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J T Slattery

Publications and source records attributed to J T Slattery.

At least 37 records · Page 2Linked to original sources
PubMed1997

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.

Aryl Hydrocarbon Hydroxylases↗
PubMed1997

Marrow transplantation for chronic myeloid leukemia: the influence of plasma busulfan levels on the outcome of transplantation.

The influence of busulfan (BU) plasma concentration on outcome of transplantation from HLA identical family members for the treatment of chronic myelogenous leukemia (CML) was examined in 45 patients transplanted in chronic phase (CP) (n = 39) or accelerated phase (AP) (n = 6). All patients received the same regimen of BU, 16 mg/kg orally and cyclophosphamide (CY), 120 mg/kg intravenously. Plasma concentrations of BU at steady state (C(SS)BU) during the dosing interval were measured for each patient. The mean C(SS)BU was 917 ng/mL (range, 642 to 1,749; median, 917; standard deviation, 213). Of patients with C(SS)BU below the median, seven (five of 18 in CP and two of four in AP) developed persistent cytogenetic relapse and three of these patients died. There were no relapses in patients with C(SS)BU above the median. The difference in the cumulative incidence of relapse between the two groups was statistically significant (P = .0003). C(SS)BU was the only statistically significant determinant of relapse in univariable or multivariable analysis. The 3-year survival estimates were 0.82 and 0.64 for patients with C(SS)BU above and below the median (P = .33). There was no statistically significant association of C(SS)BU with survival or nonrelapse mortality, although the power to detect a difference in survival between 0.82 and 0.64 was only 0.24, similarly C(SS)BU above the median was not associated with an increased risk of severe regimen-related toxicity. We conclude that low BU plasma levels are associated with an increased risk of relapse.

Adult↗
PubMed1997

Influence of polymorphic N-acetyltransferase phenotype on the inhibition and induction of acetaminophen bioactivation with long-term isoniazid.

OBJECTIVE: To determine in patients receiving isoniazid prophylaxis whether an increase in the CYP2E1 dependent formation clearance of acetaminophen (paracetamol) to N-acetyl-p-benzoquinone imine (NAPQI) occurs during a normal 24-hour isoniazid dose interval and whether the interaction is dependent on acetylation status. METHODS: Acetaminophen elimination kinetics were determined on four different occasions. Ten subjects were assigned to receive acetaminophen either simultaneously with the 8 am dose of isoniazid or 12 hours after the isoniazid dose. One week later, on the last day of isoniazid therapy, subjects received acetaminophen at the alternate time of day. The control phase acetaminophen administrations were repeated 1 and 2 weeks later, following the initial randomization. Isoniazid acetylation (NAT2) genotype was determined by analysis of genomic DNA obtained from peripheral blood leukocytes. RESULTS: The mean NAPQI formation clearance was inhibited 57% when acetaminophen and isoniazid were coadministered but was unchanged compared with time-matched control when acetaminophen was given 12 hours after the isoniazid dose. However, when data from subjects was segregated according to isoniazid (INH) acetylation phenotype, the mean ratio of NAPQI formation clearances (+INH/-INH) with 8 PM acetaminophen was significantly higher for fast acetylators compared with slow acetylators (1.36 versus 0.68; p = 0.006). CONCLUSIONS: Fast metabolizers of isoniazid appeared to clear the inducer or inhibitor from the active site of CYP2E1 more rapidly, which resulted in an increased formation of NAPQI 12 hours after the isoniazid dose. In contrast, formation of NAPQI for slow isoniazid metabolizers remained inhibited.

Acetaminophen↗
PubMed1997

Human CD34+ cells do not express glutathione S-transferases alpha.

The expression of glutathione S-transferases alpha (GST alpha) in human hematopoietic CD34+ cells and bone marrow was studied using RT-PCR and immunoblotting. The GSTA1 protein conjugates glutathione to the stem cell selective alkylator busulfan. This reaction is the major pathway of elimination of the compound from the human body. Human hematopoietic CD34+ cells and bone marrow do not express GSTA1 message, which was present at a high level in liver, an organ relatively resistant to busulfan toxicity in comparison to bone marrow. Similarly, baboon CD34+ cells and dog bone marrow do not express GSTA1. Human GSTA1 may be useful as a chemoprotective selectable marker in human stem cell gene therapy.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

Mechanism of cytochrome P450 activation by caffeine and 7,8-benzoflavone in rat liver microsomes.

