Variation in glucuronidation of lamotrigine in human liver microsomes

1. Lamotrigine (LTG), a diaminotriazine anti-epileptic, is principally metabolized at the 2-position of the triazine ring to form a quaternary ammonium glucuronide (LTGG) by uridine glucuronosyl transferease (UGT) 1A3 and UGT1A4. It has been hypothesized that glucuronidation of anti-epileptic drugs is spared with age, despite a known decrease in liver mass, based on older studies with benzodiazepines such as lorazepam. To examine this, the formation rates of LTGG formation were measured by liquid chromatography-mass spectrometry (LC-MS) in a bank of human liver microsomes (HLMs) obtained from younger and elderly donors at therapeutic concentrations.

2. The formation rate of LTGG was not significantly different in HLMs obtained from younger and elderly subjects. A four- to five-fold variation for the formation of LTGG was observed within each microsomal bank obtained from elderly and younger donors, and the range of LTGG formation was observed to be 0.15–0.78?nmoles min^?1 mg^?1 of protein across the entire set of HLMs (n?=?36, elderly and younger HLMs).

3. UGT1A4 and UGT1A3 catalysed the formation of LTGG with an intrinsic clearances of 0.28 and 0.02?μl min^?1 mg^?1 protein, respectively. UGT2B7 and UGT2B4 showed no measurable activity. No correlation was observed across the HLM bank for glucuronidation of LTG and valproic acid (a substrate for multiple UGT isoforms including UGT1A4).

     

Influence of carboxylesterase 2 genetic polymorphisms on mycophenolic acid pharmacokinetics in Japanese renal transplant recipients

1. Mycophenolic acid (MPA), converted from the prodrug mycophenolate mofetil (MMF), is generated by intestinal and hepatic esterases. The role of carboxylesterase (CES) in MMF hydrolysis was examined in vitro using human liver microsomes. V_max and K_m values of MMF hydrolysis in pooled human liver microsomes were 1368?±?44 nmol min^?1 mg^?1 protein and 1030?±?65?μM, respectively.

2. Hydrolytic activity was inhibited by the CES inhibitors phenylmethylsulfonylfluoride, bis-p-nitorophenylphosphate and diisopropylfluorophosphate, with IC₅₀ values of 77.1, 3.59 and 0.0312?μM, respectively.

3. Eighty Japanese renal transplant recipients that received repeated-doses of MMF, tacrolimus and prednisolone, were evaluated for MPA pharmacokinetics 28 days after transplantation to investigate the relationship between MPA pharmacokinetics and CES2 genetic polymorphisms.

4. No significant differences in MPA pharmacokinetics were observed between CES2 A4595G, C8721T or A-1548G genotype groups. CES2 allelic variants also did not appear to affect plasma MPA concentrations between individuals.

5. In conclusion, the study demonstrated that while CES1 and/or CES2 are involved in the hydrolysis of MMF to MPA, CES2 allelic variants appeared to make only a minor contribution to inter-personal differences in MPA pharmacokinetics.

     

Role of individual human cytochrome P450 enzymes in the in vitro metabolism of hydromorphone

1. The aim was to identify the individual human cytochrome P450 (CYP) enzymes responsible for the in vitro N-demethylation of hydromorphone and to determine the potential effect of the inhibition of this metabolic pathway on the formation of other hydromorphone metabolites.

2. Hydromorphone was metabolized to norhydromorphone (apparent K_m = 206?? 822?μM, V_max = 104 ? 834?pmol?min^?1?mg^?1 protein) and dihydroisomorphine (apparent K_m = 62 ? 557?μM, V_max = 17 ? 122?pmol?min^?1?mg^?1 protein) by human liver microsomes.

3. In pooled human liver microsomes, troleandomycin, ketoconazole and sulfaphenazole reduced norhydromorphone formation by an average of 45, 50 and 25%, respectively, whereas furafylline, quinidine and omeprazole had no effect. In an individual liver microsome sample with a high CYP3A protein content, troleandomycin and ketoconazole inhibited norhydromorphone formation by 80%.

