Inhibition of human erythrocyte and leukocyte aldehyde dehydrogenase activities by diethylthiocarbamic acid methyl ester. An in vivo metabolite of disulfiram

The inhibitory effects of diethylthiocarbamic acid methyl ester (DTC-Me), an in vivo metabolite of disulfiram (Antabuse), on the aldehyde dehydrogenase (ALDH; EC 1.2.1.3) activities in human erythrocytes and leukocytes were studied. ALDH assays were performed by incubating intact isolated blood cells in the presence of different concentrations of DTC-Me, using 3,4-dihydroxy-phenylacetaldehyde, the aldehyde derived from dopamine, as the substrate. DTC-Me was more selective as inhibitor of the leukocyte ALDH activity (which resembles the liver "mitochondrial" low Km ALDH), whereas both disulfiram and diethyldithiocarbamic acid, the reduced monomer of disulfiram, were more selective for the erythrocyte ALDH (which is similar to the "cytosolic" high-Km ALDH). Diethylthiocarbamic acid, the free acid of DTC-Me, was less potent than DTC-Me, and caused similar inactivation of the erythrocyte and leukocyte ALDH activities. The inhibition of ALDH by DTC-Me could not be completely restored by extensive dilution of intact or sonicated blood cell samples, which indicated that ALDH was irreversibly inhibited. Since the inhibition patterns with DTC-Me agrees with the previously reported patterns of inhibition of the high-Km and low-Km isozymes after the administration of disulfiram, the results suggest that DTC-Me might be the active in vivo inhibitory metabolite of disulfiram.     

Differences in kinetic characteristics and in sensitivity to inhibitors between human and rat liver alcohol dehydrogenase and aldehyde dehydrogenase

1. On the basis of kinetic properties and sensitivity to pyrazole inhibition, it is shown that liver alcohol dehydrogenase present in human mainly corresponded to class I and in rat to class ADH-3 which differed in a number of parameters. 2. Two different aldehyde dehydrogenase (ALDH) isoenzymes were detected in both human and rat liver. The human isoenzymes corresponded to the ALDH-I and ALDH-II type. 3. In the rat, one isoenzyme had low Km and showed similar activity than in human liver but differed in their sensitivity to both disulfiran and nitrofazole inhibition whereas the other presented high Km and showed greater activity than the human one. 4. Caution must be therefore paid when extrapolating to human subjects the data on ethanol metabolism obtained with rats.     

Effects of disulfiram, cyanamide and 1-aminocyclopropanol on the aldehyde dehydrogenase activity in human erythrocytes and leukocytes

The effects of the aldehyde dehydrogenase (ALDH; EC 1.2.1.3) inhibitors disulfiram, cyanamide and 1-aminocyclopropanol (ACP) on the ALDH activities in human erythrocytes and leukocytes were studied. Assays were performed by incubating intact or sonicated blood cells in the presence of different concentrations of the inhibitors, using 3,4-dihydroxyphenylacetaldehyde, the aldehyde derived from dopamine oxidation, as the substrate. The amount of acid metabolite formed was measured using high-performance liquid chromatography with electrochemical detection. The erythrocyte ALDH was extremely sensitive to disulfiram, and only about 0.5 microM was needed to cause a 50% inhibition of the activity. The leukocyte activity was less sensitive, and showed a similar degree of inhibition at an 100-fold higher concentration of disulfiram. Cyanamide and ACP were both potent inactivators of the leukocyte ALDH activity, giving a 50% inhibition at concentrations of 10 and 50 microM, respectively, whereas the erythrocyte activity was much less affected. Diethyldithiocarbamate, the reduced metabolite of disulfiram, and coprine, from which ACP is derived, were much less effective inhibitors of the erythrocyte and leukocyte ALDH activities than were disulfiram and ACP.     

