Study On The Metabolism,Pharmacokinetics,Drug-drug Interaction,and Multidrug Resistance Reversal Effect Of TPN729

TPN729 is a novel phosphodiesterase type 5(PDE5)inhibitor developed to treat erectile dysfunction,and is presenty undergoing clinical trials in China.Compared to the currently available PDE5 inhibitors on the market(sildenafil,vardenafil,tadalafil,and avanafil),TPN729 is proved to be a potent PDE5 inhibitor with a better selectivity profile of 2.5 times higher than sildenafil against PDE6 and 500 times higher than tadalafil against PDE11,thus providing fewer side effects and better compliance.The objectives of this study were to elucidate the metabolic and pharmacokinetic characteristics of TPN729 in humans,evaluate its potential for drug-drug interactions associated with drug-metabolizing enzymes and transporters,and investigate its multidrug resistance reversal effect.1.Metabolism and Pharmacokinetics in HumansA rapid and reliable analytical method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry(UPLC/Q-TOF MS)was established to investigate the metabolic profiles of TPN729 in human plasma,urine,and feces after an oral administration of 25 mg TPN729 in 3 healthy Chinese volunteers.A total of 22 metabolites were identified,of which seven were confirmed in comparison with the synthetic reference substances,including M3(N-depyrrolidine-ethyl),M8(pyrrolidine oxidative deamination),M11-1(carbonylation),M11-2(carbonylation),M12-2(monooxidation),M12-3(monooxidation),and M13(pyrrolidine ring opening).The results of the present study indicated the extensive metabolism of TPN729 in humans,including oxidative deamination,oxidative ring opening,N-dealkylation,N-oxidation,hydroxylation,dehydrogenation,lactam formation,and glucuronidation,and its pyrrolidine moiety as the metabolic soft spot.Human hepatocytes incubation of the metabolites references and aldehyde trapping experiment were further performed to elucidate the metabolic pathways of TPN729.M3 was observed to generate M1,M2-1,M2-2,M4-1,and M4-2;M11-2 was proved a starting material of M3 and M10;M12-3 corresponded to the formation of M4-2 and M11-1.Aldehyde trapping experiment with methoxylamine was adopted to explicate the formation of M13 originating from the pyrrolidine oxidative ring opening instead of lactam hydrolysis of M11-2.In plasma samples both at 1.5 h and 6 h,M3 resulting from N-dealkylation was the major circulating substance.Other drug-related components found in plasma mainly included the unchanged parent drug,N-dealkylated metabolite M2-2,and pyrrolidine lactam metabolite M11-2.In urine samples,the parent drug was identified as the major component.Minor metabolites in urine were products of N-dealkylation,oxidative deamination,and hydroxylation.In feces samples,three predominant metabolites resulting from pyrrolidine oxidative deamination or ring opening(M7,M8,and M13)were detected.The amounts of three principal metabolites were far more than the parent drug in feces,indicating that TPN729 was well absorbed in humans.Based on the metabolic profiles of TPN729 in vivo,N-dealkylation,pyrrolidine oxidative deamination,and pyrrolidine oxidative ring opening were verified as the major metabolic pathways of TPN729 in humans.The predominant drug-related component in plasma was M3 resulting from N-dealkylation.The intact parent drug was primarily eliminated via renal excretion.To elucidate detailed pharmacokinetic profiles in humans,a sensitive and selective liquid chromatography-tandem mass spectrometry(LC-MS/MS)method was developed for the simultaneous determination of TPN729 and its four major metabolites in human plasma.The analytical conditions were carefully optimized by MRM mode for highly selective identification and sensitive quantitation of TPN729 and its metabolites.The method was successfully applied to the pharmacokinetic study following oral administration of 25 mg TPN729 to 3 healthy volunteers.The pharmacokinetic results suggested that the Cmax of TPN729,M3,M8,M11-2,and M13 at 35.8,125,8.26,22.0,and 18.6 ng/m L,respectively,were reached 0.83 to 3.83 h postdose,and the AUC(0-?)values were 289,2187,51.5,74.8,and 37.0 ng·h/m L, respectively.The Cmax and systemic exposure of M3 were 3.5-and 7.6-fold those of the parent drug,respectively,whereas the AUC(0-?)values of M8,M11-2,and M13 accounted for 18%,25%,and 13% that of M0.Therefore,M3 was the