The aim of this study was to assess the influence of host genetic variations and clinical factors in relation to efavirenz level in HIV-1 infected Thai adults. A total of 100 HIV-infected subjects treated with efavirenz/lamivudine/tenofivir were prospectively enrolled. The panel of CYP2A6, CYP2B6 and CYP3A4/5 polymorphisms was genotyped. At steady state, plasma efavirenz concentrations were measured using high performance liquid chromatography. The relationship between host genetic and clinical factors in terms of efavirenz pharmacokinetics in HIV-1 infected Thai adults was analyzed. The minor allele frequency for CYP2A6 −48T>G, CYP2B6 g.18492T>C, CYP3A4*1B c.−392A>G, CYP3A4*18 c.878T>C and CYP3A5*3 c.6986A>G was 0.14, 0.27, 0.01, 0.03 and 0.38, respectively. Univariant and multivariant analysis indicated associations for CYP2B6 g.18492T>C (p < 0.001 and p = 0.001), aspartate aminotransferase (AST; p = 0.001 and p = 0.006) and blood urea nitrogen (BUN; p = 0.011 and p = 0.016) with plasma efavirenz concentration. However, CYP2A6 −48T>G, CYP3A4*1B c.−392A>G, CYP3A4*18 c.878T>C and CYP3A5*3 c.6986A>G had no significant impact on plasma efavirenz concentration in HIV-1 infected Thai adults. The CYP2B6 g.18492T>C polymorphism, AST and BUN were significantly associated with low efavirenz concentrations. The results from this study can be used to improve the prediction of efavirenz plasma concentration and to optimize its dose in antiretroviral therapy.

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Cytochrome P450 3A4 (CYP3A4) plays a crucial role in the pharmacokinetic and safety profiles of drugs. However, it is difficult to properly predict the pharmacokinetics and hepatotoxicity of drugs in humans using data from experimental animals, because the catalytic activities of CYP3A4 and other drug-metabolizing enzymes differ between human and animal organs. In order to easily generate an animal model for proper evaluation of human CYP3A4-mediated drug metabolism, we developed a human CYP3A4-expressing adenovirus (Ad) vector based on our novel Ad vector exhibiting significantly lower hepatotoxicity (Ad-E4-122aT-hCYP3A4). Intravenous administration of Ad-E4-122aT-hCYP3A4 at a dose of 2 × 10¹¹ virus particles/mouse produced a mouse exhibiting human CYP3A4 activity at a level similar to that in the human liver, as shown in the dexamethasone metabolic experiment using liver microsomes. The area under the curve (AUC) of 6βOHD was 2.7-fold higher in the Ad-E4-122aT-hCYP3A4-administered mice, compared with the mice receiving a control Ad vector. This Ad vector-expressing human CYP3A4 would thus be a powerful tool for evaluating human CYP3A4-mediated drug metabolism in the livers of experimental animals.

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The effects of genetic polymorphisms of ABCB1 C3435T, POR*28, CYP3A4*1G and CYP3A5*3 variants and gender relating to metabolism on the exposure and response of amlodipine in Chinese hypertensive patients were determined. Population pharmacokinetic analyses were performed on data which were collected prospectively from 60 Chinese patients with mild to moderate essential hypertension [age range 40–74 years, males (n = 31), females (n = 29)] receiving oral racemic amlodipine for 4 weeks. Blood pressure was evaluated at the end of weeks 0 and 4. Blood samples were collected in heparinized tubes at the following times: 0, 2, 6, and 24 h on about day 28 after administration of amlodipine. A one-compartment model with first-order elimination and absorption best described the amlodipine pharmacokinetic data. ABCB1 3435 genetic polymorphism and gender affect the amlodipine oral clearance (CL/F). CL/F (L/h) = 28.8 × (1 + GNDR)^−0.531 × (ABCB1 C3435T) where GNDR = 0 and 1 are for male and female, respectively. The CL/F value in a male patient with the ABCB1 3435CC or CT genotype is 28.8 L/h. Lower CL/F and higher exposure occurs in female subjects with the ABCB1 3435CC or CT genotype who have greater decreases in blood pressure after treatment with amlodipine. The results may help to improve the efficacy and tolerability of amlodipine in essential hypertensive patients.