4. The reduction in norhydromorphone formation by troleandomycin and ketoconazole was accompanied by a stimulation in dihydroisomorphine production.

5. Recombinant CYP3A4, CYP3A5, CYP2C9 and CYP2D6, but not CYP1A2, catalysed norhydromorphone formation, whereas none of these enzymes was active in dihydroisomorphine formation.

6. In summary, CYP3A and, to a lesser extent, CYP2C9 catalysed hydromorphone N-demethylation in human liver microsomes. The inhibition of norhydromorphone formation by troleandomycin and ketoconazole resulted in a stimulation of microsomal dihydroisomorphine formation.

     

In vitro metabolism of the epoxide substructure of cryptophycins by cytosolic glutathione S-transferase: Species differences and stereoselectivity

The enzyme kinetics of the glutathione (GSH) conjugation of cryptophycin 52 (C52, R-stereoisomer) and cryptophycin 53 (C53,?S-stereoisomer) by cytosolic glutathione S-transferases (cGSTs) from human, rat, mouse, dog and monkey liver were studied. V_max, K_m, and CL_int values for glutathione conjugation of C52 (R-stereoisomer) were 0.10?±?0.01?nmol?min^?1?mg^?1, 3.24?±?0.23?µM, and (3.15?±?0.09)?×?10^?2?ml?min^?1?mg^?1, respectively, in human cytosol. Due to limited solubility relative to the K_m, only CL_int values were determined in rat ((7.76?±?0.10)?×?10^?2?ml?min^?1?mg^?1) and mouse ((7.61?±?0.50)?×?10^?2?ml?min^?1?mg^?1) cytosol. Enzyme kinetic parameters could not be determined for C53 (S-stereoisomer). Microsomal GSH conjugation in human, rat, and mouse was attributed to cytosolic contamination. No GSH conjugation was seen in any biological matrix from dog or monkey. There was little GSH conjugation of C53 by cytosol or microsomes from any species. The metabolism of C52 and C53 by epoxide hydrolase was also investigated. No diol product was observed in any biological matrix from any species. Thus, cGSTs are primarily responsible for C52 metabolism.     

Carbonyl reduction of bupropion in human liver

1. Bupropion is metabolized extensively in humans by oxidative and reductive processes. CYP2B6 mediates oxidation of bupropion to hydroxybupropion, but the enzyme(s) catalyzing carbonyl reduction of bupropion to erythro- and threohydrobupropion in human liver is unknown. The objective of this study was to examine the enzyme kinetics of bupropion reduction in human liver.

2. In human liver cytosol, the reduction of bupropion to erythro-and threohydrobupropion was NADPH dependent with Cl_int values of 0.08 and 0.60 µL·min^?1mg^?1 protein, respectively. Bupropion reduction in liver microsomes was also NADPH dependent with Cl_int values of 10.4 and 280 µL·min^?1mg^?1 protein, respectively. Formation of erythro-and threohydrobupropion in microsomes exceeded that in cytosol by 70 and 170 fold, respectively.

3. Menadione, an inhibitor of cytosolic carbonyl reducing enzymes (e.g. CBRs), inhibited erythro-and threohydrobupropion formation in cytosol with IC₅₀ of 30 and 54 µM, respectively. In microsomes 18β-glycyrrhetinic acid, an inhibitor of microsomal carbonyl reductases (e.g. 11β-HSDs), inhibited their formation with IC₅₀ of 25 and 26?nM, respectively.

4. Our findings, in agreement with recent human placental studies, show that carbonyl reducing enzymes in hepatic microsomes are significant players in bupropion reduction. Contrary to past studies, we found that threohydrobupropion (not hydroxybupropion) is the major microsomal generated hepatic metabolite of bupropion.

     

Characterization of bropirimine O-glucuronidation in human liver microsomes

1. The antitumour agent bropirimine undergoes significant Phase II conjugation in vivo. Incubation of [¹⁴C]bropirimine with human liver microsomes resulted in the formation of a single product peak (M1) using high-performance liquid chromatography with radiochemical detection and was tentatively assigned as bropirimine glucuronide based on sensitivity to β-glucuronidase and by obtaining the expected mass of 442/444 amu with liquid chromatography/mass spectrometry. Following metabolite isolation, the structure of M1 was established as bropirimine O-glucuronide by ¹H-nuclear magnetic spectroscopy.

