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Predicting plasma concentration-time profiles of disproportionate metabolites in humans is crucial for evaluating metabolites according to the Safety Testing guidelines. We evaluated Css-MRTpo, an empirical method, using chimeric mice with humanised livers capable of generating human-disproportionate metabolites. Azilsartan and AZ-M2 were administered to humanised chimeric mice, and pharmacokinetic parameters were obtained. Pharmacokinetic data for DS-1971a and DS-M1 in humanised chimeric mice were obtained from the literature. The human plasma concentration-time profiles of these compounds were simulated using the Css-MRTpo method. Azilsartan, DS-1971a, and PF-04937319 produced human disproportionate metabolites, AZ-M2, DS-M1, and PF-M1, respectively. The predicted human pharmacokinetic profiles of PF-04937319 and PF-M1 were obtained from a previous study, and their outcomes were re-evaluated. Our findings revealed that the plasma concentrations of the three metabolites were unexpectedly underpredicted, whereas the three unchanged drugs were reasonably predicted. Further, the introduction of the empirical scaling factor of 3, obtained from six model compounds, improved the predictability of metabolites, suggesting the potential usefulness of the Css-MRTpo method in combination with humanised chimeric mice for predicting the pharmacokinetic profiles of disproportionate metabolites at the early stage of new drug development.

TPN729, a novel phosphodiesterase type 5 (PDE5) inhibitor for the treatment of erectile dysfunction (ED), is in phase II clinical trials in China. Previous studies suggested that TPN729 possesses promising therapeutic value. In previous non-radiolabeled rat excretion studies, the recovery of TPN729 and its major metabolites accounted for approximately 8.58% of the administration dose in urine and faeces by 48 h post-dose.To solve this problem and further study the metabolism of TPN729 in rats, we used the radio-isotopic tracing technique for the first time. In this study, the mass balance, tissue distribution, and metabolism of TPN729 were evaluated in rats after a single oral dose of 25 mg/kg [ 14 C]TPN729 (150 μCi/kg).At 168 h post-dose, the mean total radioactivity recovery of the dose was 92.13%. Faeces was the major excretion route, accounting for 74.63% of the dose, and urine excretion accounted for 17.50%. After oral administration of [ 14 C]TPN729, radioactivity was widely distributed in all examined tissues, and a higher radioactivity concentration was observed in the stomach, large intestine, lung, liver, small intestine, and eyes. The concentration of drug-related materials were similar in plasma and blood cells. A total of 51 metabolites were identified in rat plasma, urine, faeces, and bile, and the predominant metabolically susceptible position of TPN729 was the pyrrolidine moiety. The main metabolic pathways were N -dealkylation, oxidation, and dehydrogenation.In summary, we solved the previous problem of low drug recovery, elucidated the major excretion pathway, determined the tissue distribution patterns, and investigated the metabolism of TPN729 in rats by using a radioisotopic tracing technique.

The role that the phase-II reaction, glucuronidation, plays in the biotransformation of endo and xenobiotics is discussed with particular emphasis given to the UGT1A1 isoenzyme. This individual isoenzyme is responsible for both the mono and di-glucuronidation of bilirubin together with the glucuronidation of a number of xenobiotics of clinical interest (irinotecan, belinostat, atazanavir, pegvisomant).The review then discusses the roles that the various allelic variants of the UGT1A1 gene play in bilirubin metabolism and in particular how these allelic variants are involved in the clinical manifestation of the diseases of GS, CN1 and CN2.The review concludes with the roles that the UGT1A1*28 and UGT1A1*6 alleles play in adverse drug reactions (decreased glucuronidation of irinotecan, belinostat, atazanavir, pegvisomant) leading to increased exposure, reduced clearance and neutropenia (irinotecan, belinostat), increased risk for jaundice and hyperbilirubinaemia (atazanavir) and liver toxicity (pegvisomant) before discussing the future role of UGT1A1 in personalised medicine.

