General properties and clinical possibilities of new selective inhibitors of catechol O-methyltransferase

1. The structure of catechol O-methyltransferase (COMT) has been recently characterized and a series of new and selective COMT inhibitors developed.2. Entacapone, nitecapone and tolcapone are nitrocatechol-type potent COMT inhibitors in vitro (K_i in nanomolar range). They are also very selective for COMT and active in vivo even after oral administration. CGP 28014 is a pyridine derivative that is active only in vivo.3. In animal studies, these compounds inhibit effectively the O-methylation of l-dopa, thus improving its bioavailability and brain penetration and potentiating its behavioural effects.4. Entacapone and nitecapone have mainly a peripheral effect whereas tolcapone and CGP 28014 also inhibit O-methylation in the brain.5. In man, entacapone, nitecapone and tolcapone all inhibit dose dependently the COMT activity in erythrocytes. These COMT inhibitors also decrease the amount of COMT dependent metabolites of adrenaline and noradrenaline in plasma.6. In human volunteers, entacapone, tolcapone and CGP 28014 improve the bioavailability of l-dopa and inhibit the formation of 3-O-methyldopa.7. In the first clinical studies in patients with Parkinson's disease, both entacapone and tolcapone potentiate and prolong the therapeutic effect of l-dopa.     

The specificity of glucuronidation of entacapone and tolcapone by recombinant human UDP-glucuronosyltransferases.

The COMT inhibitors entacapone and tolcapone are rapidly metabolized in vivo, mainly by glucuronidation. In this work, the main UGT isoforms responsible for their glucuronidation in vitro were characterized by using a subset of representative cloned and expressed human UGT isoforms. Entacapone in particular was seen to be an exceptionally good substrate for UGT1A9 with an even higher reaction velocity value at 500 microM substrate concentration compared with that of the commonly used substrate, propofol (1.3 and 0.78 nmol min(-1) mg(-1), respectively). Neither entacapone nor tolcapone was glucuronidated by UGT1A6. Tolcapone was not detectably glucuronidated by UGT1A1, and the rate of glucuronidation of entacapone was also low by this isoform. However, UGT1A1 was the only UGT capable of catalyzing the formation of two glucuronides of the catecholic entacapone. Both COMT inhibitors were glucuronidated at low rates by the representative members of the UGT2B family, UGT2B7 and UGT2B15. Michaelis-Menten parameters were determined for entacapone and tolcapone using recombinant human UGT isoforms and human liver microsomes to compare the kinetic properties of the two COMT inhibitors. The kinetic data illustrates that UGT1A9 exhibited a much greater rate of glucuronidation and a far lower K(m) value for both entacapone and tolcapone than UGT2B15 and UGT2B7 whose contribution is minor by comparison. Entacapone showed a 3 to 4 times higher V(max) value and a 4 to 6 times lower K(m) value compared with those of tolcapone both in UGT1A9 cell lysates and in human liver microsomes.     

Glucuronidation Metabolic Kinetics of Valproate in Guinea Pigs: Nonlinear at Clinical Concentration Levels

Purpose. Nonlinear conjugation metabolic rate of valproic acid (VPA) has been speculated previously from plasma elimination and liver concentration of VPA in guinea pigs. The purposes of the present study were to assess our speculation by direct measurement of VPA glucuronidation rate in vitro. Methods. VPA at various concentrations (10–200 µg/ml) was incubated with guinea-pig liver-homogenate, mitochondria or microsome in the presence of cofactor, uridine 5-diphosphoglucuronic acid (UDPGA). The maximum glucuronidation rate (V_max) and Michaelis-Menten constant (K_m) of VPA were determined. Results. On a body weight basis, the V_max and the K_m values of VPA glucuronidation estimated from liver homogenate were 1.8 µmol/min/kg and 0.3 µmol/ml, respectively; and that from microsome suspension were 1.2 µmol/min/kg and 0.16 µmol/ml, respectively. These data are comparable with the primary metabolic parameters observed from previous in vivo study. The glucuronidation clearance calculated from these parameters was 0.10–0.48 fraction of total clearance, which was in agreement with the reported data observed from clinical and animal urinary recoveries of VPA-G. The glucuronidation reaction was not detectable in mitochondria suspension. Conclusions. The glucuronidation kinetics of VPA is nonlinear and saturable within clinical concentration range. Estimation of in vivo VPA glucuronidation kinetics from in vitro kinetic parameters is feasible.     

