Drug Metabolism and Disposition current issue

Expression and Characterization of CYP4V2 as a Fatty Acid {omega}-Hydroxylase [SHORT COMMUNICATIONS]

Nakano, M., Kelly, E. J., Rettie, A. E. — Fri, 16 Oct 2009 12:24:51 PDT

Bietti's crystalline dystrophy is an ocular disease that is strongly associated with polymorphisms in the CYP4V2 gene. CYP4 enzymes are typically microsomal fatty acid -hydroxylases that function together with mitochondrial and peroxisomal β-oxidation enzymes to degrade cellular lipids. Indeed, ocular and peripheral cells cultured from patients with Bietti's have been reported to exhibit abnormal lipid metabolism. However, CYP4V2 possesses low sequence homology to other members of the CYP4 family. Therefore, we cloned and expressed CYP4V2 and analyzed the functional characteristics of this new cytochrome P450 enzyme. We find that CYP4V2 is a selective -hydroxylase of saturated, medium-chain fatty acids with relatively high catalytic efficiency toward myristic acid. Moreover, N-hydroxy-N'-(4-n-butyl-2-methylphenyl formamidine) (HET0016) is a nanomolar inhibitor of the enzyme. Therefore, CYP4V2 exhibits catalytic functions typical of a human CYP4 enzyme, but with a distinctive chain-length selectivity coupled with high -hydroxylase specificity. Consequently, defective -oxidation of ocular fatty acids/lipids secondary to mutations in the CYP4V2 gene appears to be a plausible mechanism underlying Bietti's crystalline dystrophy.

Effect of N-Acetyltransferase 2 Polymorphism on Tumor Target Tissue DNA Adduct Levels in Rapid and Slow Acetylator Congenic Rats Administered 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine or 2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline [SHORT COMMUNICATIONS]

Fri, 16 Oct 2009 12:24:51 PDT

2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are suspected human carcinogens generated in well done meats. After N-hydroxylation, they are O-acetylated by N-acetyltransferase 2 (NAT2) to electrophiles that form DNA adducts. dG-C8-MeIQx and dG-C8-PhIP adducts have been identified in human tissues. In the female rat, administration of PhIP leads to mammary and colon tumors, whereas MeIQx induces liver tumors. Both humans and rats exhibit NAT2 genetic polymorphism yielding rapid and slow acetylator phenotypes. Because O-acetylation is an activation pathway, we hypothesized that MeIQx- and PhIP-induced DNA damage would be greater in tumor target tissues and higher in rapid than slow NAT2 acetylators. Adult female rapid and slow acetylator rats congenic at the Nat2 locus received a single dose of 25 mg/kg MeIQx or 50 mg/kg PhIP by gavage, and tissue DNA was isolated after 24 h. Deoxyribonucleoside adducts were identified and quantified by capillary liquid chromatography-tandem mass spectrometry using isotope dilution methods with deuterated internal standards. Major adducts were those bound to the C8 position of deoxyguanosine. dG-C8-PhIP DNA adducts were highest in colon, lowest in liver and did not significantly differ between rapid and slow acetylator congenic rats in any tissue tested. In contrast, dG-C8-MeIQx adducts were highest in liver and significantly (p < 0.001) higher in rapid acetylator liver than in slow acetylator liver. Our results are consistent with the tumor target specificity of PhIP and MeIQx and with increased susceptibility to MeIQx-induced liver tumors in rapid NAT2 acetylators.

In Vivo Evaluation of Drug-Drug Interaction via Mechanism-Based Inhibition by Macrolide Antibiotics in Cynomolgus Monkeys [ARTICLES]

Ogasawara, A., Negishi, I., Kozakai, K., Kume, T. — Fri, 16 Oct 2009 12:24:51 PDT