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Myricetin is a flavonoid that has recently been suggested to interfere with the intestinal folate transport system. The present study was conducted to examine that possibility, focusing on its inhibitory effect on proton-coupled folate transporter (PCFT) as the molecular entity of the transport system. The uptake transport of folate was first examined in the Caco-2 cell as an intestinal epithelial cell model, and its carrier-mediated component, of which the Michaelis constant (K_m) was 0.407 µM, was found to be noncompetitively inhibited by myricetin with an inhibition constant (K_i) of 61 µM. Consistent with that, folate transport by human PCFT stably expressed in Madin-Darby canine kidney II (MDCKII) cells, of which the K_m was 1.246 µM, was also noncompetitively inhibited by myricetin with a K_i of 130 µM. Thus, myricetin was suggested to inhibit intestinal folate transport by acting noncompetitively on PCFT, although the K_m and K_i were similarly shifted to some extent to be smaller in Caco-2 cells. Finally, epigallocatechin-3-gallate was also suggested to act in a noncompetitive manner as an inhibitory flavonoid. Care may need to be taken, therefore, in the ingestion of myricetin and some flavonoids to maintain the absorption of folate and antifolate drugs.

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Hypothesis: Simotinib hydrochloride (SIM6802), which is a new epidermal growth factor receptor–tyrosine kinase inhibitor (EGFR-TKI), is often prescribed for cancer patients with comorbidities and has serious adverse effects on gastrointestinal physiology. The drug-drug interactions (DDIs) between simotinib and other drugs in combination and the underlying mechanism of its gastrointestinal toxicity remain unclear. We hypothesized that the DDIs and the gastrointestinal toxicity of simotinib were related to its effects on the permeability of the intestine. Methods: To determine the intestinal absorption capacity, pharmacokinetic studies and an in situ loop assay were used. The intestinal permeability was measured by a Caco-2 Transwell model. Real time PCR and Western blots were applied to detecting the expression changes of cell junction genes. Results: Our research demonstrated that simotinib upregulated the absorption of cefaclor, valaciclovir and acyclovir. The increase of non-selective absorption was caused by the low expression of cell junction gene afadin-6 and the increase in paracellular permeability in intestinal epithelial cells after simotinib treatment. Conclusion: These findings revealed that simotinib upregulated intestinal absorption by increasing the paracellular permeability of intestinal epithelial cells. Our research provides theoretical bases for better formulation of EGFR-TKIs to alleviate adverse gastrointestinal effects and also provides guidance for clinical administration of simotinib.

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Human equilibrative nucleoside transporter 1 (hENT1) transports various nucleoside analogues into cells. Although the single hENT1 promoter region (P1) and the mRNA isoform (a1) have been characterized previously, we have recently identified additional promoter regions P2 and P3 (which primarily generate c1/2/3 mRNAs and d1/2/3/4 mRNAs, respectively) in the human liver. Therefore, this study aimed at identifying the primary hENT1 mRNA isoforms expressed in human hepatocytes, while simultaneously obtaining functional evidence of alternative hENT1 promoter usage. Our results showed that the expressions of hENT1c1, d3, and (to a lesser extent) c2 mRNAs were strikingly predominant over the other mRNA isoforms in human hepatocytes, that the abundant expression of these mRNAs was consistent with the high levels of P2 and P3 promoter activity, and that these promoters were significantly marked by transcriptionally active histone modification in hepatic cells. To summarize, our results demonstrate that, resulting from the manipulated alternative promoter usage, hENT1c1 and d3 (and c2) mRNAs are primarily expressed in human hepatocytes, which suggests that they may play important roles in controlling hENT1 expression levels in those cells. Our findings are expected to provide significant insights into the molecular machinery of hENT1 expression control.

