Tomoyuki Mizuno

Targeted Disruption of the Multidrug and Toxin Extrusion 1 (Mate1) Gene in Mice Reduces Renal Secretion of Metformin

Multidrug and toxin extrusion 1 (MATE1/SLC47A1) is important for excretion of organic cations in the kidney and liver, where it is located on the luminal side. Although its functional and regulatory characteristics have been clarified, its pharmacokinetic roles in vivo have yet to be elucidated. In the present study, to clarify the relevance of MATE1 in vivo, targeted disruption of the murine Mate1 gene was carried out. The lack of Mate1 expression in the kidney and liver was confirmed by reverse transcription-polymerase chain reaction and Western blot analysis. The mRNA levels of other organic cation transporters such as Octs did not differ significantly between wild-type [Mate1(+/+)] and Mate1 knockout [Mate1(-/-)] mice. It is noteworthy that the Mate1(-/-) mice were viable and fertile. Pharmacokinetic characterization was carried out using metformin, a typical substrate of MATE1. After a single intravenous administration of metformin (5 mg/kg), a 2-fold increase in the area under the blood concentration-time curve for 60 min (AUC0-60) of metformin in Mate1(-/-) mice was observed. Urinary excretion of metformin for 60 min after the intravenous administration was significantly decreased in Mate1(-/-) mice compared with Mate1(+/+) mice. The renal clearance (CLren) and renal secretory clearance (CLsec) of metformin in Mate1(-/-) mice were approximately 18 and 14% of those in Mate1(+/+) mice, respectively. This is the first report to demonstrate an essential role of MATE1 in systemic clearance of metformin.

ABCG2 421C>A polymorphism and high exposure of sunitinib in a patient with renal cell carcinoma

Sunitinib is an oral multitargeted tyrosine kinase inhibitor (TKI) for renal cell carcinoma (RCC) [1]. Due to severe adverse effects, patients are forced to reduce or discontinue sunitinib, and discontinuation often leads to tumor regrowth. Breast cancer resistance protein (ABCG2) expressed in the intestine functions as an efflux pump of drugs. Among single-nucleotide polymorphisms (SNPs) in the ABCG2 gene, ABCG2 421C>A is the most common mutant allele in Asians ( 30%) [2]. This SNP was reported to be associated with an increase in oral bioavailability of several drugs including TKIs [2]. In the clinical setting, we experienced one RCC patient with severe adverse events early after oral administration. We then carried out pharmacokinetic and pharmacogenetic analyses of sunitinib, focusing on ABCG2. Five RCC patients, three men and two women [mean age: 55.6 (34–67)], were analyzed. All five patients started 50 mg of sunitinib once a day. Adverse events were graded by National Cancer Institute—Common Toxicity Criteria for Adverse Effects v3.0. This study was approved by Kyoto University Graduate School and Faculty of Medicine Ethics Committee. Blood samples were collected on day 8, and plasma concentrations of sunitinib and its major metabolite SU012662 were determined by high-performance liquid chromatography (HPLC). HPLC conditions were as follows: column, GEMINI-NX 3 lm C18 (4.6 · 150 mm); mobile phase, acetate buffer in methanol at 33 : 67 (sunitinib) and 40 : 60 (SU012662) and wavelength, 423 nm. The SNP was determined by direct sequencing. Cell culture, transfection and transport experiments were carried out as described previously with a slight modification [3, 4]. One patient (patient 1) showed severe adverse events such as hypertension (grade 3), facial acne (grade 3) and elevation of amylase (grade 3). The maximum concentration and area under the concentration–time curve of sunitinib in patient 1 were 2.5-fold higher than in the other four patients (Figure 1A). Plasma concentration profiles of SU012662, a major metabolite of sunitinib, were comparable among patients (data not

OCT1 genetic variants influence the pharmacokinetics of morphine in children.

