We describe the longitudinal follow-up of calcineurin activity and its clinical relevance in 4 de novo living-donor kidney transplant recipients treated with cyclosporine (n=1) or tacrolimus (n=3). The calcineurin activity in peripheral blood mononuclear cells was measured in combination with therapeutic drug monitoring during hospitalization. Serial blood samplings were performed after the oral administration of each drug to evaluate the temporal pharmacokinetic and pharmacodynamic profiles. Significant changes in enzyme activity were evaluated in relation to clinical outcomes. A nadir of calcineurin activity occurred at the maximum blood drug concentration within 4 h post-dose in most cases. Unlike cyclosporine, tacrolimus partially suppressed calcineurin activity throughout the dosing interval compared to the pre-dose level (cyclosporine, 62-67% inhibition; tacrolimus, 13-35% inhibition). Notably, calcineurin activity rapidly increased a few days before the onset of acute rejection in 2 patients, 1 receiving cyclosporine and 1 receiving tacrolimus, despite the achievement of therapeutic trough blood concentrations. These preliminary findings indicate that therapeutic monitoring of calcineurin activity in addition to the measurement of blood drug concentrations may be helpful to evaluate the pharmacodynamic effects of cyclosporine and tacrolimus early after renal transplantation.

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  The pharmacokinetics and pharmacodynamics (PK/PD) of inhaled insulin in humans have not been modeled previously. We rationalized a model for the effects of inhaled insulin on glucose infusion rate during a euglycemic clamp study based on the mechanism of insulin action and compared parameter estimates between subcutaneous and inhaled insulin in healthy and diabetic subjects. Published data from two studies in 11 healthy volunteers and 18 type 1 diabetes patients were digitized. The subjects received four different doses of inhaled insulin and one or three different doses subcutaneously at the start of a 10 h glucose clamp. All data were modeled simultaneously using NONMEM VI. Insulin pharmacokinetics were described by a one-compartment model with one (inhaled) or two (subcutaneous insulin) first-order absorption processes and first-order elimination. Insulin effects on glucose were described by an indirect response model. A biophase direct effect equation for the glucose infusion rate was implemented. Pharmacodynamic parameter estimates were 15.1 mg/min/kg for maximal glucose infusion rate (GIR_max) and 88.0 mIU/L for SC₅₀ for diabetic patients and 62.9 mIU/L for healthy subjects. A PK/PD model based on fundamental principles of insulin action and glucose turnover suggests comparable potencies of inhaled and subcutaneous insulin.

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  The objectives of this study were to evaluate the relative contribution of the direct pathway in overall brain transport for 17 model drugs with different physicochemical properties after nasal administrations and to identify factors that govern the fraction of the dose transported to the brain via the direct pathway (F_{a, direct}). When the model drugs were nasally administered to rats, 5 of the 17 model drugs were delivered to a significant extent to the brain via the direct pathway. Multiple linear regression analyses showed that the correlation between various physicochemical properties and F_{a, direct} was not statistically significant, indicative of a lack of primary physicochemical determinants in the direct transport pathway. Transporters such as rOAT3 and rOCT2 were expressed at significant levels in rat olfactory epithelia, and uptakes of standard substrates were significantly decreased in HEK293 cells expressing rOAT3 and rOCT2 in the presence of the five model drugs that were delivered to appreciable extents to the brain via the direct pathway. Therefore, these observations indicate that carrier-mediated transport may play a role in the brain delivery of drugs from the nose via the direct transport pathway.

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  This study aims to investigate hepatic pharmacokinetics of the four most common drugs (metoprolol, omeprazole, spironolactone, and furosemide) given to patients undergoing liver transplantation before surgery. The investigation was carried out in CCl₄-induced fibrotic perfused rat livers and the results were compared to those in normal rat liver. Drug outflow fraction-time profiles were obtained after bolus injection into a single-pass-perfused normal or fibrotic rat liver. The pharmacokinetic parameters were estimated using previously developed barrier-limited and space-distributed models. The results showed a marked increase in the liver fibrosis index for CCl₄-treated rats compared to controls (p<0.05). The extraction ratios (E) for all drugs were significantly lower (p<0.05) in fibrotic than in normal livers and the decrease in E was consistent with the decrease in intrinsic clearance and permeability-surface area product. In addition, other than for furosemide, the mean transit times for all drugs were significantly longer (p<0.01) in the fibrotic livers than in normal livers. Pharmacokinetic model and stepwise regression analyses suggest that these differences arise from a reduction in both the transport of drugs across the basolateral membrane and their metabolic clearance and were in a manner similar to those previously found for another group of drugs.