2. Studies aimed at identifying the human liver UDP-glucuronosyltransferase (UGT) enzyme(s) involved in the glucuronidation of bropirimine were carried out using recombinant human UGTs and it was determined that glucuronidation of bropirimine was catalysed by UGT1A1, UGT1A3 and UGT1A9. Bropirimine O-glucuronidation followed Michaelis–Menten kinetics and the K_m and V_max (mean ± SD; n?=?3) were 1217 ± 205?μM and 667 ± 188?pmol?min^?1 mg^?1, respectively.

3. The activity of bropirimine O-glucuronidation by human liver microsomes was inhibited by bilirubin (40%) and with mefenamic acid (80%). Although buprenorphine extensively inhibited the activity of bropirimine O-glucuronidation by UGT1A3, the inhibition profile did not parallel that observed in HLMs.

4. The results demonstrate that UGT1A9 and to a lesser extent UGT1A1 are responsible for the majority of bropirimine O-glucuronidation in man.     

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.

     

Comparative inhibition of inducible and constitutive CYPs in rat hepatic microsomes by parathion

1. In microsomal fractions, the phosphorothioate pesticide parathion inhibits cytochrome P450 (CYP) enzymes by reversible and irreversible mechanisms resulting in the long-term suppression of drug oxidation. The present study evaluated the relative susceptibilities of constitutive and inducible CYP2 and CYP3 steroid hydroxylases to inhibition by the pesticide.

2. Enzyme kinetic analysis indicated that constitutive and dexamethasone (DEX)-induced androst-4-ene-3,17-dione (AD) 6β-hydroxylations were similarly susceptible to inhibition by parathion (K_m/K_i ratios 1.5–1.6). However, preincubation of parathion with NADPH-fortified microsomes intensified the extent of inhibition of CYP3A-dependent 6β-hydroxylation. Comparison of K_m/K_i ratios indicated that 6β-hydroxylation activity in fractions from DEX-pretreated rats was about twice as susceptible as the control activity to inactivation by parathion metabolites (K_m/K_i ratio of 8.0 versus 4.0).

3. The time-dependent loss of AD 6β-hydroxylation by parathion occurred more efficiently in fractions from DEX-induced liver than in control. Thus, half-times of 1.3 and 6.1?min, respectively, were determined for the inactivation of DEX-inducible and constitutive activities. Parathion concentrations required for half-maximal inactivation were 32 and 67?μM in microsomes from DEX-induced and control rats.

4. In phenobarbital (PB)-induced fractions CYP2B1-mediated AD 16β-hydroxylation was inhibited potently in a reversible fashion by parathion (K_i?=?0.37?μM; K_m/K_i ratio about 73). Inhibition was not enhanced at parathion concentrations near the K_i by a preincubation step with NADPH.

5. In control microsomes parathion elicited a type I binding interaction with oxidized CYP (K_s?=?7.7?μM, ΔA_max?=?2.2?×?10^?2?a.u.?nmol CYP^?1; ΔA_max/K_s 2.86?×?10³?a.u. nmol?CYP^?1/mM). Ligand binding was 13- and 1.6-fold more efficient in PB and DEX microsomes, respectively.

6. These findings indicate that pretreatment of rats with enzyme-inducing drugs like DEX and PB alters the profile of CYPs and their susceptibility to inhibition by parathion. Potent reversible inhibition of CYP2B1 occurred in PB-induced fractions and DEX-inducible CYPs 3A were more susceptible to mechanism-based inactivation than the corresponding constitutive CYPs from the same subfamily.

