New paradigm for expediting drug development in Asia.

Some Asian regulators currently require Phase I data in Asians before joining global Phase II/III trials. Here, we discuss inherent limitations of Phase I ethnic sensitivity studies (ESS) to identify potential interethnic differences. We review recent new drug applications (NDAs) for Japan and China to critically assess the value of separate ESSs in Asian populations. Given that the observed value of ESS was limited, we propose a new global drug development paradigm: if relevant safety, pharmacokinetic (PK), and pharmacogenetic (PG) data are available from the original Phase I study population, it might be possible to extrapolate those data to Asian populations for their inclusion in Phase II/III trials, without an ESS. This could help to streamline drug development in Asia while still addressing regulatory requirements.

Effect of Hepatic Impairment on the Pharmacokinetics of Pradigastat, a Diacylglycerol Acyltransferase 1 (DGAT1) Inhibitor.

BACKGROUND AND OBJECTIVE: Pradigastat, a novel diacylglycerol acyltransferase 1 inhibitor, is under development to treat familial chylomicronemia syndrome. The potential impact of hepatic impairment on the pharmacokinetics of pradigastat was evaluated in this study. METHODS: In this study, a single oral dose of 20 mg pradigastat was administered first to patients with mild and moderate hepatic impairment (n = 10/group) and subsequently to patients with severe hepatic impairment (n = 6). The pharmacokinetics of pradigastat were compared between each patient group and the respective matched healthy subjects. RESULTS: As compared with the respective matched healthy groups, the geometric mean ratios of the area under the plasma concentration-time curve from time zero to infinity (AUC inf) (h · ng/mL) were 1.49, 1.06 and 1.99 in mild, moderate and severe hepatic impairment patients, respectively; the observed maximum plasma concentration (C max) (ng/mL) values were 0.97, 1.28 and 2.74, respectively; and the total body clearance of the drug from plasma (CL/F) (L/h) values were 0.67, 0.95 and 0.50, respectively. The elimination half-life and plasma protein binding of pradigastat were comparable among all the patients. There were no apparent relationships between AUC inf or C max and albumin or bilirubin levels (R (2) < 0.3; p > 0.05). Overall, 19 adverse events (AEs) were reported in 13 patients. The incidence of AEs appeared to increase with increasing severity of hepatic impairment. CONCLUSION: No clinically significant differences in the pharmacokinetics of pradigastat were observed in mild and moderate hepatic impairment patients compared with healthy subjects. However, the systemic exposure of pradigastat doubled while the clearance decreased by half in patients with severe hepatic impairment compared with healthy subjects. All treatments were well tolerated in the study.

Novel deletion in glycoprotein G forms a cluster and causes epidemiologic spread of herpes simplex virus type 2 infection.

The herpes simplex virus type 2 (HSV-2) glycoprotein G (gG-2) gene of 106 clinical isolates was analyzed and six isolates were identified with 63 nucleotides comprising 21 amino acids (aa) deleted in the immunodominant region. Compared with strain HG52, variations in the gG-2 gene were found at 276 and 27 sites in nucleotide and aa sequences, respectively, in the 106 strains. Significant variations in both nucleotides and aa were accumulated in the immunodominant region rather than in the other regions (P < 0.001), indicating that the immunodominant region might be indispensable in vivo and a hot spot for variation. The frequency of 21 aa-deleted strains (HSVΔ21/gG-2) among clinical isolates was 5%, indicating the advantage of this deletion of gG-2 for epidemiological expansion. Phylogenetic analysis of the 106 strains indicated that the HSVΔ21/gG-2 strains formed a cluster among the various variations but that their genomes showed different endonuclease digestion patterns. The antibody titers to total HSV antigens of patients infected with wild HSV-2 and HSVΔ21/gG-2 were similar, but patients with HSVΔ21/gG-2 had a lower antibody titer to gG-2 than those with wild HSV-2 (P < 0.001). HSVΔ21/gG-2 might be less immnunogenic and reduce antibody production to gG-2, while its pathogenicity in humans was not distinguished in its clinical manifestations. Thus, infection with HSVΔ21/gG-2 caused genital lesions similar to wild HSV-2 infection, but evaded the immune response to gG-2 to allow epidemiological spread, indicating the importance of this deletion in the immunodominant region of gG-2 in the pathogenesis and transmission of genital herpes.

The quantitative prediction of CYP-mediated drug interaction by physiologically based pharmacokinetic modeling.

