Combinatorial chemistry and high-throughput screening have increased the possibility of finding new lead compounds at much shorter time periods than conventional medicinal chemistry. However, too much promising drug candidates often fail because of unsatisfactory ADME properties. In silico ADME studies are expected to reduce the risk of late-stage attrition of drug development and to optimize screening and testing by looking at only the promising compounds. To this end, many in silico approaches for predicting ADME properties of compounds from their chemical structure have been developed, ranging from data-based approaches such as quantitative structure-activity relationship (QSAR), similarity searches, and 3-dimensional QSAR, to structure-based methods such as ligand-protein docking and pharmacophore modelling. In addition, several methods of integrating ADME properties to predict pharmacokinetics at the organ or body level have been studied. In this article, we briefly summarize in silico ADME approaches.
The Eisai hyperbilirubinemic rat (EHBR) should be a useful animal model for studies on the toxicity of organic anions which are substrates of multidrug resistance-associated protein 2 (Mrp2), since the systemic exposure to these compounds is expected to be increased in EHBR. In this study, we tested the value of EHBR for this purpose, using pravastatin (PV) and methotrexate (MTX) as model compounds. In the case of a single oral dose of PV (200 mg/kg), Cmax in plasma was 4.0-fold higher and AUC0-∞ was 3.6-fold larger than those of normal Sprague-Dawley rats (SDR), respectively. When multiple doses of PV were given to EHBR without co-administration of any other compound, drug-induced skeletal muscle toxicity (myopathy/rhabdomyolysis) and increased creatine phosphokinase (CPK) level were observed, whereas a control experiment using SDR did not show any toxic change. When a single dose of MTX (0.6 mg/kg) was given to EHBR orally, Cmax was 1.7-fold higher and AUC0-∞ was 1.6-fold larger than those of SDR, respectively. When multiple doses of MTX were given to EHBR, the changes in bone marrow, spleen and intestines were more severe than those in SDR. These findings support the view that EHBR would be a valuable animal model for toxicity studies on organic anion compounds which are substrates of Mrp2.
The simultaneous diffusion and metabolism of ethyl nicotinate (EN) in a cultured human skin model, Living Skin Equivalent-high, was evaluated by the in vitro skin permeation and metabolism experiments, and esterase distribution was also determined. Theoretical calculations using Fick's 2nd Law of Diffusion with Michaelis-Menten kinetics were performed to obtain the permeation and metabolic parameters together with information on enzyme distribution. The obtained data was compared with the corresponding results in excised hairless rat skin. The partition coefficient of EN from the vehicle to the skin was of the same order of magnitude for the cultured human skin and hairless rat skin, but the diffusion and metabolic parameters were different. Esterase concentration in the epidermal membrane was greater than in the dermis of cultured skin, which was similar to hairless rat skin. Taking into account the similarities and differences between the membranes, the cultured human skin model can be utilized as a model membrane to rapidly predict simultaneous diffusion and metabolism of the prodrug through human skin.
L-lactic acid transport plays an important role in the regulation of L-lactic acid circulation into and out of muscle. To clarify the transport mechanism of L-lactic acid in skeletal muscle, L-lactic acid uptake was investigated using a L6 cell line. mRNAs of monocarboxylate transporter (MCT) 1, 2 and 4 were found to be expressed in L6 cells. The [14C] L-lactic acid uptake by L6 cells increased up to pH of 6.0. The [14C] L-lactic acid uptake at pH 6.0 was concentration-dependent with a Km of 3.7 mM. This process was reduced by α-cyano-4-hydroxycinnamate, a typical MCT1, 2 and 4 inhibitor. These results suggest that an MCT participates in the uptake of L-lactic acid by L6 cells. [14C] L-lactic acid uptake was markedly inhibited by monocarboxylic acids and monocarboxylate drugs but not by dicarboxylic acids and amino acids. Moreover, benzoic acid, a substrate for MCT1, competitively inhibited this process with Ki of 1.7 mM. [14C] L-lactic acid efflux in L6 cells was inhibited by α-cyano-4-hydroxycinnamate but not by benzoic acid. These results suggest that [14C] L-lactic acid efflux in L6 cells is mediated by MCT other than MCT1.