     

Sulfation of resveratrol in human liver: Evidence of a major role for the sulfotransferases SULT1A1 and SULT1E1

Sulfation of resveratrol, a polyphenolic compound present in grapes and wine with anticancer and cardioprotective activities, was studied in human liver cytosol. In the presence of 3′-phosphoadenosine-5′-phosphosulfate, three metabolites (M1–3) whose structures were identified by mass spectrometry and NMR as trans-resveratrol-3-O-sulfate, trans-resveratrol-4′-O-sulfate, and trans-resveratrol-3-O-4′-O-disulfate, respectively. The kinetics of M1 formation in human liver cytosol exhibited an pattern of substrate inhibition with a K_i of 21.3?±?8.73?µM and a V_max/K_m of 1.63?±?0.41?µL?min^?1mg^?1 protein. Formation of M2 and M3 showed sigmoidal kinetics with about 56-fold higher V_max/K_m values for M3 than for M2 (2.23?±?0.14 and 0.04?±?0.01?µL?min^?1?mg^?1). Incubation in the presence of human recombinant sulfotransferases (SULTs) demonstrated that M1 is almost exclusively catalysed by SULT1A1 and only to a minor extent by SULT 1A2, 1A3 and 1E1, whereas M2 is selectively formed by SULT1A2. M3 is mainly catalysed by SULT1A2 and 1A3. In conclusion, the results elucidate the enzymatic pathways of resveratrol in human liver, which must be considered in humans following oral uptake of dietary resveratrol.     

Inhibitory effects of commonly used herbal extracts on UGT1A1 enzyme activity

1. Commonly used herbal supplements were screened for their potential to inhibit UGT1A1 activity using human liver microsomes. Extracts screened included ginseng, echinacea, black cohosh, milk thistle, garlic, valerian, saw palmetto, and green tea epigallocatechin gallate (EGCG). Estradiol-3-O-glucuronide (E-3-G) formation was used as the index of UGT1A1 activity.

2. All herbal extracts except garlic showed inhibition of UGT1A1 activity at one or more of the three concentrations tested. A volume per dose index (VDI) was calculated to estimate the volume in which the daily dose should be diluted to obtain an IC₅₀-equivalent concentration. EGCG, echinacea, saw palmetto, and milk thistle had VDI values >2.0?L per dose unit, suggesting a higher potential for interaction.

3. Inhibition curves were constructed for EGCG, echinacea, saw palmetto, and milk thistle. IC₅₀ values were (mean ± SE) 7.8?±?0.9, 211.7?±?43.5, 55.2?±?9.2, and 30.4?±?6.9 µg/ml for EGCG, echinacea, saw palmetto, and milk thistle extracts, respectively.

4. Based on our findings, inhibition of UGT1A1 by milk thistle and EGCG and to a lesser extent by echinacea and saw palmetto is plausible, particularly in the intestine where higher extract concentrations are anticipated. Further clinical studies are warranted.

     

Almokalant glucuronidation in human liver and kidney microsomes: evidence for the involvement of UGT1A9 and 2B7

1. Almokalant, a class III antiarrythmic drug, is metabolized to form isomeric glucuronides identified in human urine. Synthesis of the total glucuronide was studied in human liver and kidney microsomes. Recombinant UDP-glucuronosyltransferases (UGTs) were screened for activity and kinetic analysis was performed to identify the isoform(s) responsible for the formation of almokalant glucuronide in man.

2. From a panel of recombinant isoforms used, both UGT1A9 and 2B7 catalysed the glucuronidation of almokalant. The K_m values in both instances were similar with 1.06?mM for the 1A9 and 0.97?mM for the 2B7. V_max for 1A9 was fourfold higher than that measured for UGT2B7, 92 compared with 21?pmol?min^?1?mg^?1, respectively, but UGT1A9 was expressed at approximately twofold higher level than the UGT2B7 in the recombinant cell lines. Therefore, the contribution of UGT2B7 to almokalant glucuronidation could be as significant as that of UGT1A9 in man.

3. Liver and kidney microsomes displayed similar K_m values to the cloned expressed UGTs, with the liver and kidney microsomes at 1.68 and 1.06?mM almost identical to the 1A9.