Caffeine and 7,8-benzoflavone activate CYP3A2 in rat liver microsomes. Both activators appear to enhance enzyme activity by an increase in Vmax and to a lesser extent a decrease in Km. Additive effect studies demonstrated that the two activators oppose one another's effect. Electron transfer steps in the cytochrome P450 cycle are involved in the mechanism of cytochrome P450 activation, as indicated by the lack of effect of caffeine or 7,8-benzoflavone on cumene hydroperoxide-supported oxidation of acetaminophen by cytochrome P450. The involvement of cytochrome b5 in the formation of N-acetyl-p-benzoquinone imine (NAPQI) was implicated through a synergistic effect of NADH on the NADPH-supported reaction. Anti-cytochrome b5, but not anti-cytochrome P450 reductase IgG, diminished the activation effect of caffeine on NAPQI formation. Neither antibody altered the effect of 7,8-benzoflavone on NAPQI formation. The impairment of NAPQI formation by cytochrome b5 antibody suggests that cytochrome P450 activation by caffeine but not 7,8-benzoflavone is mediated in part through enhancement of the transfer of the second electron to cytochrome P450 from cytochrome b5.

Animals↗
PubMed1997

Pharmacokinetic consequences of induction of CYP2E1 by ligand stabilization.

Models of the time course of the effect of P450 induction on substrate clearance have previously only considered induction through enhanced synthesis of protein. Induction of CYP2E1 does not always conform to this model, in that many chemicals induce the enzyme through stabilization of the protein apparently by binding to the active site. While such binding protects the enzyme from degradation, it also results in competitive inhibition of substrate clearance. We present a model based on experimental studies of chemical induction of CYP2E1 by ligand stabilization through which this mechanism of induction can be translated into its pharmacokinetic consequence with regard to clearance of substrate. CYP2E1 is considered to be localized in two pools: Pool 1 at which two mechanisms of degradation, fast and slow, operate and pool 2, at which only the slower mechanism operates. Binding of substrate to enzyme in pool 1 stabilizes it from degradation by the fast process, leaving only the slow process. Ligand stabilization therefore results in induction of CYP2E1 as enzyme accumulates as a consequence of unchanged synthesis. Binding of ligand to the active site results in competitive inhibition of the clearance of substrate. Model-based computer simulations show that the time course of interaction between inhibitor/inducer and substrate can be predicted from knowledge of I/Ki and S/Km and the synthesis and degradation kinetics of CYP2E1. The simulations demonstrate further that as long as inhibitor/inducer administration is not interrupted, the clearance of substrate will always be less than the value observed at low concentration of substrate even if the substrate concentration is raised to displace inhibitor/inducer from the active site. On the other hand, the degree of inhibition of clearance is less than would be seen if induction had not taken place. Clearance of substrate is observed to rise above the value observed in the absence of the inhibitor/inducer only after the inhibitor/inducer concentration declines low enough for substrate to gain access to the active site of the enzyme. The model-based simulations agree with reports of the interaction between isoniazid and acetaminophen in humans.

Acetaminophen↗
PubMed1997

Cytochrome P450 isozymes involved in lisofylline metabolism to pentoxifylline in human liver microsomes.

We describe the kinetics of pentoxifylline formation from lisofylline in human liver microsomes using selective inhibitors of cytochrome P450 isozymes, correlation studies with specific isozyme activities, and cDNA-expressed human CYP1A2 and 2E1. A biphasic model fitted the data best for the formation of pentoxifylline, Km1 = 0.282 +/- 0.135 microM, Vmax1 = 0.003 +/- 0.001 nmol/min/mg protein, Km2 = 158 +/- 42.6 microM and Vmax2 =0.928 +/- 0.308 nmol/min/mg (N = 4). Pentoxifylline formation by the low Km isoform (200 microM lisofylline) required NADPH, was not inhibited by any isozyme-specific P450 inhibitor, and was inhibited only 10% and 20%, respectively, by aminobenzotriazole and N-octamylamine. We concluded that the low Km enzyme was not a cytochrome P450. At 5 microM of lisofylline the CYP1A2 inhibitor, furafylline, inhibited pentoxifylline formation by 58.8%, and the nonspecific CYP2E1 inhibitor, diethyldithiocarbamate, inhibited pentoxifylline formation by 21.7%. When preincubated with furafylline plus diethyldithiocarbamate, inhibition of pentoxifylline formation was increased 71.4%. Microsomal CYP1A2 activity correlated with pentoxifylline formation (r2 = 0.870, p < 0.001). However, CYP2E1 activity did not correlate with pentoxifylline formation (r2 = 0.143, p = 0.181). Baculovirus insect cell expressed human CYP1A2 formed pentoxifylline at 0.987 nmol/min/nmol cytochrome P450 at 5 microM lisofylline. cDNA expressed CYP2E1 did not catalyze formation of pentoxifylline. Diethyldithiocarbamate inhibited pentoxifylline formation by 85.7% in cDNA expressed CYP1A2. We conclude that CYP1A2 is the high affinity enzyme catalyzing pentoxifylline formation from lisofylline.

Cytochrome P-450 CYP1A2↗
PubMed1996

Busulfan-glutathione conjugation catalyzed by human liver cytosolic glutathione S-transferases.

