OATP1B3 (699G>A) and CYP2C9*2, *3 significantly influenced the transport and metabolism of glibenclamide and glipizide.

Glibenclamide and glipizide show large substantial inter-individual variation in clinical efficacy, which may be resulted from the genetic differences of metabolic enzymes and transporters in individuals. This study purposed to investigate the effect of OATP1B3 and CYP2C9 genetic polymorphisms on the transport and metabolism of glibenclamide and glipizide in human. An LC-MS method was used to determine the uptake of glibenclamide and glipizide in OATP1B3, OATP1B3 (344T > G) and OATP1B3 (699G > A)-HEK293T cells and their metabolism in CYP2C9*1, *2 and *3 recombinase system. Glibenclamide can be taken in OATP1B3 (wild-type), OATP1B3 (344T > G) and OATP1B3 (699G > A)-HEK293T cells with the Vmax values of 44.91 ± 7.97, 46.08 ± 8.69, and 37.31 ± 5.04 pmol/min/mg, while glipizide was taken in with Vmax of 16.50 ± 3.64, 16.87 ± 4.23, and 13.42 ± 2.79 pmol/min/mg, respectively. The internal clearance of glibenclamide and glipizide in OATP1B3 (699G > A) was less than that in wild-type. Glibenclamide can be metabolized in CYP2C9*1, *2 and *3 recombinase system with the Vmax values of 1.58 ± 0.71, 0.69 ± 0.25, and 0.41 ± 0.13 nmol/min/mg protein, while glipizide was metabolized with Vmax of 8.82 ± 2.78, 5.99 ± 1.95, and 2.87 ± 1.03 nmol/min/mg protein, respectively. The internal clearance of glibenclamide and glipizide in CYP2C9*2 and *3 was markedly reduced compared to that in CYP2C9*1. These results collectively demonstrate that OATP1B3 (699G > A) and CYP2C9*2 and *3 have a significant influence on the transport and metabolism of glibenclamide and glipizide.

significantly to inter-individual differences in the disposition of many clinically relevant drugs [11][12][13] . SLCO1B3 334T > G (Ser112Ala) and 699G > A (Met233Ile) were commonly reported to be associated with the altered transport function in vitro compared to the expressed wild-type protein. The recent studies have shown SLCO1B3 334T > G and 699G > A influence the pharmacokinetic profile of a newly identified substrate, the glucuronide metabolite of the immunosuppressant mycophenolic acid 14,15 . CYP2C9 accounts for about 20% of the total cytochrome P450 in liver microsomes and takes part in the metabolism of approximately 10-20% of commonly used drugs 16,17 . There are marked polymorphisms in the human CYP2C9 gene that significantly affect drug metabolism 18,19 . In Caucasians, the frequencies of the CYP2C9*2 (Arg 144Cys) and CYP2C9*3 (Ile359Leu) alleles of the CYP2C9 variant are 0.08-0.125, 0.03-0.085, respectively. But in the Asian population, CYP2C9*2 is rare and the allele frequencies of *3 and *13 (Leu90Pro) were 0.043-0.077 and 0.00-0.012, respectively 20,21 . Lots of in vitro and in vivo studies have shown that CYP2C9 *2 and CYP2C9 *3 point mutation have crucial impacts on CYP enzyme activity and significant influences on drug metabolism [22][23][24][25] .
It is unclear to date how OATP1B3 and CYP2C9 polymorphisms are associated with the substantial inter-individual variation of glibenclamide and glipizide in human. In the present study, therefore, we aimed to explore the effects of OATP1B3 and CYP2C9 polymorphisms on the transport and metabolism of glibenclamide and glipizide by using HEK293T cells expressing OATP1B3 and human recombinant enzymes expressing CYP2C9 with different genotypes.