Irreversible inhibition, characterized as mechanism-based inhibition (MBI), of cytochrome P450 in drugs has to be avoided for their safe use. A comprehensive assessment of drug-drug interaction (DDI) potential is important during the drug discovery process. In the present study, we evaluated the effects of macrolide antibiotics, erythromycin (ERM), clarithromycin (CAM), and azithromycin (AZM), which are mechanism-based inhibitors of CYP3A, on biotransformation of midazolam (MDZ) in monkeys. These macrolides inhibited the formation of 1'-hydroxymidazolam in monkey microsomes as functions of incubation time and macrolide concentration. Furthermore, the inactivation potentials of macrolides (k_inact/K_I: CAM ERM > AZM) were as effective as that observed in human samples. In in vivo studies, MDZ was administered orally (1 mg/kg) without or with multiple oral dosing of macrolides (15 mg/kg, twice a day on days 1–3). On day 3, the area under the plasma concentration-time curve (AUC) of MDZ increased 7.0-, 9.9-, and 2.0-fold with ERM, CAM, and AZM, respectively, compared with MDZ alone. Furthermore, the effects of ERM and CAM on the pharmacokinetics of MDZ were also observed on the day (day 4) after completion of macrolide treatments (AUC changes: 7.3- and 7.3-fold, respectively). Because the plasma concentrations of macrolides immediately before MDZ administration on day 4 were much lower than the IC₅₀ values for reversible CYP3A inhibition, the persistent effects may be predominantly caused by CYP3A inactivation. These results suggest that the monkey might be a suitable animal model to predict DDIs caused by MBI of CYP3A.

Investigation of Long-Term Retention of Unchanged (-)-N-{2-[(R)-3-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)piperidino]ethyl}-4-fluorobenzamide, A Novel "Funny" If Current Channel Inhibitor, and Its Metabolites in the Eyeball and Thoracic Aorta of Rats [ARTICLES]

Umehara, K.-i., Nakada, N., Noguchi, K., Iwatsubo, T., Usui, T., Kamimura, H. — Fri, 16 Oct 2009 12:24:51 PDT

(–)-N-{2-[(R)-3-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)piperidino]ethyl}-4-fluorobenzamide (YM758), a novel "funny" If current channel inhibitor, was being developed as a treatment for stable angina and atrial fibrillation. After a single oral administration of ¹⁴C-YM758, extensive accumulation and long-term retention of radioactivity were observed in the eyeballs of nonalbino rats and in the thoracic aorta of albino/nonalbino rats. Radioluminograms of the eyeballs of nonalbino rats indicated that the radioactivity was localized to the uveal tract, which suggests that the radioactivity may be positively charged and bound mainly to the melanins. Treatment with a mixture of 2 mol/l hydrochloric acid and methanol (5:95, v/v) allowed for the recovery of the major portion of radioactivity from the eyeball, which suggests reversible binding. The radioactive constituents in eyeballs consisted of the unchanged drug (YM758) and three metabolites [mainly 6,7-dimethoxy-2-[(3R)-piperidin-3-ylcarbonyl]-1,2,3,4-tetrahydroisoquinoline (YM-252124)]. Using the organic solvent mixture described above, almost all of the radioactivity was not collected from the thoracic aorta, and approximately 90% was recovered by treatment with elastase, which suggests that some metabolites covalently bind to the elastin fiber localized in the tunica media.

A Possible Mechanism for the Differences in Efficiency and Variability of Active Metabolite Formation from Thienopyridine Antiplatelet Agents, Prasugrel and Clopidogrel [ARTICLES]

Fri, 16 Oct 2009 12:24:51 PDT

The efficiency and interindividual variability in bioactivation of prasugrel and clopidogrel were quantitatively compared and the mechanisms involved were elucidated using 20 individual human liver microsomes. Prasugrel and clopidogrel are converted to their thiol-containing active metabolites through corresponding thiolactone metabolites. The formation rate of clopidogrel active metabolite was much lower and more variable [0.164 ± 0.196 µl/min/mg protein, coefficient of variation (CV) = 120%] compared with the formation of prasugrel active metabolite (8.68 ± 6.64 µl/min/mg protein, CV = 76%). This result was most likely attributable to the less efficient and less consistent formation of clopidogrel thiolactone metabolite (2.24 ± 1.00 µl/min/mg protein, CV = 45%) compared with the formation of prasugrel thiolactone metabolite (55.2 ± 15.4 µl/min/mg protein, CV = 28%). These differences may be attributed to the following factors. Clopidogrel was largely hydrolyzed to an inactive acid metabolite (approximately 90% of total metabolites analyzed), and the clopidogrel concentrations consumed were correlated to human carboxylesterase 1 activity in each source of liver microsomes. In addition, 48% of the clopidogrel thiolactone metabolite formed was converted to an inactive thiolactone acid metabolite. The oxidation of clopidogrel to its thiolactone metabolite correlated with variable activities of CYP1A2, CYP2B6, and CYP2C19. In conclusion, the active metabolite of clopidogrel was formed with less efficiency and higher variability than that of prasugrel. This difference in thiolactone formation was attributed to hydrolysis of clopidogrel and its thiolactone metabolite to inactive acid metabolites and to variability in cytochrome P450-mediated oxidation of clopidogrel to its thiolactone metabolite, which may contribute to the poorer and more variable active metabolite formation for clopidogrel than prasugrel.

