by formation of a hydroxylamine metabolite by cytochromes P450. In human liver microsomes, the kinetics of P450-catalyzed N-oxidation of dapsone were biphasic, with the Michaelis-Menten constants of 0.14 +/- 0.05 and 0.004 +/- 0.003 mmol/L and the respective maximum velocities of 1.3 +/- 0.1 and 0.13 +/- 0.04 nmol/mg protein/min (mean +/- SEM). Troleandomycin (40 mumol/L) inhibited hydroxylamine formation at 100 mumol/L dapsone by 50%; diethyldithiocarbamate (150 mumol/L) and tolbutamide (400 mumol/L) inhibited at 5 mumol/L dapsone by 50% and 20%, respectively, suggesting that the low-affinity isozyme is CYP3A4 and the high-affinity isozymes are 2E1 and 2C. Disulfiram, 500 mg, 18 hours before a 100 mg oral dose of dapsone in healthy volunteers, diminished area under the hydroxylamine plasma concentration-time curve by 65%, apparent formation clearance of the hydroxylamine by 71%, and clearance of dapsone by 26%. Disulfiram produced a 78% lower concentration of methemoglobin 8 hours after dapsone.

Preferred orientations in the binding of 4'-hydroxyacetanilide (acetaminophen) to cytochrome P450 1A1 and 2B1 isoforms as determined by 13C- and 15N-NMR relaxation studies.

The widely used analgesic/antipyretic agent 4'-hydroxyacetanilide (acetaminophen, APAP) is oxidized by cytochromes P450 to a potent cytotoxin, N-acetyl-p-benzoquinone imine (NAPQI), and a nontoxic catechol, 3',4'-dihydroxyacetanilide (3-hydroxyacetaminophen, 3-OH-APAP). There are marked differences in the ratios of these two products formed from different isoforms of cytochrome P450. For example, the ratio of NAPQI to 3-OH-APAP formed by rat liver CYP1A1 was found to be approximately 3:1, whereas the ratio of the same two products formed by rat liver CYP2B1 was approximately 1:5. Investigations of the binding of APAP to CYP1A1 and CYP2B1 were carried out to assess the possibility that different preferred orientations of APAP in the active sites of these isoforms may, in part, by responsible for their different product selectivities. Although the spectral dissociation constants (Ks congruent to 0.85 mM) and UV-vis binding spectra (type I; absorption minimum congruent to 420 nm, absorption maximum congruent to 390 nm) were similar for interactions of APAP with the two P450 isoforms, NMR longitudinal relaxation times (T1) of APAP nuclei were significantly different. Two isotopically substituted analogs of APAP, [2,3',5'-13C3]-4'-hydroxyacetanilide and 4'-hydroxyacet-[15N]-anilide, were synthesized, and their binding to purified CYP1A1 and CYP2B1 was examined by NMR spectroscopy. Paramagnetic relaxation times (T1p) for each of the labeled nuclei were calculated from the T1 values obtained before (ferric) and after (ferrous-CO) treatment with Na2S2O4 and CO. The Solomon-Bloembergen equation was then used to calculate distances of the isotopically labeled nuclei from the heme iron of each P450 isoform. The results were that the amide nitrogen approaches relatively close to the heme iron in CYP1A1 (3.64 +/- 0.51 A) whereas it is significantly further away (> 4.5 A) in CYP2B1. In contrast, the aryl carbon atoms ortho to the phenolic group of APAP approach closer to the heme iron of CYP2B1 (3.19 +/- 0.12 A) than to the heme iron of CPY1A1 (3.66 +/- 0.30 A). The results are consistent with the hypothesis that CYP1A1 produces NAPQI preferentially because of closer proximity of the heme iron to the amide nitrogen, whereas CYP2B1 produces 3-OH-APAP preferentially because of closer proximity of the heme iron to the phenolic oxygen in this isoform.

Inhibition and induction of cytochrome P4502E1-catalyzed oxidation by isoniazid in humans.