We have examined the catalytic activity of glutathione S-transferases (GST) in the conjugation of busulfan with glutathione (GSH) in human liver cytosol, purified human liver GST, and cDNA-expressed GST-alpha 1-1. Human liver microsomes and cytosol were incubated with 40 microM busulfan and 1 mM GSH. Cytosol catalyzed the formation of the GSH-busulfan tetrahydrothiophenium ion (THT+) in a concentration-dependent manner, whereas microsomes lacked activity. The total and spontaneous rates of THT+ formation increased with pH (pH range, 6.50-7.75), with the maximum difference at pH 7.4. Due to the limited aqueous solubility of busulfan, a K(m) for busulfan was not determined. The intrinsic clearance (Vmax/K(m)) of busulfan conjugation was 0.167 microliter/min/mg with 50-1200 microM busulfan and 1 mM GSH. GSH Vmax and K(m) for busulfan conjugation were 30.6 pmol/min/mg and 312 microM, respectively. Ethacrynic acid (0.03-15 microM) inhibited cytosolic busulfan-conjugating activity with 40 microM busulfan and 1 mM GSH. Enzyme-mediated THT+ formation was decreased 97% by 15 microM ethacrynic acid with no effect on the spontaneous reaction. In incubations with affinity-purified liver GST and GST-alpha 1-1, the intrinsic clearance for busulfan conjugation was 0.87 and 2.92 microliters/min/mg, respectively. Busulfan is a GST substrate with a high K(m) relative to concentrations achieved clinically (1-8 microM).

Antineoplastic Agents, Alkylating↗
PubMed1996

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.

Adult↗
PubMed1996

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.

Adult↗
PubMed1996

Effects of caffeine and theophylline on acetaminophen pharmacokinetics: P450 inhibition and activation.

Metabolism of acetaminophen (APAP) to its reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI) is mediated by cytochrome P450. A pharmacokinetic study was conducted to quantitate changes in the formation clearance (Cl(f)) of NAPQI to assess in vivo the activation and inhibition of NAPQI formation by methylxanthines. Cl(f) of NAPQI was unaltered by methylxanthine administration in saline-pretreated rats. In phenobarbital-induced rats receiving a nontoxic dose of APAP (100 mg/kg i.v.), a single dose of caffeine (100 mg/kg i.p.) co-administered with APAP increased the Cl(f) of NAPQI formation from 0.58 +/- 0.47 to 2.08 +/- 1.1 1 ml/min/kg (P = .01). Unlike caffeine, theophylline (93 mg/kg i.p.) had no effect on the Cl(f) of NAPQI in phenobarbital-induced rats. The increase in the Cl(f) of NAPQI immediately after a single dose of caffeine demonstrates that P450 activation by caffeine can occur in vivo, as we observed previously in microsomes. The same dose of APAP and methylxanthines also was administered to rats induced with methylcholanthrene. The co-administration of either a single dose of caffeine or theophylline diminished the Cl(f) of NAPQI by 86% (P = .01) and 52% (P = .03), respectively. These in vivo results agree with our previous studies of the effects of the methylxanthines on the formation of NAPQI in rat liver microsomes.

Acetaminophen↗
PubMed1996

Busulfan, cyclophosphamide and fractionated total body irradiation for allogeneic marrow transplantation in advanced acute and chronic myelogenous leukemia: phase I dose escalation of busulfan based on targeted plasma levels.

A previous phase I study determined that the maximum tolerated dose (MTD) of busulfan (BU) that could be given with a fixed dose of cyclophosphamide (CY) of 50 mg/kg and total body irradiation (TBI) dose of 12.0 Gy was 7 mg/kg. A phase II study was carried out in patients with advanced myeloid malignancies receiving allogeneic transplants without improvement in outcome as compared to historical controls. In that study, steady-state concentration (Css) of BU in 13 patients receiving a fixed dose of BU varied from 209 to 735 ng/ml. In an attempt to decrease the variability of the Css of BU, a study of targeting specific plasma concentrations was performed. In this study, BU dose was adjusted up or down based on first dose pharmacokinetics. The first dose level evaluated was 7.5 mg/kg with a target BU plasma level of 460 ng/ml. Six patients were entered at this level and the median BU plasma concentration achieved was 410 (range 390-533). One of six patients developed grade 3-4 regimen-related toxicities (RRT). Dose level II was a target of 559 ng/ml with a starting oral dose of BU of 9.6 mg/kg. Twelve patients were entered at this level and median plasma BU level was 548 (range 427-689). Three of 12 (25%) patients developed grade 3-4 RRTs and this was considered to be the MTD. The actuarial probability of grade II-IV acute GVHD was 0.70. Eight of 21 evaluable patients (38%) developed chronic GVHD. Of 18 patients who died, seven died of relapse at a median of 160 days (range 65-353) and 11 (61%) died of causes other than relapse at a median of 152 days (range 18-570). The actuarial probabilities of DFS, relapse and relapse-free mortality at 2 years in all patients were 0.13, 0.50 and 0.75, respectively. This study showed that targeted BU plasma levels within 10% of target can reliably be achieved with a bias of -2.07% and mean absolute error of 7.47%. Overall, targeting made a -31.8% to 100% in plasma BU Css as compared to expected BU Css based on first dose pharmacokinetics if targeting were not performed in this study. Thus targeting avoided much of the variability in BU concentrations seen in other studies. When compared with our previous phase II experience in same group of patients receiving same regimen, dose escalation of BU based on targeted plasma levels did not improve the outcome.