Detection and Characterization of a New Metabolite of 17{alpha}-Methyltestosterone [ARTICLES]

Fri, 16 Oct 2009 12:24:51 PDT

The misuse of the anabolic steroid methyltestosterone is currently routinely monitored in doping control laboratories by gas chromatography-mass spectrometry (GC-MS) of two of its metabolites: 17-methyl-5β-androstane-3,17β-diol and 17-methyl-5-androstane-3,17β-diol. Because of the absence of any easy ionizable moiety, these metabolites are poorly detectable using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI). In this study, the metabolism of methyltestosterone has been reinvestigated by the use of a precursor ion scan method in LC-ESI-MS/MS. Two metabolites have been detected using this method. Both compounds have been confirmed in postadministration urine samples of an urokinase plasminogen activator-severe combined immunodeficiency (uPA-SCID) mouse with humanized liver and were characterized by LC-MS/MS and GC-MS using both quadrupole and time of flight analyzers. From the detailed study of the fragmentation, these metabolites were proposed to be epimethyltestosterone and a dehydrogenated compound. Epimethyltestosterone has previously been described as a minor metabolite, whereas the occurrence of the oxidized metabolite has not been reported. Comparison with the synthesized reference revealed that the structure of the dehydrogenated metabolite is 6-ene-epimethyltestosterone. A selected reaction monitoring method including three transitions for each metabolite has been developed and applied to samples from an excretion study and to samples declared positive after GC-MS analysis. 6-Ene-epimethyltestosterone was found in all samples, showing its applicability in the detection of methyltestosterone misuse.

Effects of Dose and Route of Administration on Pharmacokinetics of ({+/-})-3,4-Methylenedioxymethamphetamine in the Rat [ARTICLES]

Fri, 16 Oct 2009 12:24:51 PDT

Based on animal data, there is speculation that (±)-3,4-methylenedioxymethamphetamine (MDMA) is neurotoxic to humans. Extrapolation of MDMA findings from animals to humans requires assessment of pharmacokinetics in various species, and low-dose administration data from rats are lacking. In this study, we examine MDMA pharmacokinetics in rats given low (2 mg/kg) and high (10 mg/kg) doses of racemic MDMA via intraperitoneal, subcutaneous, and oral routes. Repeated blood specimens were collected from venous catheters, and plasma was assayed for MDMA and its metabolites, 4-hydroxy-3-methoxymethamphetamine (HMMA) and 3,4-methylenedioxyamphetamine (MDA), by gas chromatography-mass spectrometry. After 2 mg/kg, maximum MDMA concentrations (C_max) were ~200 ng/ml for intraperitoneal and subcutaneous routes, but less for the oral route. MDMA plasma half-lives were <1 h for low-dose groups, whereas HMMA and MDA half-lives were >2 h. After 10 mg/kg, MDMA areas under the curve (AUCs) were 21-fold (intraperitoneal), 10-fold (subcutaneous), and 36-fold (oral) greater than those at 2 mg/kg. In contrast, HMMA AUC values in high-dose groups were <3-fold above those at 2 mg/kg. Several new findings emerge from this report of low-dose MDMA pharmacokinetics in rats. First, 2 mg/kg MDMA in rats can produce MDMA C_max values similar to those in humans, perhaps explaining why both species discriminate 1.5 mg/kg MDMA in laboratory paradigms. Second, our data provide additional support for nonlinear kinetics of MDMA in rats, and, analogous to humans, this phenomenon appears to involve impaired drug metabolism. Finally, given key similarities between MDMA pharmacokinetics in rats and humans, data from rats may be clinically relevant when appropriate dosing conditions are used.

Effects of Dose and Route on the Disposition and Kinetics of 1-Butyl-1-methylpyrrolidinium Chloride in Male F-344 Rats [ARTICLES]

Knudsen, G. A., Cheng, Y., Kuester, R. K., Hooth, M. J., Sipes, I. G. — Fri, 16 Oct 2009 12:24:51 PDT

