Mechanism Study On The Effect Of Renal Impairment On The Pharmacokinetics Of OATs Substrates

The disease condition can influence the microenvironment in vivo and the levels of some endogenous substances,which may lead to the activity change of drug metabolic enzymes and transporters.For example,the plasma exposure of enalapril,an antihypertensive drug,increased significantly in patients with renal failure,so the dosage was adjusted in such patients clinically to avoild adverse reactions.Clarification of the mechanism of pharmacokinetic changes of drugs and metabolites in chronic renal failure（CRF）condition may provide beneficial insights for the preclinical and clinical drug research.The study took the nitromidazole antimicrobial morinidazole,cyano pyrrolidine dipeptidyl peptidase-4（DPP-4）inhibitor vildagliptin as objectives,to investigate the effect of renal impairment on the pharmacokinetics of their parent drug and metabolites,and discuss the mechanism of renal impairment on the pharmacokinetics of organic anion transporter（OAT）.The research would provide a theoretical basis for the rational drug use and treatment.1.Mechanism study on the effect of CRF on the pharmacokinetics of morinidazole and its conjugated metabolitesMorinidazole is a novel 5-nitromidazole antibacterial drug,and given by racemate clinically.Following an intravenous infusion in humans,morinidazole was primarily metabolized via N⁺-glucuronidation forming the N⁺-glucuronide of S-morinidazole M8-1 and N⁺-glucuronide of R-morinidazole M8-2,as well as O-sulfation forming the sulfate conjugate M7.Morinidazole and its conjugates M7,M8-1,and M8-2 were mainly excreted through the urine,accounting for 21.2%,13.0%,6.6%,and 28.4%of the dose.The AUC_0-t values of conjugates M7,M8-1,and M8-2 in severe renal impairment patients were 15.1-,20.4-,and 17.4-fold higher than those in healthy subjects with the same dose,whereas the AUC_0-t value of the parent drug was only 1.5-fold higher.M7 was a sensitive substrate of two renal transporters,namely,organic anion transporter 1（OAT1）and OAT3;M8-1 and M8-2 were the only substrates of OAT3.The objective of this study was to investigate the mechanism of the increased plasma exposures of morinidazole conjugates in renal failure patients.CRF was induced by two-stage 5/6 nephrectomy（5/6 Nx）,and biochemistry parameters and histopathological sections showed that the CRF model was successfully constructed.Sulfate conjugate M7 was synthesized to directly evaluate the pharmacokinetics in 5/6 Nx and control rats,excluding the influence of the metabolism from morinidazole to M7.Compared with control rats,the plasma clearance of M7 in5/6 Nx rats significantly decreased to 27.8%,whereas the MRT and AUC_0-t values increased by 3.05-and 3.61-fold,respectively.mRNA expressions of Oat1 and Oat3 of residue kidneys in control and 5/6 Nx rats were determined by qRT-PCR.In 5/6 Nx rats,mRNA expression of Oat1 and Oat3decreased by approximately 50%.To further confirm whether the decreased mRNA levels of transporters could induce the changes in the uptakes of the conjugated metabolites,experiments on fresh kidney slices from 5/6 Nx and control rats were conducted.Accumulations of the three conjugated metabolites increased over time but showed no significant difference between control and 5/6 Nx kidney slices.Although the mRNA expression decreased,the activities of two transporters presented no apparent effect on the uptakes of the conjugated metabolites.As a result,the down-regulated expression of transporters may not induce the increase in the plasma exposures of morinidazole-conjugated metabolites.Protein-bound uremic toxins indoxyl sulfate（IS）,hippuric acid（HA）increased in the plasma and kidney of 5/6 Nx rats.The concentrations of IS increased to 4.93-and5.67-fold in the plasma and kidneys of 5/6 Nx rats,whereas the concentrations of HA increased to 4.87-and 6.85-fold compared with those in control rats.Correlation was found between the AUC values of M7 and plasma concentrations of IS or HA.The concentration of indole-3-acetate acid（IAA）,another protein-bound uremic toxin,did not changed in the kidney and plasma of 5/6 Nx rats,and there was no correlation between the AUC values of M7 and plasma concentration of IAA.Therefore,IS,HA and their mixture were used to evaluate their effects on the renal uptakes of M7,M8-1,and M8-2.Individual and mixtures of IS and HA dose-dependently inhibited the uptakes of M7,M8-1,and M8-2 in normal fresh rat kidney slices.This result indicated that the increased uremic toxins indeed inhibited the renal uptake of three morinidazole conjugated metabolites.In CRF patients,except for HA,IS and IAA,another protein-bound uremic toxin,3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid（CMPF）also increased in CRF patients.In OAT3-overexpressed HEK293 cells,HA,IS and CMPF inhibited the uptake of M7,M8-1 and M8-2.CMPF exhibited the highest inhibitory effect with IC₅₀ values of 19.2（M7）,8.53（M8-1）,and 6.75μM（M8-2）.These values were lower than the CMPF plasma concentration in CRF