We studied the effect of isoniazid administration on the cytochrome P4502E1-catalyzed elimination of chlorzoxazone and acetaminophen. Isoniazid, 300 mg daily, was administered for 7 days to a group of 10 volunteer slow acetylators. Acetaminophen, 500 mg, and chlorzoxazone, 750 mg, were administered on separate occasions before isoniazid, during the period of isoniazid administration, and after the discontinuation of isoniazid. Isoniazid inhibited the clearance of chlorzoxazone by 58%, as assessed from plasma data, and inhibited the formation of acetaminophen thioether metabolites (a measure of the formation of the hepatotoxin N-acetyl-p-benzoquinone imine and catechol oxidative metabolites of acetaminophen, as determined from their recovery in urine, by 63% and 49%, respectively. Two days after the discontinuation of isoniazid, the clearance of chlorzoxazone was increased over the value before isoniazid by 56%. Acetaminophen thioether but not catechol metabolites were increased by 56% 1 day after the discontinuation of isoniazid and had returned to the pre-isoniazid value 3 days after the discontinuation of isoniazid. We conclude that the time course of the interaction with regard to chlorzoxazone elimination and formation is compatible with an inhibition-induction effect of isoniazid on cytochrome P4502E1. The mechanism of this biphasic effect is probably induction by protein stabilization, which results in inhibition of catalytic activity while isoniazid is present.

Activation of acetaminophen-reactive metabolite formation by methylxanthines and known cytochrome P-450 activators.

Previous in vitro studies suggested that caffeine enhanced acetaminophen (APAP) oxidation to N-acetyl-p-benzoquinone imine (NAPQI) by selectively activating the male-specific constitutive cytochrome P-450IIIA2. Monomethylxanthine and dimethylxanthine analogs of caffeine (also metabolites) were studied for their potential effect to accelerate NAPQI formation in various preparations of rat liver microsomes. In contrast to caffeine, none of the mono- and dimethylxanthines (2.5 mM) activated P-450. Rather, the analogs either inhibited NAPQI formation or had no effect; 1-methylxanthine (2.5 mM) was the only compound which consistently inhibited (25-70%) APAP oxidation in all microsomal preparations. Thus, all three methyl groups appear to be required for P-450 activation by methylxanthines. Because of the highly selective activation effect of caffeine, it was of particular interest to determine whether other known P-450 activators could enhance APAP oxidation. Both acetone (400 mM) and flavone (50 microM) accelerated NAPQI formation in all microsomal preparations, whereas metyrapone caused only inhibition. Flavone (50 microM) caused a pattern of activation similar to that observed with 5 mM caffeine (maximal activation of 125-300%), except that NAPQI formation was increased approximately 40% by flavone in microsomes prepared from adult females, whereas no activation was caused by caffeine. Acetone yielded a pattern of P450 activation very different from that of either caffeine or flavone; the maximal degree of activation (3 times control) was observed in microsomes prepared from adult females. In contrast to caffeine and flavone, the degree of activation by acetone in microsomes prepared from juvenile animals was considerably lower (50%) than that observed in adult microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition and activation of acetaminophen reactive metabolite formation by caffeine. Roles of cytochromes P-450IA1 and IIIA2.

Caffeine has previously been shown to diminish or potentiate acetaminophen (APAP) hepatotoxicity in rats, depending on induction state. To elucidate the P-450 forms involved in these divergent effects, rat liver microsomes, prepared after pretreatment with various inducers, were used to examine the effect of caffeine on N-acetyl-p-benzoquinone imine (NAPQI) formation. The addition of caffeine to incubations with 3-methylcholanthrene (MC)-induced microsomes resulted in a biphasic effect on the formation of NAPQI. A 43% decrease in NAPQI formation was observed as caffeine concentration was increased from 0 to 0.5 mM; however, NAPQI formation was accelerated as caffeine concentration increased, exceeding the control (no caffeine) value, at caffeine concentrations greater than 2.5 mM. Incubations with purified P-450IA1 showed that as caffeine concentration increased from 0 to 5 mM, a 50% inhibition was observed with no evidence of acceleration. In contrast to MC microsomes, the addition of caffeine to incubations with uninduced and phenobarbital-induced adult rat microsomes resulted in a marked (3- to 4-fold) acceleration of NAPQI formation with no evidence of inhibition. Caffeine (5 mM) also accelerated NAPQI formation in microsomes isolated from diabetic rats, but to a substantially lesser extent (120%), suggesting a modest (if any) effect on P-450IIE1, a form previously shown to form NAPQI from APAP. Interestingly, caffeine caused a 3- to 4-fold increase in NAPQI formation by juvenile male and female rat microsomes, but no activation was observed with adult female rat microsomes. These results suggested that caffeine activated a member of the cytochrome P-450IIIA subfamily.(ABSTRACT TRUNCATED AT 250 WORDS)