Studies were conducted to characterize the effects of dose and route of administration on the disposition of 1-butyl-1-methylpyrrolidinium (BmPy-Cl) in male Fischer-344 rats. After a single oral administration of [¹⁴C]BmPy-Cl (50 mg/kg), BmPy-Cl in the blood decreased rapidly after C_max of 89.1 min with a distribution half-life (t_1/2) of 21 min, an elimination half-life (t_1/2_β) of 5.6 h, and a total body clearance of 7.6 ml/min. After oral administration (50, 5, and 0.5 mg/kg), 50 to 70% of the administered radioactivity was recovered in the feces, with the remainder recovered in the urine. Serial daily oral administrations of [¹⁴C]BmPy-Cl (50 mg/kg/day for 5 days) did not result in a notable alteration in disposition or elimination. After each administration, 88 to 94% of the dose was eliminated in a 24-h period, with 63 to 76% of dose recovered in the feces. Intravenous administration of [¹⁴C]BmPy-Cl (5 mg/kg) resulted in biphasic elimination. Oral systemic bioavailability was 43.4%, approximately equal to the dose recovered in urine after oral administration (29–38%). Total dermal absorption of [¹⁴C]BmPy-Cl (5 mg/kg) was moderate when it was applied in dimethylformamide-water (34 ± 13%), variable in water (22 ± 8%), or minimal in ethanol-water (13 ± 1%) vehicles. Urine was the predominant route of elimination regardless of vehicle. Only parent [¹⁴C]BmPy-Cl was detected in the urine after all doses and routes of administration. BmPy-Cl was found to be a substrate for (K_t = 37 µM) and inhibitor of (IC_{50/tetraethylammonium} = 0.5 µM) human organic cation transporter 2. In summary, BmPy-Cl is moderately absorbed, extracted by the kidney, and eliminated in the urine as parent compound, independent of dose, number, or route of administration.

Tissue Distribution, Ontogeny, and Hormonal Regulation of Xenobiotic Transporters in Mouse Kidneys [ARTICLES]

Cheng, X., Klaassen, C. D. — Fri, 16 Oct 2009 12:24:51 PDT

Kidneys play important roles in the elimination of numerous endogenous and exogenous chemicals. In recent years, at least 37 xenobiotic transporters have been identified in mammalian kidneys. Although much progress has been made, information on 14 of these transporters (ATP-binding cassette [Abc] a1, apical sodium bile acid transporter [Asbt], breast cancer resistance protein, concentrative nucleoside transporter 1, equilibrative nucleoside transporter [Ent] 2, Ent3, sodium-phosphate cotransporter [Npt] 1, Npt2a, Npt2b, Npt2c, organic anion transporter [Oat] 5, organic anion-transporting polypeptide [Oatp] 4c1, peptide transporter 2, and uric acid transporter [Urat] 1) in kidneys is quite limited. Therefore, the purpose of the present study was to examine the tissue distribution, ontogeny, and hormonal regulation of these 14 transporters in kidneys of mice. Other than in kidneys, Npt2b is also highly expressed in liver and lung, Npt2c in liver and colon, Asbt in ileum, and Abca1 in liver, lung, testis, ovary, and placenta of mice. Most of these (13 of 14) transporters are lowly expressed in mouse kidneys until 15 days of age, which in part contributes to the immaturity of excretory function in fetal and newborn kidneys. One exception is Ent2, which is highly expressed before birth and gradually decreases after birth until reaching adult levels at 15 days of age. Gender-divergent expression of male-predominant (Urat1 and Oatp4c1) and female-predominant (Oat5) transporters in mouse kidneys is primarily due to stimulatory effects of androgens and estrogens, respectively. In conclusion, the mRNA expression of xenobiotic transporters in kidneys is determined by tissue, age, and sex hormones.

Different Effects of Ketoconazole on the Stereoselective First-Pass Metabolism of R/S-Verapamil in the Intestine and the Liver: Important for the Mechanistic Understanding of First-Pass Drug-Drug Interactions [ARTICLES]

Fri, 16 Oct 2009 12:24:51 PDT

In this acute study a pig jejunal intestinal perfusion model with multiple plasma sampling sites and three different administration routes was used to investigate the quantitative contribution of the intestine versus liver to the first-pass extraction of each enantiomer of verapamil (VER). A subclinical dose of ketoconazole (8 mg) was coadministered in the perfusion solution to selectively inhibit gut wall CYP3A. Both enantiomers of VER and its main metabolite norverapamil (NOR) as well as the inhibitor ketoconazole were quantified in all plasma compartments by liquid chromatography-tandem mass spectrometry. The overall first-pass metabolic extraction of VER and the metabolite NOR was shown to be stereoselective with the S-isomer being more extensively extracted. For VER the ratio of R- enantiomer to S-enantiomer was greater in the hepatic vein than in the portal vein (~2.2 versus 1.4), indicating that the stereoselective metabolism of VER in pigs mainly occurs on the first pass through the liver and not in the intestine. Ketoconazole increased the area under the curve from time 0 to 6 h and C_max of R- and S-VER at least 3-fold in the portal vein, most likely explained by inhibition of gut wall metabolism. Conversely, hepatic extraction was increased because the effect of gut wall metabolism was not observed at the peripheral sampling sites. In conclusion, this study provided novel and more direct information on the contribution of the intestine and the liver, respectively, to the overall first-pass extraction of racemic VER.