AIM Large interindividual variability in morphine disposition could contribute to unpredictable variability in morphine analgesia and adverse events. Caucasian children have more adverse effects and slower morphine clearance than African-American children. To study variations in intravenous morphine pharmacokinetics in children, we examined the influence of genetic polymorphisms in OCT1. METHODS In 146 children undergoing adenotonsillectomy, 146 concentration-time profiles (2-4 measurements per patient) were available. Population pharmacokinetic analysis characterized the profiles in NONMEM(®) and tested OCT1 variants as covariates. RESULTS Allometrically scaled post hoc Bayesian morphine clearance in homozygotes of loss-of-function OCT1 variants (n = 9, OCT1*2-*5/*2-*5) was significantly lower (20%) than in wild-type (n = 85, OCT1*1/*1) and heterozygotes (n = 52, OCT1*1/*2-*5; p < 0.05). CONCLUSION Besides bodyweight, OCT1 genotypes play a significant role in intravenous morphine pharmacokinetics. Relatively high allelic frequencies of defective OCT1 variants among Caucasians may explain their lower morphine clearance and possibly higher frequencies of adverse events compared with African-American children. Original submitted 21 December 2012; Revision submitted 7 May 2013.

The effect of ABCG2 genotype on the population pharmacokinetics of sunitinib in patients with renal cell carcinoma.

Background: Sunitinib, a multitargeted tyrosine kinase inhibitor, offers favorable therapeutic outcomes to patients with advanced renal cell carcinoma. However, to maximize the clinical benefits, an effective therapeutic management strategy with dose optimization is essential. The objectives of this analysis were to describe the pharmacokinetics (PK) of sunitinib by a population PK approach and to quantitatively evaluate the effect of potential predictive factors including ABCG2 genotype on the PK of sunitinib. Methods: Plasma concentration–time profiles at 3 consecutive days including a total of 245 sunitinib plasma concentrations were available from 19 Japanese patients with renal cell carcinoma. Blood samples were collected on days 2, 8, and 15 after the start of the therapy. Population PK analysis was performed using NONMEM 7.2. Body weight, gender, and genotype of ABCG2 421C>A were evaluated as potential covariates. Interoccasion variability (IOV) among the 3 sampling days was also assessed as a random effect parameter. Results: The sunitinib PK profiles were best described by a 1-compartment model with first-order absorption. The ABCG2 421C>A genotype was identified as a significant covariate for the prediction of oral clearance (CL/F). No significant improvement in model fit was observed by including body weight and/or gender. A systematic difference in estimated population CL/F was observed between days 2 and 8, which was quantified as approximately 30% decrease over time. This difference was described as a covariate for CL/F in the model. IOV included as a random effect parameter significantly improved the model fit. Conclusions: This analysis provides a population PK model of sunitinib with the ABCG2 421C>A genotype as a predictive covariate for CL/F. It also suggests that IOV and change of CL/F over time need to be considered to predict the sunitinib PK more accurately. These findings will be implemented to optimize the pharmacotherapy of sunitinib.

ABCC3 and OCT1 genotypes influence pharmacokinetics of morphine in children

AIM Large interindividual variability in morphine pharmacokinetics could contribute to variability in morphine analgesia and adverse events. METHODS Influence of weight, genetic polymorphisms, race and sex on morphine clearance and metabolite formation from 220 children undergoing outpatient adenotonsillectomy was studied. A nonlinear mixed effects model was developed in NONMEM to describe morphine and morphine glucuronide pharmacokinetics. RESULTS Children with ABCC3 -211C>T polymorphism C/C genotype had significantly higher levels of morphine-6-glucuronide and morphine-3-glucuronide formation (∼40%) than C/T+T/T genotypes (p < 0.05). In this extended cohort similar to our earlier report, OCT1 homozygous genotypes (n = 13, OCT1*2-*5/*2-*5) had lower morphine clearance (14%; p = 0.06), and in addition complementing lower metabolite formation (∼39%) was observed. ABCB1 3435C>T TT genotype children had lower levels of morphine-3-glucuronide formation though no effect was observed on morphine and morphine-6-glucuronide pharmacokinetics. CONCLUSION Our data suggest that besides bodyweight, OCT1 and ABCC3 genotypes play a significant role in the pharmacokinetics of intravenous morphine and its metabolites in children.

Pharmacokinetics of Oral Methadone in the Treatment of Neonatal Abstinence Syndrome: A Pilot Study.