Assessment of UDP-glucuronosyltransferase catalyzed formation of Picroside II glucuronide in microsomes of different species and recombinant UGTs

1. This study compared the hepatic glucuronidation of Picroside II in different species and characterized the glucuronidation activities of human intestinal microsomes (HIMs) and recombinant human UDP-glucuronosyltransferases (UGTs) for Picroside II.

2. The rank order of hepatic microsomal glucuronidation activity of Picroside II was rat > mouse > human > dog. The intrinsic clearance of Picroside II hepatic glucuronidation in rat, mouse and dog was about 10.6-, 6.0- and 2.3-fold of that in human, respectively.

3. Among the 12 recombinant human UGTs, UGT1A7, UGT1A8, UGT1A9 and UGT1A10 catalyzed the glucuronidation. UGT1A10, which are expressed in extrahepatic tissues, showed the highest activity of Picroside II glucuronidation (K_m?=?45.1 μM, V_max?=?831.9 pmol/min/mg protein). UGT1A9 played a primary role in glucuronidation in human liver microsomes (HLM; K_m?=?81.3 μM, V_max?=?242.2 pmol/min/mg protein). In addition, both mycophenolic acid (substrate of UGT1A9) and emodin (substrate of UGT1A8 and UGT1A10) could inhibit the glucuronidation of Picroside II with the half maximal inhibitory concentration (IC₅₀) values of 173.6 and 76.2 μM, respectively.

4. Enzyme kinetics was also performed in HIMs. The K_m value of Picroside II glucuronidation was close to that in recombinant human UGT1A10 (K_m?=?58.6 μM, V_max?=?721.4 pmol/min/mg protein). The intrinsic clearance was 5.4-fold of HLMs. Intestinal UGT enzymes play an important role in Picroside II glucuronidation in human.

     

Cis-resveratrol glucuronidation kinetics in human and recombinant UGT1A sources

1. It was hypothesized that cis-resveratrol glucuronidation contributes to a greater extent to in-vitro disposition of total resveratrol than previously assumed. To this end, the kinetic data for cis-resveratrol glucuronidation are reported.

2. Glucuronidation assays were conducted in human liver and intestinal microsomes and in uridine diphosphate-glucuronosyltransferases (UGTs) UGT1A1, UGT1A6, UGT1A9, and UGT1A10. Kinetic parameters were estimated for the major cis-resveratrol-3-O-glucuronide (cis-R3G). Substrate inhibition was observed with apparent V_max, K_m and K_i of 6.1?±?0.3/27.2?±?1.2 nmol min^?1 mg^?1, 415?±?48.1/989.9?±?92.8 and 789.6?±?76.3/1012?±?55.9?μM in human intestinal microsomes (HIMs) and UGT1A6, respectively (estimate?±?standard error (SE)). Biphasic kinetics were observed in human liver microsomes (HLMs), while sigmoidal kinetics were seen in UGT1A9 (V_max?=?11.92?±?0.2 nmol min^?1 mg^?1; K_m?=?360?μM; n?=?1.27?±?0.07). The 4′-O-glucuronide (cis-R4′G) exhibited atypical kinetics in HLM, HIM, UGT1A1, and UGT1A10. UGT1A9 catalysed cis-R4′G formation at high substrate concentrations (V_max?=?0.33?±?0.015 nmol min^?1 mg^?1; K_m?=?537.8?±?67.8?μM).

3. In conclusion, although the rates of formation of cis-R3G in HLM and UGT1A9 were higher than those for trans-R3G, the contribution to total resveratrol disposition could not be determined fully due to atypical kinetics observed.