To elucidate the metabolism of pazopanib, a metabolomics approach was performed based on ultra-performance liquid chromatography coupled with electrospray ionization quadrupole mass spectrometry. A total of 22 pazopanib metabolites were identified in vitro and in vivo. Among these metabolites, 17 were novel, including several cysteine adducts and aldehyde derivatives. By screening using recombinant CYPs, CYP3A4 and CYP1A2 were found to be the main forms involved in the pazopanib hydroxylation. Formation of a cysteine conjugate (M3), an aldehyde derivative (M15) and two N-oxide metabolites (M18 and M20) from pazopanib could induce the oxidative stress that may be responsible in part for pazopanib-induced hepatotoxicity. Morphological observation of the liver suggested that pazopanib (300 mg/kg) could cause liver injury. The aspartate transaminase and alanine aminotransferase in serum significantly increased after pazopanib (150, 300 mg/kg) treatment; this liver injury could be partially reversed by the broad-spectrum CYP inhibitor 1-aminobenzotriazole (ABT). Metabolomics analysis revealed that pazopanib could significantly change the levels of L-carnitine, proline and lysophosphatidylcholine 18:1 in liver. Additionally, drug metabolism-related gene expression analysis revealed that hepatic Cyp2d22 and Abcb1a (P-gp) mRNAs were significantly lowered by pazopanib treatment. In conclusion, this study provides a global view of pazopanib metabolism and clues to its influence on hepatic function.

1. Belinostat is a histone deacetylase inhibitor that has been approved for the treatment of peripheral T-cell lymphoma. This study aimed to identify the UDP-glucuronosyltransferase (UGT) enzymes responsible for belinostat glucuronidation through kinetic determination using recombinant enzymes with determined enzyme concentrations. 2. The rate of glucuronidation was determined by incubation of belinostat with enzyme preparations. Kinetic parameters such as K m and V max were derived by fitting an appropriate model to the glucuronidation data. The role of active UGT enzymes to belinostat metabolism was evaluated using inhibition experiments and activity correlation analyses. 3. Human liver microsomes generated a glucuronide metabolite (i.e. belinostat glucuronide) from belinostat. The glucuronide structure was confirmed by high-resolution mass spectrometry as well as the fragmentation pattern. Of 12 test UGT enzymes, only four (UGT1A1, 1A3, 2B4, and 2B7) showed metabolic activities toward belinostat. UGT1A1 was the most active enzyme, followed by UGT2B7, 1A3, and 2B4. Kinetic profiles for UGT1A1, 1A3, 2B4, and 2B7 were well described by Michaelis-Menten, Michaelis-Menten, Hill equation, and substrate inhibition equation, respectively. 4. Glucuronidation of belinostat was markedly inhibited by emodin and apigenin (two potent inhibitors of UGT1A1), and by quinidine and diclofenac sodium (two selective inhibitors of UGT2B7). Belinostat glucuronidation was found to be significantly correlated with β-estradiol 3-O-glucuronidation and zidovudine glucuronidation. 5. It was concluded that in addition to UGT1A1, UGT2B7 was also an important contributor to belinostat glucuronidation.

1. Following oral administration of [ 14 C]TAK-438, the radioactivity was rapidly absorbed in rats and dogs. The apparent absorption of the radioactivity was high in both species. 2. After oral administration of [ 14 C]TAK-438 to rats, the radioactivity in most tissues reached the maximum at 1-hour post-dose. By 168-hour post-dose, the concentrations of the radioactivity were at very low levels in nearly all the tissues. In addition, TAK-438F was the major component in the stomach, whereas TAK-438F was the minor component in the plasma and other tissues. High accumulation of TAK-438F in the stomach was observed after oral and intravenous administration. 3. TAK-438F was a minor component in the plasma and excreta in both species. Its oxidative metabolite (M-I) and the glucuronide of a secondary metabolite formed by non-oxidative metabolism of M-I (M-II-G) were the major components in the rat and dog plasma, respectively. The glucuronide of M-I (M-I-G) and M-II-G were the major components in the rat bile and dog urine, respectively, and most components in feces were other unidentified metabolites. 4. The administered radioactive dose was almost completely recovered. The major route of excretion of the drug-derived radioactivity was via the feces in rats and urine in dogs.

1. 4'-(p-Toluenesulfonylamide)-4-hydroxychalcone (TSAHC) is a synthetic sulfonylamino chalcone compound possessing anti-cancer properties. The aim of this study was to elucidate the metabolism of TSAHC in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of TSAHC. 2. TSAHC was incubated with HLMs or recombinant P450 isoforms (rP450) in the presence of an nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-regenerating system. The metabolites were identified and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). P450 isoforms, responsible for TSAHC metabolite formation, were characterized by chemical inhibition and correlation studies in HLMs and enzyme kinetic studies with a panel of rP450 isoforms. 3. Two hydroxyl metabolites, that is M1 and M2, were produced from the human liver microsomal incubations (K(m) and V(max) values were 2.46 µM and 85.1 pmol/min/mg protein for M1 and 9.98 µM and 32.1 pmol/min/mg protein for M2, respectively). The specific P450 isoforms responsible for two hydroxy-TSAHC formations were identified using a combination of chemical inhibition, correlation analysis and metabolism by expressed recombinant P450 isoforms. The known P450 enzyme activities and the rate of TSAHC metabolite formation in the 15 HLMs showed that TSAHC metabolism is correlated with CYP2C and CYP3A activity. The P450 isoform-selective inhibition study in HLMs and the incubation study of cDNA-expressed enzymes also showed that two hydroxyl metabolites M1 and M2 biotransformed from TSAHC are mainly mediated by CYP2C and CYP3A, respectively. These findings suggest that CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 isoforms are major enzymes contributing to TSAHC metabolism.

