Construction Of Electrotransfection Cell Models With High OAT1/3 Expression And Its Application In Clinical Pharmacokinetic Study Of Azlocillin And Piperacillin In Children

BackgroundBoth azlocillin and piperacillin are semisynthetic penicillin,which are often used as first-line drugs in the treatment of severe infection and septicemia in children due to their wide antibacterial spectrum,high antibacterial activity and low incidence of adverse reactions.Under conventional dosing regimens,blood concentrations vary widely in children,leading to poor response or nephrotoxicity,the mechanism of which remains unknown.Azlocillin and piperacillin are mostly eliminated by the kidney after administration.Multiple transporters including organic anionic transporter 1(OAT1)and organic anionic transporter 3(OAT3)distributed in the kidney,widely involved in drug interactions.However,few studies have explored renal transporter transport of azlocillin and piperacillin and the effect of transporter gene polymorphism on clinical pharmacokinetics in children.Therefore,this study intends to construct cell models with high expression of transporters to screen out key transporters and clarify their effects on the clinical pharmacokinetics of azlocillin and piperacillin in children.MethodsElectroporation transfection was used to construct cell models with high expression of transporters.Higher transfection efficiency and stable transfection effect were obtained by optimizing electrical parameters.Methods for the detection of intracellular azlocillin and piperacillin concentrations used liquid chromatography-tandem mass spectrometry(LC-MS/MS)was established and verified.The uptake of azlocillin and piperacillin was studied using the electrotransfection cell models,and the key transporters were screened out.Rats were given inhibitors of key transporters to investigate the effects of key transporters on the in vivo processes of azlocillin and piperacillin.Clinical samples of azlocillin and piperacillin in pediatric patients were obtained by opportunistic sampling strategy.Plasma concentrations were determined by high-performance liquid chromatography-ultraviolet detection(HPLC-UV).Pharmacokinetic parameters were calculated by nonlinear mixed effect model(NONMEM).Genome wide association study(GWAS)was used to identify gene loci that significantly affected drug metabolism.ResultsThe optimized electroporation transfection conditions were as follows:HEK293 cell density 5×106/mL,voltage 240 V,capacitance 500 μF,positive infinite ∞ resistance,4 mm shock cup,plasmid concentration 24 μg/mL.A method for the detection of intracellular drug concentration by LC-MS/MS was established.The results of selectivity,matrix effect and linearity in the detection range of 0.0005-0.2 nmol/mL all met the requirements.At the concentration of 5 μM,uptake of azlocillin by HEK293-OAT3 cells was significantly higher than HEK293-NC cells(p<0.0001),and the intracellular concentration was about 6 folds of the latter.At the concentration of 250 μM,uptakes of piperacillin by HEK293-OAT3 and HEK293-OAT1 cells were significantly higher than HEK293-NC cells(p<0.0001 and p<0.05),and the intracellular concentrations were about 4 folds and only 1.5 folds of the latter.The Michaelis constant(Km)of azlocillin and piperacillin in HEK293-OAT3 are 31.30 μM and 95.46 μM,respectively.The clearance(CL)of azlocillin was significantly decreased from 0.022±0.005 L/min/kg to 0.007±0.004 L/min/kg in(p<0.001)Wistar rats given 500 mg/kg of probenecid.The CL of piperacillin was significantly decreased from 0.006±0.002 L/min/kg to 0.002±0 L/min/kg(p<0.001).102 clinical samples of azlocillin were included in the study and GWAS results showed that there was no significant correlation between OAT3-related single nucleotide polymorphism(SNP)sites and CL.52 clinical samples of piperacillin were included in the study,and GWAS results suggested that rs10792368 genotype may be closely related to CL.ConclusionIn this study,OAT 1/3 plasmid and electrotransfection cell models with high expression of OAT 1/3 transporters were successfully constructed.OAT3,a key transporter that significantly increased uptakes of azlocillin and piperacillin was screened out.After inhibiting the function of OAT3,the CL of azlocillin and piperacillin in rats was significantly reduced,the blood drug concentration and area under drug-time curve(AUC)were significantly increased,and the half-life and mean residence time(MRT)were prolonged.OAT3-related SNP were associated with the pharmacokinetic process in children,among which the genotype of rs10792368 locus significantly affected the CL of piperacillin.The CL of piperacillin was increased successively in children with AA,AC and CC genotypes.