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MicroRNAs (miRNAs) post-transcriptionally regulate mRNA expression, controlling global cell function. Altered expression or function of miRNAs causes various diseases. Chemically induced changes in miRNA expression in human tissues are not fully understood. We investigated the changes in miRNA expression by rifampicin, which modulates the expression of various genes related to drug metabolism and pharmacokinetics, in human hepatocytes, and evaluated the relationship with the gene expression changes. We found that 23 miRNAs were increased (>2-fold) and 17 miRNAs were decreased (<0.5-fold) among 150 detected miRNAs, whereas 60 genes were increased and 105 genes were decreased among 22,673 detected genes upon treatment with 10 µM rifampicin. Changes in 17 intragenic miRNAs out of 40 altered miRNAs did not occur in parallel with alterations in their host genes. We searched for the target mRNAs of the miRNAs altered by rifampicin and found that the changes in expression of 16 mRNA/miRNA pairs were inversely associated. Thus, some mRNA expression altered by rifampicin may result from miRNA regulation. In conclusion, we found that rifampicin altered miRNA expression in human hepatocytes. We obtained new insight on the mechanism of the miRNA expression changes and the complicated relationship with gene transcripts.

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A retrospective analysis suggested that blood tacrolimus concentrations were consistent among patients with a body mass index (BMI) that was lean (<18.5), normal (≥18.5 and <25) or overweight/obese (≥25). The average maintenance dose of tacrolimus in patients with BMI ≥ 25 was significantly lower compared with that in patients with a BMI of less than 25. Lean and obese Zucker rats fed a normal diet were given tacrolimus intravenously or orally. The blood concentrations of tacrolimus in obese rats were significantly higher than those in lean rats after administration via both routes. The moment analysis has suggested that CL_tot and Vd_ss of tacrolimus were not significantly different between lean and obese rats. The bioavailability was higher in obese rats, compared with that in lean rats. The protein expression of Cyp3a2 in the liver was significantly decreased in obese rats, compared with lean rats, while P-gp in the small intestine was also significantly decreased in obese rats. These results suggested that the steady-state trough concentration of tacrolimus in obese patients was well maintained by a relatively low dose compared with that in normal and lean patients, presumably due to increased bioavailability.

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Aquaporin 7 (AQP7) is an aquaglyceroporin that has recently been found to operate as a facilitative carrier rather than a channel for glycerol, although its primary function is as a water channel. To probe into its substrate specificity, we examined the inhibitory effect of a series of acyl glycerol derivatives on glycerol transport mediated by human AQP7 stably expressed in Madin-Darby canine kidney II cells. According to kinetic analyses, AQP7-mediated glycerol transport was found to be competitively inhibited by monoacetin, monobutyrin and diacetin. Therefore, it may be possible that they all could be recognized as substrates by AQP7. The inhibition constant (K_i) of monoacetin (134 µM) was smaller than that of diacetin (420 µM), but greater than the Michaelis constant for glycerol (11.8 µM). Considering another finding that inhibition by triacetin was insignificant, it is likely that a decrease in the number of hydroxyl groups in the glycerol molecule by acetyl derivatization leads to a decrease in affinity for AQP7. The K_i of monobutyrin (80 µM) was, on the other hand, comparable with that of monoacetin, suggesting that the extension of the acyl chain by two hydrocarbon units does not have an impact on affinity for AQP7.

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When developing new drugs appropriate markers for detecting induction and inhibition of cytochrome P450 3A enzymes (CYP3A) are needed. The aim of the present study was to evaluate the quinine/3-hydroxyquinine metabolic ratio (quinine MR) with other suggested markers for CYP3A induction: endogenously formed 4β-hydroxycholesterol, midazolam clearance in plasma and the 6β-hydroxycortisol/cortisol ratio in urine. We have previously performed a clinical trial in which 24 healthy subjects were randomized to take 10, 20 or 100 mg daily doses of rifampicin for 14 days (n = 8 in each group) to achieve a low and moderate CYP3A induction. In newly analyzed data from this study we can show that the quinine MR could detect CYP3A-induction even at the lowest dose of rifampicin (10 mg) (p < 0.01), comparable to a 4β-hydroxycholesterol/cholesterol ratio and midazolam clearance. The median fold-induction for the quinine MR compared to baseline was 1.7, 1.8 and 2.6 for the three dosing groups (10, 20 and 100 mg). In conclusion, in this study the quinine MR was comparable to midazolam clearance as a measure of CYP3A activity but easier to determine since only a single blood sample needs to be drawn.

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