Incorporation of Gene-Environment Interaction Terms Improved the Predictive Accuracy of Tacrolimus Stable Dose Algorithms in Chinese Adult Renal Transplant Recipients.

The narrow therapeutic window of tacrolimus necessitates daily monitoring and predictive algorithms based on genetic and nongenetic factors. In this study, we constructed predictive algorithms for tacrolimus stable dose in a retrospective cohort of 1045 Chinese renal transplant recipients. All patients were genotyped for CYP3A4 20230T>C (rs2242480), CYP3A4 T>C (rs4646437), CYP3A5*3 6898A>G (rs776746), ABCB1 129T>C (rs3213619); ABCB1 c.1236C>T (rs01128503), ABCB1 c.2677G>T/A (rs2032582) and ABCB1 c.3435C>T (rs1045642) polymorphisms, and the effects of gene-gene and gene-environment interactions on the predictive accuracy of algorithm were evaluated. In wild-type CYP3A4 rs2242480 (TT) carriers, patients who took calcium channel blockers had lower tacrolimus stable doses than those without the concomitant medications (P < 1 × 10-4 ). In contrast, there was no significant difference in mutant type patients. Similarly, the tacrolimus stable doses in wild-type CYP3A5 rs776746 carriers who had hypertension were higher than those without hypertension (P = 4.10 × 10-3 ). More importantly, dose-predictive algorithms with interaction terms showed higher accuracy and better performance than those without interaction terms. Our finding suggested that wild-type CYP3A4 rs2242480 (TT) carriers should be more cautious to take tacrolimus when they are coadministrated with calcium channel blockers, and CYP3A5 rs776746 (AA) carriers may need higher tacrolimus dosage when they are in combination with hypertension.

Corrigendum: Application of Machine-Learning Models to Predict Tacrolimus Stable Dose in Renal Transplant Recipients.

This corrects the article DOI: 10.1038/srep42192.

Long-Term Influence of CYP3A5, CYP3A4, ABCB1, and NR1I2 Polymorphisms on Tacrolimus Concentration in Chinese Renal Transplant Recipients.

BACKGROUND: The highly pharmacokinetic variability of tacrolimus makes it difficult to adjust the dose. In the current study, we investigated the influence of gene polymorphisms and other clinical factors on long-term tacrolimus dosing in Chinese renal transplant recipients. METHODS: A total of 276 renal transplant recipients were enrolled. The tacrolimus trough concentration and other clinical variables were recorded for 5 years following transplantation. Eight single nucleotide polymorphisms in four genes (CYP3A5, CYP3A4, ABCB1, and NR1I2) were genotyped using polymerase chain reaction-restriction fragment length polymorphism analysis and sequencing. The dose-adjusted tacrolimus trough concentrations were calculated and compared among patients according to allelic status. RESULTS: The alleles CYP3A5*3 and CYP3A4*18B were significantly associated with dose-adjusted tacrolimus blood trough concentrations and had a strong time-genotype interaction with tacrolimus pharmacokinetics. NR1I2 g.7635A>G had a significant interaction with time, but the dose-adjusted tacrolimus concentration did not significantly differ over 5 years posttransplantation, except for the GG genotype of NR1I2 g.7635A>G. Sex differences had an important influence on tacrolimus concentration during the later post-transplantation period. CONCLUSIONS: The interindividual variability of tacrolimus concentration appears to be due in part to the effects of these identified genetic variants and clinical characteristics. Thus, genotyping of the CYP3A4 and CYP3A5 genes should be considered with respect to determining tacrolimus dose regimens during the post-transplantation period.

Application of Machine-Learning Models to Predict Tacrolimus Stable Dose in Renal Transplant Recipients.

Tacrolimus has a narrow therapeutic window and considerable variability in clinical use. Our goal was to compare the performance of multiple linear regression (MLR) and eight machine learning techniques in pharmacogenetic algorithm-based prediction of tacrolimus stable dose (TSD) in a large Chinese cohort. A total of 1,045 renal transplant patients were recruited, 80% of which were randomly selected as the "derivation cohort" to develop dose-prediction algorithm, while the remaining 20% constituted the "validation cohort" to test the final selected algorithm. MLR, artificial neural network (ANN), regression tree (RT), multivariate adaptive regression splines (MARS), boosted regression tree (BRT), support vector regression (SVR), random forest regression (RFR), lasso regression (LAR) and Bayesian additive regression trees (BART) were applied and their performances were compared in this work. Among all the machine learning models, RT performed best in both derivation [0.71 (0.67-0.76)] and validation cohorts [0.73 (0.63-0.82)]. In addition, the ideal rate of RT was 4% higher than that of MLR. To our knowledge, this is the first study to use machine learning models to predict TSD, which will further facilitate personalized medicine in tacrolimus administration in the future.

