Prediction of drug-drug interactions for AUCoral of high clearance drug from in vitro data: utilization of a microtiter plate assay and a dispersion model.

The purpose of this study was to propose a new method to predict in vivo drug-drug interactions (DDIs) for a high clearance drug from in vitro data. As the high clearance drug, NE-100 (N, N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride) was used. First, approach based on I(u)/K(i) value was used for the prediction of DDIs between NE-100 and concomitant drugs. When the K(i) values (K(i-cal)) obtained from the microtiter plate (MTP) assay and the reported K(i) values (K(i-rep)) for these drugs were used to predict increases at levels of NE-100 AUC(oral) (AUC(oral) ratio), the AUC(oral) ratios from the I(u)/K(i-cal) correlated with those from the I(u)/K(i-rep). This result suggests that the K(i-cal) from the MTP assay can be used for prediction of DDIs instead of the K(i-rep) value. Second, a new approach combining the inhibition rate (R) calculated from the MTP assay and two physiological models was used to predict DDIs. When the AUC(oral) ratios of NE-100 by various drugs were predicted using the R value and the well-stirred model, the ratios were similar to those predicted using the I(u)/K(i). However, after co-administration of drugs such as quinidine, propafenone and thioridazine (potent inhibitors of CYP2D6), the NE-100 AUC(oral) ratios predicted from the dispersion model was much greater than those from well-stirred model. This result shows that application of the dispersion model to the prediction method using the R value might sensitively and precisely predict the increased levels of AUC(oral) by DDIs for high clearance drug, compared with the prediction method using I(u)/K(i) value.

The in vitro metabolism of desglymidodrine, an active metabolite of prodrug midodrine by human liver microsomes.

The human cytochrome P450 (CYP) isoforms catalyzing the oxidation metabolism of desglymidodrine (DMAE), an active metabolite of midodrine, were studied. Recombinant human CYP2D6, 1A2 and 2C19 exhibited appreciable catalytic activity with respect to the 5'-O-demethylation of DMAE. The O-demethylase activity by the recombinant CYP2D6 was much higher than that of other CYP isoforms. Quinidine (a selective inhibitor of CYP2D6) inhibited the O-demethylation of DMAE in pooled human microsomes by 86%, while selective inhibitors for other forms of CYP did not show any appreciable effect. Although the activity of CYP2D6 was almost negligible in the PM microsomes, the O-demethylase activity of DMAE was found to be maintained by about 25% of the pooled microsomes. Furafylline (a selective inhibitor of CYP1A2) inhibited the M-2 formation in the PM microsomes by 57%. The treatment of pooled microsomes with an antibody against CYP2D6 inhibited the formation of M-2 by about 75%, whereas that of the PM microsomes did not show drastic inhibition. In contrast, the antibody against CYP1A2 suppressed the activity by 40 to 50% in the PM microsomes. These findings suggest that CYP2D6 have the highest catalytic activity of DMAE 5'-O-demethylation in human liver microsomes, followed by CYP1A2 to a small extent.

Differences in cytochrome P450 forms involved in the metabolism of N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a novel sigma ligand, in human liver and intestine.

N,N-Dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100) has been developed to treat subjects with schizophrenia. This drug is mainly excreted in the form of oxidative metabolites. In the present study, identification of p450 forms involved in the metabolism was carried out using human livers and intestinal microsomes (HLM and HIM). Eadie-Hofstee plots for NE-100 disappearance in HLM were biphasic, thus indicating the involvement of at least two p450 forms. The metabolism of NE-100 was mediated with recombinant CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. A significant correlation was observed between activities of NE-100 metabolism and dextromethorphan O-demethylation (a specific activity for CYP2D6) or testosterone 6beta-hydroxylation (a specific activity for CYP3A4) in HLM. The activity of NE-100 metabolism was inhibited by approximately 80% by an anti-CYP2D6 antibody and only by quinidine among the p450-selective inhibitors at a low substrate concentration (0.1 microM). In contrast, with a high substrate concentration (10 microM), the activity was inhibited by an anti-CYP3A4 antibody and by ketoconazole. On the other hand, in HIM, the Eadie-Hofstee plots for NE-100 disappearance were monophasic, and the metabolism was strongly inhibited by an anti-CYP3A4 antibody and by ketoconazole but not by other inhibitors used. These results strongly suggest that NE-100 has different profiles regarding metabolism between liver and intestine. During absorption, NE-100 is mainly metabolized by CYP3A4 in the intestine and thereafter by CYP2D6 in the liver in the presence of therapeutic doses.

Metabolism of N,N-dipropyl-2-[4-methoxy-3-(2-phenyl-ethoxy)-phenyl]-ethyl-amine-monohydrochloride (NE-100), a novel sigma ligand: contribution of cytochrome P450 forms involved in the formation of individual metabolites in human liver and small intestine.