patients（250μM）.The IC50 values of HA were87.4（M7）,39.4（M8-1）,and 24.1μM（M8-2）,and the IC50 values of IS were 222（M7）,161（M8-1）,and 78.3μM（M8-2）;these values were also lower than or similar to their plasma concentrations（HA:1400μM;IS:210μM）in CRF patients.The inhibition results indicated that the increased concentrations of CMPF,HA,and IS in CRF patients caused the inhibition of renal clearance of three conjugated metabolites,thereby boosting the plasma exposures of M7,M8-1,and M8-2.In OAT1-overexpressed cells,the IC₅₀ values for M7 were as high as 200（IS）,187（CMPF）,162（HA）,and 197μM（IAA）.These data indicated that inhibition of four uremic toxins on the OAT1-mediated M7 uptake was weak.This also means that CMPF,HA and IS inhibited the renal uptake of M7 mainly because of their inhibitory effects on OAT3 but not OAT1.This could explain the similar elevated folds of AUC values between M7 and M8-1 or M8-2 in renal impairment patients,although M7 was the substrate of OAT1 and OAT3 and M8-1 or M8-2 was substrate of only OAT3.The typical OAT inhibitor,probenecid,was proved to be able to inhibit the uptake of M7 in renal transporters.To investigate whether the inhibition of transporter could influence pharmacokinetics of M7,different doses of M7 were intravenous administered in probenecid-treated rats.With the inhibitory effect of probenecid,the plasma exposure differences between control and probenecid-treated rats were similar in the three dosage level of M7,all increased to 3-fold.This change was also similar to that of 5/6 Nx rats.The result indicated that the inhibition of transporter could lead to equal pharmacokinetic alteration in CRF rats.In conclusion,although the mRNA expression levels of Oat1 and Oat3 decreased in 5/6 Nx rats,their activities were not significantly affected.Accumulations of CMPF（only detected in humans）,HA and IS,which were caused by renal impairment in humans and rats,inhibited the OAT3-mediated uptake of sulfate or N⁺-glucuronides in the kidney,leading to deceleration of renal excretion and consequent elevation in plasma exposure.Therefore,in disease conditions,we should not solely focus on the expression changes of transporters or metabolizing enzymes but should also consider the influence of altered endogenous substances on the activities of these transporters or metabolizing enzymes.2.Mechanism study on the effect of CRF on the pharmacokinetics of vildagliptin and its carboxylic acid metabolitesVildagliptin is a cyano pyrrolidine DPP-4 inhibitor and primarily metabolized to carboxylic acid,which are predominantly excreted by the kidney.It has been reported that in patients with mild,moderate and severe renal impairment and ESRD（end-stage renal disease）,the mean plasma exposure of vildagliptin increased by 32%to 134%,and C_maxax increased by 8%to 66%,compared with healthy subjects.However,the plasma exposure of its carboxylic acid metabolite（Vildagliptin-COOH）increased by1.6-.2.4-,5.4-and 6.7-fold in patients with mild,moderate and severe renal impairment and ESRD.The objective of this study was to investigate the mechanism of the increased plasma exposures of vildagliptin and its carboxylic acid metabolite in renal failure patients using 5/6 Nx rats,and in vitro experiments such as kidney slices and transporter overexpressed cells.Uptake studies using HEK293 cells individually expressing OAT1,OAT3,and OCT2 showed that vildagliptin was not the substrate of the examined transporters,but vildagliptin-COOH was a substrate of OAT3.The uptake of vildagliptin-COOH was dose-and time-dependent,which accorded with stature kinetic.The K_m value was 128μΜ,and V_maxax value was 186 pmol/min/mg protein.The uptake clearance,V_max/K_m value was 1.45μL/min/mg protein.After an oral administration of vildagliptin,the AUC value of the parent drug in5/6 Nx rats increased by 45.8%,but the AUC value of metabolic vildagliptin-COOH in5/6 Nx rats increased to 7.5-fold,compared with control rats.The other pharmacokinetic parameters changed similarly to those in CRF patients.The total cumulative excretion of vildagliptin-COOH in 5/6 Nx rats was slightly elevated by approximate 10%of dose,possibly due to the slightly increased metabolism in 5/6 Nx rat liver.The increase amount of metabolized vidgliptin-COOH was little,so it was not considered as the main reason of its boosted plasma exposures in 5/6 Nx rats.To exclude the influence of the metabolism from vildagliptin to its carboxylic acid,vildagliptin-COOH was directly administered intravenously.Compared with control rats,the plasma clearance of vildaglitin-COOH in 5/6 Nx rats significantly decreased to 30.1%,whereas the MRT and AUC_0-t values increased by 2.25-and 3.34-fold,respectively.It indicated that CRF decelerated the clearance of vildagliptin-COOH thereby increasing the plasma exposures.Expression of Oat3 mRNA in 5/6 Nx rat kidney decreased by 50%compared with control,and there is a correlation between the AUC values of vildagliptin-COOH and the expression.The uptake of vildagliptin-COOH was studied in kidney slices of 5/6Nx rats