Reduced renal clearance of cefotaxime in asians with a low-frequency polymorphism of OAT3 (SLC22A8)

Abstract

Organic anion transporter 3 (OAT3, SLC22A8), a transporter expressed on the basolateral membrane of the proximal tubule, plays a critical role in the renal excretion of organic anions including many therapeutic drugs. The goal of this study was to evaluate the in vivo effects of the OAT3-Ile305Phe variant (rs11568482), present at 3.5% allele frequency in Asians, on drug disposition with a focus on cefotaxime, a cephalosporin antibiotic. In HEK293-Flp-In cells, the OAT3-Ile305Phe variant had a lower maximum cefotaxime transport activity, Vmax , [159 ± 3 nmol*(mg protein)(-1) /min (mean ± SD)] compared with the reference OAT3 [305 ± 28 nmol*(mg protein)(-1) /min, (mean ± SD), p < 0.01], whereas the Michaelis-Menten constant values (Km ) did not differ. In healthy volunteers, we found volunteers that were heterozygous for the Ile305Phe variant and had a significantly lower cefotaxime renal clearance (CLR ; mean ± SD: 84.8 ± 32.1 mL/min, n = 5) compared with volunteers that were homozygous for the reference allele (158 ± 44.1 mL/min, n = 10; p = 0.006). Furthermore, the net secretory component of cefotaxime renal clearance (CLsec ) was reduced in volunteers heterozygous for the variant allele [33.3 ± 31.8 mL/min (mean ± SD)] compared with volunteers homozygous for the OAT3 reference allele [97.0 ± 42.2 mL/min (mean ± SD), p = 0.01]. In summary, our study suggests that a low-frequency reduced-function polymorphism of OAT3 associates with reduced cefotaxime CLR and CL(sec) .

Keywords: OAT3; cefotaxime; organic anion transporter; pharmacogenomic; pharmacokinetics; polymorphisms; renal clearance; tubular secretion.

Figure 1

Uptake of cefotaxime in HEK293 Flp-In cells expressing organic anion transporters (OATs). (a) Uptake of cefotaxime (50 µM and 100 µM) in HEK293 Flp-In cells expressing human OAT1, OAT2, OAT3 and OAT4. The cells were exposed to cefotaxime for 5 minutes. Significantly greater cefotaxime (50 µM and 100 µM) uptake was observed in HEK293-Flp-In cells transfected with OAT3 compared to cells transfected with empty vector (EV), OAT1, OAT2 and OAT4 (p< 0.005). Asterisks indicate significantly different uptake values from EV controls. Note: * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.005, n.s. not-significant. Multiple comparisons were analyzed using one-way analysis of variance followed by Dunnett’s two-tailed test. Data are shown as mean ± SD from 2 repeated experiments, and each experiment was performed in triplicate. Results are expressed as the percent of activity of the OAT3-reference. (b) Uptake of cefotaxime (100 µM) with and without the OAT inhibitor, probenecid (100 µM), in HEK293 Flp-In cells expressing OAT3 reference (OAT3-REF) and OAT3 variant, Ile305Phe. The cells were exposed to cefotaxime with or without probenecid for 5 minutes. The result showed significantly reduced cefotaxime uptake in cells transfected with OAT3 Ile305Phe compared to OAT3 reference, ** p-value < 0.01. In the presence of probenecid, cefotaxime uptake in cells transfected with empty vector (EV), OAT3 reference (OAT3-REF) or OAT3 variant (OAT3-Ile305Phe), were significantly reduced compared to cells without probenecid, ** p-value < 0.01, *** p-value < 0.005. Data are shown as mean ± SD from 2 repeated experiments and each experiment was performed in triplicate. Results are expressed as the percent of activity of the OAT3-reference (OAT3-REF).

Figure 2

Cefotaxime kinetics in HEK293 Flp-In cells expressing OAT3 reference (OAT3-REF) and its variant (OAT3-Ile305Phe). In the kinetic studies, uptake rates were determined at 5 minutes. The mean ± SEM K_m and V_max of the OAT3-reference were 717 ± 141 µM and V_max = 305 ± 28 nmol/mg protein/min respectively; the K_m and V_max of the OAT3-Ile305Phe were 549 ± 21 µM and 159 ± 3 nmol/mg protein/min respectively. The V_max for the variant was significantly lower than that of reference (p < 0.01), while the K_m value was not significantly different between the reference and the variant. Data points in the figure represent the mean ± SD from triplicate wells in a representative experiment. Two separate experiments were performed and the overall results showed that the V_max for the variant was significantly lower than that of reference (p<0.05) and no significant difference between the K_m values.

Figure 3

Effect of OAT3 genotype on (a) plasma concentrations (b) renal clearance (CL_R) and (c) net secretory clearance (CL_sec) of cefotaxime in healthy volunteers. A single 2 gram dose of cefotaxime was administered as an intravenous bolus dose over 5 min to volunteers homozygous for the reference (Ile305/Ile305, n=10) or heterozygous for the variant (Ile305/Phe305, n=5). CL_R parameters were determined using non-compartmental analyses. Data shown represent mean ± SEM (Figure 3(a)) or mean ± SD (Figure 3(b) and (c)). Significant differences between genotype groups as assessed by unpaired two-tailed t-test) are indicated as, * p<0.05, ** p<0.01 versus volunteers homozygous for the OAT3 reference allele. The pharmacokinetic parameters are shown in Table 1.

Figure 3

Effect of OAT3 genotype on (a) plasma concentrations (b) renal clearance (CL_R) and (c) net secretory clearance (CL_sec) of cefotaxime in healthy volunteers. A single 2 gram dose of cefotaxime was administered as an intravenous bolus dose over 5 min to volunteers homozygous for the reference (Ile305/Ile305, n=10) or heterozygous for the variant (Ile305/Phe305, n=5). CL_R parameters were determined using non-compartmental analyses. Data shown represent mean ± SEM (Figure 3(a)) or mean ± SD (Figure 3(b) and (c)). Significant differences between genotype groups as assessed by unpaired two-tailed t-test) are indicated as, * p<0.05, ** p<0.01 versus volunteers homozygous for the OAT3 reference allele. The pharmacokinetic parameters are shown in Table 1.

Figure 3

Effect of OAT3 genotype on (a) plasma concentrations (b) renal clearance (CL_R) and (c) net secretory clearance (CL_sec) of cefotaxime in healthy volunteers. A single 2 gram dose of cefotaxime was administered as an intravenous bolus dose over 5 min to volunteers homozygous for the reference (Ile305/Ile305, n=10) or heterozygous for the variant (Ile305/Phe305, n=5). CL_R parameters were determined using non-compartmental analyses. Data shown represent mean ± SEM (Figure 3(a)) or mean ± SD (Figure 3(b) and (c)). Significant differences between genotype groups as assessed by unpaired two-tailed t-test) are indicated as, * p<0.05, ** p<0.01 versus volunteers homozygous for the OAT3 reference allele. The pharmacokinetic parameters are shown in Table 1.