     

Synthesis and Evaluation of Heterocyclic Catechol Mimics as Inhibitors of Catechol-O-methyltransferase (COMT)

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Pharmacokinetics and pharmacodynamics after oral and intravenous administration of tolcapone, a novel adjunct to Parkinson's disease therapy

Objective: To evaluate fully the pharmacokinetics and pharmacodynamics of tolcapone, a novel inhibitor of catechol-O-methyltransferase (COMT), after oral and intravenous administration. Methods: Sixteen healthy male volunteers were given tolcapone in single doses of 200 mg orally and 50 mg intravenously, separated by a washout period of 7 days or more, in a single-center, open-label, randomized, cross-over study. Pharmacokinetic parameters were calculated using both compartmental and non-compartmental methods; pharmacodynamics were evaluated from erythrocyte COMT activity. Results: After an initial lag time of 0.5 h, tolcapone was rapidly absorbed (peak plasma concentrations were reached within approximately 2 h) following either zero-or first-order absorption kinetics. The absolute bioavailability of an oral dose was approximately 60%. The volume of distribution was approximately 9 l, and the total clearance was approximately 7 l · h⁻¹, resulting in a mean plasma half-life of 1.8 h. The degree of erythrocyte COMT inhibition was closely related to tolcapone plasma concentration; a rebound in COMT activity was observed after tolcapone withdrawal. Both oral and intravenous tolcapone were well tolerated. Discussion: Because of its relatively low systemic clearance, tolcapone exhibits only a small degree of first-pass metabolism and a relatively good oral bioavailability, which explains the higher plasma concentrations and stronger COMT inhibition observed with tolcapone compared with entacapone, another COMT inhibitor. The pharmacokinetic and pharmacodynamic profile of tolcapone obtained in this study underlines the potential of the agent to be used as an adjunct to levodopa in the treatment of Parkinson's disease. Received: 9 February 1998 / Accepted in revised form: 9 April 1998     

Kinetic properties of UDP-glucuronosyltransferase(S) in different membranes of rat liver cells

1. Glucuronidation of 4-nitrophenol, borneol and morphine occurred in rough and smooth endoplasmic reticulum, Golgi apparatus and plasma membranes of rat liver cells.

2. In all fractions, prior fixation of either substrate (UDP-glucuronic acid or the aglycone) enhanced the affinity for the second substrate.

3. Whatever the membrane, glucuronidation of 4-nitrophenol was characterized by high V_max and high affinity for UDP-glucuronic acid. On the other hand, glucuronidation of borneol exhibited a lower V_max and a lower affinity for UDP-glucuronic acid.

4. In the endoplasmic reticulum, conjugation of morphine had a low V_max but the enzyme had high affinities for both UDP-glucuronic acid and the aglycone.     

Molecular cloning and functional analysis of minipig UDP-glucuronosyltransferase 1A6

Abstract

1.?UDP-glucuronosyltransferase 1A6 (UGT1A6) plays important roles in the glucuronidation of numerous drugs, environmental pollutants, and endogenous substances. Minipigs have been used as experimental animals in pharmacological and toxicological studies because many of their physiological characteristics are similar to those of humans. The aim of the present study was to examine similarities and differences in the enzymatic properties of UGT1A6 between humans and minipigs.

2.?Minipig UGT1A6 (mpUGT1A6) cDNA was cloned by the RACE method, and the corresponding proteins were expressed in insect cells. The enzymatic function of mpUGT1A6 was analyzed by the kinetics of serotonin glucuronidation.

3.?Amino acid homology between human UGT1A6 (hUGT1A6) and mpUGT1A6 was 79.9%. The kinetics of serotonin glucuronidation by recombinant hUGT1A6 and mpUGT1A6 enzymes fit the Michaelis–Menten equation. The K_m, V_max, and CL_int values of hUGT1A6 were 10.5?mM, 4.04?nmol/min/mg protein, and 0.39?µL/min/mg protein, respectively. The K_m value of mpUGT1A6 was similar to that of hUGT1A6, whereas the V_max and CL_int values of mpUGT1A6 were approximately 2-fold higher than those of hUGT1A6.

4.?These results suggest that the enzymatic properties of UGT1A6 enzymes are moderately different between humans and minipigs.     