PURPOSE: The objective is to confirm if the prediction of the drug-drug interaction using a physiologically based pharmacokinetic (PBPK) model is more accurate. In vivo Ki values were estimated using PBPK model to confirm whether in vitro Ki values are suitable. METHOD: The plasma concentration-time profiles for the substrate with coadministration of an inhibitor were collected from the literature and were fitted to the PBPK model to estimate the in vivo Ki values. The AUC ratios predicted by the PBPK model using in vivo Ki values were compared with those by the conventional method assuming constant inhibitor concentration. RESULTS: The in vivo Ki values of 11 inhibitors were estimated. When the in vivo Ki values became relatively lower, the in vitro Ki values were overestimated. This discrepancy between in vitro and in vivo Ki values became larger with an increase in lipophilicity. The prediction from the PBPK model involving the time profile of the inhibitor concentration was more accurate than the prediction by the conventional methods. CONCLUSION: A discrepancy between the in vivo and in vitro Ki values was observed. The prediction using in vivo Ki values and the PBPK model was more accurate than the conventional methods.

Effects of organic anion transporting polypeptide 1B1 haplotype on pharmacokinetics of pravastatin, valsartan, and temocapril.

OBJECTIVE: Recent reports have shown that genetic polymorphisms in organic anion transporting polypeptide (OATP) 1B1 have an effect on the pharmacokinetics of drugs. However, the impact of OATP1B1*1b alleles, the frequency of which is high in all ethnicities, on the pharmacokinetics of substrate drugs is not known after complete separation of subjects with OATP1B1*1a and *1b. Furthermore, the correlation between the clearances of OATP1B1 substrate drugs in individuals has not been characterized. We investigated the effect of genetic polymorphism of OATP1B1, particularly the *1b allele, on the pharmacokinetics of 3 anionic drugs, pravastatin, valsartan, and temocapril, in Japanese subjects. METHODS: Twenty-three healthy Japanese volunteers were enrolled in a 3-period crossover study. In each period, after a single oral administration of pravastatin, valsartan, or temocapril, plasma and urine were collected for up to 24 hours. RESULTS: The area under the plasma concentration-time curve (AUC) of pravastatin in *1b/*1b carriers (47.4 +/- 19.9 ng.h/mL) was 65% of that in *1a/*1a carriers (73.2 +/- 23.5 ng.h/mL) (P = .049). Carriers of *1b/*15 (38.2 +/- 15.9 ng.h/mL) exhibited a 45% lower AUC than *1a/*15 carriers (69.2 +/- 23.4 ng.h/mL) (P = .024). In the case of valsartan we observed a similar trend as with pravastatin, although the difference was not statistically significant (9.01 +/- 3.33 microg.h/mL for *1b/*1b carriers versus 12.3 +/- 4.6 microg.h/mL for *1a/*1a carriers [P = .171] and 6.31 +/- 3.64 microg.h/mL for *1b/*15 carriers versus 9.40 +/- 4.34 microg.h/mL for *1a/*15 carriers [P = .213]). The AUC of temocapril also showed a similar trend (12.4 +/- 4.1 ng.h/mL for *1b/*1b carriers versus 18.5 +/- 7.7 ng.h/mL for *1a/*1a carriers [P = .061] and 16.4 +/- 5.0 ng.h/mL for *1b/*15 carriers versus 19.0 +/- 4.1 ng.h/mL for *1a/*15 carriers [P = .425]), whereas that of temocaprilat (active form of temocapril) was not significantly affected by the haplotype of OATP1B1. Interestingly, the AUC of valsartan and temocapril in each subject was significantly correlated with that of pravastatin (R = 0.630 and 0.602, P < .01). The renal clearance remained unchanged for each haplotype for all drugs. CONCLUSION: The major clearance mechanism of pravastatin, valsartan, and temocapril appears to be similar, and OATP1B1*1b is one of the determinant factors governing the interindividual variability in the pharmacokinetics of pravastatin and, possibly, valsartan and temocapril.

Drug-drug interaction between pitavastatin and various drugs via OATP1B1.

