Single-nucleotide polymorphism of human concentrative nucleoside transporter and mizoribine absorption in treating childhood-onset nephrotic syndrome

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1 Acta Med Kindai Univ Vol.43, No , Single-nucleotide polymorphism of human concentrative nucleoside transporter and mizoribine absorption in Tomohiro Nagata Department of pediatrics, Kindai University Faculty of Medicine Abstract Mizoribine (MZR) is an immunosuppressant used to treat frequently relapsing nephrotic syndrome (FRNS). We often encounter patients in whom the serum MZR concentration is difficult to maintain. Even when the same dose is administered, concentrations in patients 10 years of age are often significantly lower than those in patients 9 years of age. Even the administration of MZR at high doses ( 7 mg/kg/day) can fail to increase the serum concentrations in some patients with FRNS, leading to a relapse, and individual variation in MZR absorption may contribute to these issues. We previously linked a human concentrative nucleoside transporter (hcnt)2- related transport mechanism to MZR absorption. The MZR absorption significantly differed between patients expressing and those not expressing the hcnt2 gene. In the present study, we examined whether or not single nucleotide polymorphisms (SNPs) of the three hcnt genes are involved in the viability of MZR absorption. No significant associations between the serum MZR concentrations and SNPs of any of the three hcnt genes were evident. I also found no association between the MZR absorption and the SNPs of individual patients hcnt genes considered in combination. Other absorptive or excretory mechanisms may therefore determine the maintenance of serum MZR concentrations. Key words:mizoribine, absorption, hcnt, SNP Introduction Mizoribine (MZR) is an immunosuppressant drug active against several conditions, including nephrotic syndrome, various glomerular diseases, rejection following kidney transplantation, and collagen diseases such as systemic lupus erythematosus and rheumatoid arthritis. 1-5 MZR is an imidazole nucleotide that exerts selective inhibitory effects on inosine-5-monophosphate (IMP) dehydrogenase and guanosine synthesis, which suppress the T and B lymphocyte proliferation and activation. Several studies have found that a peak serum MZR concentration of at least 3 µg/ml is required to maintain remission in children with frequently relapsing nephrotic syndrome (FRNS). 6,7 However, a previous study failed to detect any increase in the serum concentration after a dose of MZR in some children, even when they had been administered the same dose found to be effective in children of the same age and physical status, which suggests possible individual variation in the MZR absorption. 8 Furthermore, another study reported that MZR administration at 150mg/day (50 mg 3 times daily), which is approved by the Japanese health insurance system for the treatment of nephrotic syndrome (NS), cannot reliably achieve or maintain remission unless a considerable increase in the serum MZR concentration is achieved, especially in older children. 9 Our group previously reported the involvement of the human concentrative nucleoside transporter (hcnt)2 transport system in the MZR absorption and detection of hcnt2 mutations in Received September 26, 2017; Accepted October 20,