Species-dependent enantioselective glucuronidation of carprofen

1. The stereoselective glucuronidation of carprofen, a non-steroidal anti-inflammatory drug, was investigated in vitro using microsomes prepared from liver of different species (rat, dog, horse, sheep and man) or UGT2B1 expressed in fibroblasts. 2. The K_m towards the drug was very similar among these species and for the two menantiomers, whereas the V_max varied substantially according to the animal used. The rat exhibited a high stereoselective glucuronidation whereas other species, including man, presented a low stereoselectivity. The R-enantiomer was glucuronidated at a more efficient rate than its enantiomorph, and was a better substrate (in terms of V_max/K_m). 3. To explain the enantioselective disposition of carprofen in man and in the different species,the ratio of the enzymatic efficacies (V_max/K_m) were compared with the ratio of the max m pharmacokinetic parameters AUCs. The basic hypothesis that the intrinsic clearance reflect the enantioselective behaviour of carprofen seemed substantiated when we focused onmanand rat glucuronidation,but the in vivo-in vitro correlationwas not possibleinother species. 4. In conclusion, the chiral pharmacokinetics of carprofen is less dependent on the stereoselective glucuronidationthanotherstereoselective processes such as protein binding of carprofen, enzymatic hydrolysis, or renal elimination of glucuronides.     

Expression and activities of sulfotransferase in rat brain

1. Sulfotransferase (SULT) has been found in the brain; however, the details of its function remain unclear. The present study aimed to elucidate the regional differences in the expression of SULT1 and SULT2 mRNA and SULT activities in the eight functional regions of the rat brain (cerebellum, cortex, hippocampus, medulla oblongata, midbrain, olfactory bulb, striatum, and thalamus).

2. All SULT1 isoforms were detected in the medulla oblongata and thalamus. SULT2A1 mRNA was not observed in any of the eight regions, whereas SULT2B1a and SULT2B1b were found in all regions. The SULT2B1b mRNA expression level in the medulla oblongata was 1.7-fold higher than that in the liver. The sulfonation of p-nitrophenol and pregnenolone was detected in all regions. The kinetics of p-nitrophenol sulfonation in the cerebellum fitted to the substrate inhibition model (K_m?=?37.6?nM, V_max?=?2.72?pmol/min/mg, V_inh =?1.60?pmol/min/mg, and K_i?=?0.87?μM). The pregnenolone sulfonation also exhibited substrate inhibition kinetics (K_m?=?0.99?μM, V_max?=?1.53?pmol/min/mg, and K_i?=?54.67?μM).

3. We clarified that SULT1 and SULT2 were expressed and had metabolizing capacities in the rat brain, suggesting that brain SULTs may be involved in metabolism of endogenous compounds and drugs.

     

The role of physicochemical properties of entacapone and tolcapone on their efficacy during local intrastriatal administration

Aqueous solubility, apparent partition coefficient (log P_app) and catechol-O-methyltransferase (COMT, EC 2.1.1.6) inhibiting potency of entacapone and tolcapone were compared in vitro. Both drugs (at 10 and 100 μM) were also delivered directly into rat striatum via a microdialysis probe. Extracellular 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) concentrations were measured to evaluate the inhibition of striatal COMT in vivo. Although entacapone had 15-fold better aqueous solubility than tolcapone at pH 7.4, also tolcapone had sufficient aqueous solubility to remain in solution at 100 μM. The log P_app of tolcapone was higher than that reported for entacapone in the pH range from 5.0 to 7.4. Entacapone and tolcapone inhibited equally rat striatal COMT in vitro with K_i values of 1.86 and 2.50 nM, respectively. Both drugs had similar outflow from the microdialysis probe in vitro. Perfusion of 100 μM entacapone increased significantly extracellular DOPAC levels compared to the control group. Both entacapone and tolcapone (at 10 and 100 μM) decreased significantly HVA levels, but entacapone was significantly more effective than tolcapone at 100 μM. In conclusion, entacapone and tolcapone are equally potent COMT inhibitors against rat striatal COMT in vitro. After local intrastriatal administration, entacapone appeared to inhibit COMT faster and more effectively than the more lipophilic tolcapone. Thus, intrastriatal administration led to opposite results compared to those reported in the brain after systemic administration. The present results also suggest that the local distribution of entacapone and tolcapone differ when the drugs are delivered directly into the brain.     