Adolescent↗
PubMed1996

Busulfan, cyclophosphamide and fractionated total body irradiation for autologous or syngeneic marrow transplantation for acute and chronic myelogenous leukemia: phase I dose escalation of busulfan based on targeted plasma levels.

In a previous phase I study, it was concluded that tolerable doses of busulfan (BU), cyclophosphamide (CY) and total body irradiation (TBI) were 8 mg/kg, 60 mg/kg and 12.0 Gy, respectively, for autologous marrow transplant recipients. In an attempt to decrease the variability of BU steady-state concentration (Css) following oral dosing, a BU dose escalation study based on targeted plasma levels was performed in patients receiving autologous transplants for AML or syngeneic transplants for CML. In this study, the BU dose was adjusted up or down based on observed plasma concentration. All patients received a fixed dose of CY 60 mg/kg and TBI of 12 Gy. The first dose level evaluated was 8.6 mg/kg with a target BU Css of 511 ng/ml. Eight patients were entered at this level and the median BU Css achieved was 441 (range 253-566). One of eight patients developed grade 3-4 regimen-related toxicities (RRT). The oral dose of BU for dose level II was 10.6 mg/kg with a target Css of 632 ng/ml. Six patients were entered at this level and median BU Css achieved was 642 (range 566-674). One of six patients developed grade 3-4 RRT. The oral dose for dose level III was 12.6 mg/kg with a target BU Css of 754 ng/ml. Five patients with AML were entered at this dose level and the median plasma BU Css was 733 ng/ml (682-900). Two of five (40%) patients at dose level III developed grade 3-4 RRT which was considered excessive making dose level II the MTD. This study showed that targeted BU Css can reliably be achieved with a bias of -5.23% and mean absolute error of 11.3%. Overall, targeting made a -32.5% to 158.3% change in plasma BU Css as compared to expected BU Css based on first dose pharmacokinetics if targeting were not performed in this study. Thus, targeting avoided much of the variability in BU Css seen in other studies and appears to have allowed for an increase in oral dosing from 8 mg/kg to 10.6 mg/kg. Despite achieving higher and more uniform BU Css, there was no apparent effect on relapse or survival, although the number of patients evaluated was small.

Acute Disease↗
PubMed1996

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.

Animals↗
PubMed1996

Busulfan conjugation by glutathione S-transferases alpha, mu, and pi.

Busulfan is eliminated by glutathione S-transferase (GST)-catalyzed conjugation with glutathione (GSH). We have characterized the busulfan-conjugating activity of purified human liver GSTA1-1, GSTA1-2, GSTA2-2, GSTM1-1, and placental GSTP1-1. Isoforms were purified from cytosol by GSH-affinity chromatography and chromatofocusing. In addition, the busulfan-conjugating activity of cDNA-expressed GTH1 and GTH2, corresponding to GSTA1-1 and GSTA2-2, were characterized. The major product of busulfan conjugation, a thiophenium ion (THT+), was assayed by GC/MS after conversion to tetrahydrothiophene (THT). THT+ formation rate increased linearly with busulfan concentration up to its solubility limit for all GST isoforms. Because Vmax and KM could not be determined separately, the slope of the velocity vs. substrate concentration plot, Vmax/KM was used to compare isoform activities. Vmax/KM for GSTA1-1 was 7.95 microliters/min/mg protein, the highest busulfan-conjugating activity of all human liver and placenta isoforms evaluated. GSTM1-1 and GSTP1-1, respectively, had 46% and 18% of the activity of GSTA1-1. Since the polymorphic mu-class GST catalyzed busulfan conjugation, we examined busulfan clearance in 50 patients undergoing high-dose busulfan before bone marrow transplantation. Busulfan clearance was normally distributed, suggesting that GSTM1-1 does not contribute significantly to the elimination of busulfan from the body. We conclude that GSTA1-1 is the major isoform catalyzing busulfan conjugation, whereas GSTM1-1 and GSTP1-1 may be important in the protection of specific cells.

Adolescent↗
PubMed1996

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.

Chromatography, High Pressure Liquid↗