1. Asunaprevir (ASV, BMS-650032), a highly selective and potent NS3 protease inhibitor, is currently under development for the treatment of chronic hepatic C virus infection. This study describes in vivo biotransformation in humans and the identification of metabolic enzymes of ASV. 2. Following a single oral dose of [(14)C]ASV to humans, the majority of radioactivity (>73% of the dose) was excreted in feces with <1% of the dose recovered in urine. Drug-related radioactivity readily appeared in circulation and the plasma radioactivity was mainly attributed to ASV. A few minor metabolites were observed in human plasma and are not expected to contribute to the pharmacological activity because of low levels. The area under the curve (AUC) values of each circulating metabolite in humans were well below their levels in animals used in the long-term toxicological studies. In bile and feces, intact ASV was a prominent radioactive peak suggesting that both metabolism and direct excretion played important roles in ASV clearance. 3. The primary metabolic pathways of ASV were hydroxylation, sulfonamide hydrolysis and the loss of isoquinoline. In vitro studies with human cDNA expressed CYP enzymes and with human liver microsomes (HLM) in the presence of selective chemical inhibitors demonstrated that ASV was primarily catalyzed by CYP3A4 and CYP3A5.

1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR). 2. Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (fu > 0.8) to extensive (fu < 0.1). Of the compounds screened, the highest binding to both HSA and LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for β-estradiol (a polar, neutral compound). 3. The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP. 4. Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP.

1. In early discovery stages, 2-methyl-N-(2'-(pyrrolidinyl-1-ylsulfonyl)-[1,1'-biphenyl]-4-yl)propan-1-amine (PBPA) demonstrated monoamine oxidase A (MAO-A) and cytochrome P450 (CYP)-mediated clearance. While human liver microsomes predicted low CL(b) PBPA demonstrated a moderate CL(p)/F in humans. The plasma pharmacokinetic (PK) of PBPA was characterized by unexpected high inter-individual variability. Hence, a retrospective analysis was undertaken to understand the disposition processes of PBPA, by applying in vitro mechanistic tools. 2. The in vitro-to-in vivo of rat CL(b) of PBPA was calculated as similar to that of human, suggesting rat to be a better predictor of a MAO-A/CYP substrate, but not dog or monkey; this is consistent with differences in expression of MAO-A in rat, dog, monkey and human. Fraction metabolized (f(m)) of human MAO A (hMAO-A) (50%), CYP3A4 (8%), CYP3A5 (16%) and CYP2D6 (29%) was determined, in vitro. 3. While the fm of CYP3A5 was <50%, Michaelis-Menten kinetics demonstrated that it was a higher capacity pathway compared with MAO-A, 2D6 and 3A4. This was consistent with strong association of dose-normalized plasma C(max) and area under the plasma concentration time curve (AUC(0-tlast)) of PBPA with CYP3A5 genotype, but not with genotype of CYP2D6. 4. This investigation demonstrates the value of integrating in vitro mechanistic tools to gain comprehensive understanding of disposition properties of drug candidates, in a discovery paradigm and prior to the investment in clinical trials.

Upamostat (Mesupron®) is a new small molecule serine protease inhibitor. The drug candidate was developed to inhibit the urokinase-type plasminogen activator (uPA) system, which plays a major role in tumor invasion and metastasis. Upamostat is currently in clinical development as an anti-metastatic and non-cytotoxic agent against pancreatic and breast cancer. Upamostat is the orally available amidoxime- (i.e. hydroxyamidine-) prodrug of the pharmacologically active form, WX-UK1. In this study, the reductive enzymatic activation of upamostat to its corresponding amidine WX-UK1 was analyzed. The recently discovered molybdenum enzyme "mitochondrial Amidoxime Reducing Component" (mARC) catalyses together with its electron transport proteins cytochrome b₅ and NADH cytochrome b₅ reductase the reduction of N-hydroxylated prodrugs. In vitro biotransformation assays with porcine subcellular fractions and the reconstituted human enzymes demonstrate an mARC-dependent N-reduction of upamostat.