2 T. Nagata two of eight children with NS treated with MZR. 10 We also detected no significant differences in the urinary excretion of MZR between children with and without hcnt2 mutations. 10 I therefore hypothesized that single-nucleotide polymorphisms (SNPs) of the hcnt genes might affect the MZR absorption. In the present study, I examined the association between SNPs of hcnt and the serum MZR concentration. Patients and Methods MZR doses and serum concentrations I retrospectively investigated 53 children with FRNS who had received MZR in our hospital. The serum concentration of MZR was measured at 2 hours after the oral administration. The guidelines for the Diagnosis of Childhood Idiopathic Nephrotic Syndrome established in 2013 defined a high dose of MZR as at least 7mg/kg/day and an effective serum concentration as at least 3μg/mL. 7 Under the definition of FRNS, patients with relapse within one year after initiating MZR therapy were considered to have relapse. 7 Detection and evaluation of hcnt SNPs From database for SNPs (dbsnp (NCBI) https// we selected 3 SNPs of the hcnt1, 2, and 3 genes known to occur in Japan: hcnt1(slc28a1) G565A, hcnt2(slc28a2)c65t, and hcnt3 (SLC28A3)A338G. The SNPs were detected using an i-densy IS-5320 gene analyzer (Arkray, Kyoto, Japan) in whole-blood samples from the patients. 11 Written informed consent regarding participation in this study was obtained from all patients or their parents. The protocol of this study was approved by the Ethics Review Board of Kindai University (27-055). Statistical analyses The t-test was used to examine the associations between the SNPs of hcnt2 and 3 and the serum concentrations of MZR. An analysis of variance (ANOVA) was conducted to investigate the differences in the serum concentration of MZR with respect to the SNP of hcnt1 in combination with the SNP of each hcnt. The relationships of the serum MZR levels with the SNPs were then compared between children with a serum MZR concentration 3.0μg/mL and those with a concentration <3.0μg/mL using the chi-squared test. All statistical analyses were performed using for EZR software (Saitama Medical Center, Jichi Medical University). P-values below 0.05 were considered to indicate significance. 12 Results Patients background characteristics I examined a total of 53 patients (35 males, 18 females). The median age was 9 years old (range, 0 to 31). The median body weight was 37 kg (range, 8.1 to 69.5; Table 1). Based on the findings from the initial renal biopsy specimens, we diagnosed 41 patients with minimal change nephrotic syndrome (MCNS); 3 with focal segmental glomerulosclerosis (FSGS), and 2 with membranoproliferative glomerulonephritis (MPGN). Before MZR administration, all 53 patients received corticosteroids, and 27 received cyclosporine; 8, cyclophosphamide; and 8, mycophenolate mofetil. Table 1 Patients characteristics Total (male to female ratio) 53 (35:18) Age (years) Median (range) 9 (0-31) Age of onset (years) Median (range) 3 (0-14) MZR dose (mg) Median (range) 300 ( ) Body weight (kg) Median (range) 37 ( ) Serum creatinine (mg/dl) Median (range) 0.39 ( ) Number of relapses 3 (1-20) The associations of the serum MZR concentration with the age, dose, and hcnt SNPs in children with FRNS A positive correlation was evident between the MZR dose per kilogram of body weight (mg/kg) and the serum MZR concentration (µg/ml; r=0.40; p=0.0029; Fig.1). In 5 children, serum MZR concentrations did not increase 3 µg/ml even when high-dose MZR was administered, leading to relapse; this may be due to individual variation in the MZR absorption (Table 2). The mean serum MZR concentrations were 2.95±0.21 µg/ml in 31 children with hcnt1 G/A, 20

3 Single-nucleotide polymorphism of human concentrative nu-cleoside transporter and mizoribine absorption in 2.62±0.56 µg/ml in 7 with hcnt1 A/A, and 3.42±0.25 µg/ml in 15 with hcnt1 G/G. The mean concentrations were 3.04±0.17 µg/ml in 47 children with hcnt2 C/C, 3.04±0.34 µg/ml in 6 with hcnt2 C/T, 3.13±0.16 µg/ml in 46 with hcnt3 A/A, and 2.42±0.52 µg/ml in 7 with hcnt3 A/G. Thus, no significant differences in the serum concentration of MZR were apparent among the various SNPs of hcnt1, 2, and 3 (hcnt1, P=0.25; hcnt2: P=0.99; hcnt3, P=0.13; Figs. 2 to 4). In addition, we examined whether or not the serum MZR concentrations differed with respect to hcnt SNP combinations