Hemoglobin Vesicles, Polyethylene Glycol (PEG)ylated Liposomes Developed as a Red Blood Cell Substitute, Do Not Induce the Accelerated Blood Clearance Phenomenon in Mice [ARTICLES]

Fri, 16 Oct 2009 12:24:51 PDT

The hemoglobin vesicle (HbV) is an artificial oxygen carrier encapsulating a concentrated hemoglobin solution in a liposome of which the surface is covered with polyethylene glycol (PEG). It was recently reported that repeated injections of PEGylated liposomes induce the accelerated blood clearance (ABC) phenomenon, in which serum anti-PEG IgM plays an essential role. To examine this issue, we investigated whether HbV induces the ABC phenomenon in mice at a dose of 0.1 mg Hb/kg, a dose that is generally known to induce the ABC phenomenon, or at 1400 mg Hb/kg, which is proposed for clinical use. At 7 days after the first injection of nonlabeled HbV (0.1 mg Hb/kg), the mice received HbV in which the Hb had been labeled with ¹²⁵I. After a second injection, HbV was rapidly cleared from the circulation, and uptake clearances in liver and spleen were significantly increased. In contrast, at a dose of 1400 mg Hb/kg, the pharmacokinetics of HbV was negligibly affected by repeated injection. It is interesting to note that IgM against HbV was produced 7 days postinjection at both of the above doses, and their recognition site was determined to be 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-PEG in HbV. These results suggest that a clinical dose of HbV does not induce the ABC phenomenon, and that suppression of ABC phenomenon is caused by the saturation of phagocytic processing by the mononuclear phagocyte system. Thus, we conclude that induction of the ABC phenomenon would not be an issue in the dose regimen used in clinical settings.

Molecular Properties and CYP2D6 Substrates: Central Nervous System Therapeutics Case Study and Pattern Analysis of a Substrate Database [ARTICLES]

Chico, L. K., Behanna, H. A., Hu, W., Zhong, G., Roy, S. M., Watterson, D. M. — Fri, 16 Oct 2009 12:24:51 PDT

CYP2D6 substrate status is a critical Go/No Go decision criteria in central nervous system (CNS) drug discovery efforts because the polymorphic nature of CYP2D6 can lead to variable patient safety and drug efficacy. In addition, CYP2D6 is disproportionately involved in the metabolism of CNS drugs compared with other drug classes. Therefore, identifying trends in small molecule properties of CNS-penetrant compounds that can help discriminate potential CYP2D6 substrates from nonsubstrates would allow additional prioritization in the synthesis and biological evaluation of new therapeutic candidates. We report here the conversion of the CNS drug minaprine from substrate to nonsubstrate, as well as the conversion of the related CNS drug minozac from nonsubstrate to substrate, through the use of analog synthesis and CYP2D6 enzyme kinetic analyses. No single molecular property strongly correlated with substrate status for this 3-amino-4-methyl-6-phenylpyridazine scaffold, although molecular volume and charge appeared to be indirectly related. A parsed database of CYP2D6 substrates across diverse chemical structures was assembled and analyzed for physical property trends correlating with substrate status. We found that a complex interplay of properties influenced CYP2D6 substrate status and that the particular chemical scaffold affects which properties are most prominent. The results also identified an unexpected issue in CNS drug discovery, in that some property trends correlative with CYP2D6 substrates overlap previously reported properties that correlate with CNS penetrance. These results suggest the need for a careful balance in the design and synthesis of new CNS therapeutic candidates to avoid CYP2D6 substrate status while maintaining CNS penetrance.