Kinetics of in vitro nitro reduction of 2,4-dinitrophenol gy rat liver homogenates

The in vitro metabolism of [¹⁴C]-2,4-dinitrophenol (DNP) was examined in rat liver homogenates (300–500 g males). DNP and the metabolites, 4-amino-2-nitrophenol (4A2NP) and 2-amino-4-nitrophenol (2A4NP) were identified and quantified. The Michaelis-Menten constant, K_m, and the maximum velocity, V_max, were determined for the reduction reaction using a Hofstee plot. The K_m and V_max for DNP reduction were, respectively: 1.8 × 10^?4m and 1.13 nmol/mg protein/min. p-Nitrobenzoic acid (PNBA), o-nitrophenol (ONP), p-nitrophenol (PNP) and 2,4-dinitro-6-sec-butylphenol (DNBP) were examined as inhibitors of DNP nitro reduction. PNBA had no effect, but PNP, ONP and DNBP significantly inhibited DNP nitro reduction. ONP was a competitive inhibitor of DNP reduction while PNP and DNBP were noncompetitive. The K_t values calculated from the regression lines, for ONP, PNP and DNBP respectively, were 5.1 × 10^?4m, 7.1 × 10^?4m.     

Biotransformation of nitrosobenzene,phenylhydroxylamine, and aniline in the isolated perfused rat liver

1. Haemoglobin-free single-pass perfusion of isolated rat liver with [¹⁴C]aniline, [¹⁴C]phenylhydroxylamine, and [¹⁴C]nitrosobenzene was carried out.

2. Perfusion with aniline revealed apparent enzyme kinetics for 4-aminophenol formation with K_m = 144μM, V_max = 51 nmol/min per g liver wet; for 2-aminophenol K_m = 144μM, V_max = 16nmol/min per g; for acetanilide K_m = 33μM, V_max = 25 nmol/min per g. Formation of phenylhydroxylamine and nitrosobenzene was observed at a rate of 1.5 nmol/min per g provided that these metabolites had been trapped within red cells.

3. Perfusion with phenylhydroxylamine displayed a metabolic pattern similar to aniline with apparent phenylhydroxylamine reduction kinetics of K_m = 260μM and V_max = 600 nmol/min per g. In addition an acid-labile phenylhydroxylamine glucuronide was formed.

4. Perfusion with nitrosobenzene showed very rapid reduction to phenylhydroxylamine and to the metabolites observed with phenylhydroxylamine. In postmicrosomal supernatant, enzymic reduction of nitrobenzene by NADH and NADPH showed K_m = 12μM nitrosobenzene and V_max = 5000nmol/min per g.

5. Three per cent of nitrosobenzene was irreversibly bound to liver proteins. After 20min perfusion with nitrosobenzene, 0.95 μmol of liver glutathione was lost per 10μmol nitrosobenzene infused; 0.16μmol of glutathione was released with effusate and bile, 0.46μmol of glutathionesulphinanilide was produced, the rest, 0.33μmol, may have formed mixed disulphides.     

Catechol-O-methyltransferase inhibitors: Clinical potential in the treatment of Parkinson's disease

Substitution therapy with levodopa and a peripheral inhibitor of aromatic L-amino acid decarboxylase (AADC) is the cornerstone in the treatment of Parkinson's disease. Chronic therapy with levodopa, however, is associated with the occurrence of motor complications that are partially related to the pharmacokinetics of levodopa. The formation of 3-O-methyldopa (3-OMD), a long-lived metabolite that confers no clinical benefit, is catalyzed by catechol-O-methyltransferase (COMT), an enzyme that is particularly abundant in the gastrointestinal tract, liver, and kidneys. Potent, reversible, selective, and orally active COMT inhibitors have become available. The nitrocatechols, entacapone and tolcapone, have undergone extensive clinical testing as adjuncts to levodopa in the therapy of Parkinson's disease. Studies in healthy subjects and parkinsonian patients have shown the inhibitory effect of both drugs on the formation of 3-OMD, leading to an increase in levodopa bioavailability and elimination half-life, without a change in peak plasma concentrations (C_max) or time to reach C_max (t_max). In patients, COMT inhibitors significantly prolong and smooth, i.e., reduce peak-trough fluctuations, the effects of levodopa, as reflected in improved motor scores and lengthened duration of ON time (periods of good response) without an influence on the time to onset of action or the peak effect. Entacapone 200 mg is given in conjunction with each levodopa dose, whereas tolcapone is administered in a fixed 100 to 200 mg t.i.d. dosing regimen. The tolerability profiles of the two drugs are similar, with urine discoloration and an increase in dopaminergic side effects such as nausea, orthostatic hypotension, and dyskinesias being most prominent. Tolcapone induces a higher incidence of diarrhea and liver enzyme abnormalities. It is anticipated that COMT inhibitors will prove beneficial in extending and smoothing the effects of levodopa in patients with Parkinson's disease and end-of-dose wearing-off fluctuations. Drug Dev. Res. 42:1–25, 1997. © 1997 Wiley-Liss, Inc.     