It has already been demonstrated that pitavastatin, a novel potent HMG-coenzyme A reductase inhibitor, is taken up into human hepatocytes mainly by organic anion transporting polypeptide (OATP) 1B1. Because OATP2B1 is also localized in the basolateral membrane of human liver, we took two approaches to further confirm the minor contribution of OATP2B1 to the hepatic uptake of pitavastatin. Western blot analysis revealed that the ratio of the band density of OATP2B1 in human hepatocytes to that in our expression system is at least 6-fold lower compared with OATP1B1 and OATP1B3. The uptake of pitavastatin in human hepatocytes could be inhibited by both estrone-3-sulfate (OATP1B1/OATP2B1 inhibitor) and estradiol-17beta-D-glucuronide (OATP1B1/OATP1B3 inhibitor). These results further supported the idea that OATP1B1 is a predominant transporter for the hepatic uptake of pitavastatin. Then, to explore the possibility of OATP1B1-mediated drug-drug interaction, we checked the inhibitory effects of various drugs on the pitavastatin uptake in OATP1B1-expressing cells and evaluated whether the in vitro inhibition was clinically significant or not. As we previously reported, we used the methodology for estimating the maximum unbound concentration of inhibitors at the inlet to the liver (I(u,in,max)). Judging from I(u,in,max) and inhibition constant (K(i)) for OATP1B1, several drugs (especially cyclosporin A, rifampicin, rifamycin SV, clarithromycin, and indinavir) have potentials for interacting with OATP1B1-mediated uptake of pitavastatin. The in vitro experiments could support the clinically observed drug-drug interaction between pitavastatin and cyclosporin A. These results suggest that we should pay attention to the concomitant use of some drugs with pitavastatin.

Involvement of BCRP (ABCG2) in the biliary excretion of pitavastatin.

Pitavastatin, a novel potent 3-hydroxymethylglutaryl coenzyme A reductase inhibitor, is distributed selectively to the liver and excreted into bile in unchanged form in rats. We reported previously that the hepatic uptake is mainly mediated by organic anion transporting polypeptide (OATP) 1B1, whereas the biliary excretion mechanism remains to be clarified. In the present study, we investigated the role of breast cancer resistance protein (BCRP) in the biliary excretion of pitavastatin. The ATP-dependent uptake of pitavastatin by human and mouse BCRP-expressing membrane vesicles was significantly higher compared with that by control vesicles with Km values of 5.73 and 4.77 microM, respectively. The biliary excretion clearance of pitavastatin in Bcrp1-/- mice was decreased to one-tenth of that in control mice. The biliary excretion of pitavastatin was unchanged between control and Eisai hyperbilirubinemic rats, indicating a minor contribution of multidrug resistance-associated protein (Mrp) 2. This observation differs radically from that for a more hydrophilic statin, pravastatin, of which biliary excretion is largely mediated by Mrp2. These data suggest that the biliary clearance of pitavastatin can be largely accounted for by BCRP in mice. In the case of humans, transcellular transport of pitavastatin was determined in the Madin-Darby canine kidney II cells expressing OATP1B1 and human canalicular efflux transporters. A significant basal-to-apical transport of pitavastatin was observed in OATP1B1/MDR1 and OATP1B1/MRP2 double transfectants as well as OATP1B1/BCRP double transfectants, implying the involvement of multiple transporters in the biliary excretion of pitavastatin in humans. This is in contrast to a previous belief that the biliary excretion of statins is mediated mainly by MRP2.

Bile salt export pump (BSEP/ABCB11) can transport a nonbile acid substrate, pravastatin.

Pravastatin is a well known 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor. Cumulative studies have shown that pravastatin is taken up into hepatocytes by the organic anion transporting polypeptide family transporters and excreted into the bile as an intact form by multidrug resistance-associated protein 2 (MRP2). It is generally accepted that the bile salt export pump (BSEP/ABCB11) mainly transports bile acids and plays an indispensable role in their biliary excretion. Interestingly, we found that BSEP could accept pravastatin as a substrate. Significant ATP-dependent uptake of pravastatin by human BSEP (hBSEP)- and rat BSEP (rBsep)-expressing membrane vesicles was observed, and the ratio of the uptake activity of pravastatin to that of taurocholic acid (TCA) by hBSEP was 3.3-fold higher than that by rBsep. The K(m) value of pravastatin for hBSEP was 124 muM. A mutual inhibition study between TCA and pravastatin revealed that they competitively interact with hBSEP. Several statins inhibited the hBSEP- and rBsep-mediated uptake of TCA; however, the specific uptake of other statins (cerivastatin, fluvastatin, and pitavastatin) by hBSEP and rBSEP was not detected. The inhibitory effects of hydrophilic statins (pravastatin and rosuvastatin) on the uptake of TCA by BSEP were relatively lower than those of lipophilic statins. These data suggest that BSEP may be partly involved in the biliary excretion of pravastatin in both rats and humans.