IL-3 and CTLA4 gene polymorphisms may influence the tacrolimus dose requirement in Chinese kidney transplant recipients.

The highly variable pharmacokinetics and narrow therapeutic window of tacrolimus (TAC) has hampered its clinical use. Genetic polymorphisms may contribute to the variable response, but the evidence is not compelling, and the explanation is unclear. In this study we attempted to find previously unknown genetic factors that may influence the TAC dose requirements. The association of 105 pathway-related single nucleotide polymorphisms (SNPs) with TAC dose-adjusted concentrations (C0/D) was examined at 7, 30 and 90 d post-operation in 382 Chinese kidney transplant recipients. In CYP3A5 non-expressers, the patients carrying the IL-3 rs181781 AA genotype showed a significantly higher TAC logC0/D than those with the AG genotype at 30 and 90 d post-operation (AA vs AG, 2.21±0.06 vs 2.01±0.03, P=0.004; and 2.17±0.06 vs 2.03±0.03, P=0.033, respectively), and than those with the GG genotype at 30 d (AA vs GG, 2.21±0.06 vs 2.04±0.03, P =0.011). At 30 d, the TAC logC0/D in the grouped AG+GG genotypes of CTLA4 rs4553808 was significantly lower than that in the AA genotype (P =0.041) in CYP3A5 expressers, but it was higher (P=0.008) in the non-expressers. We further validated the influence of CYP3A5 rs776746, CYP3A4 rs2242480 and rs4646437 on the TAC C0/D; other candidate SNPs were not associated with the differences in TAC C0/D. In conclusion, genetic polymorphisms in the immune genes IL-3 rs181781 and CTLA4 rs4553808 may influence the TAC C0/D. They may, together with CYP3A5 rs776746, CYP3A4 rs2242480 and rs4646437, contribute to the variation in TAC dose requirements. When conducting individualized therapy with tacrolimus, these genetic factors should be taken into account.

Effects of berberine on pharmacokinetics of midazolam and rhodamine 123 in rats in vivo.

AIM: To evaluate whether berberine hydrochloride (BBR) could modify the pharmacokinetic profiles of midazolam (MDZ), a substrate of CYP3A, and rhodamine 123 (Rh123), a substrate of P-glycolprotein (P-gp), in male rats. METHODS: The rats were given with varied does of BBR or 75 mg/kg ketoconazole as a positive control for 10 days by intragastric administration. Single-pass duodenum perfusion of 20 mg/kg MDZ and inguinal artery canulated rats were used in the study. Plasma concentrations of MDZ and 1'-hydroxymidazolam (1'-OH-MDZ) were analyzed by high performance liquid chromatography (HPLC). The rats were given with varied does of BBR or 4 mg/kg verapamil as a positive control for 10 days by intragastric administration. Blood was obtained from the caudal vein of rats after single-pass intragastric administration of 5 mg/kg Rh123. HPLC was used to analyze the plasma concentrations of Rh123. RESULTS: BBR produced similar results as the ketoconazole (positive control group) with a dose-dependent increase in the AUC(0-t) and AUMC (0-t) of midazolam except at the dose of 50 mg/kg (p < 0.01). And BBR could significantly increase the peak plasma concentrations (Cmax) of MDZ (p < 0.01), but reduce the clearance rate (CLz) and the apparent volume of the distribution (Vz) of MDZ (p < 0.05). The results also indicated that BBR had no significant impact on the half-life period (t1/2) and the time to reach peak concentration (tmax). Meanwhile, BBR could dose-dependently decrease AUC(0-t) and AUMC(0-t) of 1'-OH-MDZ significantly (p < 0.05), and expedite the clearance rate of 1'-OH-MDZ while gaining its apparent volume of distribution (p < 0.05), but had no significant impact on t1/2 and Tmax. The result also showed that BBR, except at the dose of 50 mg/kg, and the positive verapamil group could significantly increase the AUC(0-t) and AUC(0-∞) of Rh123 (p < 0.001), meanwhile raise Cmax of Rh123 and shorten its Vz inversely (p < 0.05). Additionally, pre-treatment with BBR had no significant influence with the half-life period of Rh123, while significantly reduced its clearance rate (p < 0.05). CONCLUSION: The metabolism of MDZ and Rh123 was controlled by BBR. The results were most likely due to the inhibition by BBR on CYP3A enzymes and P-gp transporter.