including all three genes present in a given patient. However, no significant differences were found: G/A-C/C-A/A, 2.95±0.23 µg/ml (24 children); G/A-C/C-A/G, 2.75±1.15 µg/ml (3 children); G/A-C/T-A/A, 3.14±0.38 µg/ml (4 children); A/A-C/C-A/A: 2.88±0.85 µg/ml (4 children); A/A-C/C-A/G, 1.47±0.40 µg/ml (2 children); A/A-C/T-A/A, 3.77 µg/ml (1 child); G/G-C/C- A/A, 3.63±0.27 µg/ml (12 children); G/G-C/C- A/G, 2.88±0.40 µg/ml (2 children); and G/G- C/T-A/A, 1.95 µg/ml (1 child; P=0.38, Table 3). In the 41 cases of MCNS, no association was noted between the serum MZR concentrations and the SNPs of hcnt (hcnt1, P=0.21; hcnt2: P=0.42; hcnt3, P=0.32). The SNPs were also compared between children with serum MZR concentrations of 3 µg/ml and those with concentrations <3 µg/ml. No significant differences in the SNPs of hcnt1, 2, or 3 were present between the 2 groups (hcnt1, P=0.98; hcnt2, P=0.74; hcnt3, P=0.94). Fig.1 Association between MZR dose (mg/kg) and serum MZR concentration (µg/ml) Table 2 Five children, not attaining effective serum MZR concentrations while receiving high doses Sex Age MZR dose At onset At serum concentration measurement Serum MZR concentration SNP (mg/ kg ) (µg/ml) hcnt1 hcnt2 hcnt3 Renal Pathology Male G/A C/C A/A MC Male G/A C/C A/A - Male G/A C/C A/A MC Male G/A C/C A/G MC Male G/A C/C A/A - MZR, mizoribine; SNP, single-nucleotide polymorphism; MC, minimal change nephrotic syndrome 21

4 T. Nagata Table 3 Patient SNP and serum MZR concentration Values are mean ±SE. SNP hcnt1 - hcnt2 - hcnt3 Number of Patients MZR concentration(µg/ml) G/A - C/C - A/A ±0.23 G/A - C/C - A/G ±1.15 G/A - C/T - A/A ±0.38 A/A - C/C - A/A ±0.85 A/A - C/C - A/G ±0.40 A/A - C/T - A/A G/G - C/C - A/A ±0.27 G/G - C/C - A/G ±0.40 G/G - C/T - A/A Fig.2 Association between single-nucleotide polymorphism of hcnt1 and serum MZR concentration(p=0.25) 22

5 Single-nucleotide polymorphism of human concentrative nu-cleoside transporter and mizoribine absorption in Fig.3 Association between SNP of hcnt2 and serum MZR concentration(p=0.99) Fig.4 Association between SNP of hcnt3 and serum MZR concentration(p=0.13) 23

6 T. Nagata Discussion A previous study compared the effects of MZR therapy (4 mg/kg/day) with those of placebo administration for FRNS in children, finding that, especially in children 10 years of age, the recurrence rate in the MZR group was significantly lower than in older children. To maintain remission of FRNS, combination therapy with a corticosteroid and MZR is commonly used. 2 However, recent studies have concluded that these agents potent immunosuppressive effects were insufficient when the peak serum concentration of MZR at 2 hours after the oral administration was 3.0 µg/ml, suggesting that high-dose MZR (7 to 10 mg/kg/day) may be required in non-responders to other immunosuppressive agents. 13,14 In some patients matched for age and body weight treated with the same dose of MZR, the serum MZR concentration was not maintain, leading to relapse; this may reflect individual variation in the bioavailability of MZR. I should therefore explore the presence of individual differences in the factors suspected of being related to the MZR absorption. Several studies have reported the involvement of hcnt, a nucleoside transporter carrying out secondary active transport of nucleosides accompanying the transport of sodium, in the MZR absorption via cotransport with Na+. This protein is expressed along the brush border membrane of intestinal epithelial cells. Three molecular types have been identified: CNT1, CNT2, and CNT3. CNT1 selectively transports pyrimidine nucleoside; CNT2, purine nucleoside; and CNT3, both nucleosides. 15,16 The gastrointestinal tract shows no CNT3-derived nucleoside uptake activity, but CNT1 and CNT2 activities are observed. 