The kinetics of the enzymatic O-methylation of catechols and catecholamines

The enzymaticO-methylation of twelve catecholamines and two catechols was studied. From initial reaction rates K_m and V_max were determined. Under the reaction conditions applied the enzymaticO-methylation appears to be first order in the concentration of catechol(amine). For all twelve catecholamines and the two catechols the K_m and V_max based on initial reaction rates are equal to those from the ln(c/c_o) plots (= first-order kinetics). This fact and the variation of k_obs (reaction rate constant in the first-order kinetics) with the starting concentration comply with the formulae derived for these kinetics. The variation of k_obs is caused by product inhibition. From the formulae it follows that K_i (inhibition constant) = K_m; this is confirmed by the experimental data. The polarity of the compounds seems to contribute to the reaction rate at low substrate concentration; the linear relationship between this value and log D may support an apolar region in the binding part of catechol-O-methyltransferase, without denying other contributing factors.     

Characterization of catechol glucuronidation in rat liver.

Catechols are a class of substances from natural or synthetic origin that contain a 1,2-dihydroxybenzene group. We have characterized the glucuronidation by rat liver microsomes and by the rat liver recombinant UDP-glucuronosyltransferase isoforms UGT1A6 and UGT2B1 of a series of 42 structurally diverse catechols, including neurotransmitters, polyphenols, drugs, and catechol estrogens. Small catechols (4-nitrocatechol, 2,3-dihydroxybenzaldehyde, 4-methylcatechol, and tetrachlorocatechol), tyrphostine A23, and octylgallate were glucuronidated at the highest rate by rat liver microsomes and the recombinant enzymes. By contrast, polyphenols from green tea (catechin and related compounds), 3,5-dinitrocatechol, the catechol-O-methyltransferase inhibitor drugs (entacapone, nitecapone, and tolcapone), the carboxyl catechols (gallic acid and dihydroxybenzoic acid derivatives), and the neurotransmitters and dopaminergic drugs, except dobutamine, were glucuronidated at low rate. Glucuronidation of most catechols was increased upon treatment of rats by 3-methylcholanthrene (3-MC) or Aroclor 1254. No induction was observed after administration of phenobarbital and clofibrate or treatment with catechols. Partial least-squares modeling was carried out to explain the variations of glucuronidation activity by liver microsomes of nontreated and 3-MC-treated rats. The model developed explained 82% and predicted 61% of the variations of glucuronidation activities. Among the 17 electronic and substructure parameters used that characterize the catechols, the hydrophobicity/molar volume ratio of catechols showed a strong positive correlation with the glucuronidation rate. The effect of the pK(a) of the catechol group was modeled to be nonlinear, the optimal pK(a) value for glucuronidation being between 8 and 9. Hydrogen bonding and steric effects also were important to account for to predict the glucuronidation rates.     

Deuterium isotope effect on the metabolism of N-nitrosodimethylamine and related compounds by cytochrome P4502E1

1. Deuteration of N-nitrosodimethylamine (NDMA) decreases its carcinogenicity, and produces an isotope effect on its metabolism in vivo. Consistent with these results are the observations that deuteration caused a 5-fold increase in the apparent K_m, but not the V_max the demethylation and denitrozation of NDMA in acetone-induced rat liver microsomes. These microsomes are a good source of cytochrome P4502E1.