Tetrahydropalmatine (THP), with one chiral centre, is one of the major constituents of Rhizoma corydalis. THP is considered to possess analgesic, sedative, hypnotic actions and cardiac protection. The aim of this study was to elucidate the stereoselective interaction between THP and ABC transporters. The present study investigated three most important ABC transporters, including P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1) and breast cancer resistance protein (BCRP). The intracellular accumulation and bidirectional transport suggested THP enantiomers were inhibitors of P-gp, but not of MRP1 or BCRP. The IC(50) values of (-)-THP and (+)-THP on rhodamine 123 (P-gp substrate) efflux were 48.6 and 20.0 µM, respectively, which showed obvious stereoselective difference. In the bidirectional transport, THP enantiomers showed high passive permeability and the contribution of P-gp could not be testified. The western blot and real-time RT-PCR assays showed that THP enantiomers reduced the protein expression of P-gp, but did not affect its mRNA expression. In in vitro cytotoxicity test, THP enantiomers showed the potential of increasing the cytotoxicity of doxorubicin in P-gp-mediated multidrug resistant tumour cells. The present study showed the stereoselective interaction between THP enantiomers and P-gp, which should be considered in clinical practice.

Macitentan is a tissue-targeting, dual endothelin receptor antagonist, currently under phase 3 investigation in pulmonary arterial hypertension. In this study the disposition and metabolism of macitentan were investigated following administration of a single oral 10 mg dose of (14)C-macitentan to six healthy male subjects. The total radioactivity in matrices was determined using liquid scintillation counting. The proposed structure of metabolites was based on mass spectrometry characteristics and, when available, confirmed by comparison with reference compounds. Mean (± SD) cumulative recovery of radioactivity from faeces and urine was 73.6% (± 6.2%) of the administered radioactive dose, with 49.7% (± 3.9%) cumulative recovery from urine, and 23.9% (± 4.8%) from faeces. In plasma, in addition to parent macitentan, ACT-132577, a pharmacologically active metabolite elicited by oxidative depropylation and the carboxylic acid metabolite ACT-373898 were identified. In urine, four entities were identified, with the hydrolysis product of ACT-373898 as the most abundant one. In faeces, five entities were identified, with the hydrolysis product of macitentan and ACT-132577 as the most abundant one. Concentrations of total radioactivity in whole blood were lower compared to plasma, which indicates that macitentan and its metabolites poorly bind to or penetrate into erythrocytes.

Genetic variants of Na(+)-taurocholate co-transporting polypeptide (NTCP; SLC10A1) and ileal apical sodium-dependent bile acid transporter (ASBT; SLC10A2), which greatly contribute to bile acid homeostasis, were extensively explored in the Korean population and functional variants of NTCP were compared among Asian populations. From direct DNA sequencing, six SNPs were identified in the SLC10A1 gene and 14 SNPs in the SLC10A2 gene. Three of seven coding variants were non-synonymous SNPs: two variants from SLC10A1 (A64T, S267F) and one from SLC10A2 (A171S). No linkage was analysed in the SLC10A1 gene because of low frequencies of genetic variants, and the SLC10A2 gene was composed of two separated linkage disequilibrium blocks contrary to the white population. The stably transfected NTCP-A64T variant showed significantly decreased uptakes of taurocholate and rosuvastatin compared with wild-type NTCP. The decreased taurocholate uptake and increased rosuvastatin uptake were shown in the NTCP-S267F variant. The allele frequencies of these functional variants were 1.0% and 3.1%, respectively, in a Korean population. However, NTCP-A64T was not found in Chinese and Vietnamese subjects. The frequency distribution of NTCP-S267F in Koreans was significantly lower than those in Chinese and Vietnamese populations. Our data suggest that NTCP-A64T and -S267F variants cause substrate-dependent functional change in vitro, and show ethnic difference in their allelic frequencies among Asian populations although the clinical relevance of these variants is remained to be evaluated.

A model that predicts human metabolism and disposition of drug candidates would be of value in early drug development. In this study, a chimeric (uPA+/+)/SCID mouse model was evaluated with three structurally distinct compounds (GW695634, a benzophenone, SB-406725, a tetrahydroisoquinoline and GW823093, a fluoropyrrolidine) for which human metabolism and disposition was characterized. Human metabolite profiles in plasma and/or urine were compared to those of chimeric (uPA+/+)/SCID and control CD-1 or (uPA+/+)/SCID) mice. GW695634 and SB-406725 exhibited primarily hepatic metabolism and were chosen as probes to assess which human metabolites would likely circulate systemically. GW823093 exhibited a combination of hepatic and extrahepatic metabolism such that renal excretion of drug-related material was ~2-fold greater in humans than in mice, and thus chosen as a probe to assess if the chimeric (uPA+/+)/SCID mouse would predict the urinary excretion of human metabolites. We observed that human metabolism and disposition was well represented for GW695634, somewhat represented for GW823093 and minimally represented for SB-406725. Collectively, the results of this and other studies suggest that while limitations for prediction of human metabolism and disposition exist, humanized chimeric mouse models can potentially represent informative new tools in drug discovery and development.

The purpose of this study was to investigate the effect of genetic variations in organic anion-transporting polypeptide 1B1 (OATP1B1) and Na(+)/taurocholate co-transporting polypeptide (NTCP) on the uptake of various statins having different affinities for these transporters. The functional activities and simultaneous expression of NTCP and OATP1B1 were confirmed by the uptake of taurocholate and estrone-3-sulphate as representative substrates for NTCP and OATP1B1, respectively, and by an immunofluorescence analysis. The substrate specificities of NTCP and OATP1B1 for statins and the effects of genetic variations on the uptake of rosuvastatin, pitavastatin, and atorvastatin were measured. Based on the K(m) values and intrinsic clearances of the three statins, pitavastatin was taken up more efficiently than rosuvastatin and atorvastatin by OATP1B1. Consequently, the cellular accumulation of pitavastatin was modulated according to the genetic variation of OATP1B1 (OATP1B1*15), rather than NTCP*2. In contrast, NTCP*2 displayed greater transport of atorvastatin and rosuvastatin, compared with NTCP wild type. Thus, the measurements of decreased rosuvastatin and atorvastatin transport by OATP1B1*15 were confounded by the presence of NTCP and its genetic variant, NTCP*2. In conclusion, the functional consequences of genetic variants of NTCP and OATP1B1 may be different for various statins, depending on the substrate specificity of the OATP1B1 and NTCP transporters.

AZD2624 was pharmacologically characterized as a NK3 receptor antagonist intended for treatment of schizophrenia. The metabolic drug-drug interaction potential of AZD2624 was evaluated in in vitro studies. CYP3A4 and CYP3A5 appeared to be the primary enzymes mediating the formation of pharmacologically active ketone metabolite (M1), whereas CYP3A4, CYP3A5, and CYP2C9 appeared to be the enzymes responsible for the formation of the hydroxylated metabolite (M2). The apparent K(m) values were 1.5 and 6.3 µM for the formation of M1 and M2 in human liver microsomes, respectively. AZD2624 exhibited an inhibitory effect on microsomal CYP3A4/5 activities with apparent IC(50) values of 7.1 and 19.8 µM for midazolam and testosterone assays, respectively. No time-dependent inactivation of CYP3A4/5 activity (midazolam 1'-hydroxylation) by AZD2624 was observed. AZD2624 demonstrated weak to no inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP2D6. AZD2624 was not an inducer of CYP1A2 or CYP2B6. Although AZD2624-induced CYP3A4 activity in hepatocytes, the potential of AZD2624 to cause inductive drug interactions of this enzyme was low at relevant exposure concentration. Together with targeted low efficacious concentration, the results of this study demonstrated AZD2624 has a relatively low metabolic drug-drug interaction potential towards co-administered drugs. However, metabolism of AZD2624 might be inhibited when co-administrated with potent CYP3A4/5 inhibitors.

GW695634 is the prodrug of GW678248, a novel non-nucleoside reverse transcriptase inhibitor with potent antiviral activity against HIV/AIDS efavirenz- and nevirapine-resistant viruses. In mice, rats, and monkeys following oral administration of [(14)C]GW695634, the primary pathway of metabolic clearance was by amide hydrolysis and the main route of elimination (46%-75% of the dose) was in the feces. The primary metabolic pathway of clearance for GW695634 and GW678248 in the preclinical species was by amide hydrolysis. At least six metabolites were observed that were the products of GW695634 and GW678248 amide hydrolysis.

The area under the curve (AUC) of mirodenafil after intravenous administration in diabetes mellitus induced by streptozotocin (DMIS) rats was significantly smaller (by 28.0 %) than the control value, and the AUC(SK3541)/AUC(mirodenafil) ratio was significantly greater (by 130 %) in DMIS rats. This may be explained by the significantly faster hepatic CL(int) of mirodenafil, owing to increased hepatic CYP1A, CYP2B1/2, CYP2D, and CYP3A expression, and a faster hepatic blood flow rate, compared with control values. The AUC of mirodenafil after oral administration was comparable between DMIS and control rats, possibly because of the comparable intestinal CL(int), which may be attributable to increased CYP1A2 expression and decreased CYP2D expression in the intestines of DMIS rats.

The purpose of this paper is to characterize the cytochrome P450 (CYP) enzymes involved in the metabolism of a new oral erectogenic, mirodenafil, to a major circulating active metabolite, N-dehydroxyethyl-mirodenafil, and to investigate the inhibitory potential of mirodenafil on seven CYP enzymes in human liver microsomes. CYP3A4 was identified as the major enzyme and CYP2C8 as a minor enzyme responsible for mirodenafil N-dealkylation based on correlation analysis, inhibition studies, and cDNA-expressed CYP enzyme activities. Plasma concentrations of mirodenafil and its N-dealkylated metabolite could therefore change with co-administration of known CYP3A4 inducers or inhibitors. Mirodenafil inhibited CYP3A4, CYP2C19 and CYP2D6 activities with IC50 values of 15.6, 38.2 and 77.0 microM, respectively, in human liver microsomes. However, it is very unlikely that mirodenafil will significantly alter the clearance of other compounds metabolized by CYPs 1A2, 2A6, 2C8, 2C9, 2C19, 2D6 and 3A4 because the maximum plasma concentration of mirodenafil is 0.55 microM after oral dosing of mirodenafil (100 mg) in male volunteers.

1. Methods for the co-expression in Escherichia coli of human cytochrome P450 (CYP) 2C8 and CYP2C9 with NADPH-cytochrome P450 reductase (OxR) to produce a catalytically active system were compared. 2. Approaches assessed were expression of a CYP:OxR fusion construct, bicistronic plasmids, simultaneous transformation with CYP and OxR plasmids, and separate expression of CYP and OxR with reconstitution of activity by mixing the bacterial membranes. Two N-terminal modifications (Delta3-20 and 17alpha-leader) of the individual P450s were additionally investigated. 3. Each approach gave efficient expression of CYP2C8 and CYP2C9, but the bicistronic constructs under the expression conditions used gave low OxR expression and low catalytic activity. CYP expression was higher with the Delta3-20 construct for CYP2C9 and with the 17alpha-presequence construct for CYP2C8. 4. Using torsemide as substrate, all methods gave catalytically active systems with K(m) values similar to human liver microsomes. Mixing bacterial membranes containing separately expressed CYP and OxR reconstituted a catalytically active system with the Delta3-20 construct for CYP2C9 but not for CYP2C8, and with neither of the 17alpha- presequence constructs. OxR co-expressed with CYP in the same membrane interacted with CYP to reconstitute activity more effectively than addition of exogenous OxR membranes. 5. Expression construct and OxR co-expression strategy should be individualized for CYP isoforms.

1. The liver is the target organ for the lipid-regulating effect of rosuvastatin, a new 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, and liver-selective uptake of this drug is therefore a desirable property. The uptake kinetics of rosuvastatin were investigated and compared with those of pravastatin using isolated rat hepatocytes. 2. Uptake for both drugs involved both active transport and passive diffusion processes. The Michaelis constant (K(m)) of uptake rate for rosuvastatin (9.17 micro M) was approximately half that for pravastatin (16.5 micro M). However, the maximum uptake rate (V(max)) and carrier-mediated uptake clearance (V(max)/K(m)) of rosuvastatin were significantly (p < 0.01) greater than those of pravastatin, and a larger contribution of carrier-mediated uptake clearance to total uptake clearance was shown for rosuvastatin (contribution ratio 0.903 versus pravastatin 0.654). 3. Sodium and chloride ions did not play a significant role in the uptake of rosuvastatin and pravastatin, but the uptake of both drugs was inhibited both by depletion of cellular ATP and by organic anions such as bromosulfophthalein. 4. Rosuvastatin competitively inhibited the uptake of pravastatin, with an inhibition constant (K(i)) (2.75 micro M) relatively similar to its K(m). 5. The results suggest that an organic anion transport protein is the main mediator of the hepatic uptake of rosuvastatin and pravastatin, which occurs in an ATP-dependent manner. Our results indicated that rosuvastatin was taken up by the hepatocytes via the same transport systems as pravastatin, but with a greater affinity and efficiency than pravastatin.

1. The in vitro human liver metabolism of the alpha1-adrenoceptor blocker tamsulosin was investigated. When 14C-tamsulosin was incubated with human liver microsomes, it was converted to five known urinary metabolites and at least three unknown metabolites. Of the former group, the predominant metabolite was the O-deethylated metabolite (M-1), followed by the o-ethoxyphenoxy acetic acid (AM-1) and the m-hydroxylated metabolite (M-3). 2. There was a good linear relationship between AM-1 formation and testosterone 6beta-hydroxylase activity in microsomes from each of 10 individual donors. The rate of M-1 formation also correlated with the same activity, albeit the correlation curve did not pass through the origin. By contrast, the rates of M-3 and the O-demethylated metabolite (M-4) formation correlated with dextromethorphan O-demethylase activity. 3. Ketoconazole strongly inhibited AM-1 formation and reduced that of M-1 by c. 60%. Immunoinhibition studies using anti-rat antibodies supported these results. The formation of M-3 and M-4 was inhibited by quinidine and sparteine. 4. It is concluded that formation of tamsulosin metabolites, AM-1 and M-1, is catalysed by CYP3A4 whereas that of M-3 and M-4 is catalysed by CYP2D6. However, minor contributions from other CYPs cannot be excluded.

1. The tissue distribution and metabolism of a new filaricidal agent P903 (N-[(2-phenylethynyl)sulfonyl]morpholine) were studied in rat. 2. After s.c. administration of 14C and 13C P903, the Tmax in the blood was observed on day 2. Elimination was slow and > 95% was bound to protein. Radioactivity was distributed in the whole organism but particularly in erythrocytes and the lymphatic channel. Four days later, > 60% of the radioactivity was excreted in urine and faeces at equal amounts and 15% remained at the injection point. 3. In all biological fluids tested no P903 was found but only its metabolites. 4. One principal metabolite, the N-[(2-phenyloxo-2-ethane) sulphonyl] morpholine or oxosulphonamide was identified in blood, urine and faeces as compared with the reference compound by GC/MS and NMR. This latter molecule was detected following hydrolysis by hydrochloric acid but not with beta glucuronidase/sulphatase. 5. Unconjugated and conjugated oxosulphonamide represented > 85% of the radioactivity at all times tested in blood but only 38 and 35% respectively of urinary and faecal radioactivity on day 1 after the administration of the labelled drug. 6. Thus, P903 is rapidly converted to a reactive metabolite, probably an oxirene, which is then conjugated with endogenous components to form conjugated oxosulphonamide and an unknown metabolite. The role of this reactive metabolite in antifilarial activity seems to be very important in understanding the mechanism of action of P903.

1. The antiepileptic agent zonisamide, 1,2-benzisoxazole-3-methanesulphonamide, was metabolized reductively to 2-sulphamoyl-acetylphenol (SMAP) not only under anaerobic conditions but also under aerobic conditions in liver microsomes of rat pretreated with phenobarbital or dexamethasone. 2. NADPH was required for the formation of SMAP from zonisamide under aerobic conditions. In addition, the reductive metabolism of zonisamide under these conditions was substantially inhibited by carbon monoxide, ketoconazole, and cimetidine, known inhibitors of cytochrome P450. 3. The formation of SMAP under aerobic conditions in liver microsomes was increased by pretreatment of rat with triacetyloleandomycin (TAO) and was increased by the treatment of the microsomes with ferricynaide. 4. These results imply that zonisamide is metabolized reductively to SMAP by a cytochrome P450 belonging to the 3A subfamily under aerobic conditions as well as anaerobic conditions.

1. The metabolism of tamsulosin hydrochloride (TMS), a potent alpha 1-adrenoceptor blocking agent, was studied after a single oral administration to rat and dog. 2. Eleven metabolites (1, 2, 3, 4 and their glucuronides, sulphates of 1 and 3, and A-1) were identified from the urine and bile of rat and dog administered TMS. 3. Unchanged drug and metabolites in urine and bile were quantified in rat and dog dosed with 14C-TMS(1 mg/kg). In rat the main metabolic routes were de-ethylation of the o-ethoxyphenoxy moiety, demethylation of the methoxybenzenesulphonamide moiety, and conjugation of the resultant metabolites by glucuronic acid and sulphuric acid. In dog the main pathways were de-ethylation of the ethoxyphenoxy moiety, conjugation of the de-ethylated product by sulphuric acid, and oxidative deamination of the side chain. 4. The organ responsible for the metabolism of TMS in rat was estimated using 9000g supernatants of liver, kidney, small and large intestine homogenate and plasma. The drug was rapidly metabolized in liver but hardly metabolized in the other organs or plasma.

1. 14C-Sulphamethazine (4-amino-N-(4,6-dimethyl-2-pyrimidinyl)benzene-[U-14C]-sulphonamide; 220 mg/kg of body weight) was given orally or i.v. to lactating dairy cows. Milk collected from 0-48 h after dosing accounted for 2.0% (oral dose) and 1.1% (i.v. dose) of the total 14C-activity administered. 2. Sulphamethazine accounted for 70-79% (oral dose) and 54-75% (i.v. dose) of the total 14C in milk samples collected from 0-48 h after dosing. N4-acetylsulphamethazine accounted for 1-2% (oral dose) and 1-4% (i.v. dose) of the 14C in milk. 3. The major 14C-labelled metabolite in the milk was isolated and identified as the N4-lactose conjugate of sulphamethazine, a unique type of metabolite not previously reported. This metabolite accounted for 10-14% (oral dose) and 9-20% (i.v. dose) of the 14C-activity in the milk collected from 0-48 h after dosing with 14C-sulphamethazine. 4. N4-lactose conjugates of sulphapyridine, sulphamerazine, sulphathiazole, sulphadimethoxine and sulphaquinoxaline were present in the milk from cows orally dosed with these five sulphonamide drugs.

1. The activity of glutathione (GSH) transferases in rat liver cytosol was inhibited by the (-) enantiomer of a uricosuric diuretic (6,7-dichloro-5[N,N-dimethylsulphamoyl]-2,3-dihydrobenzofuran-2-ca rboxylic acid, DBCA) in a concentration-dependent manner. Although the DBCA (+) enantiomer inhibited the activity of liver cytosol GSH transferases, it was less effective. 2. Among four purified GSH transferase isozymes obtained from rat liver cytosol, isozyme 3-3 showed stereoselective interactions with the enantiomers of DBCA. This isozyme most actively and preferentially catalysed the transfer of GSH to DBCA (-) enantiomer.

1. A glutathione (GSH) conjugate of the uricosuric diuretic DBCA 6,7-dichloro-5(N,N-dimethylsulphamoyl)-2,3-dihydrobenzofuran-2-car boxylic acid, formed enzymically in rat liver 100,000g supernatant fortified with GSH, was purified by t.l.c. and h.p.l.c. 2. According to mass spectrometry the composition of the GSH-conjugate corresponded to C21H29N4O11S2Cl2. 1H- and 13C-n.m.r. studies gave the structure of the conjugate as 2-glutathionyl-3-[3,4-dichloro-5-(N,N-dimethyl sulphamoyl)2-hydroxyphenyl]-propionic acid, which indicates that GSH was bound to the chiral centre carbon and caused scission of the C-O bond in the dihydrofuran ring.

Oral doses (20 mg/kg) of [Me-14C]toluene-2-sulphonamide were rapidly eliminated by rats (92% of dose in 24 h). Most of the 14C (88%) was recovered in the urine within 7 days with litte (5%) in the faeces. Larger oral doses (125 and 200 mg/kg) were eliminated more slowly (70 and 43% respectively in 24 h) but the overall distribution of 14C between urine and faeces was unchanged. 2. Low oral doses (0.2--0.4 mg/kg) of [Me-14C]toluene-2-sulphonamide were excreted more slowly in man than in the rat, with about 50% recovered in the urine in 24 h and 80% in 48 h. Negligible 14C (less than 1%) appeared in the faeces. 3. The main metabolites of toluene-2-sulphonamide were 2-sulphamoylbenzyl alcohol and its sulphate and glucuronic acid conjugates (80% of the 14C in the urine of rats and 35% in man) and saccharin (35% in man and 3% in the rat). Other metabolities found in the urine were 2-sulphamoylbenzoic acid (2% in the rat and 4% in man) and N-acetyltoluene-2-sulphonamide (6% in rat and 2% in man) together with unchanged compound (5% in rat and 3% in man).

1. 4-Sulphamoyl[carboxy-14C]benzoic acid was rapidly eliminateda after oral administration to rats (94% dose in 24 h). After 6 days most of the 14C (73-83% dose) was recovered in the urine with significant amounts (18-32% dose) in the faeces due to incomplete absorption. 2. The 14C in the urine and faeces was unchanged 4-sulphamoylbenzoic acid. No 14CO2 was detected in the expired air. 3. After oral administration of [methyl-14C]toluene-4-sulphonamide to rats the label was rapidly eliminated largely in the urine (66-89% dose) with little in the faeces (2-8% dose). The 14C in the faeces was 4-sulphamoylbenzoic acid, which probably originated in the tissues since the gut flora was unable to effect this biotransformation. 4. The urine of rats given [14C]toluene-4-sulphonamide contained 4-sulphamoylbenzoic acid as the major metabolite (93% of the urinary 14C) together with small amounts of unchanged compound (1.5-2.3% of urinary 14C), 4-sulphamoylbenzyl alcohol (2.0-3.9%), 4-sulphamoylbenzaldehyde (0-1.5%) and at higher doses N-acetyltoluene-4-sulphonamide (2.1-2.3%).