17 Accordingly, the MZR absorption in the digestive tract is mediated by CNT1 and CNT2. With respect to the MZR absorption, some studies of healthy Japanese men have reported that the bioavailability of MZR in subjects with the hcnt1 565-A/A gene was significantly lower than that in those with the G/G gene, while the serum MZR concentrations in children with the hcnt1 A/A gene were slightly lower than those with the G/G gene. 18,19,20 I examined the influence of SNPs of hcnt on the serum MZR concentrations in children 10 years of age versus 11 to 20 years of age. In the 28 children 10 years of age, no significant association between the serum MZR concentrations and the SNPs of hcnt was found. Similarly, in the 19 children 11 to 20 years of age, no significant association between the serum MZR concentrations and the SNPs of hcnt was found, but the serum MZR concentrations of the hcnt1 A/A gene (2 cases) were lower than those of the G/G gene (6 cases) (P=0.04). However, the number of cases was small, and a study involving a larger number of cases is therefore needed in the future. Age may be associated with individual differences in the bioavailability of MZR. A previous study reported that the MZR absorption in younger children was actually lower than that in adults. 21 Few reports have examined the factors affecting the bioavailability of MZR, although extracellular fluid volume and the expression of hcnt or an active age difference have been suggested. Individual differences in the MZR absorption between patients with childhood-onset FRNS are likely to have different origins. Our group previously reported that sodium was necessary for the uptake of MZR by hcnt and that inosinic acid, the nucleotide of a nucleic acid component extracted from dried bonito, inhibited the MZR absorption. 10 In the present study, some patients had been administrated other immunosuppressants before MZR administration; these drugs might have influenced the MZR absorption. Nucleoside transporters aside from CNT include equilibrative nucleoside transporter (ENT). 22 Individual variation in the SNPs affecting the MZR absorption mechanisms, including those in ENT, or MZR excretion mechanisms may underlie difficulties in readily achieving high serum concentrations of MZR. It is necessary to examine the difference in the expression of intestinal hcnt by age, diet, and immunosuppressive drugs given before MZR administration. Conclusion Even when children with FRNS received the same dose of MZR, the serum concentration of MZR was significantly lower among older children, leading to relapse. I hypothesized that differences in SNPs of the hcnt gene, which is involved in MZR absorption, may contribute to differences in the serum MZR concentrations. However, no association was noted between these variable factors. Factors other than SNP of hcnt may underlie the observed differences in the MZR absorption. 24

7 Single-nucleotide polymorphism of human concentrative nu-cleoside transporter and mizoribine absorption in Acknowledgement I am grateful to Dr.Tsukasa Takemura, Dr.Mitsuru Okada, and Dr.Tomoki miyazawa (Department of Pediatrics, Kindai University Faculty of Medicine, Osaka, Japan) for technical assistance and useful comments. Conflict of interest The author declares that I have no conflicts of interest. References 1.Sugitani A, et al. (2006) Revival of effective and safe high-dose mizoribine for the kidney transplantation. Clin Transplant 20: Yoshioka K, et al. (2000) A multicenter trial of mizoribine compared with placebo in children with frequently relapsing nephritic syndrome. Kidney Int 58: Kawasaki Y, et al. (2004) Efficacy of prednisolone and mizoribine therapy for diffuse IgA nephropathy. Am J Nephrol 24: Tanaka H, Tsugawa K, Suzuki K, Nakahata T, Ito E (2006) Long-term mizoribine intermittent pulse therapy for young patients with flare of lupus nephritis. Pediatr Nephrol 21: Tanaka E, et al. (2006) Acceptability