predominant drug-related component in plasma,followed by the parent drug.The elimination half-life of TPN729,M3,M8,M11-2,and M13 averaged 7.75 to 11.6 h.The systemic clearance for M3 was slightly slower than that for the parent compound.2.Drug-drug Interactions Associated with Drug-metabolizing Enzymes and TransportersDrug-drug interaction studies associated with drug-metabolizing enzymes began with in vitro phenotyping experiments,including human recombinant CYP isoforms incubation and liver microsomes incubation with selective chemical inhibitors.The depletion of TPN729 after incubation with or without specific inhibitors was detected to identify the metabolic enzymes for TPN729.The results suggested that CYP3A4 was principally responsible for the metabolism of TPN729 as the normalized contribution of CYP3A4 for TPN729 depletion was over 70%.Consistent with this,ketoconazole(a typical selective inhibitor for CYP3A)and 1-aminobenzotriazole(inhibitor for all the cytochrome enzymes)significantly inhibited TPN729 metabolism in liver microsomes,whereas other chemical inhibitors had little effect on its metabolism.For animal studies,pretreatment with ketoconazole or rifampicin(a typical inducer of CYP3A)remarkably affected the plasma concentrations of TPN729 in dogs after its oral administration: ketoconazole increased the AUC0-? and Cmax by 6.8 and 1.5 folds,respectively,whereas rifampicin decreased these parameters by 75.1% and 63.7%,respectively.The similar pharmacokinetic interaction occurred in rats after its oral administration and pretreatment with ketoconazole or dexamethasone(a typical inducer of CYP3A).However,pharmacokinetic interaction was not observed following the intravenous administration of TPN729 and pretreatment with CYP3 A modulators,which indicated that CYP3 A inhibitors or inducers interfered the first-pass metabolism of TPN729 and caused pharmacokinetic interactions.As TPN729 is administrated orally in its clinical application,attention should be paid to the high potential of TPN729 as a victim for drug-drug interactions with CYP3 A.On the contrary,incubations with CYP450 probe substrates were conducted to evaluate the inhibition potency of TPN729 on various drug-metabolizing enzymes.TPN729 exhibited moderate time-dependent inhibition on CYP2C8,CYP2C9,CYP2C19,and CYP3A4(IC50 = 3.3 ~ 9.2 ?M),and demonstrated little inhibition to CYP1A2,CYP2B6,and CYP2D6(IC50 > 100 ?M).Since the predictable plasma level of TPN729 after its oral administration of the highest dose(100 mg)was far below the aforementioned IC50 values,there is little potential for TPN729 to be a perpetrator for drug-drug interactions with drug-metabolizing enzymes.Drug-drug interaction studies associated with drug transporters employed various cell lines,including HEK293-OATP1B1/OATP1B3/OATP2B1/OAT1/OAT3/OCT2,Caco-2,and MDCKII-MDR1 cell lines to evaluate the participation of uptake or efflux transporters in the disposition of TPN729 in vivo.Uptake studies using HEK293 cells showed that TPN729 was neither the substrate of uptake transporters nor the potent inhibitor of them(IC50 = 17 ~ 193 ?M).Hence,there is little potential for TPN729 to be involved in drug-drug interactions associated with uptake transporters.To investigate the permeability of TPN729,the concentrations of TPN729 at apical and basolateral side were determined after incubation with Caco-2 cells.The results suggested good permeability of TPN729,and potential inhibition on efflux transporters.Afterwards,efflux studies using MDCKII-MDR1 cells were further performed to investigate the efflux properties of TPN729.Despite a substrate of P-gp(efflux ratio = 30.8),coadministration of selective P-gp inhibitor,tariquidar,exerted little influence on the pharmacokinetics of TPN729 in vivo owing to its good permeability.However,TPN729 exhibited potent inhibition on P-gp-mediated imatinib transport(IC50 = 0.75 ?M).Based on the Drug Ineraction Studies Guidance for Industry published by FDA,an in vivo drug interaction study using digoxin as P-gp positive substrate was performed to evaluate the drug interaction potential of TPN729 via P-gp inhibition.The results demonstrated that the P-gp mediated gastrointestinal efflux of digoxin was significantly inhibited by the clinically relevant 5 mg/kg TPN729,manifesting as 1.95-and 3.7-fold increase for AUC0-? and Cmax,respectively.Since digoxin is a commonly