Identification of the hepatic efflux transporters of organic anions using double-transfected Madin-Darby canine kidney II cells expressing human organic anion-transporting polypeptide 1B1 (OATP1B1)/multidrug resistance-associated protein 2, OATP1B1/multidrug resistance 1, and OATP1B1/breast cancer resistance protein.

Until recently, it was generally believed that the transport of various organic anions across the bile canalicular membrane was mainly mediated by multidrug resistance-associated protein 2 (MRP2/ABCC2). However, a number of new reports have shown that some organic anions are also substrates of multidrug resistance 1 (MDR1/ABCB1) and/or breast cancer resistance protein (BCRP/ABCG2), implying MDR1 and BCRP could also be involved in the biliary excretion of organic anions in humans. In the present study, we constructed new double-transfected Madin-Darby canine kidney II (MDCKII) cells expressing organic anion-transporting polypeptide 1B1 (OATP1B1)/MDR1 and OATP1B1/BCRP, and we investigated the transcellular transport of four kinds of organic anions, estradiol-17beta-d-glucuronide (EG), estrone-3-sulfate (ES), pravastatin (PRA), and cerivastatin (CER), to identify which efflux transporters mediate the biliary excretion of compounds using double-transfected cells. We observed the vectorial transport of EG and ES in all the double transfectants. MRP2 showed the highest efflux clearance of EG among these efflux transporters, whereas BCRP-mediated clearance of ES was the highest in these double transfectants. In addition, two kinds of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, CER and PRA, were also substrates of all these efflux transporters. The rank order of the efflux clearance of PRA mediated by each transporter was the same as that of EG, whereas the contribution of MDR1 to the efflux of CER was relatively greater than for PRA. This experimental system is very useful for identifying which transporters are involved in the biliary excretion of organic anions that cannot easily penetrate the plasma membrane.

In vitro and in vivo correlation of the inhibitory effect of cyclosporin A on the transporter-mediated hepatic uptake of cerivastatin in rats.

Previously, we have shown that the inhibition of the transporter-mediated hepatic uptake of cerivastatin (CER) by cyclosporin A (CsA) could, at least partly, explain a pharmacokinetic interaction between these drugs in humans. In the present study, we have examined the effect of CsA on the in vivo disposition of CER in rats and the in vitro uptake of [14C]CER in isolated rat hepatocytes in an attempt to evaluate the effect of inhibition of transporter-mediated hepatic uptake on the in vivo CER disposition. The steady-state plasma concentration of CER increased 1.4-fold when coadministered with CsA up to a steady-state blood concentration of 4 microM. Studies of [14C]CER uptake into isolated rat hepatocytes showed saturable transport, with the saturable portion accounting for more than 80% of the total uptake. CsA competitively inhibited the uptake of [14C]CER with a Ki of 0.3 microM. The IC50 for the uptake of [14C]CER in the absence and presence of rat plasma was 0.2 and 2.3 microM, respectively. The in vivo hepatic uptake of [14C]CER evaluated by the liver uptake index method was also inhibited by CsA in a dose-dependent manner. On the other hand, CsA did not inhibit the metabolism of [14C]CER in rat microsomes. The in vitro and in vivo correlation analysis revealed that this pharmacokinetic interaction between these drugs in rats could be quantitatively explained by the inhibition of transporter-mediated hepatic uptake. Thus, this drug-drug interaction in rats is predominantly caused by the transporter-mediated uptake process.

Gemfibrozil and its glucuronide inhibit the organic anion transporting polypeptide 2 (OATP2/OATP1B1:SLC21A6)-mediated hepatic uptake and CYP2C8-mediated metabolism of cerivastatin: analysis of the mechanism of the clinically relevant drug-drug interaction between cerivastatin and gemfibrozil.