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  Nonsteroidal anti-inflammatory drugs (NSAIDs) delay renal excretion of antifolate methotrexate by inhibiting human organic anion transporters hOAT1 (SLC22A6) and hOAT3 (SLC22A8). In this study, we performed uptake experiments using Xenopus laevis oocytes to assess the inhibitory effect of selective cyclooxygenase-2 inhibitors on hOAT1 and hOAT3. The uptake of methotrexate into oocytes was increased by the injection of hOAT1 and hOAT3 cRNA, and transport was strongly inhibited by lumiracoxib. The apparent 50% inhibitory concentrations of lumiracoxib were estimated to be 3.3 μM and 1.9 μM for uptake of p-aminohippurate by hOAT1 and of estrone sulfate by hOAT3, respectively. Eadie-Hofstee plot analysis showed that lumiracoxib inhibited hOAT1 and hOAT3 in a competitive manner. For other cyclooxygenase-2 inhibitors celecoxib, etoricoxib, rofecoxib and valdecoxib, slight to moderate inhibition of hOAT3 only was observed. These findings show that lumiracoxib has inhibitory potential toward hOAT1 and hOAT3, comparable to that of nonselective NSAIDs.

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  In this study, ³H- or ¹³C₂,D₂-sphingosine (SPH) was orally administered to mice to assess absorption, mass balance, tissue distribution, and metabolites in the skin. The blood concentration of ³H-SPH showed a Tmax of 10.7 hr. The radioactivity in the skin reached 763.4 ng eq./g tissue at 12 hr, and decreased to 181.7 ng eq./g tissue at 168 hr. The concentration of radioactivity at 12 hr was 577.6 and 100.7 ng eq./g tissue in the dermis and epidermis, respectively. Thereafter, the dermis concentration decreased to 158.5 ng eq./g tissue, while the epidermis concentration increased to 298.8 ng eq./g tissue, suggesting that radioactivity moves from the dermis to the epidermis. Unchanged SPH along with lipophilic metabolites was detected in the skin of mice exposed orally to ³H- or ¹³C₂,D₂-SPH. Moreover, in an in vitro study using human skin keratinocytes, a ¹³C₂,D₂-SPH-treatment resulted in the intracellular production of glucosylceramides (GlcCer) and ceramides (Cer) containing labeled-SPH. These results indicate the followings: first, that SPH is absorbed through the digestive tract and distributed to the skin; second, it is transferred from the dermis to the epidermis; and third, SPH is partly distributed to the skin in an unchanged form, and some of the distributed compounds are converted into GlcCer and Cer by biosynthesis.

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  The aim of this study was to examine in vitro, by means of kinetic analysis and molecular docking simulations, the effects of the flavone diosmetin and its flavanone analog hesperetin on CYP (cytochrome P450) 2C9-mediated drug metabolism. To this purpose, the conversion of diclofenac to 4′-hydroxydiclofenac by human liver microsomes was used as a model assay for assessing the CYP2C9 inhibitory activity of these two flavonoids. Kinetic analyses showed that diosmetin and hesperetin were reversible, dead-end inhibitors of 4′-hydroxydiclofenac formation; their mean K_i (inhibitor dissociation constant) values were 1.71±0.58 and 21.50±3.62 μM, respectively. Diosmetin behaved as a competitive inhibitor, since it increased markedly the K_m (substrate concentration yielding 50% of V_max) of the reaction without affecting the V_max (maximum velocity of reaction). Hesperetin modified markedly K_m and to a lesser extent also modified V_max, thus acting as a mixed competitive-noncompetitive inhibitor. The results of molecular docking simulations were consistent with those of kinetic analysis, since they showed that the putative binding sites of both diosmetin and hesperetin coincided with the CYP2C9 substrate binding site.
   The demonstration that diosmetin and hesperetin inhibit CYP2C9-mediated diclofenac metabolism at low micromolar concentrations is of potential clinical relevance because CYP2C9 is responsible for the biotransformation of various therapeutically important drugs that have narrow therapeutic indexes.

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  Cycloastragenol (CAG) is the aglycone derivative of astragaloside IV which has recently been demonstrated to activate telomerase and represents a potential drug candidate for the treatment of degenerative diseases. In the present study, intestinal absorption and metabolism of CAG were examined using the Caco-2 model and liver microsomes, respectively. The results showed that CAG rapidly passes through the Caco-2 cell monolayer by passive diffusion. Four different glucuronide conjugates and two oxidized CAG metabolites were found in the apical and basolateral sides of Caco-2 monolayer, suggesting that first-pass intestinal metabolism of CAG might occur upon passage through the intestinal epithelium. CAG underwent extensive metabolism in rat and human liver microsomes with only 17.4% and 8.2%, respectively, of the starting amount of CAG remaining after 30 min of incubation. Monohydroxylation of the parent and oxidization of the hydroxylated CAG were found in the liver samples. The present study indicates that CAG is efficiently absorbed through intestinal epithelium. However, extensive first-pass hepatic metabolism would limit the oral bioavailability of this compound.