Western blot analysis. The OATP1B3 expression in OATP1B3, (344T > G), (699G > A)-HEK293T cells was detected by western blot. Cell extracts were prepared in lysis buffer. The cell debris was removed by centrifugation at 12,000 × g at 4 °C for 15 min and the total protein concentration was measured using a BCA Protein Assay Kit (Vazyme Biotech Co., Ltd, Nanjing, China). Protein samples (50 μg) were subjected to SDS-PAGE and electrophoretically transferred to PVDF membranes (EMD Millipore, Bedford, MA, USA). Immunoblots were probed with mouse polyclonal OATP1B3 antibody (diluted 1:5000) (Proteintech, Chicago, IL, USA) and with mouse polyclonal anti-GAPDH (diluted 1:4000) (Sigma Co., Ltd, USA) antibody as the loading control. After incubation with HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), signals were detected by Super Signal West Dura (Pierce, Rockford, IL, USA) using a Bio-Rad ChemiDoc XRS imaging system (Bio-Rad Laboratories), and densitometry analysis was performed using Image Lab Sofware (Bio-Rad Laboratories).

Quantification of glibenclamide and glipizide in HEK293T cells. Firstly, the cells were adjusted
to an appropriate density (7.0 × 10 5 /ml) and then were added to 12-well culture plates at 1.0 ml per well. For time-dependent studies, the uptake of glibenclamide and glipizide in HEK293T cells were measured at 2, 5, 10, 15, 20, and 30 min. For concentration-dependent studies, the uptake of glibenclamide and glipizide in HEK293T cells were measured in a series of concentrations of 2.5, 5, 10, 20, 40, and 80 μM. Experiments contained MOCK-HEK293T cells group, OATP1B3-HEK293T cells group, OATP1B3 (344T > G)-HEK293T cells group and OATP1B3 (699G > A)-HEK293T cells group. Cells were incubated at 37 °C for 20 min with HBSS containing 10 mM HEPES (HBSS-HEPES, 99:1, v/v). At the end of the incubation period, indicated concentrations of glimepiride or gliclazide were added, cells were then placed in the incubator at 37 °C for 15 min or for specified times. The experiment was terminated by removing the medium and immediately washing cells with ice-cold HBSS four times and then adding 1 ml sterile water. Cells were disrupted by 4 cycles of freeze-thawing at −80 °C, and 200 μl of cell lysate was placed in a 1.5 ml centrifuge tube with 20 μl of 5 μM gliquidone internal standard, 30 μl of glacial acetic acid, and 800 μl ethyl acetate. After vortexing for 5 min, the tube was centrifuged at 15,000 × g for 10 min, and 700 μl of the supernatant was concentrated by vacuum drying at 55 °C for 30 min. The resulting concentrate was dissolved in 200 μl of acetonitrile, vortexed for 5 min, and centrifuged at 15,000 × g for 10 min, 100 μl of supernatant was finally used for LC/MS analysis 3 . The protein concentration of each sample was determined using a BCA Protein Assay Kit (Vazyme Biotech Co., Ltd, Nanjing, China). For the standardization of the OATP1B3 protein content in HEK293T cells, the total protein content (determined by BCA Protein Assay Kit) was divided by OATP1B3, OATP1B3 (344T > G), OATP1B3 (699G > A) relative protein content (determined by western blot).   Quantification of glibenclamide and glipizide in CYP2C9 recombinant enzyme incubation system. The final volume of incubation system was 200 μl, containing 50 mM sodium phosphate buffer (pH = 7.4) and 0.4 mg/ml human recombinant CYP2C9 (CYP2C9*1, CYP2C9*2, and CYP2C9*3). A series of concentrations of glibenclamide and glipizide (1-80 μM) were prepared in methanol, and the final concentration of methanol was no more than 0.5% (v/v). The reactions took place at 37 °C in a shaker. After pre-warming for 5 min, the reaction was initiated by adding a NADPH regenerating system containing 1.3 mM NADP + , 3.3 mM glucose-6-phosphate, 3.3 mM MgCl 2 , and 0.4 U/ml glucose-6-phosphate dehydrogenase. In the negative controls, the NADPH regenerating system was not added. The reactions were stopped at 30 min by adding 100 μl ice-cold Statistical analysis. All data are presented as means with their standard deviations (mean ± SD). Kinetic parameters were obtained using GraphPad Prism (GraphPad Software 5.01, San Diego, CA, US) followed by non-linear, least-squares regression analysis via the Michaelis-Menten equation. Statistical analysis was conducted using SPSS 12.0. A one-way analysis of variance (ANOVA) was performed to determine whether the differences among all the groups were statistically significant, and then Dunnett's post hoc test was carried out to statistically. P < 0.05 was considered to be statistically significant.