4. The results suggest a significant role for UGT1A9 and 2B7 in the catalysis of almokalant glucuronidation.     

Pharmacokinetics of the prodrug thiamphenicol glycinate and its active parent compound thiamphenicol in beagle dogs following intravenous administration

1. This study investigated the pharmacokinetics of thiamphenicol glycinate (TG) and thiamphenicol (TAP) in beagles (n?=?6) after intravenous administration of 50?mg/kg TG hydrochloride. Plasma concentrations of TG and TAP were measured by a HPLC-UV method.

2. Two-compartment model was selected to describe the pharmacokinetic characteristics of TG and TAP in vivo. Main parameters were as follows: AUC_0–∞ of TAP and TG were 16,328?±?1682 µg·min/mL and 3943?±?546 µg·min/mL, respectively. The total plasma clearance (CL) of TG and TAP were 12.7?±?2.0?mL/min/kg and 2.5?±?0.3?mL/min/kg, respectively. Mean residence time (MRT) of TG and TAP were 27.5?±?3.5 and 207.2?±?20.2?min, respectively. The transformative rate constant (k_1M) from TG to TAP was 0.0477?±?0.0028?min^?1. The elimination rate constant (k_M10) from TAP was 0.0238?±?0.0044?min^?1. Coefficients of variation (CV) between observed values and predicted ones were 5.9% and 18.2%, respectively. The volume of distribution of the central compartment for TG (V_C) and TAP (V_CM) were 0.264?±?0.022?L/kg and 0.127?±?0.023?L/kg, respectively.

3. Pharmacokinetic parameters suggested that TG was presumably cleaved quickly by tissue esterase to release TAP for effectiveness in beagles after administration.

     

Biotransformation and pharmacokinetics of the antiplasmodial naphthylisoquinoline alkaloid dioncophylline A

The biotransformation of the antiplasmodial naphthylisoquinoline alkaloid dioncophylline A by rat liver microsomes and its pharmacokinetics in male rats were studied. Incubation of dioncophylline A with rat liver microsomes resulted in the formation of the major metabolite 5′-O-demethyldioncophylline A, and a second minor metabolite, corresponding to the mass of an as yet unknown 4-hydroxydioncophylline A. Kinetic constants of the formation of 5′-O-demethyldioncophylline A were K_m?=?32?nmol and V_max?=?20?pmol?min^?1?mg^?1). Administration of dioncophylline A at a dose of 6.67?mg?kg^?1 body weight to rats intravenously and orally (n?=?4 per group) resulted in peak plasma levels of 0.84 and 0.11?µg?ml^?1, respectively. Levels of metabolites were below the limit of quantitation (LOQ). The following pharmacokinetic parameters of dioncophylline A were determined: oral bioavailability of 25%, plasma half-life of 2.5?h and partition volume of 8?l?kg^?1 body weight. Concentrations of dioncophylline A metabolites in all plasma and urine samples were below the limit of detection (LOD) and recovery of dioncophylline A in urine was very low, suggesting distribution into lipid rich tissues.     

Inhibition of phenol sulfotransferase (SULT1A1) by quercetin in human adult and foetal livers