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  Rapid determination of in vitro metabolic stability and metabolite profiling of new chemical entities using microsomes or other liver preparations is one of the most important steps in drug discovery. In this paper, we report the use of liquid chromatography-hybrid triple quadrupole/linear ion trap mass spectrometry for the simultaneous analysis of metabolic stability, metabolite profiling, and the kinetics of metabolite formation of praziquantel and three structural analogs. Multiple reaction monitoring (MRM) scans were used to quantify the disappearance of parent compounds and the formation of metabolites. MRM-information dependent acquisition-enhanced product ion (MRM-IDA-EPI) scans were used for the identification of the metabolites formed. Metabolic stability of these anthelmintics were studied in human liver microsomes (HLM) using MRM as a survey scan, which resulted in the identification of a higher number of metabolites compared to neutral loss (NL), precursor ion (PI), and enhanced mass spectrometry (EMS) scans. MRM-IDA-EPI scans resulted in the generation of similar calibration curves to MRM-only quantitative analysis. Therefore, the quantitative capabilities of the method was not affected by the additional qualitative information obtained during the same run. The formation of major metabolites was also simultaneously monitored, which could be used to understand the kinetics and mechanism of metabolite formation. Finally, our data demonstrate that the three analogs had higher metabolic stability than the anthelmintic prototype (praziquantel).

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  To investigate the pharmacokinetics and mechanism of intestinal absorption of JBP485 in rats, the pharmacokinetics of JBP485 were investigated in vivo both intravenously and orally. The effects of glycylsarcosine (Gly-Sar) on the uptake and transepithelial transport of JBP485 were examined in everted intestinal sacs, in situ jejunal perfusion, Caco-2 cells and PEPT1 transfected Hela cells. The gastrointestinal absorption of JBP485 was rapid. T_1/2β was 2.25±0.06 h, CL_plasma was 2.99±0.002 ml/min/kg, V_d was 0.22±0.05 l/kg and bioavailability was about 30% at a dosage of 25 mg/kg. JBP485 underwent rapid distribution in the tissues. Gly-Sar significantly decreased JBP485 uptake and transport in these models. A kinetic study showed that JBP485 was transported by PEPT1 in Caco-2 cells with Km and Vmax values of 0.33±0.13 mM and 0.72±0.06 nmol/mg protein/10 min, respectively. JBP485 appeared to have linear pharmacokinetics at intravenous doses of 6.25-100 mg/kg with minor first-pass effect, and JBP485 was mainly distributed in the kidney; JBP485 is a substrate for PEPT1 which is involved in the absorption of JBP485 in rat intestine.

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  Organic anion transporting polypeptide 1B1 (OATP1B1) and OATP1B3 are human hepatocyte transporters that mediate the uptake of various endogenous and exogenous substances. Genetic variations in solute carrier transporter 1B1 (SLCO1B1) and SLCO1B3 genes, which encode OATP1B1 and OATP1B3 proteins, could affect the pharmacokinetics of drugs leading to interindividual differences in drug responses. The full extent of SLCO1B1 and SLCO1B3 polymorphisms in white Canadians was analyzed using DNA sequencing procedures. We identified 49 and 41 nucleotide sequence variants leading to 10 and 9 major haplotypes in SLCO1B1 and SLCO1B3 genes, respectively. We report several novel mutations within regulatory and coding regions that could affect gene transcription, translation and function. Comparison with other studies revealed that the distribution of SLCO1B1 and SLCO1B3 polymorphisms and haplotypes differs widely across populations. Data from this survey will ultimately contribute to the design of pharmacogenetic studies in the Canadian population.

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  Dihydrofolate reductase gene (DHFR) 19-bp deletion polymorphisms result in varied DHFR enzymatic activity affecting the risk for preterm delivery, spina bifida, and the efficacy of methotrexate (MTX). Ethnic differences in DHFR 19-bp polymorphisms may be responsible for the divergent findings in previous genetic studies. We compared genotype and allele frequency of DHFR intronic 19-bp deletion polymorphisms in ethnically homogenous East Asians (from Japan) and others by polymerase chain reaction assay conducted on 277 healthy Japanese individuals. The genotype distribution was as follows: wild/wild, 11.9% (n=33); wild/deletion, 40.1% (n=111); deletion/deletion, 48.0% (n=133). The frequencies of wild type and deletion alleles were 0.32 and 0.68, respectively. The obtained genotype distribution was consistent with those calculated by Hardy-Weinberg equilibrium. The genotype distribution and allele frequencies in the Japanese population were significantly different from those previously reported for other ethnic populations. Determination of intronic 19-bp deletion polymorphisms of DHFR may be useful for monitoring the efficacy and side effects of MTX for the treatment of diseases such as rheumatoid arthritis and childhood acute leukemia in the Japanese population because the frequency of the deletion allele is higher.

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