A serious pharmacokinetic interaction between cerivastatin (CER) and gemfibrozil (GEM) has been reported. In the present study, we examined the inhibitory effects of GEM and its metabolites, M3 and gemfibrozil 1-O-beta-glucuronide (GEM-1-O-glu), on the uptake of CER by human organic anion transporting polypeptide 2 (OATP2)-expressing cells and its metabolism in cytochrome P450 expression systems. Uptake studies showed that GEM and GEM-1-O-glu significantly inhibited the OATP2-mediated uptake of CER with IC(50) values of 72 and 24 microM, respectively. They also inhibited the CYP2C8-mediated metabolism of CER with IC(50) values of 28 and 4 microM, respectively, whereas M3 had no effects. GEM and GEM-1-O-glu minimally inhibited the CYP3A4-mediated metabolism of CER. The IC(50) values of GEM and GEM-1-O-glu for the uptake and the metabolism of CER obtained in the present study were lower than their total, and not unbound, plasma concentrations. However, considering the possibly concentrated high unbound concentrations of GEM-1-O-glu in the liver and its relatively larger plasma unbound fraction compared with GEM itself, the glucuronide inhibition of the CYP2C8-mediated metabolism of CER appears to be the main mechanism for the clinically relevant drug-drug interaction. Previously reported clinical drug interaction studies showing that coadministration of GEM with pravastatin or pitavastatin, both of which are known to be cleared from the plasma by the uptake transporters in the liver, only minimally (less than 2-fold) increased the area under the plasma concentration-time curve of these statins, also supported our present conclusion.

Contribution of OATP2 (OATP1B1) and OATP8 (OATP1B3) to the hepatic uptake of pitavastatin in humans.

Pitavastatin, a novel potent 3-hydroxymethylglutaryl-CoA reductase inhibitor, is selectively distributed to the liver in rats. However, the hepatic uptake mechanism of pitavastatin has not been clarified yet. In the present study, we investigated the contribution of organic anion transporting polypeptide 2 (OATP2/OATP1B1) and OATP8 (OATP1B3) to pitavastatin uptake using transporter-expressing HEK293 cells and human cryopreserved hepatocytes. Uptake studies using OATP2- and OATP8-expressing cells revealed a saturable and Na(+)-independent uptake, with K(m) values of 3.0 and 3.3 microM for OATP2 and OATP8, respectively. To determine which transporter is more important for its hepatic uptake, we proposed a methodology for estimating their quantitative contribution to the overall hepatic uptake by comparing the uptake clearance of pitavastatin with that of reference compounds (a selective substrate for OATP2 (estrone-3-sulfate) and OATP8 (cholecystokinin octapeptide) in expression systems and human hepatocytes. The concept of this method is similar to the so-called relative activity factor method often used in estimating the contribution of each cytochrome P450 isoform to the overall metabolism. Applying this method to pitavastatin, the observed uptake clearance in human hepatocytes could be almost completely accounted for by OATP2 and OATP8, and about 90% of the total hepatic clearance could be accounted for by OATP2. This result was also supported by estimating the relative expression level of each transporter in expression systems and hepatocytes by Western blot analysis. These results suggest that OATP2 is the most important transporter for the hepatic uptake of pitavastatin in humans.

Interaction between fibrates and statins--metabolic interactions with gemfibrozil.

An in vitro study was carried out in order to examine the metabolic basis of the interaction between fibrates and statins. Metabolic inhibition of statins was noted in the presence of gemfibrozil. However, increase in the unchanged form was fairly small for pitavastatin, compared with other statins. Several CYP enzymes were shown to be principally responsible for the metabolism of gemfibrozil in contrast to other fibrates. In the presence of gemfibrozil, a focal point was obtained in Dixon plots, demonstrating that there was inhibition of CYP2C8-, CYP2C9- and CYP3A4-mediated metabolism. We propose that the increase of plasma concentration caused by co-administration of gemfibrozil and statins is at least partially due to CYP-mediated inhibition.

Studies on the interaction between fibrates and statins using human hepatic microsomes.

To gain a better understanding of the mechanism of drug-drug interaction between fibrates and statins, several in vitro experiments were performed. On coincubation with several fibrates, pitavastatin (CAS 147526-32-7) did not displace fibrates from their protein binding in human plasma. The presence of gemfibrozil (CAS 25812-30-0) inhibited the metabolism of statins (cerivastatin (CAS 145599-86-6) and atorvastatin (CAS 134523-00-5)) remarkably. However, the increase of the unchanged form was fairly small for pitavastatin. The metabolic profile of gemfibrozil was also investigated. The cytochrome P (CYP) enzyme CYP2C9 plays a major role in the metabolism of gemfibrozil. Gemfibrozil showed a high affinity for CYP enzymes and a relatively high metabolism velocity. Moreover, several inhibitory effects of gemfibrozil on CYP-mediated metabolism were detected--in contrast to other fibrates. Although the mechanism of the drug-drug interaction was not completely clarified, it is suggested that the increase of plasma concentration caused by the co-administration of gemfibrozil and statins is at least partially due to the inhibition of the CYP-mediated metabolism.