The Role of Human UDP-Glucuronyltransferases on the Formation of the Methylenedioxymethamphetamine (Ecstasy) Phase II Metabolites R- and S-3-Methoxymethamphetamine 4-O-Glucuronides [ARTICLES]

Schwaninger, A. E., Meyer, M. R., Zapp, J., Maurer, H. H. — Fri, 16 Oct 2009 12:24:51 PDT

Different pharmacokinetic properties have been observed for the two enantiomers of the entactogen 3,4-methylendioxy-methamphetamine, most probably a result of enantioselective metabolism. The aim of the present work was to study the involvement of human UDP-glucuronyltransferase (UGT) isoforms in the glucuronidation of the enantiomers of its major metabolite 4-hydroxy-3-methoxymethamphetamine (HMMA). First, the reference standards of R- and S-HMMA-O-glucuronide were synthesized semipreparatively using the enzymes of rat liver microsomes, followed by isolation with semipreparative high-performance liquid chromatography and identification using mass spectrometry and NMR. Racemic HMMA was then incubated using heterologously expressed human UGTs and pooled human liver microsomes (HLMs), and the glucuronides were quantified by liquid chromatography-linear ion trap-mass spectrometry. UGT1A1, UGT1A3, UGT1A8, UGT1A9, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 were involved in the glucuronidation of HMMA. UGT2B15, UGT2B17, and HLM revealed classic Michaelis-Menten kinetics, whereas UGT1A9 and UGT2B7 showed sigmoidal curves and the respective Eadie-Hofstee plots indicated autoactivation kinetics. UGT2B15 showed the highest affinity and activity. UGT2B15, UGT2B17, and HLMs were not considerably enantioselective but showed slight preferences for S-HMMA. Marked enantioselectivity could only be observed for UGT1A9 with respect to the S-enantiomer and for UGT2B7 with respect to the R-enantiomer. In conclusion, the O-glucuronidation of HMMA in vivo should not be expected to be enantioselective, and the different pharmacokinetic properties may not be caused directly by glucuronidation.

UDP-Glucuronosyltransferases in Conjugation of 5{alpha}- and 5{beta}-Androstane Steroids [ARTICLES]

Sten, T., Kurkela, M., Kuuranne, T., Leinonen, A., Finel, M. — Fri, 16 Oct 2009 12:24:51 PDT

We have examined the glucuronidation of androsterone (5-androstane-3-ol-17-one), etiocholanolone (5β-androstane-3-ol-17-one), 5-androstane-3-,17β-diol (5-diol), and 5β-androstane-3-, 17β-diol (5β-diol) by 19 recombinant human UDP-glucuronosyltransferases (UGTs). The results reveal large differences in stereo- and regioselectivity between UGT2B7, UGT2B15, and UGT2B17. UGT2B7 conjugated all four androgens at the 3-OH but not at the 17-OH that is available in both diols. UGT2B7 exhibited a higher glucuronidation rate toward the steroids with a flat backbone, androsterone and 5-diol, compared with etiocholanolone and 5β-diol, which have a bent backbone. UGT2B17 readily glucuronidated androsterone and, particularly, etiocholanolone at the 3-OH, but in the two diols it exhibited high preference for the 17-OH and low glucuronidation rate at the 3-OH. UGT2B15 did not glucuronidate any of the studied four androgens at the 3-OH, but it did conjugate both diols at the 17-OH, with a clear preference for 5-diol. Of the UGT1A subfamily, only UGT1A4 catalyzed the glucuronidation of androsterone and 5-diol at measurable rates, even if low. UGT2A1 and UGT2A2 glucuronidated most compounds in this study, but mostly at rather low rates. An exception was the glucuronidation of etiocholanolone by UGT2A1 that revealed a very low substrate affinity in combination with very high V_max value. The results shed new light on the substrate selectivity of individual UGTs in steroid glucuronidation. In addition they bear implications for doping analyses and its dependence of genetic polymorphism because testosterone is a precursor in the biosynthesis of these four androgens, whereas the contribution of UGT2B17 to their glucuronidation varies greatly.

Differential Regulation of Drug Transporter Expression by Hepatocyte Growth Factor in Primary Human Hepatocytes [ARTICLES]

Le Vee, M., Lecureur, V., Moreau, A., Stieger, B., Fardel, O. — Fri, 16 Oct 2009 12:24:51 PDT