1. Quercetin is one of the most abundant flavonoids in edible vegetables, fruit and wine. The aim was to study the type of inhibition of SULT1A1 by quercetin in the human adult and foetal livers. 2. The activity of SULT1A1 was measured with 4 µM 4-nitrophenol and 0.4 µM 3'-phosphoadenosine-5'-phosphosulphate-[³⁵S], and its mean (± SD) and median were 769 ± 311 and 740 pmol min^?1 mg^?1, respectively (adult liver, n = 10), and 185 ± 98 and 201 pmol min^?1 mg^?1, respectively (foetal liver, n = 8, p < 0.0001). 3. In non-inhibited samples, K_m for SULT1A1 (mean ± SD) was 0.31 ± 0.14 µM (adult liver) and 0.49 ± 0.17 µM (foetal liver, n.s.). V_max for SULT1A1 (mean ± SD) was 885 ± 135 pmol min^?1 mg^?1 (adult liver) and 267 ± 93 pmol min^?1 mg^?1 (foetal liver, p = 0.007). 4. The IC₅₀ of quercetin for SULT1A1 was measured in three samples of adult and foetal livers and was 13 ± 2.1 and 12 ± 1.4 nM, respectively. 5. The type of inhibition was mixed non-competitive in adult and foetal livers and K_i was 4.7 ± 2.5 nM (adult liver) and 4.8 ± 1.6 nM (foetal liver). 6. In the adult liver, the intrinsic clearance (mean ± SD) was 3.3 ± 1.5 ml min^?1 mg^?1 (non-inhibited samples), 0.9 ± 0.4 ml min^?1 mg^?1 (12.5 nM quercetin) and 0.5 ± 0.06 ml min^?1 mg^?1 (25 nM quercetin). In the foetal liver, the intrinsic clearance (mean ± SD) was 0.5 ± 0.2 ml min^?1 mg^?1 (non-inhibited samples), 0.12 ± 0.01 ml min^?1 mg^?1 (12.5 nM quercetin) and 0.2 ± 0.09ml min^?1 mg^?1 (25nM quercetin). 7. In conclusion, quercetin is a potent inhibitor of human adult and foetal liver SULT1A1. It reduces the sulphation rate and intrinsic clearance of 4-nitrophenol in both human adult and foetal livers. This suggests that quercetin may inhibit the sulfation rate of those drugs sulphated by SULT1A1. The inhibition of SULT1A1 is complex and not due solely to competition at the catalytic site of SULT1A1.     

Validation of cell-based OATP1B1 assays to assess drug transport and the potential for drug–drug interaction to support regulatory submissions

1. Transporters are carrier proteins that may influence pharmacokinetic, pharmacodynamic, and toxicological characteristics of drugs. The development of validated in vitro transporter models is imperative to support regulatory submissions of drug candidates. This study is focused on utilizing human embryonic kidney (HEK) 293 cell cultures genetically transfected with the human organic anion transporting polypeptides (OATP) 1B1 transporter to identify substrates and inhibitors in drug development.

2. The kinetics of OATP1B1-mediated uptake of [³H]-oestradiol 17β-glucuronide and inhibition of uptake by rifamycin SV were used to determine K_m, V_max, and IC₅₀ values over a range of passage numbers to investigate accuracy and precision. The mean K_m and V_max values were found to be 6.3?±?1.2 μM and 460?±?96 pmol min^?1 mg^?1, respectively. The mean IC₅₀ value for rifamycin SV was 0.23?±?0.07 μM on uptake of 1 μM [³H]-oestradiol 17β-glucuronide. These data were similar to previously reported values (accuracy greater than 82%), reproducible (precision less than 29%) and exhibited low standard deviations (SDs) obviating the need to study test compounds on more than one occasion.

3. [³H]-oestrone 3-sulfate and [³H]-pravastatin exhibited concentration-dependent OATP1B1 uptake, and statistically significant differences were observed at each concentration between uptake rates of HEK293-OATP1B1 and HEK293-MOCK cells (uptake ratios greater than or equal to 3). Propranolol showed no positive uptake ratio. Bezafibrate and gemfibrozil exhibited concentration-dependent inhibition of OATP1B1-mediated uptake of [³H]-oestradiol 17β-glucuronide with mean IC₅₀ values of 16 and 27 μM, respectively.

4. Based on the validation results, acceptance criteria to identify a test compound as a substrate and/or inhibitor using these specific cell lines were determined. These validated OATP1B1 assays were robust, reproducible, and suitable for routine in vitro evaluation of candidate drugs.

     

In vitro glucuronidation of aprepitant: a moderate inhibitor of UGT2B7

Abstract

1.?Aprepitant, an oral antiemetic, commonly used in the prevention of chemotherapy-induced nausea and vomiting, is primarily metabolized by CYP3A4. Aprepitant glucuronidation has yet to be evaluated in humans. The contribution of human UDP-glucuronosyltransferase (UGT) isoforms to the metabolism of aprepitant was investigated by performing kinetic studies, inhibition studies and correlation analyses. In addition, aprepitant was evaluated as an inhibitor of UGTs.