Results
Uptake of glibenclamide and glipizide in OATP1B3-HEK293T cells. Uptake experiments of glibenclamide and glipizide were carried out as described with the addition of different concentrations of glibenclamide and glipizide (2.5-80 μM). The time-dependent experiments showed that the uptake of glibenclamide and glipizide nearly attained a steady-state at 15 min (Fig. 3). Concentration-dependent experiments showed that the uptake of glibenclamide and glipizide in OATP1B3-HEK293T cells was nearly saturated at 40 μM (Figs 4 and 5). The transport of glibenclamide and glipizide by OATP1B3 was related to the genotype. Compared with the wildtype OATP1B3, mutants OATP1B3 (699G > A) showed a decreased transport capacity, while mutants OATP1B3 (344T > G) had no influence on the transport capacity. The uptake of glibenclamide and glipizide in OATP1B3 (699G > A)-HEK293T cells was less than that in OATP1B3-HEK293T cells (Fig. 4). For glibenclamide, the V max and intrinsic clearance (CL int , defined as V max /K m ) in OATP1B3 (699G > A)-HEK293T cells was decreased to 85% and 65% of that in OATP1B3-HEK293T cells, respectively (Fig. 5, Table 1). For glipizide, the V max and CL int in OATP1B3 (699G > A)-HEK293T cells was decreased to 80% and 64% of that in OATP1B3-HEK293T cells, respectively (Fig. 5, Table 2).  Metabolism of glibenclamide and glipizide by recombinant CYP2C9. The catalytic activities of wild-type CYP2C9*1 and two allelic variants CYP2C9*2 and *3 were assessed by using glibenclamide and glipizide as substrates. Michaelis-Menten plots for each of the CYP2C9 variants are shown in (Fig. 6), and the corresponding kinetic parameters are summarized in (Tables 3 and 4). As shown in (Tables 3 and 4), the two variants exhibited changed V max and CL int values of glibenclamide and glipizide as compared to the wild-type protein. The two variants, CYP2C9*2 and CYP2C9*3, caused the V max of glibenclamide decreased to 43% and 25%, respectively and the CL int of glibenclamide decreased to 44% and 26%, respectively, compared with the wild-type  Table 3. Glibenclamide metabolic kinetic parameters in human recombinase CYP2C9*1/*2/*3 (n = 3, mean ± SD). **p < 0.01, *p < 0.05 (vs CYP2C9*1 group), CL int (intrinsic clearance) = V max /K m .  Table 4. Glipizide metabolic kinetic parameters in human recombinase CYP2C9*1/*2/*3 (n = 3, mean ± SD). **p < 0.01, *p < 0.05 (vs CYP2C9*1 group), CL int (intrinsic clearance) = V max /K m .  (Table 3). For glipizide, the V max was decreased to 68% and 32%, respectively, and the CL int was decreased to 55% and 23% respectively, in CYP2C9*2 and *3 recombinant compared to CYP2C9*1 recombinant as shown in (Table 4). These results revealed that point mutations in CYP2C9*2 and CYP2C9*3 had vital impacts on CYP enzyme activity and significantly affected the metabolism of glibenclamide and glipizide.

Discussion
OATP1B3 is a liver-specific transporter that has an important role in transporting certain endogenous and exogenous substrates into the liver. Consequently, this protein is suspected to play a central function in the disposition of drugs for both metabolism and hepatobiliary elimination 26,27 . OATP1B3 is encoded by the (SLCO) 1B3 gene and several SLCO1B3 nucleotide sequence variations have been identified. The OATP1B3 gene polymorphisms T334G (Ser112Ala) and G699A (Met233Ile) show a complete linkage disequilibrium with an allele frequency of 0.728. (112Ala/233Ile), the linkage disequilibrium of these mutants will lead to a decrease in OATP1B3 transport activity 9 . Changes in the transport function of OATP1B3 caused by mutation affect the transfer of substrate drugs, which eventually changes the blood drug concentration of a drug and affects drug treatment 28,29 . Two common variants, SLCO1B3 334T > G (Ser112Ala) and 699G > A (Met233Ile), were found to be associated with altered transport function in vitro compared to the expressed wild-type protein. Schwarz et al. 9 . found that the translocation of cholecystokinin-8 (CCK8) and rosuvastatin in the highly expressed OATP1B3-Met233Ile (699G > A) HeLa cells was significantly lower than that in wild-type OATP1B3 HeLa cells.