Association of CYP3A4*18B and CYP3A5*3 polymorphism with cyclosporine-related liver injury in Chinese renal transplant recipients.

OBJECTIVE: The purpose of this study was to investigate the associations between CYP3A4*18B and CYP3A5*3 polymorphism and cyclosporine-related liver injuries in Chinese renal transplant recipients. METHODS: We genotyped 339 renal transplant recipients treated with a triple immunosuppressive regimen including cyclosporine for CYP3A4*18B and CYP3A5*3 polymorphism using the polymerase chain reaction restriction fragment length polymorphism assay. RESULTS: The incidence of liver injury in the study population was 36.9% (125/339). At 1 month after transplantation, the trough concentration of cyclosporine (C0) in the group with CYP3A4*1/*1(GG alleles) was significantly higher than in the group with CYP3A4*18B/*1 8B(AA alleles) (p < 0.05). At 3 months after transplantation, the C0 in the group with CYP3A4*1/*1 and group with CYP3A4*1/*18B was markedly higher than in the group with CYP3A4*18B/*18B (p < 0.05). The GG genotypes of CYP3A4*18B were more common in the liver injury group compared with the control group (p < 0.05). Univariate logistic regression analysis showed that subjects carrying the GG genotypes had a 5.136- and 2.528-fold higher risk of developing cyclosporine-related liver injury than those with the AA and GA genotypes. When adjusted for sex, the risk of the CYP3A4*18B genotypes was OR = 4.969 for GG compared to AA (p = 0.030), and OR = 2.634 for GG compared to GA (p = 0.025). However, no association was observed between CYP3A5*3 polymorphisms with cyclosporine-related liver injury. CONCLUSIONS: These results suggested that the wild type of CYP3A4*18B is a risk factor for the development of cyclosporine- related liver injuries in Chinese renal transplant recipients.

In vivo effect of Schisandrin B on cytochrome P450 enzyme activity.

To investigate the possible drug interaction, this study is designed to evaluate the ability of Schisandrin B (Sch B) to modulate cytochrome P450 3A activity (CYP3A) in vivo and to alter the pharmacokinetic profiles of CYP3A substrate (midazolam) in treated rats. Rats were repeated administered with physiological saline (negative control group), ketoconazole (75 mg/kg, positive control group) or varied doses of Sch B (experimental groups) for three consecutive days. Subsequently, changes in hepatic microsomal CYP3A activity and the pharmacokinetic profiles of midazolam and 1'-hydroxy midazolam in plasma were studied to evaluate CYP3A activity. The results indicated that Sch B significantly dose-dependently inhibited rat hepatic microsomal CYP3A activity with Ki value of 16.64 mg/kg and showed the characteristic of a noncompetitive inhibitor. Oral administration of Sch B for 3 days in rats produced significant effect on the pharmacokinetics of oral midazolam. Sch B resulted in a significant, dose-dependent increase in midazolam AUC0-∞ except at the dose of 2 mg/kg, while AUC0-∞ increased by 26.1% (8 mg/kg) and 60.6% (16 mg/kg), respectively. In the pharmacokinetic profiles of 1'-hydroxy midazolam, the significant, dose-dependent decrease in AUC0-∞ was observed except at the dose of 2 mg/kg, while AUC0-∞ reduced by 44.5% (8 mg/kg) and 49.2% (16 mg/kg), respectively. These results suggested that 3-day treatment of Sch B could increase concentration and oral bioavailability of drug metabolized by CYP3A. When the drug, consisting of Sch B, is used in the clinic for more than 3 days, the possible drug-drug interactions should be taken into consideration.