Distribution of disulfiram and its chief metabolites over erythrocyte cell membranes and inactivation of erythrocyte aldehyde dehydrogenase activity

The distribution of disulfiram (Antabuse over erythrocyte cell membranes and the inhibitory action of the parent drug and its metabolites on a disulfiram sensitive erythrocyte isozyme of aldehyde dehydrogenase (ALDH) was investigated in intact and haemolyzed human erythrocytes. These studies showed that not only disulfiram but also its bis(diethyldithiocarbamato) copper complex (Cu(DDC)2) were distributed over the erythrocyte cell membranes. In addition, disulfiram was the only substance examined that inactivaed erythrocyte ALDH, a reaction which was dependent on the concentration of disulfiram added.     

Activities of xenobiotic metabolizing enzymes in rat placenta and liver in vitro

In order to assess whether the placental metabolism of xenobiotic compounds should be taken into consideration for physiologically-based toxicokinetic (PBTK) modelling, the activities of seven phase I and phase II enzymes have been quantified in the 18-day placenta of untreated Wistar rats. To determine their relative contribution, these activities were compared to those of untreated adult male rat liver, using commonly accepted assays. The enzymes comprised cytochrome P450 (CYP), flavin-containing monooxygenase (FMO), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), esterase, UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST). In contrast to liver, no activities were measurable for 7-ethylresorufin-O-dealkylase (CYP1A), 7-pentylresorufin-O-dealkylase (CYP2B), 7-benzylresorufin-O-dealkylase (CYP2B, 2C and 3 A), UGT1, UGT2 and GST in placenta, indicating that the placental activity of these enzymes was well below their hepatic activity. Low activities in placenta were determined for FMO (4%), and esterase (8%), whereas the activity of placental ADH and ALDH accounted for 35% and 40% of the hepatic activities, respectively. In support of the negligible placental CYP activity, testosterone and six model azole fungicides, which were readily metabolized by rat hepatic microsomes, failed to exhibit any metabolic turnover with rat placental microsomes. Hence, with the possible exception of ADH and ALDH, the activities of xenobiotic-metabolizing enzymes in rat placenta are too low to warrant consideration in PBTK modelling.     

The metabolism of acetaldehyde and not acetaldehyde itself is responsible for in vivo ethanol-induced lipid peroxidation in rats

A single oral administration of ethanol (5 g/kg) to rats induced a marked increase in lipid peroxidation, in the liver and kidney within 9 hr, as assessed by malondialdehyde accumulation. The pretreatment with alcohol dehydrogenase (ADH) inhibitor, 4-methylpyrazole (1 mmol/kg) caused approximately 50% inhibition of the hepatic ADH activity and abolished this ethanol-induced lipid peroxidation. The disulfiram treatment (100 mg/kg) significantly inhibited 63% of the hepatic low Km aldehyde dehydrogenase (ALDH) but not the high Km ALDH. The cyanamide treatment (15 mg/kg) effectively decreased 83% of the low Km and 70% of the high Km ALDH in the liver. Although there was more than a 20-fold elevation of acetaldehyde levels by the inhibition of acetaldehyde metabolism with disulfiram or cyanamide, the ethanol-induced lipid peroxidation was significantly suppressed by pretreatment with these drugs. More than 90% inhibition of xanthine oxidase and dehydrogenase by the pretreatment with allopurinol (100 mg/kg), with no effect on the hepatic ADH and ALDH activities, did not alter the enhancement of lipid peroxidation following ethanol administration. We propose that the metabolism of acetaldehyde (probably via the low Km ALDH) and not acetaldehyde itself is responsible for the ethanol-induced lipid peroxidation in vivo and that the contribution of xanthine oxidase, as an initiator of lipid peroxidation through acetaldehyde oxidation is minute during acute intoxication.     