Hepatocyte growth factor (HGF) is known to down-regulate expression of drug-detoxifying proteins such as cytochromes P450 (P450s) in human hepatocytes. The present study was designed to determine whether HGF may also impair expression of uptake and efflux drug transporters, which constitute important determinants of the liver detoxification pathway, such as P450s. Exposure of primary human hepatocytes to 20 ng/ml HGF for 48 h was found to down-regulate mRNA levels of major sinusoidal uptake transporters, including sodium taurocholate-cotransporting polypeptide (NTCP), organic anion-transporting polypeptide (OATP) 2B1, OATP1B1, organic cation transporter (OCT) 1, and organic anion transporter 2. HGF concomitantly reduced NTCP, OATP2B1, and OATP1B1 protein expression and NTCP, OATP, and OCT1 transport activities. With respect to efflux pumps, HGF decreased mRNA expression of the canalicular bile salt export pump, whereas that of the multidrug resistance (MDR) 1 gene was transiently increased. Moreover, Western blot analysis indicated that HGF up-regulated expressions of MDR1/P-glycoprotein and breast cancer resistance protein in human hepatocytes, whereas those of multidrug resistance gene-associated protein (MRP) 2 and MRP3 were unchanged. However, HGF prevented constitutive androstane receptor-related up-regulation of MRP2 occurring in phenobarbital-treated hepatocytes. Taken together, these data demonstrate that HGF differentially regulates transporter expression in human hepatocytes, i.e., it represses most of the sinusoidal uptake transporters, whereas expression of most of the efflux transporters is unchanged or increased. Such changes probably contribute to alterations of pharmacokinetics in patients with diseases associated with increased plasma levels of HGF such as fulminant hepatitis.

In Vitro Glucuronidation of Fenofibric Acid by Human UDP-Glucuronosyltransferases and Liver Microsomes [ARTICLES]

Fri, 16 Oct 2009 12:24:51 PDT

Fenofibric acid (FA), the active moiety of fenofibrate, is an agonist of the peroxisome proliferator-activated nuclear receptor that modulates triglyceride and cholesterol profiles. Lipid response to fenofibrate and FA serum concentrations is highly variable. Although FA is reported to be almost exclusively inactivated by UDP-glucuronosyltransferases (UGTs) into FA-glucuronide (FA-G), the contribution of UGT isoenzymes has never been systematically assessed. Heterologously expressed human UGT1A and UGT2B and their coding variants were tested for FA glucuronidation using liquid chromatography/mass spectrometry. Recombinant UGT2B7 presented the highest V_max/K_m value (2.10 µl/min/mg), 16-fold higher than the activity of other reactive UGTs, namely, UGT1A3, UGT1A6, and UGT1A9 (0.13, 0.09, and 0.02 µl/min/mg, respectively). UGT2B7.1 (His²⁶⁸) and UGT2B7.2 (Tyr²⁶⁸) enzyme activity was similar, whereas UGT1A3.2 (R¹¹A⁴⁷), UGT1A3.3 (Trp¹¹), and UGT1A9.3 (Thr³³) showed 61 to 96% reduced V_max/K_m values compared with the respective (1) reference proteins. FA-G formation by a human liver bank (n = 48) varied by 10-fold, but the rate of formation was not associated with common genetic variations in UGT1A3, UGT1A6, UGT1A9, and UGT2B7. Correlation with activities for the probe substrates zidovudine (UGT2B7; r² = 0.75), mycophenolic acid (UGT1A9; r² = 0.42), fulvestrant (UGT1A3; r² = 0.36), but not serotonin (UGT1A6; r² = 0.06) indicated a primary role for UGT2B7 and lesser roles of UGT1A9 and UGT1A3 in hepatic FA glucuronidation. This was confirmed by a strong correlation of FA-G formation with UGT2B7 protein content and inhibition by fluconazole, a known UGT2B7 selective inhibitor. Additional studies are required to identify genetic factors contributing to the observed FA glucuronidation variability.

Inoculation of Human Tumor Cells Alters the Basal Expression but Not the Inducibility of Cytochrome P450 Enzymes in Tumor-Bearing Mouse Liver [ARTICLES]

Sugawara, M., Okamoto, K., Kadowaki, T., Kusano, K., Fukamizu, A., Yoshimura, T. — Fri, 16 Oct 2009 12:24:51 PDT