Review of the use of defined daily dose concept in drug utilisation research in China.

PURPOSE: This study aimed to understand the characteristics of drug utilisation researches (DURs) using concepts of defined daily dose in China and to provide further suggestion for future DURs in China. METHODS: DURs using concepts of defined daily dose published in China were identified from China Journal Full-text Database, and in-depth data analysis was conducted for DURs published in every even-numbered year. RESULTS: In total, 2,911 DURs published between 1989 and 2009 were identified, of which 1,268 were included for further data analysis. All studies were hospital-based. Types of drugs commonly assessed in DURs were Anti-infectives for systemic use (34.1%), drugs for Nervous system (25.5%) and drugs for Alimentary tract and metabolism (14.3%). In addition, 63 DURs published in even-numbered year focusing on Chinese Herbal Medicine (CHM) were identified. Commonly used sources of defined dose were Xin Bian Yao Wu Xue/New Materia Medica (83.9%), drug information leaflets (66.8%) and Chinese Pharmacopoeia (52.0%). Common indicators used in DURs include defined daily doses (DDDs), drug utilisation index (DUI) and daily dose cost (DDC). CONCLUSION: DUR is a popular method to explore the use of both pharmaceutical drugs and CHM in China. The definition of defined daily dose and its related indicators presented in the DURs were highly varied. From this, it follows that DURs with more consistent methodology are highly needed for a thorough understanding of drug utilisation in China. Apart from DURs focusing on the hospital setting, more DURs from other health settings are needed.

Inhibitory effects of continuous ingestion of Schisandrin A on CYP3A in the rat.

The objective of this study was to evaluate the ability of schisandrin A (SchA) to inhibit the P450 enzyme CYP3A in vivo. Male Sprague-Dawley rats were intragastrically administered with varied doses of SchA (8 mg/kg or 16 mg/kg or 32 mg/kg) or 75 mg/kg ketoconazole for three consecutive days. Ketoconazole, a chemical inhibitor of CYP3A, was used as positive control. Subsequently, changes in hepatic microsome CYP3A activity and the pharmacokinetic profiles of midazolam (MDZ), a specific CYP3A substrate, were studied as indicators of rat hepatic microsomal activity of CYP3A. Differences in the plasma concentrations of MDZ and its related metabolites and the hepatic microsome concentrations of 1'-hydroxymidazolam were analysed by high-performance liquid chromatography. The current results provide direct and explicit evidence that SchA produced concentration-dependent inhibition of MDZ metabolite formation in rat liver microsomes (p < 0.01 or p < 0.001). Regular SchA consumption also caused concentration-dependent increase in Cmax and area under the concentration-time curve (AUC0-t and AUC0-∞ ) of peroral MDZ (p < 0.05 or p < 0.01) compared to vehicle-treated rats, whereas those of its metabolites (1'-hydroxymidazolam) were reduced (p < 0.05 or p < 0.01). Analysis of the data suggests that changes in the pharmacokinetic profiles of peroral MDZ in the rat model were contributed mainly to SchA inhibition of CYP3A activity. These results suggest that SchA, as an inhibitor of CYP3A, possesses a clinically beneficial property of altering the disposition of drugs metabolized by CYP3A.

Association between inosine triphosphate pyrophosphohydrolase deficiency and azathioprine-related adverse drug reactions in the Chinese kidney transplant recipients.

Azathioprine (AZA) is a thiopurine prodrug commonly used in patients with kidney transplantation. The aim of this study is to explore in patients with kidney transplantation whether AZA-related side effects can be explained by the inosine triphophate pyrophosphatase (ITPA) or thiopurine S-methyltransferase (TPMT) polymorphisms using both pheno-and genotyping. Erythrocyte ITPA and TPMT activity of 155 patients with kidney transplantation and AZA therapy was determined by HPLC. The frequencies of ITPA and TPMT polymorphisms were detected. Among 155 patients, three cases with zero activity were homozygote for 94C>A. The allele frequency of the 94C>A polymorphism was 0.12. Allele for the IVS2+21A>C mutation in the patients of this study was not found. Thirty-five cases had stopped azathioprine medication or were on reduced dose due to AZA-related side effects, including hematotoxicity (n = 12), hepatotoxicity (n = 18), gastrointestinal toxicity (n = 5, one patient developed hepatotoxicity simultaneously) and flu-like symptoms (n = 1). No statistical significant associations between ITPA 94C>A phenotype or genotype and AZA-related hematotoxicity or hepatotoxicity could be detected. However, five patients who developed gastrointestinal disturbance, two patients were homozygote for 94C>A and other three patients had 94C>A heterozygous allele. The patient who experienced flu-like symptoms were the remaining homozygote for 94C>A. This study demonstrates that ITPA activity reduced in patients with 94C>A mutation (P < 0.01). Patients with ITPA 94C>A homozygous allele are at high risk to develop AZA-related gastrointestinal toxicity and flu-like symptoms (P < 0.01). TPMT wild-type/ITPA variant (homozygote) is closely related to the AZA-induced side effects (P < 0.01).