and usefulness of mizoribine in the management of rheumatoid arthritis in methotrexate-refractory patients and elderly patients, based on analysis of date from a large-scale observa-tional cohort study. Mod Reumatol 16: Goto M, et al. (2006) Beneficial and adverse effects of high-dosage MZR therapy in the management of children with frequently relapsing nephrotic syndrome. Japanese Society of Nephrology 48: Iijima K, et al. (2013) Clinical guidelines for idiopatic nephrotic syndrome in children. Japanese society for pediatric nephrology 8.Kuroda T, et al. (2007) Mizoribine therapy for patients with lupus nephritis: the association between peak mizoribine concentration and clinical efficacy. Mod Rheumatol 17: Yoshioka K, et al. (2000) A multicenter trial of mizoribine compared with placebo in children with frequently relapsing nephronic syndrome. Kidney Int 58: Okada M, et al. (2008) Examination of human Na+/nucleoside cotransporter (hcnt2) in mizoribine ab-sorption. Japanese Journal of Pediatric Nephrology 21: Shunichi Suzuki, Mariko Komori, Mitsuharu Hirai, Norio Ureshino, Shinya Kimura (2012) Development of a novel, fully-automated genotyping system: principle and applications. Sensors 12: Kanda Y (2013) Technical report, Bone Marrow Transplantation 48: Fujinaga S, et al. (2012) Maintenance therapy with single-daily, high-dose mizoribine after cyclophosphamide therapy for prepubertal boys with severe steroiddependent nephrotic syndrome. Clin Nephrol 78: Ohtomo Y, et al. (2005) High-dose mizoribine therapy for childhood-onset frequently relapsing ster-oiddependent nephrotic syndrome with cyclosporin nephrotoxicity. Pediatr Nephrol 12: Casado FJ, et al. (2002) Nucleoside transporters in absorptive epithelia. J Physiol Biochem 58: Lu H,Chen C,Klaassen C.Tissue (2004) distribution of concentrative and equilibrative nucleoside transport-ers in male and female rats and mice. Drug Metab Dispos 32: Patil SD, Unadkat JD (1997) Functional expression of human intestinal Na+-independent nucleoside transporters in Xenopus laevis oocytes. Am.J.Physiol 272: G Naito T, et al. (2010) Impact of concentrative nucleoside transporter 1 gene polymorphism on oral bioavailability of mizoribine in stable kidney transplant recipients. Basic Clin Pharmacol Toxicol 106: Ishida K, et al. (2013) Effect of salt intake on bioavailability of mizoribine in healthy Japanese males. Drug Metab Pharmacokinet 28: Fukao M, et al. (2011) Effect of genetic polymorphisms of SLC28A1, ABCG2, and ABCC4 on bioavailabil-ity of mizoribine in healthy Japanese males. Drug Metab Pharmacokinet 26: Uemura O, Ushijima K, Yamada T, Kinpara Y (2007) Blood concentration and urinary excretion of mizoribine for internal use in pediatric kidney disease patients -bio-availability varies with age. Japanese Journal of Pediatric Nephrology 20: Ito N, et al. (2008) The serum concentration and pharmacokinetic factors of mizoribine in children with chronic renal diseases. Japanese Journal of Pediatric Nephrology 21: Takuma A, et al. (2012) What is the reason why does mizoribine ineffectiveness for the patient with ster-oidresistant nephrotic syndrome? Japanese Journal of Pediatric Nephrology 25: Fuke T, et al. (2012) Mizoribine requires individual dosing due to variation bioavailability. Pediatrics Interna-tional 54: Nagai T, et al. (2016) The true distribution volume and bioavailability of mizoribine in children with chronic kidney disease. Clin Exp Nephrol 26.Kaneda H, et al. (2016) Population pharmacokinetics of mizoribine in pediatric patients with kidney disease. Clin Exp Nephrol 20: Young JD, Yao SY, Baldwin JM, Cass CE, Baldwin SA (2013) The human concentrative and equilibrative nucleoside transporter families, SLC28 and SLC29. Mol Aspects Med 34: Yoshida K, Sakai Y, Fukao M, Hashimoto Y (2016) Intestinal absorption mechanisms of mizoribine without sodium intake. Clin Pharmacy 42: