Changes in power generation performance of an undershot cross-flow-type hydraulic turbine in an irrigation channel due to snow masses passing through the rotor.

This study is focused on the development of a microhydraulic turbine that can stably and efficiently generate electricity even in channel where snow masses frequently flow down. A hydraulic turbine of an undershot cross-flow type was installed in an irrigation channel, and the change in the turbine performance was measured when spherical snowballs were released one by one from the upstream. The observation of the snowballs passing through the turbine was also conducted. Consequently, the variations in the power generated by the rotor were classified into three modes based on the motion of the snowballs, and could be organized by the ratio of the snowballs' cross-sectional area to the product of the rotor width and blade interval. Furthermore, the emergence of the power output overshoot phenomenon, in which the power output temporarily increases compared to clear water when the rotor restarts after stopping, was identified, and the relationship between the amount of loss when the rotor stops and that of electric energy gained during the overshoot was clarified. Certain guidelines for the installation of the undershot cross-flow type in irrigation channels of snowy regions was successfully obtained.

Inhibition of Fatty Acid Synthesis Induces Apoptosis of Human Pancreatic Cancer Cells.

Cancer cells tend to have a high requirement for lipids, including fatty acids, cholesterol and triglyceride, because of their rapid proliferative rate compared to normal cells. In this study, we investigated the effects of inhibition of lipid synthesis on the proliferation and viability of human pancreatic cancer cells. Of the inhibitors of lipid synthesis that were tested, 5-(tetradecyloxy)-2-furoic acid (TOFA), which is an inhibitor of acetyl-CoA carboxylase, and the fatty acid synthase (FAS) inhibitors cerulenin and irgasan, significantly suppressed the proliferation of MiaPaCa-2 and AsPC-1 cells. Treatment of MiaPaCa-2 cells with these inhibitors significantly increased the number of apoptotic cells. In addition, TOFA increased caspase-3 activity and induced cleavage of poly (ADP-ribose) polymerase in MiaPaCa-2 cells. Moreover, addition of palmitate to MiaPaCa-2 cells treated with TOFA rescued cells from apoptotic cell death. These results suggest that TOFA induces apoptosis via depletion of fatty acids and that, among the various aspects of lipid metabolism, inhibition of fatty acid synthesis may be a notable target for the treatment of human pancreatic cancer cells.

[Recent findings regarding the mechanism of idiosyncratic drug toxicity].

Animal experiments cannot predict the probability of idiosyncratic drug toxicity; consequently, an important goal of the pharmaceutical industry is to develop a new methodology for preventing this form of drug reaction. Although the mechanism remains unclear, immune reactions are likely involved in the toxic processes underlying idiosyncratic drug toxicity: the drug is first activated into a chemically reactive metabolite that binds covalently to proteins and then acts as an immunogen. Therefore, screening tests to detect chemically reactive metabolites are conducted early during drug development and typically involve trapping with glutathione. More quantitative methods are then used in a later stage of drug development and frequently employ (14)Cor (3)H-labeled compounds. It has recently been demonstrated that a zone classification system can be used to separate risky drugs from likely safe drugs: by plotting the amount of each protein-bound reactive metabolite in vitro against the dose levels in vivo, the risk associated with each drug candidate can be assessed. A mechanism for idiosyncratic drug-induced hepatotoxicity was proposed by analogy to virus-induced hepatitis, in which cytotoxic T lymphocytes play an important role. This mechanism suggests that polymorphism in human leukocyte antigens is involved in idiosyncrasy, and a strong correlation with a specific genotype of human leukocyte antigens has been found in many cases of idiosyncratic drug toxicity. Therefore, gene biomarkers hold promise for reducing the clinical risk and prolonging the life cycle of otherwise useful drugs.

Idiosyncratic drug hepatotoxicity: strategy for prevention and proposed mechanism.

Idiosyncratic drug toxicity has led to the market withdrawal of many drugs in the past. Since animal experiments are not predictive of such toxicity, the pharmaceutical industry continues to seek new methodologies for the prevention of such effects. Although the mechanism of idiosyncratic drug toxicity remains unclear, immune reactions are likely involved. Although drugs with low molecular weights are typically not themselves immunogenic, these drugs may become haptens after being converted to chemically reactive metabolites and becoming covalently cross-linked to proteins. Therefore, screening tests to detect chemically reactive metabolites, most typically by trapping with glutathione, are carried out at early stages of drug development. More quantitative methods are used in later stages of drug development; radioassays for covalent binding (using (14)Cor (3)H-labeled compounds) are most frequently employed. A zone classification system created by combining previous assessment criteria for the chemically reactive metabolites in vitro (<50 pmole/mg-protein) and for the dose levels in vivo (<10 mg/day) could be used for risk assessment of drug candidates. A mechanism for idiosyncratic, drug-induced hepatotoxicity is proposed by analogy to virus-induced hepatitis, where cytotoxic T lymphocytes play an important role; we suggest that idiosyncrasy reflects the involvement of polymorphisms in the human leucocyte antigen-encoding loci. In fact, a strong correlation has been found between of idiosyncratic drug toxicity and specific human leucocyte antigen genotypes. Therefore, screening of patients for gene biomarkers is expected to reduce the clinical risk of idiosyncratic drug toxicity, thereby prolonging the life cycle of otherwise useful drugs.

Involvement of reactive oxygen species in the enhancement of membrane lipid peroxidation by sonodynamic therapy with functionalized fullerenes.

BACKGROUND/AIM: Sonodynamic cancer therapy is based on the preferential uptake and/or retention of a sonosensitizing drug (sonosensitizer) in tumor tissues and subsequent activation of the drug by ultrasound irradiation. In the present study, we investigated the participation of lipid peroxidation in the mechanism of the sonodynamically-induced antitumor effect with functionalized fullerenes, such as polyhydroxy fullerene (PHF. MATERIALS AND METHODS: Ultrasonically-induced cell damage and lipid peroxidation with PHF were compared in the same in vitro insonation setup. Sarcoma 180 cells suspended in PBS were exposed to 2 MHz ultrasound in the presence and absence of PHF. Cell viability was determined by the Trypan Blue exclusion test. Lipid peroxidation in cell membranes was estimated by measuring the amount of malondialdehyde as the thiobarbituric acid-reactive-substances. RESULTS: Significant enhancement of the rates of both ultrasonically-induced cell damage and lipid peroxidation was observed in the presence of PHF, both of which were positively correlated with PHF. The enhancement of cell damage and lipid peroxidation with PHF was suppressed by reactive oxygen scavengers such as histidine and tryptophan. CONCLUSION: The good correlation observed in the presence of PHF suggests that membrane lipid peroxidation is one of the important intermediary events in sonodynamically-induced cellular damage. The inhibitory effects of histidine and tryptophan also provide evidence that singlet oxygen plays an important role in PHF-mediated sonosensitization of membranes and that this moiety may be an important mediator of cell destruction in sonodynamic therapy associated with PHF and ultrasound.

Hepatic microsomal thiol methyltransferase is involved in stereoselective methylation of pharmacologically active metabolite of prasugrel.

Prasugrel, a thienopyridine antiplatelet drug, is converted in animals and humans to the pharmacologically active metabolite R-138727 [(2Z)-{1-[(1RS)-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-sulfanylpiperidin-3-ylidene}ethanoic acid], which has two chiral centers, occurring as a mixture of four isomers. The RS and RR isomers are more active than the SS and SR isomers (RS > RR > > SR = SS). The pharmacologically active metabolite is further metabolized to an S-methylated metabolite that is the major identified inactive metabolite in humans. In rat, dog, and human liver microsomes supplemented with S-adenosyl methione, the SS and SR isomers of the active metabolite were extensively S-methylated while the RS and RR isomers were not. Addition of 2,3-dichloromethyl benzylamine (50 µM) completely inhibited the S-methylation reaction, indicating that the microsomal and cytosolic thiol methyltransferase but not the cytosolic thiopurine S-methyltransferase is involved in the methylation. The hepatic intrinsic clearance values for methylation of the RS, RR, SS, and SR isomers (ml/min/kg) were 0, 0, 40.4, and 37.6, respectively, in rat liver microsomes, 0, 0, 11.6, and 2.5, respectively, in dog liver microsomes, and 0, 0, 17.3, and 17.7, respectively, in human liver microsomes, indicating that the RS and RR isomers are not methylated in vitro and that the methylation of SS and SR isomers is high with rat > human > dog. This finding in vitro agreed well with the in vivo observation in rats and dogs, where the S-methylated SS and SR isomers were the major metabolites in the plasma whereas negligible amounts of S-methylated RS and RR isomers were detected after intravenous administration of the pharmacologically active metabolites.

Sonodynamically-induced anticancer effects by functionalized fullerenes.

BACKGROUND: Functionalized fullerenes, such as polyhydroxy fullerenes (PHF), have attracted particular attention due to their water solubility and their potential application in tumor imaging and therapy as carbon nanomaterials. In this study, the sonodynamically-induced antitumor effect of PHF was investigated. MATERIALS AND METHODS: Sonodynamically-induced antitumor effects of PHF in combination with ultrasound were investigated using isolated sarcoma 180 cells and solid tumor from colon 26 carcinoma cells. RESULTS: The cell damage induced by sonication was enhanced by two-fold in the presence of 80 µM PHF. Histidine significantly inhibited this enhancement. This inhibitory effect suggests that the sonodynamically-induced antitumor effect was mediated by sonodynamically-generated reactive oxygen species. The combined treatment of ultrasonic exposure with PHF suppressed the growth of implanted colon 26 tumors. The destruction of tumor tissue was observed with the ultrasonic treatment in combination with PHF, while neither the treatment with PHF alone nor that with ultrasound alone caused necrosis. CONCLUSION: These results suggest that PHF is a potential sonosensitizer for sonodynamic treatment of solid tumors.

Microdose pharmacogenetic study of ¹4C-tolbutamide in healthy subjects with accelerator mass spectrometry to examine the effects of CYP2C9∗3 on its pharmacokinetics and metabolism.

Microdose study enables us to understand the pharmacokinetic profiles of drugs in humans prior to the conventional clinical trials. The advantage of microdose study is that the unexpected pharmacological/toxicological effects of drugs caused by drug interactions or genetic polymorphisms of metabolic enzymes/transporters can be avoided due to the limited dose. With a combination use of accelerator mass spectrometry (AMS) and (14)C-labaled compounds, the pharmacokinetics of both parent drug and its metabolites can be sensitively monitored. Thus, to demonstrate the usability of microdose study with AMS for the prediction of the impact of genetic polymorphisms of CYP enzyme on the pharmacokinetics of unchanged drugs and metabolites, we performed microdose pharmacogenetic study using tolbutamide as a CYP2C9 probe drug. A microdose of (14)C-tolbutamide (100 µg) was administered orally to healthy volunteers with the CYP2C9(∗)1/(∗)1 or CYP2C9(∗)1/(∗)3 diplotype. Area under the plasma concentration-time curve for the (14)C-radioactivity, determined by AMS, or that for the parent drug, determined by liquid chromatography/mass spectrometry, was about 1.6 times or 1.7 times greater in the CYP2C9(∗)1/(∗)3 than in the CYP2C9(∗)1/(∗)1 group, which was comparable to the previous reports at therapeutic dose. In the plasma and urine, tolbutamide, carboxytolbutamide, and 4-hydroxytolbutamide were detected and practically no other metabolites could be found in both diplotype groups. The fraction of metabolites in plasma radioactivity was slightly lower in the CYP2C9(∗)1/(∗)3 group. Microdose study can be used for the prediction of the effects of genetic polymorphisms of enzymes on the pharmacokinetics and metabolic profiles of drugs with minimal care of their pharmacological/toxicological effects.

Cost-effectiveness analysis of microdose clinical trials in drug development.

Microdose (MD) clinical trials have been introduced to obtain human pharmacokinetic data early in drug development. Here we assessed the cost-effectiveness of microdose integrated drug development in a hypothetical model, as there was no such quantitative research that weighed the additional effectiveness against the additional time and/or cost. First, we calculated the cost and effectiveness (i.e., success rate) of 3 types of MD integrated drug development strategies: liquid chromatography-tandem mass spectrometry, accelerator mass spectrometry, and positron emission tomography. Then, we analyzed the cost-effectiveness of 9 hypothetical scenarios where 100 drug candidates entering into a non-clinical toxicity study were selected by different methods as the conventional scenario without MD. In the base-case, where 70 drug candidates were selected without MD and 30 selected evenly by one of the three MD methods, incremental cost-effectiveness ratio per one additional drug approved was JPY 12.7 billion (US$ 0.159 billion), whereas the average cost-effectiveness ratio of the conventional strategy was JPY 24.4 billion, which we set as a threshold. Integrating MD in the conventional drug development was cost-effective in this model. This quantitative analytical model which allows various modifications according to each company's conditions, would be helpful for guiding decisions early in clinical development.

Involvement of reactive oxygen species in sonodynamically induced apoptosis using a novel porphyrin derivative.

In this study, we investigated the induction of apoptosis by ultrasound in the presence of the novel porphyrin derivative DCPH-P-Na(I). HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of DCPH-P-Na(I), and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Reactive oxygen species were measured by means of ESR and spin trapping technique. Cells treated with 8 µM DCPH-P-Na(I) and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas significant morphologic changes were not observed in cells exposed to either ultrasound or DCPH-P-Na(I) alone. Also, DNA ladder formation and caspase-3 activation were observed in cells treated with both ultrasound and DCPH-P-Na(I) but not in cells treated with ultrasound or DCPH-P-Na(I) alone. In addition, the combination of DCPH-P-Na(I) and the same acoustical arrangement of ultrasound substantially enhanced nitroxide generation by the cells. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. These results indicate that the combination of ultrasound and DCPH-P-Na(I) induced apoptosis in HL-60 cells. The significant reduction in sonodynamically induced apoptosis, nitroxide generation, and caspase-3 activation by histidine suggests active species such as singlet oxygen are important in the sonodynamic induction of apoptosis. These experimental results support the possibility of sonodynamic treatment for cancer using the induction of apoptosis.

Glutaredoxin is involved in the formation of the pharmacologically active metabolite of clopidogrel from its GSH conjugate.

Clopidogrel is a thienopyridine antiplatelet agent that is converted to the active metabolite, R-361015, in vivo. Clopidogrel is first oxidized to a thiolactone intermediate R-115991. R-115991 is thought to be metabolized to a GSH conjugate of R-361015 (R-361015-SG) and then is reduced to R-361015 in the presence of GSH. In this study, we investigated the enzyme-mediated formation of R-361015 from R-361015-SG in human liver microsomes and cytosols. After incubation of R-115991 in human liver microsomes, the formation of R-361015-SG, and subsequently of R-361015, was observed. The apparent formation rate of R-361015-SG was markedly decreased when human liver cytosols were added. Fitting the data to the kinetic model showed that the rate constant of R-361015-SG reduction to R-361015 in human liver microsomes was approximately 20-fold higher in the presence of human liver cytosols (6.56 min⁻¹) than in the absence of cytosols (0.326 min⁻¹). In addition, the formation rate of R-361015 from R-361015-SG was higher in human liver cytosols (2843 ± 1176 pmol · min⁻¹ · mg⁻¹) compared with in human liver microsomes (508 ± 396 pmol · min⁻¹ · mg⁻¹). The formation of R-361015 from R-361015-SG in human liver microsomes or cytosols was inhibited by anti-human glutaredoxin antibody in a concentration-dependent manner. Recombinant human glutaredoxin mediated the formation of R-361015 from R-361015-SG with the K(m) and V(max) values of 30.0 ± 1.3 µM and 381.6 ± 209.8 pmol · min⁻¹ · µg⁻¹, respectively. The intrinsic clearance value (V(max)/K(m)) was 12.9 ± 7.5 µl · min⁻¹ · µg⁻¹. In conclusion, we found that human glutaredoxin is a main contributor to the formation of the pharmacologically active metabolite of clopidogrel from its GSH conjugate in human liver.

Paraoxonase 1 as a major bioactivating hydrolase for olmesartan medoxomil in human blood circulation: molecular identification and contribution to plasma metabolism.

Olmesartan medoxomil (OM) is a prodrug-type angiotensin II type 1 receptor antagonist. The OM-hydrolyzing enzyme responsible for prodrug bioactivation was purified from human plasma through successive column chromatography and was molecularly identified through N-terminal amino acid sequencing, which resulted in a sequence of 20 amino acids identical to that of human paraoxonase 1 (PON1). Two recombinant allozymes of human PON1 (PON1(192QQ) and PON1(192RR)) were constructed and were clearly demonstrated to hydrolyze OM; hydrolysis by the latter allozyme was slightly faster than that by the former. In addition, we evaluated the contribution of PON1 to OM bioactivation in human plasma. Enzyme kinetic studies demonstrated that OM was hydrolyzed more effectively by the recombinant PON1 proteins than by purified albumin. The OM-hydrolyzing activities of the recombinant PON1 proteins and diluted plasma were greatly reduced in the absence of calcium ions. Immunoprecipitation with anti-PON1 IgG completely abolished the OM-hydrolyzing activity in human plasma, whereas the activity was partially inhibited with anti-albumin IgG. The distribution pattern of the OM-hydrolyzing activity in human serum lipoprotein fractions and lipoprotein-deficient serum was examined and showed that most of the OM-hydrolyzing activity was located in the high-density lipoprotein fraction, with which PON1 is closely associated. In conclusion, we identified PON1 as the OM-bioactivating hydrolase in human plasma on a molecular basis and demonstrated that PON1, but not albumin, plays a major role in OM bioactivation in human plasma.

Involvement of different human glutathione transferase isoforms in the glutathione conjugation of reactive metabolites of troglitazone.

Null mutation of glutathione transferase (GST) M1 and GSTT1 was reported to correlate statistically with an abnormal increase in the plasma levels of alanine aminotransferase or aspartate aminotransferase caused by troglitazone in diabetic patients (Clin Pharmacol Ther, 73:435-455, 2003). This clinical evidence leads to the hypothesis that GSH conjugation catalyzed by GSTT1 and GSTM1 has a role in the elimination of reactive metabolites of troglitazone. However, the contribution of GST isoforms expressed in human liver to the detoxification of reactive metabolites of troglitazone has not yet been clarified. We investigated the involvement of human GST isoforms in the GSH conjugation of reactive metabolites of troglitazone using recombinant GST enzymes. Five reported GSH conjugates of reactive metabolites were produced from troglitazone after incubation with liver microsomes, NADPH, and GSH in a GSH concentration-dependent manner. Addition of human recombinant GSTA1, GSTA2, GSTM1, or GSTP1 protein to the incubation mixture further increased the GSH conjugates. However, the addition of GSTT1 did not show any catalytic effect. It is of interest that one of the reactive metabolites with a quinone structure was predominantly conjugated with GSH by GSTM1. Thus, we demonstrated that the GST isoforms contributed differently to the GSH conjugation of individual reactive metabolites of troglitazone, and GSTM1 is the most important GST isoform in the GSH conjugation of a specific reactive metabolite produced from the cytotoxic, quinone-form metabolite of troglitazone.

Clinical relevance of liquid chromatography tandem mass spectrometry as an analytical method in microdose clinical studies.

PURPOSE: To investigate the potency of LC-MS/MS by means of sensitivity and the applicability for cassette dosing in microdose clinical trials. METHODS: Thirty one top-selling 31 drugs were spiked to human plasma, extracted, and analyzed by LC-MS/MS. RESULTS: The lower limits of quantification for each drug varied from 0.08 to 50 pg/mL, and were lower than one eighth of the assumed maximum plasma concentration at microdose in all drugs except for losartan, indicating the high performance in acquisition of full pharmacokinetic profiles at microdose. We also succeeded in simultaneous analysis of multiple compounds, assuming a situation of cassette dosing in which multiple drug candidates would be administrated simultaneously. CONCLUSIONS: Together with the features of LC-MS/MS, such as immediate verification, the utilization of non-radiolabeled drugs and no special facilities, we suppose that LC-MS/MS analysis would be widely applicable in conducting microdose clinical studies.

Sonodynamically-induced antitumor effect of mono-l-aspartyl chlorin e6 (NPe6).

BACKGROUND: The sonodynamically-induced in vitro and in vivo antitumor effects of mono-l-aspartyl chlorin e6 (NPe6) was investigated. MATERIALS AND METHODS: Both in vitro and in vivo antitumor effects were tested in combination with ultrasound at 2 MHz. RESULTS: The rate of ultrasonically-induced damage on isolated sarcoma 180 cells in air-saturated suspension was enhanced two-fold with 80 µM NPe6. The co-administration of 25 mg/kg NPe6 followed by ultrasonic exposure at 2 MHz suppressed the growth of implanted colon 26 cell tumors at an intensity at which ultrasound alone showed only a slight antitumor effect. CONCLUSION: These in vitro and in vivo results suggest that NPe6 is a potential sensitizer for sonodynamic tumor treatment. The enhancement of cell damage by NPe6 was significantly inhibited by histidine, which may suggest reactive oxygen species plays a primary role in sonodynamically-induced antitumor effect.

HA-33 facilitates transport of the serotype D botulinum toxin across a rat intestinal epithelial cell monolayer.

A large size botulinum toxin complex (L-TC) is composed of a single neurotoxin (BoNT), a single nontoxic nonhaemagglutinin (NTNHA) and a haemagglutinin (HA) complex. The HA complex is comprised of three HA-70 molecules and three arm structures of HA-33/HA-17 that consist of two HA-33 and a single HA-17. In addition to the mature L-TC, smaller TCs are present in cultures: M-TC (BoNT/NTNHA), M-TC/HA-70 and immature L-TCs with fewer HA-33/HA-17 arms than mature L-TC. Because L-TC displays higher oral toxicity than pure BoNT, it was presumed that nontoxic proteins are critical for food poisoning. In this study, the absorption of TCs across intestinal epithelial cells was assessed by examining the cell binding and monolayer transport of serotype D toxins in the rat intestinal epithelial cell line IEC-6. All TCs, including pure BoNT, displayed binding and transport, with mature L-TC showing the greatest potency. Inhibition experiments using antibodies revealed that BoNT, HA-70 and HA-33 could be responsible for the binding and transport. The findings here indicate that all TCs can transport across the cell layer via a sialic acid-dependent process. Nonetheless, binding and transport markedly increased with number of HA-33/HA-17 arms in the TC. We therefore conclude that the HA-33/HA-17 arm is not necessarily required for, but facilitates, transport of botulinum toxin complexes.

Drug-induced idiosyncratic hepatotoxicity: prevention strategy developed after the troglitazone case.

Troglitazone induced an idiosyncratic, hepatocellular injury-type hepatotoxicity in humans. Statistically, double null genotype of glutathione S-transferase isoforms, GSTT1 and GSTM1, was a risk factor, indicating a low activity of the susceptible patients in scavenging chemically reactive metabolites. CYP3A4 and CYP2C8 were involved in the metabolic activation and CYP3A4 was inducible by repeated administrations of troglitazone. The genotype analysis, however, indicated that the metabolic idiosyncrasy resides in the degradation of but not in the production of the toxic metabolites of troglitazone. Antibody against hepatic aldolase B was detected in the case patients, suggesting involvement of immune reaction in the toxic mechanism. Troglitazone induced apoptotic cell death in human hepatocytes at a high concentration, and this property may have served as the immunological danger signal, which is thought to play an important role in activating immune reactions. Hypothesis is proposed in analogy to the virus-induced hepatitis. After the troglitazone-case, pharmaceutical companies implemented screening systems for chemically reactive metabolites at early stage of drug development, taking both the amount of covalent binding to the proteins in vitro and the assumed clinical dose level into consideration. At the post-marketing stage, gene analyses of the case patients, if any, to find pharmacogenetic biomarkers could be a powerful tool for contraindicating to the risky patients.

The intestine as an important contributor to prasugrel active metabolite formation in vivo.

Prasugrel [2-acetoxy-5-(α-cyclopropylcarbonyl-2-fluorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine], a thienopyridine antiplatelet agent, undergoes rapid hydrolysis in vivo to a thiolactone intermediate, 2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone (R-95913), which is further converted to a pharmacologically active metabolite, 2-[1-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene acetic acid (R-138727), by oxidation via cytochromes P450. In this study, we investigated how much the intestine and liver contribute to the formation of R-95913 and R-138727 after intraduodenal administration of prasugrel (1 mg/kg) to portal vein- and hepatic vein-cannulated dogs. The areas under the plasma concentration-time curve up to 2 h of R-95913 in the portal, hepatic, and systemic veins were 525, 32, and 17 ng · h/ml, respectively, and those of R-138727 were 564, 529, and 495 ng · h/ml, respectively. The dose of prasugrel was absorbed and then converted to R-95913 and R-138727 by 93 and 13%, respectively, in the intestine. In the liver, 23% of the R-95913, which passed through the intestine, was converted to R-138727. In conclusion, this is the first report to directly demonstrate that the conversion of prasugrel to R-138727 in the intestine is comparable to that converted in the liver of dogs.

Glutaredoxin and thioredoxin can be involved in producing the pharmacologically active metabolite of a thienopyridine antiplatelet agent, prasugrel.

A thienopyridine antiplatelet agent, prasugrel, is rapidly hydrolyzed to a thiolactone metabolite (R-95913, 2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone). R-95913 is oxidized by hepatic cytochromes P450 to the pharmacologically active metabolite R-138727 (2-[1-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid). One possible intermediate in the in vitro bioactivation pathway is a glutathione conjugate, R-133490, which could be reduced to generate R-138727 in the presence of a reducing agent such as glutathione. In this study, enzymes in human liver cytosols were found to accelerate reduction of R-133490 leading to the formation of R-138727. To explore the possible reductive enzymes, we separated the various proteins in human liver cytosol based on size using gel filtration chromatography. Two active peaks were detected and found to contain thioredoxin and glutaredoxin, respectively. In addition, recombinant human glutaredoxin and thioredoxin promoted the formation of R-138727 from R-133490 with much higher activity for glutaredoxin than for thioredoxin. This study is the first in vitro observation indicating that glutaredoxin and thioredoxin in human liver are active in reducing the mixed disulfide formed between xenobiotics and glutathione.