2. For demethylation of Z-[²H₃]NDMA and E-[²H₃)NDMA, the K_m values were indistinguishable, and were between the values for those of NDMA and [²H₆]NDMA. Almost all the formaldehyde formed was derived from the non-deuterated methyl group. indicating a lack of stereoselectivity in the demethylation of NDMA.

3. NDMA and [²H₆]NDMA displayed apparent K_i values of 59 and 441 μM, respectively, for N-nitrosodiethylamine deethylase, showing an apparent isotope effect of 0.13, and displayed an isotope effect of 0.21 in the K_i values for p-nitrophenol hydroxylase.

4. With acetone and deuterated acetone as inhibitors for p-nitrophenol hydroxylase, the isotope effect on the K_i was 0.11. Similar deuterium isotope effects were also observed with acetone and dimethylformamide as competitive inhibitors for NDMA demethylase.

5. In the microsomal oxidation of ethanol, a deuterium isotope effect of about five was observed in the V_max/K_m when carbon-1 was deuterated, but was not observed in the V_max.

6. Results illustrate a unique deuterium isotope effect on the K_m values of reactions catalysed by P4502E1.     

Identification of the human liver UDP-glucuronosyltransferase involved in the metabolism of p-ethoxyphenylurea (dulcin)

Dulcin (DL), now banned, was once a widely used artificial sweetener. DL possesses an ureido group that is metabolized by direct glucuronidation in rabbit liver microsomes. Dulcin N-glucuronide (DNG) is the only type of ureido N-glucuronide known to date; ureido glucuronidation in humans has not been previously reported. Accordingly, the glucuronidation of DL was studied using human liver microsomes (HLM) and expressed human UDP-glucuronosyltransferase (UGT) enzymes. The average K _m and V _max values from nine HLM samples were 2.10 mM and 0.156 nmol/mg/min, respectively. Of the six human UGT isoforms screened for their ability to glucuronidate DL, only UGT1A1 and UGT1A9 showed activity. The apparent K _m values using UGT1A1 and UGT1A9 were 5.06 and 6.99 mM, and the apparent V _max values were 0.0461 and 0.106 nmol/min/mg, respectively. Phenolphthalein, a substrate for UGT1A9, inhibited DL glucuronidation in HLM competitively (K _i = 0.356 mM), but bilirubin, a substrate for UGT1A1, did not. These results suggest that UGT1A9 is a key enzyme catalyzing the glucuronidation of DL.     

Glucuronidation in the rat intestinal wall: Comparison of isolated mucosal cells,latent microsomes and activated microsomes

Glucuronidation and sulphation of 1-naphthol and 7-hydroxycoumarin was studied in isolated rat intestinal epithelial cells and in microsomes prepared from these cells. In the isolated cells formation of 1-naphthol sulphate could not be detected. Sulphate conjugates of 7-hydroxycoumarin constitute a minor portion of total conjugates formed. Maximum glucuronidation rates for 1-naphthol and 7-hydroxycoumarin do not differ significantly from each other (approximately 12.5 nmoles/min · g intestine). The intestinal microsomal UDP-glucuronosyltransferase, prepared from isolated cells, could be activated in vitro by Triton X-100 and MgCl₂. Activation increased both K_m^app and V_max for 1-naphthol; K_m^app for UDP-glucuronic acid was decreased by activation with MgCl₂ but increased again by further addition of Triton X-100. In fully activated microsomes K_m^app for 1 naphthol was 69.7±13.9 μM and V_max was 70.0 ± 3.9 nmoles/min · mg microsomal protein; K_m^app for UDP-glucuronic acid was 0.67 ± 0.06 mM. The glucuronidation rate (expressed as nmoles/min · g intestine) in microsomes is substantially higher than in isolated cells. It appears that glucuronidation in intact cells is limited by factors other than the extracellular substrate concn. Both cellular uptake of the substrate and availability of UDP-glucuronic acid can play a significant role. It is concluded that isolated mucosal cells are more suitable for predicting intestinal first-pass metabolism of phenolic xenobiotics than intestinal microsomes, because cellular substrate uptake and cosubstrate availability appear to be important determinants of the maximum glucuronidation rate.