The athymic nude mouse is often used to grow tumors for in vivo oncology research, including the identification of anticancer drugs, whereas wild-type mice are usually used to assess the pharmacokinetics (PK) of new chemical entities. The relationship between PK and pharmacodynamics (PD) provides useful mechanistic information and helps guide of the clinical regimen. The aim of this study was to assess whether the inoculation of human hepatocellular carcinoma cells (PLC/PRF/5) into athymic nude mice alters the expression of genes encoding the drug-metabolizing enzymes and transporters in host liver. The livers from nontumor- and tumor-bearing mice were initially subjected to drug metabolism gene microarray analysis. Microarray analysis indicated that tumor inoculation had little effect on drug metabolism-related genes, including several cytochrome P450s: Cyp1a, Cyp2b, and Cyp3a. This result was further confirmed by reverse transcription-polymerase chain reaction (RT-PCR). However, immunoreactive proteins of Cyp1a, Cyp2b, and Cyp3a were suppressed by tumor inoculation. RT-PCR and Western immunoblotting analysis showed that the inducibility of Cyp1a, Cyp2b, and Cyp3a by 3-methylcholanthrene, phenobarbital, and dexamethasone, respectively, was similar between nontumor- and tumor-bearing mice. These results suggest that inoculation of human tumor cells into athymic nude mice suppresses the expression of certain drug-metabolizing enzymes, which may alter the PK and PD of antitumor drugs.

Quantitative Evaluation of the Expression and Activity of Five Major Sulfotransferases (SULTs) in Human Tissues: The SULT "Pie" [ARTICLES]

Riches, Z., Stanley, E. L., Bloomer, J. C., Coughtrie, M. W. H. — Fri, 16 Oct 2009 12:24:51 PDT

Expression levels of the major human sulfotransferases (SULTs) involved in xenobiotic detoxification in a range of human tissues (i.e., SULT "pies") are not available in a form allowing comparison between tissues and individuals. Here we have determined, by quantitative immunoblotting, expression levels for the five principal human SULTs—SULT1A1, SULT1A3/4, SULT1B1, SULT1E1, and SULT2A1—and determined the kinetic properties toward probe substrates, where available, for these enzymes in cytosol samples from a bank of adult human liver, small intestine, kidney, and lung. We produced new isoform-selective antibodies against SULT1B1 and SULT2A1, which were used alongside antibodies against SULT1A3 and SULT1A1 previously produced in our laboratory or available commercially (SULT1E1). Expression levels were derived using purified recombinant enzymes to construct standard curves for each individual isoform and immunoblot. Substantial intertissue and interindividual differences in expression were observed. SULT1A1 was the major enzyme (>50% of total, range 420-4900 ng/mg cytosol protein) in the liver, followed by SULT2A1, SULT1B1, and SULT1E1. SULT1A3 was completely absent from this tissue. In contrast, the small intestine contained the largest overall amount of SULT of any of the tissues, with SULT1B1 the major enzyme (36%), closely followed by SULT1A3 (31%), and SULT1A1, SULT1E1, and SULT2A1 more minor forms (19, 8, and 6% of total, respectively). The kidney and lung contained low levels of SULT. We provide a unique data set that will add value to the study of the role and contribution of sulfation to drug and xenobiotic metabolism in humans.

Identification of New CYP2C19 Variants Exhibiting Decreased Enzyme Activity in the Metabolism of S-Mephenytoin and Omeprazole [ARTICLES]

Lee, S.-J., Kim, W.-Y., Kim, H., Shon, J.-H., Lee, S. S., Shin, J.-G. — Fri, 16 Oct 2009 12:24:51 PDT

Although many cases of interindividual variation in the metabolism of CYP2C19 drugs are explained by the CYP2C19*2, *3, and *17, a wide range of metabolic variation still occurs in people who do not carry these genetic variants. The objectives of this study were to identify new genetic variants and to characterize functional consequences of these variants in metabolism of CYP2C19 substrates. In total, 21 single-nucleotide polymorphisms including three new coding variants, V394M, E405K, and D256N, were identified by direct DNA sequencing in 50 randomly selected subjects and in individuals who exhibited an outlier phenotype response in the omeprazole study. Recombinant proteins produced from the coding variants V394M, E405K, and D256N were prepared by using an Escherichia coli expression system and purified. Metabolism of S-mephenytoin and omeprazole by V394M was comparable with that of the wild-type protein. E405K showed a moderate decrease in metabolism of the substrates. However, D256N exhibited a significantly decreased activity in S-mephenytoin metabolism, resulting in 50 and 76% decreases in V_max and intrinsic clearance, respectively, compared with the wild type. This variant also exhibited a significant decrease in omeprazole metabolism in vivo. CYP2C19 D256N and E405K were assigned as CYP2C19*26 and *2D, respectively, by the Cytochrome P450 Nomenclature Committee. In summary, this report characterizes the allele frequency and haplotype distribution of CYP2C19 in a Korean population and provides functional analysis of new coding variants of the CYP2C19 gene. Our findings suggest that individuals carrying CYP2C19*26 would have lower activity for metabolizing CYP2C19 substrate drugs.