Effects of Schisandra sphenanthera extract on the pharmacokinetics of midazolam in healthy volunteers.

AIMS: To assess the effect of Schisandra sphenanthera extract (SchE) on the pharmacokinetics of midazolam, a probe drug of CYP3A, and its metabolite 1'-hydroxy midazolam in healthy volunteers. METHODS: Twelve healthy male volunteers were orally treated with SchE, three capsules twice daily for 7 days. Pharmacokinetic investigations of oral midazolam administration at 15 mg were performed both before and at the end of the SchE treatment period. The plasma midazolam and 1'-hydroxy midazolam concentrations were determined by high-performance liquid chromatography-tandem mass spectrometry. Estimated pharmacokinetic parameters before and with SchE were calculated with noncompartmental techniques. RESULTS: Following administration of SchE, the average increases (%) of individual increases in AUC, AUMC and C(max) of midazolam were 119.4% [95% confidence interval (CI) 83.9, 155.0], 183.4% (95% CI 120.5, 246.2) and 85.6% (95% CI 14.4, 156.9), respectively (P < 0.01 or 0.05). On average, there was a 133.3% (95% CI 8.9, 257.7) increase in midazolam t(max) (P < 0.01). The average decrease (%) in CL/F was 52.1% (95% CI 44.9, 59.4) (P < 0.01). No significant changes were seen in midazolam half-life. After co-administration of SchE, the average increase (%) in t(max) of 1'-hydroxy midazolam was 150.0% (95% CI 22.2, 277.8) (P < 0.05). No significant differences were observed in the other pharmacokinetic parameters of 1'-hydroxy midazolam. CONCLUSIONS: SchE can markedly increase the oral bioavailability of midazolam in healthy volunteers. SchE is an inhibitor of CYP3A and has a high susceptibility to alter the disposition of drugs metabolized by CYP3A.

Relationships between thiopurine S-methyltransferase polymorphism and azathioprine-related adverse drug reactions in Chinese renal transplant recipients.

OBJECTIVE: To systematically investigate the relationships between thiopurine S-methyltransferase (TPMT) polymorphisms and azathioprine-related adverse drug reactions in patients with kidney transplantation. METHODS: Erythrocyte TPMT activity of 150 patients with kidney transplantation and AZA therapy was determined by HPLC. The frequency of four common TPMT mutant alleles, TPMT*2, *3A, *3B, and *3C was determined by allele-specific PCR and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. RESULTS: Thirty cases (20%) had stopped azathioprine medication or were on reduced dose due to azathioprine-related side effects. The TPMT activity range of cases who never experienced side effects was 16.63-68.25 U, the mean of the controls was 38.43 +/- 11.59 U. The mean value of 12 cases with hematotoxicity was 23.50 +/- 10.33 U, much lower than the control mean (P < 0.05). No significant difference between the mean value of 18 cases with hepatotoxicity and the control mean (P > 0.05) was seen. No case with TPMT deficiency was found in all patients studied, and TPMT*2, *3A, and *3B were not detected in any of them. TPMT*3C heterozygous alleles were found in 4.7% (seven cases) of these patients, all seven cases had intermediate TPMT activity, and the mean was 16.75 +/- 2.09 U, much lower than other TPMT wild-type patients (P < 0.05). In the seven TPMT*3C patients, four cases experienced side effects (hematotoxicity, n = 2; hepatotoxicity, n = 2). CONCLUSIONS: This study demonstrates that TPMT activity is reduced in patients with TPMT*3C mutation. AZA-induced hematotoxicity is related to the reduced TPMT activity.