OBJECTIVE To characterize the population pharmacokinetics of oral methadone in neonates requiring pharmacologic treatment of neonatal abstinence syndrome and to develop a pharmacokinetic (PK) model toward an evidence-based treatment protocol. STUDY DESIGN Based on a methadone dosing protocol, serum concentrations of methadone and its metabolites were assessed by high performance liquid chromatography-tandem mass spectrometry from dried blood spots. Population PK analysis was performed to determine the volume of distribution and clearance of oral methadone. Methadone plasma concentration-time profiles were simulated from the deduced PK model to optimize the dosing regimen. RESULTS There was substantial interindividual variability in methadone concentrations. Blood concentrations of methadone were best described by a 1-compartment model with first-order absorption. The population mean estimates (coefficient of variation percentage) for oral clearance and volume of distribution were 8.94 (103%) L/h/70 kg and 177 (133%) L/70 kg, respectively. Optimized dosing strategies were developed based on the simulated PK profiles. We suggest a starting dose of 0.1 mg/kg per dose every 6 hours for most patients requiring pharmacologic treatment of neonatal abstinence syndrome followed by an expedited weaning phase. CONCLUSIONS The proposed dosing regimen may reduce the cumulative dose of opioid and shorten the length of hospitalization. Future studies should aim to validate the simulated dosing schemes with clinical data and expand our understanding of the between-patient PK variability. TRIAL REGISTRATION ClinicalTrials.gov: NCT01754324.

Developmental trajectory of intestinal MDR1/ABCB1 mRNA expression in children.

Infants and children are unique patient populations compared with adults as they not only differ physiologically and anatomically but also experience rapid changes in growth and development over the course of their childhood. These dynamic and variable developmental changes can have major impact on the pharmacokinetic and pharmacodynamic profile of a drug from its absorption, distribution, metabolism and elimination (ADME) properties to its effects [1]. Since oral administration is one of the primary routes for giving a drug to children, many studies have been implemented to provide evidence for developmental differences in the absorption of drugs [1, 2]. As is well known, the bioavailability of orally administered drugs is regulated by various metabolic enzymes and transporters expressed in the small intestine. Therefore, the developmental changes in activity of metabolic enzymes and transporters could explain developmental differences in absorption. To date, several studies have characterized the ontogeny of metabolic enzymes in tissues including the small intestine [1–3]. However, there is little information on the ontogeny of drug transporters in the human intestine. P-glycoprotein (MDR1/ABCB1), an ATP-binding cassette transporter, located in the small intestine mediates the efflux of substrates from the epithelial cells to the intestinal lumen [4]. Thus, the inter-individual variability in the expression of intestinal ABCB1 is an important factor which determines the bioavailability of ABCB1 substrates, as has been shown for tacrolimus [5]. Since many drugs used in neonates, infants and children are substrates of ABCB1, it is important to characterize the effects of developmental changes of intestinal ABCB1 on drug disposition in children [1, 2]. Here, we evaluate ABCB1 mRNA expression patterns in human small intestine tissues collected from children to provide fundamental information on the age-dependent expression. mRNA expression data were obtained as part of a previous study at Kyoto University Hospital, Japan [5]. In short, 206 paediatric patients undergoing living donor liver transplantation were enrolled in the study. All mucosal specimens of the upper jejunum were obtained from a part of the Roux-en-Y limb for biliary reconstruction or around the bile drainage tube during surgery. Samples were immediately frozen in liquid nitrogen and stored at −80°C until analysis. ABCB1 mRNA expression was determined by quantitative RT-PCR as previously described [5]. The amount was determined with a standard curve generated using known amounts of standard plasmid DNA. To verify the quality of the total cellular RNA extracted, GAPDH mRNA expression was also quantified as an internal standard. Demographics of patients were as follows: median age (range) 1.27 years (62 days–18.9 years), median body weight (range) 8.5 kg (3.7–70 kg), 85 male and 121 female patients. The study was approved by the Institutional Review Board and all participants, patients and/or care givers, gave written informed consent or assent. Large inter-individual variability in intestinal ABCB1 expression was observed across all ages (Figure 1). Over the studied age range from 62 days to 18.9 years, no clear maturation trajectory of ABCB1 expression was observed. This may indicate that the expression of intestinal ABCB1 has reached at the certain level by at least 2 months after the birth. A most recent report showed no significant difference in intestinal ABCB1 mRNA expression between neonates and adults [6]. Although our data are not completely comparable due to the lack of samples in neonates, the report indirectly supports our observation. On the other hand, there is an earlier study demonstrating a very weak or no expression of ABCB1 in the fetal intestine [7]. Furthermore, a study using enterocyte cell lines demonstrated that breast milk induces ABCB1 expression in a dose-and time-dependent manner [8]. An animal study also demonstrated that the ABCB1 expression in the small intestine of neonatal rodents dramatically increased shortly after initiation of breastfeeding [8]. Since lactation is generally started right after the birth, the expression of intestinal ABCB1 is anticipated to initiate at the same time and rapidly reach levels comparable with those seen in adults. Figure 1 Age related changes of intestinal ABCB1 mRNA expression. Data represent individual ABCB1 mRNA expression As with the small intestine, developmental changes in hepatic ABCB1 expression has been of great interest as an important consideration in the development of age-appropriate dosing strategies. In a recent preliminary report, it was noted that hepatic ABCB1 mRNA expression in fetuses, neonates, infants and children was 25–60-fold lower compared with adult values [9]. In addition, in a recent review article, Klaassen & Aleksunes also reported that mRNA expression of ABCB1 in perinatal human liver samples was very low and gradually increased over time until the age of 7 years [10]. The maturation pattern of ABCB1 expression is most likely organ-specific and needs to be studied in each organ. In conclusion, this analysis suggests that the maturation of intestinal ABCB1 mRNA expression is completed relatively soon after birth. This finding contributes to an improved understanding of the age dependent maturation of ABCB1 transporter mRNA expression and its potential effect on the disposition of drug substrates.