2.?Glucuronidation of aprepitant was catalyzed by UGT1A4 (82%), UGT1A3 (12%) and UGT1A8 (6%) and K_ms were 161.6?±?15.6, 69.4?±?1.9 and 197.1?±?28.2?µM, respectively. Aprepitant glucuronidation was significantly correlated with both UGT1A4 substrates anastrazole and imipramine (r_s?=?0.77, p?<?0.0001 for both substrates; n?=?44), and with the UGT1A3 substrate thyroxine (r_s?=?0.58, p?<?0.0001; n?=?44).

3.?We found aprepitant to be a moderate inhibitor of UGT2B7 with a K_i of ～10?µM for 4-MU, morphine and zidovudine. Our results suggest that aprepitant can alter clearance of drugs primarily eliminated by UGT2B7. Given the likelihood for first-pass metabolism by intestinal UGT2B7, this is of particular concern for oral aprepitant co-administered with oral substrates of UGT2B7, such as zidovudine and morphine.     

Kinetic analysis of bile acid sulfation by stably expressed human sulfotransferase 2A1 (SULT2A1)

1. Human sulfotransferase 2A1 (SULT2A1) is a member of the hydroxysteroid sulfotransferase (SULT2) family that mediates sulfo-conjugation of a variety of endogenous molecules including dehydroepiandrosterone (DHEA) and bile acids. In this study, we have constructed a stable cell line expressing SULT2A1 by transfection into HEK293 cells. The expression system was used to characterize and compare the sulfation kinetics of DHEA and 15 human bile acids by SULT2A1.

2. Formation of DHEA sulfate demonstrated Michaelis–Menten kinetics with apparent K_m and V_max values of 3.8?μM and 130.8 pmol min^?1 mg^?1 protein, respectively. Sulfation kinetics of bile acids also demonstrated Michaelis–Menten kinetics with a marked variation in apparent K_m and V_max values between individual bile acids.

3. Sulfation affinity was inversely proportional to the number of hydroxyl groups of bile acids. The monohydroxy- and most toxic bile acid (lithocholic acid) had the highest affinity, whereas the trihydroxy- and least toxic bile acid (cholic acid) had the lowest affinity to sulfation by SULT2A1. Intrinsic clearance (CL_int) of ursodeoxycholic acid (UDCA) was approximately 1.5- and 9.0-fold higher than that of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA), respectively, despite the fact that all three are dihydroxy bile acids.

     

Comparison of intrinsic metabolic clearance in fresh and cryopreserved human hepatocytes

1. We compared the intrinsic clearance (CL_int) of a number of substrates in suspensions of fresh and cryopreserved human hepatocytes from seven donors.

2. CL_int values for a cocktail incubation of phenacetin, diclofenac, diazepam, bufuralol, midazolam, and hydroxycoumarin were 4.9?±?3.4, 18?±?7.2, 5.1?±?4.9, 6.3?±?3.3, 9.8?±?5.8 and 22?±?14?μl min^?1/10⁶ cells, respectively, and they correlated well with corresponding CL_int values using cryopreserved hepatocytes from 25 different donors.

3. CL_int values of each cocktail substrate and 20 AstraZeneca new chemical entities were compared in fresh and cryopreserved hepatocytes from the same three donors. There was a statistically significant correlation between CL_int in fresh and cryopreserved hepatocytes for each of the three livers (p?int values was 1.03.

4. In conclusion, the results add further support to the use of cryopreserved human hepatocytes as a screening model for the intrinsic clearance of new chemical entities.

     

Identification of cytochrome P450 enzymes involved in the metabolism of 4′-methoxy-α-pyrrolidinopropiophenone (MOPPP), a designer drug,in human liver microsomes

1. The metabolism of 4′-methoxy-α-pyrrolidinopropiophenone (MOPPP), a novel designer drug, to its demethylated major metabolite 4′-hydroxy-pyrrolidinopropio-phenone (HO-PPP) was studied in pooled human liver microsomes (HLM) and in cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes.