As the substrate for the uptake of OATP1B3, glibenclamide and glipizide, the blood plasma concentration may be influenced by OATP1B3 genetic polymorphism. In the present study, we found that the uptake of glibenclamide and glipizide in OATP1B3 (699G > A)-HEK293T cells was significantly reduced compared with OATP1B3-HEK293T cells (Fig. 5). For glibenclamide in OATP1B3 (699G > A) -HEK293T cells, the V max was decreased to 85% and CL int was decreased to 65% of that in OATP1B3-HEK293T cells. For glipizide, the V max was decreased to 80% and CL int was decreased to 64%. Thus, it can be deduced that the uptake of glibenclamide and glipizide are affected by mutation 699G > A. However, the mutants OATP1B3 (344T > G) had no influence on the transport capacity. Further in vivo studies are needed to perform in the future for confirming the vitro results.
It is well-known that metabolic activity changes due to CYP2C9 polymorphisms play an important role in adverse drug reactions. The changes of CYP2C9 activity may increase the risk of adverse drug reactions for patients when they are administered with CYP2C9-specific substrates such as S-warfarin, phenytoin, glipizide, and tolbutamide which have narrow therapeutic windows 30 . Niemi et al. 25 . reported that the AUC of glibenclamide in individuals heterozygous for the CYP2C9*3 allele (n = 52) was 280% of the respective value in the CYP2C9*1/*1 subjects (n = 55). Yin et al. 22 . also demonstrated that the oral plasma AUC of glibenclamide in the CYP2C9*1/*3 subjects (n = 56) of the Chinese population was increased by approximately 100% compared with that in the CYP2C9*1/*1 subjects. These clinical studies appear to suggest that CYP2C9 contributes significantly to glibenclamide metabolism in vivo. At the some time, glipizide oral clearance in one homozygous carrier of the CYP2C9*3 allele was only 20% of that in carriers of the CYP2C9 wild-type 31 . In vivo studies have shown that CYP2C9 gene polymorphism has a significant effect on the sulfonylurea metabolism. However, in vivo studies are often affected by many other factors, such as physiology, age, and food. Of note, the significant changes in metabolism associated with the CYP2C9*2 and *3 alleles demonstrated in vitro and in vivo for a variety of substrates do not appear to be consistent for all substrates evaluated 19,32 . Therefore, we study the impact of CYP2C9 gene polymorphisms of drug metabolism by recombinant enzyme in vitro model. In the present study, we found that CYP2C9*2 was less efficient than CYP2C9*1 in metabolizing glibenclamide and glipizide. However, this was not due to differences in the apparent K m , but was the result of a decreased V max and CL int in the metabolism of glibenclamide and glipizide by CYP2C9*2 when compared with CYP2C9*1. Similarly, the allelic variant CYP2C9*3 was also less efficient than CYP2C9*1 in metabolizing glibenclamide and glipizide. This may be explained by the increased K m and decreased V max in the metabolism of glibenclamide and glipizide by CYP2C9*2 (Table 4). It is possible that CYP2C9*2 and CYP2C9*3 are risk factors for adverse drug reactions such as hypoglycemia or allergic reactions due to sulphonylurea.
In summary, glibenclamide and glipizide can be transported by OATP1B3 and metabolized by CYP2C9. The mutants OATP1B3 (699G > A) can significantly decrease their transport capacity, while CYP2C9*2 and *3 mutants show significantly reduced metabolism toward glibenclamide and glipizide. Taken together with our previous results 7 that the mutations of OATP1B1*5, *15 and CYP2C9*2, *3 significantly affect the transport and metabolism of glimepiride and gliclazide, it can be deduced that OATP1B1/1B3 and CYP2C9 play a vital role in the disposition of these oral hypoglycemic agents in humans and their pharmacokinetic behaviors can be markedly influenced by OATP1B1/1B3 and CYP2C9 gene polymorphisms. We should pay much attention to the potential impacts of OATP1B1/1B3 and CYP2C9 gene polymorphisms on the pharmacokinetics and pharmacodynamics when using these oral hypoglycemic drugs in clinical settings.