Effects of Schisandra sphenanthera extract on the pharmacokinetics of tacrolimus in healthy volunteers.

AIM: To assess the effect of Schisandra sphenanthera extract (SchE) on the pharmacokinetics of tacrolimus in healthy volunteers. METHODS: Twelve healthy male volunteers were orally treated with SchE, three capsules twice daily for 13 days. Pharmacokinetic investigations of oral tacrolimus administration at 2 mg were performed both before and at the end of the SchE treatment period. Whole blood tacrolimus concentrations were determined by enzyme-linked immunosorbent assay. Estimated pharmacokinetic parameters before and with SchE were calculated with noncompartmental techniques. RESULTS: Following administration of SchE, the average percentage increases of individual increases in AUC, AUMC and C(max) of tacrolimus were 164.2% [95% confidence interval (CI) 70.1, 258.4], 133.1% (95% CI 49.5, 261.3) and 227.1% (95% CI 155.8, 298.4), respectively (P < 0.01 or 0.05). On average, there was a 36.8% (95% CI 13.4, 60.2) increase in tacrolimus t(max) (P < 0.01). The average percentage decreases in CL/F and V/F were 49.0% (95% CI 31.1, 66.9) and 53.7% (95% CI 40.1, 67.4), respectively (P < 0.01). CONCLUSIONS: SchE can increase the oral bioavailability of tacrolimus. The results of this study will add important information to the interaction area between drugs and herbal products.

Transport studies with 5-aminosalicylate.

OBJECTIVE: It is generally accepted that 5-aminosalicylate (5-ASA; mesalamine), widely used in inflammatory bowel disease therapy, exerts its action from the intraluminal site of the intestine. In addition to local metabolism of 5-ASA, it has been assumed that therapeutic mucosal concentrations of 5-ASA depend on transporter-mediated secretion back to the lumen. METHODS: We tested the hypothesis that 5-ASA represents a substrate of P-glycoprotein (P-gp) and/or multidrug resistance-associated protein 2 (MRP2), thereby possibly contributing to variable therapeutic effects. Polarized, basal-to-apical transport of [(3)H]5-ASA was studied in monolayers of Caco-2 and L-MDR cells, both of which express P-gp in their apical membrane, as well as in MDCK cells transfected with human MRP2. Moreover, we investigated the influence of 5-ASA on transport of digoxin in Caco-2 cells. RESULTS: In Caco-2 cells a P-gp-mediated efflux of 5-ASA (5-500 muM) could be excluded. Likewise, in L-MDR1 and MRP2 cells no transport differences in either the basal-to-apical or apical-to-basal direction were measurable. 5-ASA (50 muM to 5 mM) had no effect on the transport of digoxin. CONCLUSION: From these in-vitro experiments one can conclude that intestinal secretion of 5-ASA is apparently not mediated by P-gp or MRP2. Further studies are needed to identify the nature of the involved active carrier system(s).

Thiopurine S-methyltransferase as a target for drug interactions.

OBJECTIVE: The present study was undertaken to investigate the possible effects of various agents on thiopurine methyltransferase (TPMT) activity in red blood cells (RBCs) from patients with chronic inflammatory bowel disease (IBD). METHODS: In three groups of patients with very high, normal and intermediate TPMT activity (each n=6), the inhibitory potential of furosemide, piretanide, azathioprine (AZA) and testosterone was assessed by ex vivo measurements of TPMT activity in RBCs. From individual concentration-response curves, IC50 values have been determined. RESULTS: Independent of the basal TPMT activity, lowest IC50 values were calculated for furosemide (15-19 microM), followed by testosterone (30-72 microM), piretanide (300-313 microM) and AZA (430-532 microM). Compared with reported plasma concentration achieved during treatment, only furosemide would have the potential to inhibit TPMT also in vivo, whereas the IC50 values of the other agents are far above the corresponding plasma levels. CONCLUSIONS: Our ex vivo study revealed that only furosemide has the potential to inhibit TPMT activity in patients with IBD. This possibility should be taken into consideration if the diuretic and AZA or 6-mercaptopurine are coadministered. However, the extrapolation to the clinical setting remains open.