2. CYP2C19 catalysed the demethylation with apparent K_m and V_max values of 373.4 ± 45.1?μM and 6.0 ± 0.3?pmol?min^?1?pmol^?1 CYP, respectively (mean ± SD). Both CYP2D6 and HLM exhibited clear biphasic profiles with apparent K_m,1 values of 1.3 ± 0.4 and 22.0 ± 6.5?μM, respectively, and V_max,1 values of 1.1 ± 0.1 pmol?min^?1?pmol^?1 CYP and 169.1 ± 20.5?pmol?min^?1?mg^?1 protein, respectively.

3. Percentages of intrinsic clearances of MOPPP by particular CYPs were calculated using the relative activity factor (RAF) approach with (S)-mephenytoin-4′-hydroxylation or bufuralol-1′-hydroxylation as index reactions for CYP2C19 or CYP2D6, respectively.

4. MOPPP, HO-PPP and the standard 3′,4′-methylenedioxy-pyrrolidinopropio-phenone (MDPPP) were separated and analysed by liquid chromatography–mass spectrometry in the selected-ion monitoring (SIM) mode.

5. The CYP2D6 specific chemical inhibitor quinidine (3?μM) significantly (?p<0.0001) inhibited HO-PPP formation by 91.8 ± 0.5% (mean ± SEM) in incubation mixtures with HLM and 2?μM MOPPP.

6. It can be concluded from the data obtained from kinetic and inhibition studies that polymorphically expressed CYP2D6 is the enzyme mainly responsible for MOPPP demethylation.     

Pharmacokinetics,disposition and lipid-modulating activity of 5-{2-[4-(3,4-difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid,a potent and subtype-selective peroxisome proliferator-activated receptor α agonist in preclinical species and human

1. 5-{2-[4-(3,4-Difluorophenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid (1) is a novel, potent, and selective agonist of the peroxisome proliferator-activated receptor alpha (PPAR-α).

2. In preclinical species, compound 1 demonstrated generally favourable pharmacokinetic properties. Systemic plasma clearance (CL_p) after intravenous administration was low in Sprague–Dawley rats (3.2?±?1.4?ml min^?1 kg^?1) and cynomolgus monkeys (6.1?±?1.6?ml min^?1 kg^?1) resulting in plasma half-lives of 7.1?±?0.7?h and 9.4?±?0.8?h, respectively. Moderate bioavailability in rats (64%) and monkeys (55%) was observed after oral dosing. In rats, oral pharmacokinetics were dose-dependent over the dose range examined (10 and 50?mg kg^?1).

3. In vitro metabolism studies on 1 in cryopreserved rat, monkey, and human hepatocytes revealed that 1 was metabolized via oxidation and phase II glucuronidation pathways. In rats, a percentage of the dose (approximately 19%) was eliminated via biliary excretion in the unchanged form.

4. Studies using recombinant human CYP isozymes established that the rate-limiting step in the oxidative metabolism of 1 to the major primary alcohol metabolite M1 was catalysed by CYP3A4.

5. Compound 1 was greater than 99% bound to plasma proteins in rat, monkey, mouse, and human.

6. No competitive inhibition of the five major cytochrome P450 enzymes, namely CYP1A2, P4502C9, P4502C19, P4502D6 and P4503A4 (IC₅₀’s?>?30 μM) was discerned with 1.

7. Because of insignificant turnover of 1 in human liver microsomes and hepatocytes, human clearance was predicted using rat single-species allometric scaling from in vivo data. The steady-state volume was also scaled from rat volume after normalization for protein-binding differences. As such, these estimates were used to predict an efficacious human dose required for 30% lowering of triglycerides.

8. In order to aid human dose projections, pharmacokinetic/pharmacodynamic relationships for triglyceride lowering by 1 were first established in mice, which allowed an insight into the efficacious concentrations required for maximal triglyceride lowering. Assuming that the pharmacology translated in a quantitative fashion from mouse to human, dose projections were made for humans using mouse pharmacodynamic parameters and the predicted human pharmacokinetic estimates.

9. First-in-human clinical studies on 1 following oral administration suggested that the human pharmacokinetics/dose predictions were in the range that yielded a favourable pharmacodynamic response.