Ontogeny of the activity of alcohol dehydrogenase and aldehyde dehydrogenases in the liver and placenta of the guinea pig

The objectives of this study were to elucidate the ontogeny of the activity of alcohol dehydrogenase (ADH), low Km aldehyde dehydrogenase (ALDH) and high Km ALDH in the liver and placenta of the guinea pig, and to determine the relationship between the relative activity of each enzyme in the guinea pig maternal-placental-fetal unit and the disposition of ethanol and its proximate metabolite, acetaldehyde. The enzyme activities were determined in maternal liver, fetal liver, and placenta of the guinea pig at 34, 50, 60 and 65 days of gestation (term, about 66 days), in the liver of the 2-day-old neonate, and in adult liver. There was low ADH activity in fetal liver and placenta throughout gestation and in neonatal liver. The fetal liver low Km ALDH activity increased progressively and, at 60 days of gestation, was similar to adult liver activity, as was also the case for neonatal liver enzyme activity. Placental low Km ALDH activity was less than adult liver activity throughout gestation. Fetal hepatic high Km ALDH activity increased during gestation, but was less than adult liver activity, as was also the case for neonatal liver enzyme activity. Placental high Km ALDH activity was low throughout gestation. For oral administration of 0.5 g ethanol/kg maternal body weight to pregnant guinea pigs at mid-gestation (34 days), the maternal blood and fetal body ethanol concentration-time curves were similar. Acetaldehyde was measurable in maternal blood and fetal body at similar concentrations, which were 100- to 1000-fold less than the respective ethanol concentrations. The major difference in the disposition of ethanol and acetaldehyde at near-term pregnancy, compared with mid-gestation, was the lack of measurable acetaldehyde in fetal blood. These results indicate that the guinea pig fetus throughout gestation has virtually no capacity to oxidize ethanol, and its duration of exposure to ethanol is regulated by maternal hepatic ADH-catalyzed biotransformation of ethanol. The fetus, however, appears to have increasing low Km ALDH-dependent capacity to oxidize ethanol-derived acetaldehyde during development, and would appear to be increasingly protected from exposure to acetaldehyde as gestation progresses.     

A micromethod for the determination of the activities of kininases in rat plasma. Kinetics and inhibitory characteristics.

Kininase II (EC 3.4.15.1) (KII) and kininase I (KI) (EC 3.4.12.7) activities of rat plasma were characterized by the hydrolysis of hippuryl-L-histidyl-L-leucine (HHL), hippuryl-L-arginine (HLA) [expressed as carboxypeptidase N1 (CN1) activity] and hippuryl-L-lysine (HLL) [expressed as carboxypeptidase N2 (CN2) activity]. Using a spectrophotometric assay, biochemical characteristics of the three enzymes were investigated. The Michaelis-Menten constants were as follows: KII: Km 2.55 +/- 0.22 mM, Vmax 0.357 +/- 0.017 mumol/min/mL; CN1: Km 6.93 +/- 0.32 mM, Vmax 0.748 +/- 0.019 mumol/min/mL; and CN2: Km 35.8 +/- 1.52 mM, Vmax 13.11 +/- 0.40 mumol/min/mL. EDTA and O-phenanthroline inhibited the three enzyme assays at the same Ki, whereas captopril and 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MERGETPA), allowed for the demonstration of the specificity of each assay. Furthermore, Ki values of MERGETPA against both CN1 (4.75 microM) and CN2 (2.36 microM) activities do not support the hypothesis that KI activity may be accounted for by the presence of isoenzymes in rat plasma.     

Disulfiram metabolism as a requirement for the inhibition of rat liver mitochondrial low Km aldehyde dehydrogenase.

In humans and animals, disulfiram produces a disulfiram-ethanol reaction after an ethanol challenge, the basis of which is the inhibition of liver aldehyde dehydrogenase (ALDH). Disulfiram and the metabolites diethyldithiocarbamate (DDTC), diethyldithiocarbamate-methyl ester (DDTC-Me), and S-methyl-N,N-diethylthiolcarbamate (DETC-Me) were studied in order to determine the role of bioactivation in disulfiram's action as an inhibitor of rat liver mitochondrial low Km ALDH (RLM low Km ALDH). In in vitro studies, disulfiram and DDTC (0.01 to 2.0 mM) both inhibited RLM low Km ALDH in a concentration-dependent manner. The addition of rat liver microsomes to the mitochondrial incubation did not further increase disulfiram-induced RLM low Km ALDH inhibition. However, DDTC-induced RLM low Km ALDH inhibition was increased further, but only at DDTC concentrations less than 0.05 mM. DDTC-Me and DETC-Me (2.0 mM) similarly exhibited an increased RLM low Km ALDH inhibition after the addition of liver microsomes. In in vivo studies, disulfiram (75 mg/kg), DDTC (114 mg/kg), DDTC-Me (41.2 mg/kg) or DETC-Me (18.6 mg/kg) administered i.p. to female rats inhibited RLM low Km ALDH. Inhibition of drug metabolism by pretreatment of rats with the cytochrome P450 inhibitor N-octylimidazole (NOI) (20 mg/kg, i.p.) prior to either disulfiram, DDTC, DDTC-Me or DETC-Me administration blocked the inhibition of RLM low Km ALDH. The in vitro and in vivo data support the conclusion that bioactivation of disulfiram to a reactive chemical species is required for RLM low Km ALDH inhibition and a disulfiram-ethanol reaction.     