and control rats.The uptake at 37°C was significantly higher than that of 4°C in both groups,certifying the active transport of vildagliptin-COOH.The uptake of vildagliptin-COOH elevated with the increased substrate concentration and incubation time extension.However,accumulations of vildagliptin-COOH showed no significant difference between control and 5/6 Nx kidney slices.Although the mRNA expression decreased,the activity of Oat3 presented no apparent effect on the uptake of the vildagliptin-COOH.Plasma concentration of IS and HA increased to about 4-fold in 5/6 Nx rats and they both highly correlated with the elevation of vildagliptin-COOH AUC value.Individual and mixtures of IS and HA dose-dependently inhibited the uptakes of vildagliptin-COOH in normal fresh rat kidney slices.This result indicated that the increased uremic toxins indeed inhibited the renal uptake of vildagliptin-COOH.The inhibitory effects of uremic toxins were further identified in OAT3-overexpressed HEK293 cells.Mixtures of CMPF,IS,HA and IAA showed strong inhibitory effect on the uptake of vildagliptin-COOH in OAT3-overexpressed HEK293cells,and mixture with the lowest concentration（10,10,10 and 1μΜ）inhibited the uptake by 63.7%.Higher level mixture（>30,30,30 and 3μΜ）of the four uremic toxins inhibited the uptake by over 90%,similar to the inhibitory effect of typical OAT inhibitor,probenecid.In the study of inhibitory effect of individual uremic toxin,CMPF exhibited the strongest inhibitory effect with the IC₅₀ value of 5.75μΜ,much lower than its plasma concentration（250μM）in CRF patients.HA and IS also showed the inhibitory effect with IC₅₀ value of 29.0μΜand 69.5μΜrespectively,which were both lower than their plasma concentration（1400μΜand 210μΜ）in CRF patients.The result prompted that the accumulated uremic toxins in CRF patients could strongly inhibited the uptake of vildagliptin-COOH in proximal tubule epithelial cells,thereby decelerating its excretion in the kidney and increasing its plasma exposures.The Dixon plot and Cornish-Bowden plot showed the inhibition types of IS,CMPF,and HA were predominantly mixed inhibition.Probenecid was the typical OAT inhibitor and proved to inhibit the uptake of vildagliptin-COOH in both kidney slice and OAT3-overexpressed HEK293 cells.To investigate whether the inhibition of Oat3 would influence the pharmacokinetics of its substrate,vildagliptin-COOH was directly administered intravenously into the control and probenecid-treated rats.In probenecid-treated rats,the clearance of vildagliptin-COOH was decelerated by 27.5%and the plasma exposure increased by 41.5%,significantly.The inhibitory effects of probenecid in vivo and in vitro were consistent.In conclusion,chronic renal failure has weak effect on vildagliptin and its slight plasma exposure was changed by GFR.However,the plasma exposure of vildagliptin-COOH increased with the severity of renal impairment,possibly because it was the substrate of OAT3.Although the mRNA expression of Oat3 in 5/6 Nx rats decreased,the activity did not influence the uptake of vildagliptin-COOH.The accumulated protein-bound uremic toxins,HA,IS and CMPF（only detected in human）in CRF condition,directly inhibited the uptake of vildagliptin-COOH in OAT3,leading to the deceleration of its renal clearance,thereby resulting in the elevation of its plasma exposure.3.ConclusionsThis thesis demonstrated the critical role of protein-bound uremic toxins in the change of the pharmacokinetics of renal transporter substrates.In chronic renal failure,the accumulated protein-bound uremic toxins,IS,HA,and CMPF showed strong inhibitory effect on the uptake in OAT3,but weak inhibitory effect on OAT1.This may cause the dramatic increase in plasma exposure of OAT3 substrates,such as morinidazole conjugated metabolites and vildagliptin carboxylic acid metabolite.Morinidazole and vildagliptin were not substrates of renal uptake transporter,therefore their pharmacokinetics changed slightly in patients with chronic renal failure.It has been reported that chronic high level of uremic toxins may influence the transporters by regulating their expression indirectly.In this study,however,the altered expression did not change the uptake.The study result also indicated that in disease conditions,we should not solely focus on the expression changes of transporters or metabolizing enzymes but should also consider the influence of altered endogenous substances on the activities of these transporters or metabolizing enzymes.Protein-bound uremic toxins could not be easily removed by conventional dialysis due to the high degree of protein binding.Thus,for organic anion drugs and metabolites,especially substrates of OAT3,clinicians should consider their pharmacokinetic change,such as deceleration in kidney clearance and increase in plasma exposure,which may influence the safety and efficiency.The current results also suggested that in renal failure patients,decreasing uremic toxin levels might help to reverse the altered pharmacokinetics of organic anion drugs and metabolites.