In the present study, human cytochrome P450 (CYP) forms involved in producing the primary metabolites of NE-100 were identified. Major metabolites of NE-100 in human liver microsomes (HLM) were N-depropylation of NE-100 (NE-098), p-hydroxylation of phenyl group of NE-100 (NE-152), m-hydroxylation of phenyl group of NE-100 (NE-163) and O-demethylation of NE-100 (NE-125). Judging from the correlation and inhibition studies, NE-125 and NE-152+163mix formations were predominantly mediated by CYP2D6 and NE-098 formation was mediated by multiple CYP forms at a low NE-100 concentration (0.1 microM) in the HLM. According to relative activity factor (RAF) approaches, all these reactions were predominantly catalyzed by CYP2D6 at a substrate concentration assuming a plasma level of NE-100 (K(m)>>S) in case of the human liver. Depending on the increase in NE-100 concentrations, the rate of contribution for NE-098 and NE-152+163mix formations increased in CYP3A4, although the predominant contribution of CYP2D6 for NE-125 formation did not change. In human intestinal microsomes (HIM), NE-100 was mainly metabolized to NE-098 and NE-152+163mix by CYP3A4. The intrinsic clearance for their formations in HIM was 3.2 and 14.9 times less than those in HLM, respectively, and no formation of NE-125 was observed in HIM. These results strongly suggest that CYP2D6 is the predominant form for NE-100 metabolism in the human liver in in vivo conditions (K(m)>>S) and the liver plays a more important role than does the small intestine in the first pass metabolism.

Effects of clarithromycin and amoxicillin on gastric emptying in rats.

The effects of oral administration of clarithromycin (CLR), amoxicillin (AMX), and lansoprazole (LPZ) on gastric emptying in rats were investigated by a glass powder method and a phenol red method. By both test methods, no significant effects on gastric emptying were observed when CLR, AMX, or LPZ was administered alone or when the three drugs were administered concomitantly. The levels of gastrointestinal absorption of [(14)C]CLR and [(14)C]AMX were measured. Four hours after injection of [(14)C]CLR or [(14)C]AMX into the stomach and duodenum loops of rats, 86.63 and 1.27% of the original amount of [(14)C]CLR administered were recovered in the contents of the stomach and duodenum loops, respectively, and 80.01 and 55.88% of the original amount of [(14)C]AMX administered were recovered in the contents of the stomach and duodenum loops, respectively.

mRNA expression and amino acid transport characteristics of cultured human brain microvascular endothelial cells (hBME).

An in vitro cell culture system for estimating the human blood-brain barrier (BBB) permeability of drugs is required for the development of drugs with effects on the central nervous system. In this study, cultured human brain microvascular endothelial cells (hBME) were characterized. hBME cells exhibited concentration-dependent uptake of L-Leu, L-Glu and L-Lys with K(m) values of 51.1+/-23.1 microM, 163.3+/-79.8 microM and 72.4+/-56.6 microM, respectively. The cellular accumulation of rhodamine123 in hBME cells was unaffected by P-glycoprotein (P-gp) substrates (cyclosporin A, quinidine and verapamil), while the accumulation in human P-gp-overexpressing cells was significantly increased in the presence of these P-gp substrates. RT-PCR revealed that hBME cells expressed large neutral amino acid transporter 1 (LAT1) and its associated molecule (4F2hc), excitatory amino acid transporter 3 (EAAT3), cationic amino acid transporter 1 (CAT1), glucose transporter 1 (GLUT1), monocarboxylic acid transporter 1 (MCT1) and multidrug resistance-associated protein 1 (MRP1). However, no expression of multidrug resistance protein 1 (MDR1) was detected. The results suggest that these amino acid transporters are functionally expressed at the human BBB, and that hBME cells retain the in vivo BBB transport functions and expression characteristics. Consequently, hBME cells should be a useful tool for studies of the human BBB.

Application of microtiter plate assay to evaluate inhibitory effects of various compounds on nine cytochrome P450 isoforms and to estimate their inhibition patterns.

Using a microtiter plate (MTP) assay consists of recombinant cytochromes P450 and fluorescent probes, we evaluated inhibitory effects of commercially available model-compounds, 18 typical substrates and 8 selective inhibitors, on nine cytochromes P450 (CYPs) activities. The IC(50) values obtained from the assay were used to estimate inhibition constant (Ki) values, assuming competitive inhibition. The Ki values calculated from IC(50) (the Ki(-cal)) with the MTP assay using recombinant CYPs were compared with the Ki values (the Ki(-rep)), reported for human liver microsomes (HLM). Regarding all the inhibitory effects of the 26 test compounds on each CYP activity, a good correlation (r(2)=0.7306) was found between Ki(-cal) and Ki(-rep). The inhibitory patterns of some compounds on the five major CYP isoforms were estimated, using the MTP assay with the preincubation method. Furafylline and erythromycin, both mechanism based inhibitors, strongly inhibited CYP1A2 and CYP3A4 activity, respectively and their inhibitory effects increased depending on the preincubation time. In contrast, the inhibitory effects of phenacetin, diclofenac, S-mephenytoin, dextromethorphan, bufuralol and terfenadine, typical substrates for CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, respectively, on each recombinant CYP activity decreased after preincubation. Therefore, the MTP assay is a useful high throughput screening method to evaluate inhibitory effects of new drug candidates on 9 CYP isoforms in HLM. In addition, the MTP assay with the preincubation method might be beneficial to estimate inhibitory patterns on CYP isoforms of new drug candidates and to estimate main CYP isoforms responsible for metabolism of these compounds.