Comparative subcellular distribution of aldehyde dehydrogenase in rat,mouse and rabbit liver

The subcellular distribution of hepatic aldehyde dehydrogenase (ALDH) activity was determined in Buffalo, Fischer 344, Long-Evans, Sprague-Dawley, Wistar and Purdue/Wistar rats. These subcellular distributions were compared to the distribution of mouse and rabbit liver ALDH. For the six rat strains, at millimolar propionaldehyde concentrations, NAD-dependent ALDH activity was associated primarily with mitochondria (51%) and microsomes (30%). At millimolar acetaldehyde concentrations, NAD-dependent ALDH was primarily mitochondrial (up to 80%). Less than 1% of total NAD-dependent aldehyde dehydrogenase was found in the cytosol. The highly inbred Purdue/ Wistar line possessed significantly less acetaldehyde-NAD ALDH activity as well as less total NADP-dependent ALDH activity than the other strains. In CD-1 mouse liver, millimolar K_m NAD-dependent ALDH activity was found in mitochondria (60%), microsomes (23%) and cytosol (5%). In rabbit liver, millimolar K_m NAD-dependent ALDH was also distributed among mitochondria (36%), microsomes (19%) and cytosol (28%). At micromolar substrate concentrations, mitochondria possessed the majority of rat, mouse and rabbit liver ALDH activity. In all three species, NADP-dependent ALDH activity was found predominantly in the microsomal fraction (up to 65%). The cytosol possessed little NADP-dependent ALDH in any species. We conclude that there are significant species differences in the subcellular distribution of aldehyde dehydrogenase between rat, mouse and rabbit liver. In all three species, mitochondria and microsomes possessed the majority of hepatic aldehyde dehydrogenase activity. However, the cytosol of mouse and rabbit liver also made a significant contribution to total ALDH activity. For the six rat strains examined, liver cytosol possessed little or no ALDH activity.     

A comparative study on the effects of disulfiram and beta-lactam antibiotics on the acetaldehyde-metabolizing system in rats

Several beta-lactam antibiotics, especially those containing N-methyltetrazolylthiomethyl groups at the 3-position of the cephalosporin nucleus, affect the alcohol-metabolizing system in rats. These effects were compared those with disulfiram, well-known as a potent inhibitor of aldehyde dehydrogenase (ALDH). Both disulfiram and antibiotics containing the N-methyltetrazolylthiomethyl group inhibited both mitochondrial low Km ALDH and acetaldehyde oxidation in rat livers. The high Km ALDH and alcohol dehydrogenase activities in livers were not affected by these treatments. When ethanol was given to rats pretreated with disulfiram or these antibiotics, the blood acetaldehyde concentration increased markedly concomitant with a decrease in activity of the low Km ALDH. Administration of N-methyltetrazolethiol alone suppressed the low Km enzyme activity and also increased the blood acetaldehyde level; both effects were pronounced and observed several hours after administration. beta-Lactam antibiotics without N-methyltetrazolethiol in their molecule did not affect the liver mitochondrial enzyme activity or the blood acetaldehyde level.     

7-Alkoxyquinoline O-dealkylation by microsomes from human liver and placenta.