Human organic anion transporters hOAT1 (SLC22A6) and hOAT3 (SLC22A8) are responsible for renal tubular secretion of an antifolic acid methotrexate, and are considered to be involved in drug interaction of methotrexate with nonsteroidal anti-inflammatory drugs (NSAIDs). In our hospital, a delay of methotrexate elimination was experienced in a patient with Hodgkin's disease, who took loxoprofen, a commonly used NSAID in Japan, which suggested a cause. In this study, we examined the drug interaction via hOAT1 and hOAT3, using Xenopus laevis oocytes. hOAT1 and hOAT3 mediated the methotrexate transport with low affinity (Km of 724.0 μM) and high affinity (Km of 17.2 μM), respectively. Loxoprofen and its trans-OH metabolite, an active major metabolite, markedly inhibited the methotrexate transport by both transporters. Their inhibition concentrations (IC50) were in the range of the therapeutic levels. These findings suggest that loxoprofen retards the elimination of methotrexate, at least in part, by inhibiting hOAT1 and hOAT3.
The effect of genetic polymorphism of human organic anion transporting polypeptide C (OATP-C) on the lipid-lowering response to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors was assessed. A retrospective study was conducted on 66 patients who underwent treatment of hyperlipidemia with HMG-CoA reductase inhibitors in a municipal hospital in a community-based cohort of Ehime prefecture in the southern part of Japan. Plasma lipid concentrations before and after administration were analyzed in patients in relation to the 521T/C (Val-174→Ala) polymorphism in the OATP-C gene (TT: n=44 (66.7%), TC: n=20 (30.3%), CC: n=0 (0.0%), undetermined: n=2 (3.0%)). Total cholesterol level was significantly lowered after treatment with HMG-CoA reductase inhibitors in all patients (p<0.001); moreover, subjects with the 521C allele showed an attenuated total-cholesterol-lowering effect compared with those homozygous for the 521T allele (-22.3±8.7% vs. -16.5±10.5%, p<0.05). These data suggest that the 521T/C polymorphism of the OATP-C gene modulates the lipid-lowering efficacy of HMG-CoA reductase inhibitors.
A population pharmacokinetic substudy design of a new chemical entity was evaluated based on the bias in parameter estimates and the power of detecting a specific subpopulation showing different clearance using a clinical trial simulation approach. The effect of analysis algorithms on type I error was also assessed. The design factors included the number of patients (n=100-300) and the number of sampling points per patient (n=2-6). Simulation data were generated from a model developed based on a Phase I study. The power was evaluated for a percentile of test statistics obtained by the simulation study. The clearance (CL) related parameters were estimated with sufficient accuracy in all study designs and all analysis algorithms: the first order (FO), first order conditional estimation (FOCE) and first order conditional estimation with interaction (FOCE-INTER) methods. With the FO and FOCE methods, the type I error rate increased as the frequency of sampling from each patient became higher, but such increase was hardly observed with the FOCE-INTER method. The power tended to depend on the size of the subpopulation. A large difference was found in the power of detecting a specific subpopulation showing a clearance decrease of 30% or 50%. Therefore, the most dominant factors controlling power would be the size of the subpopulation and the decreasing ratio of CL in the subpopulation. These findings obtained by the clinical trial simulation approach are useful for optimization of study design and determination of the limits of evaluation.
Lansoprazole fast disintegrating tablet (LFDT) has been developed as a multiple unit formulation to increase the QOL of patients, i.e., easy intake without water. However, there is a possibility that patients intake LFDT in accordance with clarithromycin and amoxicillin with water. To study the effect of water on the absorption of lansoprazole (LPZ), the study was carried out using human volunteers. After selected by phenotype of LPZ metabolism, extensive metabolizers (EMs) of LPZ were used in this study. Twelve healthy male EMs intook LFDT containing 30 mg LPZ with 150 mL of water and without-water, i.e., with saliva, to study the pharmacokinetics of LPZ from the gastrointestinal tract by a cross-over manner with one-week washout period under fasted condition in the morning. The mean AUC0-24s were 2004.4±973.6 ng·h/mL in without-water experiment and 2018.5±1159.6 ng·h/mL in the case of with-water experiment. Mean Cmaxs were 851.9±450.8 ng/mL in without-water experiment and 830.8±456.8 ng/mL in with-water experiment, respectively. ANOVA was applied to the log-transformed AUC0-24 and Cmax values. The 90% two sided confidence intervals for log-transformed AUC0-24 was 0.78-1.22 and that for log-transformed Cmax was 0.67-1.37, respectively. By comparing these pharmacokinetic parameters, we may state that there was no significant difference between the two administration modes.