Effect of P-Glycoprotein and Breast Cancer Resistance Protein Inhibition on the Pharmacokinetics of Sunitinib in Rats

The aim of this study was to elucidate the roles of P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) in the plasma concentration, biliary excretion, and distribution to the liver, kidney, and brain of sunitinib. The pharmacokinetics of sunitinib was examined in rats treated with PSC833 (valspodar) and pantoprazole, potent inhibitors of P-gp and BCRP, respectively. The sunitinib concentrations in plasma, bile, liver, kidney, and brain were determined by liquid chromatography–tandem mass spectrometry. It was found that the area under the concentration-time curve for 4 hours (AUC0–4) and maximum concentration (Cmax) of sunitinib administered intraintestinally were significantly increased by pretreatment with PSC833 or pantoprazole. Each inhibitor markedly reduced the biliary excretion of sunitinib for 60 minutes after an intravenous administration and significantly increased the distribution of sunitinib to the liver as well as kidney. In addition, the brain distribution of sunitinib was significantly increased by PSC833 but not pantoprazole, and coadministration of both inhibitors further enhanced the accumulation of sunitinib in the brain. These results demonstrate that plasma concentrations of sunitinib and the biliary excretion and distribution to the kidney, liver, and brain of sunitinib are influenced by pharmacologic inhibition of P-gp and/or BCRP.

Age-dependent changes in sirolimus metabolite formation in patients with neurofibromatosis type 1.

Background: Sirolimus is an inhibitor of mammalian target of rapamycin, which exhibits large interindividual pharmacokinetic variability. We report sirolimus pharmacokinetic data collected as part of a concentration-controlled multicenter phase II clinical trial in pediatric patients with neurofibromatosis type 1. The purpose of this study was to explore the effect of growth on age-dependent changes in sirolimus clearance with a focus on cytochrome P450 3A (CYP3A) subfamily mediated metabolism. Methods: Predose blood samples were obtained at steady state from 18 patients with neurofibromatosis type 1. Sirolimus and its 5 CYP3A-dependent primary metabolites were quantified by HPLC–UV/MS. Concentration ratios of metabolites to sirolimus (metabolic ratio) were calculated as an index of metabolite formation. Results: Metabolic ratios of the main metabolites, 16-O-demethylsirolimus (16-O-DM) and 24-hydroxysirolimus (24OH), were significantly correlated with sirolimus clearance, whereas this was not the case for the other 3 metabolites (25-hydroxysirolimus, 46-hydroxysirolimus, and 39-O-demethylsirolimus). The ratios for the 16-O-DM and 24OH metabolites were lower in children than adults. No significant difference in allometrically scaled metabolic ratios of 16-O-DM and 24OH was observed between children and adults. Conclusions: This study suggests that the age-dependent changes in sirolimus clearance can be explained by size-related increases in CYP3A metabolic capacity, most likely due to liver and intestinal growth. These findings will help facilitate the development of age-appropriate dosing algorithms for sirolimus in infants and children.

Developmental pharmacokinetics of sirolimus: Implications for precision dosing in neonates and infants with complicated vascular anomalies

Sirolimus has recently been shown to be efficacious and tolerable in pediatric patients with complicated vascular anomalies. Nevertheless, dosing information remains very limited especially for neonates and infants. The purpose of this study was to develop an age‐appropriate sirolimus starting dosing regimen based on the developmental changes in drug elimination capacity using data collected in neonates and infants.