1. The O-dealkylation of seven 7-alkoxyquinoline derivatives by human hepatic and placental microsomes and the effect of maternal cigarette smoking on placental 7-alkoxyquinoline metabolism was studied. 2. None of several monoclonal antibodies to isoenzymes of cytochrome P450 had a clear effect on metabolism of the compounds by liver microsomes. 3. Maternal cigarette smoking induced the O-dealkylation of all of the 7-alkoxyquinoline derivatives, being greatest for 7-butoxy- and 7-benzyloxyquinoline. 4. Placental 7-alkoxyquinoline metabolism induced by smoking was partially inhibited by the monoclonal antibody 1-7-1 raised against 3-methylcholanthrene-induced rat liver P450. 5. None of the 7-alkoxyquinoline O-dealkylations could be assigned specifically to any known P450 isoenzyme in human liver or placenta.     

Dose-effect relationship of disulfiram in human volunteers. II: A study of the relation between the disulfiram-alcohol reaction and plasma concentrations of acetaldehyde, diethyldithiocarbamic acid methyl ester, and erythrocyte aldehyde dehydrogenase activity.

The study was designed to elucidate the basic pharmacological and biochemical effects of the disulfiram dose (Antabus) provoking disulfiram-alcohol reaction (DAR) in 52 human volunteers after ethanol challenge. Disulfiram was given daily in increasing doses (1, 100, 200, and 300 mg) in successive 14 day periods, with ethanol challenge at the end of each period, until a DAR was achieved. Irrespective of dose (except the 1 mg dose), the DAR was always accompanied by almost complete inactivation (about 97%) of aldehyde dehydrogenase (ALDH) activity in erythrocytes, plasma concentrations of diethyldithiocarbamic acid methyl ester (Me-DDC) in the range of 8-472 nmol/l and accumulated plasma concentrations of acetaldehyde in the range of 7-197 mumol/l. In four of the volunteers, the cardiovascular effects of the DAR were recorded as a decrease in diastolic blood pressure (14-47 mmHg) and an increase in pulse rate (9-40 beats/min.), accompanied by a two- to fourfold increase in the plasma concentrations of adrenaline and noradrenaline. The enzyme kinetics of ALDH in erythrocytes were regularly analysed in eight volunteers during DSF intake. In addition to the expected decrease in oxidizing capacity, the Km values were also impaired, which suggests that the inhibitor is implicated in an active site directed reaction.     

In vitro and in vivo inhibition of rat liver aldehyde dehydrogenase by S-methyl N,N-diethylthiolcarbamate sulfoxide, a new metabolite of disulfiram.

In summary, these data provide the first evidence that DETC-MeSO is a natural metabolite of disulfiram, and a potent inhibitor of rat liver mitochondrial low Km ALDH both in vitro and in vivo. It is therefore proposed that, based upon evidence to date, DETC-MeSO appears to be the chemical species to which disulfiram must be bioactivated, and is the metabolite most likely responsible for disulfiram's inhibition of rat liver mitochondrial low Km ALDH in vivo. Characterization of the properties of DETC-MeSO as the metabolite responsible for disulfiram's action as an ALDH inhibitor is presently in the process of being completed.     

Catalytic activity and inhibition of carbonic anhydrase of rat tissues

Carbonic anhydrase activity was studied in stomach and kidney homogenates, and isoenzymes were purified from erythrocytes and livers of male and female rats. Two liver isoenzymes of male and female rats and one erythrocyte isoenzyme had low CO₂ hydration activity. The enzymes of stomach and kidney, one isoenzyme of erythrocytes and one of female rat liver had high CO₂ hydration activity. The esterase activity toward p-nitrophenyl acetate paralleled the CO₂ hydration activity of all the isoenzymes. However, the esterase activity toward β-naphthyl acetate was either absent or did not show any correlation with the CO₂ hydration activity of isoenzymes. Male rat liver carbonic anhydrases were 1000 times less sensitive to sulfonamides than female rat liver carbonic anhydrases for the inhibition both of CO₂ hydration and esterase activity. Male rat liver carbonic anhydrases were as sensitive to inhibition by monovalent anions as were the female rat liver carbonic anhydrases. It is concluded that the active site of carbonic anhydrases from male rat liver is more hydrophilic than the active site of carbonic anhydrase from female rat liver or other tissues of the rat.     

Glucuronidation of DRF-6574, hydroxy metabolite of DRF-4367 (a novel COX-2 inhibitor) by pooled human liver, intestinal microsomes and recombinant human UDP-glucuronosyltransferases (UGT): role of UGT1A1, 1A3 and 1A8.

DRF-4367 is a novel COX-2 inhibitor, which showed good efficacy in several animal models of inflammation. In a comparative in vitro metabolism in various liver microsomes, DRF-4367 forms a hydroxy metabolite (DRF-6574) mediated by CYP2D6 and 2C19 isoenzymes. DRF-6574 readily undergoes Phase-II metabolism and forms glucuronide and sulfate conjugates both in vitro and in vivo. The objective of the present study was two folds: to study the glucuronidation of DRF-6574 in human liver and intestinal microsomes and to identify the recombinant human liver and intestinal UDP-glucuronosyltransferase (UGT) enzymes responsible for glucuronidation of DRF-6574. Of twelve recombinant UGTs tested, two hepatic UGTs viz., UGT1A1 and 1A3 and an extra hepatic UGT i.e., UGT1A8 showed the catalytic activity. The enzyme kinetics in pooled human liver, intestinal and recombinant UGT microsomes showed a typical Michaelis-Menten plot. The apparent Km and Vmax value for DRF-6574 was found to be 116 +/- 24 microM and 2.07 +/- 0.12 microg/min/mg protein and 142 +/- 17 microM and 3.83 +/- 0.15 microg/min/mg protein in pooled human liver and intestinal microsomes, respectively. The intrinsic clearance (Vmax/Km) value for DRF-6574 was estimated to be 0.043 and 0.065 ml/min/mg protein, respectively in pooled human liver and intestinal microsomes. Moreover we have determined the Km and Vmax and intrinsic clearance values for specific UGTs viz., UGT 1A1, 1A3 and 1A8. The apparent Km and Vmax values are 23 +/- 7.2 microM, 3.44 +/- 0.17 microg/min/mg protein for UGT1A1, 60 +/- 7.9 microM, 3.67 +/- 0.11 microg/min/mg protein for UGT1A3, 96 +/- 8.0 microM, 2.95 +/- 0.06 microg/min/mg protein for UGT1A8. The intrinsic clearance values (Vmax/Km) estimated were 0.367, 0.148, 0.074 ml/min/mg protein for UGT1A1, 1A3 and 1A8, respectively. The intrinsic clearance value in UGT1A8 was very close to that in human intestinal and liver microsomes. The formation of DRF-6574 glucuronide by human liver, intestinal and UGT1A1, 1A3 and 1A8 microsomes was effectively inhibited by phenylbutazone.     

Species difference in stereoselective involvement of CYP3A in the mono-N-dealkylation of disopyramide.

1. To determine which CYP isoenzyme is involved in the N-dealkylation of disopyramide (DP) metabolism in human and dog, and to determine the stereoselectivity of DP metabolism with human CYP and dog CYP isoenzymes, the following in vitro metabolism studies of DP were conducted: correlation between human CYP isoenzyme activities and DP metabolism with human liver microsomes; inhibition of DP metabolism in human and dog liver microsomes with chemical inhibitors of CYP isoenzymes; inhibition of DP metabolism in human microsomes with human CYP antibodies; inhibition of DP metabolism in dog liver microsomes with human and dog CYP antibodies; metabolism of DP with human (CYP3A4) and dog (CYP3A12) cDNA-expressed isoenzymes; determination of Km and Vmax of DP enantiomers by using cDNA-expressed CYP3A4 and CYP3A12. 2. In human liver microsomes, the formation of the mono-N-dealkylated disopyramide (MNDP) metabolite was best correlated with CYP3A4 activities. DP metabolism was substantially inhibited by ketoconazole, troleandomycin (TA) and human CYP3A4 antibody. DP was metabolized by cDNA-expressed CYP3A isoenzymes. In dog liver microsomes, DP metabolism was inhibited by ketoconazole, TA and dog anti-CYP3A12. DP was also metabolized by cDNA-expressed CYP3A12. 3. CYP3A4 and CYP3A12 are the principal isoenzymes involved in DP metabolism in human and dog respectively. There was no stereoselectivity in N-dealkylation of DP by human CYP3A4. However, there was notable stereoselectivity in the N-dealkylation by dog CYP3A12.     

Micha?lis--Menten kinetics of phenazone elimination in the perfused pig liver

The purpose of the present study was to define the elimination kinetics of phenazone (NFN) in the isolated perfused pig liver. In five experiments phenazone was administered as constant infusion to obtain steady-state periods over a wide range of concentrations. The elimination of phenazone followed saturation kinetics (concentrations 0.1-12 mmol x 1(-1) and the maximal elimination rate (Vmax) was on average 102 mumol x min-1 x kg-1 liver and the Micha?lis-constant (Km) of 2.6 mmol x 1(-1). Estimates of Vmax and Km for the microsomal phenazone hydroxylase activity measured in liver biopsies found to be considerably lower than in the perfused liver. The hepatic elimination of phenazone during perfusion of pig liver at phenazone concentrations corresponding to human therapeutic doses follows first-order kinetics.     

Relative contributions of CYP2C9 and 2C19 to phenytoin 4-hydroxylation in vitro: inhibition by sulfaphenazole, omeprazole, and ticlopidine

OBJECTIVES: To determine the relative contribution of cytochromes P450 (CYP) 2C9 and 2C19 to the formation of 5-(-4-hydroxyphenyl)-5-phenylhydantion (HPPH) from phenytoin (PPH). DESIGN: Hydroxylation of PPH to form HPPH was studied in vitro using human liver microsomes and microsomes from cDNA-transfected human lymphoblastoid cells. RESULTS: Formation of HPPH from PPH in liver microsomes had a mean (+/- SEM) apparent Km [substrate concentration corresponding to 50% of maximal reaction velocity (Vmax)] of 23.6 +/- 1.8 mumol/l. Coincubation with the CYP2C9 inhibitor, sulfaphenazole (SPA), at 5 mumol/l reduced reaction velocity to less than 15% of control values. The mean inhibitor concentration at which 50% inhibition is achieved (IC50 value) for SPA versus PPH hydroxylation (0.49 microM) was similar to the SPA IC50 versus flurbiprofen hydroxylation (0.46 microM) and tolbutamide hydroxylation (0.7-1.5 microM). In contrast, the CYP2C19 inhibitor omeprazole (OME) at 10 mumol/l produced only a small degree of inhibition. Incubation of PPH with microsomes from cDNA-transfected human lymphoblastoid cells containing CYP1A2, 2A6, 2B6, 2C8, 2D6, 2E1, or 3A4 yielded no detectable formation of HPPH. Only CYP2C9 and 2C19 had PPH hydroxylation activity, with apparent Km values for the high-affinity component of 14.6 mumol/l and 24.1 mumol/l, respectively. Based on Vmax values in liver microsomes, the Vmax and Km values in expressed CYPs and the relative abundance of the two isoforms in human liver, CYP2C9, and 2C19 were estimated to have relative contributions of 90% and 10%, respectively, to net intrinsic clearance. CONCLUSIONS: Formation of HPPH from PPH is mediated exclusively by CYP2C9 and 2C19, with CYP2C9 playing the major role.     

Effect of cefclidin and E1077, new cephalosporins, on the alcohol-metabolizing system in rats]

Administration of latamoxef and cefoperazone, reported to act like disulfiram in humans, caused a depression of mitochondrial low Km aldehyde dehydrogenase (low Km ALDH) activity in rats. In addition, a marked increase of blood acetaldehyde concentration was observed when rats were given alcohol orally at 18 hours after administration of these cephalosporins. However, mitochondrial low Km ALDH activity and blood acetaldehyde level were not altered by repeated administration of 300 mg and 1,000 mg of cefclidin (CFCL, E1040) or E1077 per kg. From these results, it was concluded that neither CFCLn or E1077 affected the alcohol metabolizing-system.