prescribed cardiovascular drug with narrow therapeutic range,close attention should be paid to the combination therapy of TPN729 and digoxin.The potential of TPN729 as a perpetrator in drug-drug interactions associated with other P-gp substrates should also be considered in clinical practice.3.Reversal of Multidrug ResistanceMultidrug resistance(MDR)is one of the major obstacles in successful chemotherapy of cancer,and the overexpression of P-gp is a major contributor to multidrug resistance,responsible for pumping the drugs out of the tumor cells.So I investigated whether TPN729 could reverse the multidrug resistance by inhibiting P-gp.As the previous tissue distribution results in rats suggested a highest level of TPN729 in lung tissue,approximately 100 times higher than the plasma concentration,a P-gp-overexpressing paclitaxel-resistant human lung adenocarcinoma cell line A549/T was selected in this study to evaluate the multidrug resistance reversal effect of TPN729 in vitro and in vivo xenograft mouse model.Based on the observed pharmacokinetic data of TPN729 in humans and plasma-lung ratio in rats,the concentration of TPN729 in lung in humans was expected to reach 10 ?M,much higher than the IC50 detected in vitro.The reversal of multidrug resistance by TPN729 was first investigated in sensitive A549 and P-gp-overexpressing resistant A549/T cell lines in vitro,using paclitaxel and doxorubicin as positive P-gp substrates.Furthermore,A549/T xenograft mouse model was established to assess the multidrug resistance reserval activity of TPN729 in vivo.In the CCK-8 assay,TPN729 could effectively decrease the IC50 values of paclitaxel and doxorubicin in A549/T cells and resensitize the resistant cells to antitumor agents.The reversal folds for paclitaxel and doxorubicin were 22.9 and 5.35,respectively.For cisplastin,which is not a P-gp substrate,TPN729 did not alter its sensitivity in A549/T cells.In addition,TPN729 did not alter the sensitivity of parental A549 cells to these drugs.These results illustrated that TPN729 resensitized the resistant tumor cells to P-gp substrates.In the cell apoptosis assay,TPN729 increased paclitaxel-induced apoptosis in A549/T cells by 30%,further proving the reversal of drug resistance by TPN729 via potentiation of apoptosis.Mechanistic studies were designed to investigate the impact of TPN729 on intracellular accumulation of chemotherapeutic drugs and P-gp expression of resistant cells.10 ?M TPN729 was observed to increase the concentration of paclitaxel and doxorubicin in A549/T cells by 3.1 and 5.8 folds,respectively.And the intracellular accumulation of chemotherapeutic drugs could be further increased by increasing TPN729 concentration.The results of western blot suggested that TPN729 didn't alter the expression of P-gp and exterted its MDR reversal effect by direct inhibition on the function of P-gp.As a result,the P-gp-mediated efflux of antitumor drugs was reduced and drug accumulation in resistant tumor cells was increased.A549/T xenograft model in nude mice was finally established to examine the in vivo MDR reversal effect of TPN729.The tumor growth rates were compared between the paclitaxel group and the combination group of TPN729 and paclitaxel.After two weeks of drug treatment,the combination group displayed a remarkable tumor growth retardation compared to the the paclitaxel group.On the 14 th day,the average tumor weights of the paclitaxel group and combination group were 89% and 34% of the average tumor weight of the control group,respectively,corresponding to the average tumor volumes of 83% and 37% compared to the control group.Cell apoptosis in tumors was determined by TUNEL assay.The result was consistent with the in vitro data,co-treatment of TPN729 and paclitaxel exhibiting considerably enhanced apoptosis.The treatment of TPN729 in A549/T xenograft mouse model confirmed its MDR reversal actitivity in vivo,which supported the development of TPN729 as a novel potential MDR reversal agent in the future.4.ConclusionsTPN729 is well absorbed in humans and undergoes extensive metabolism,including oxidative deamination,oxidative ring opening,N-dealkylation,N-oxidation,hydroxylation,dehydrogenation,lactam formation,and glucuronidation.The metabolite resulting from N-dealkylation is the predominant drug-related substance in plasma.TPN729 is a sensitive substrate of CYP3A4,suffering from extensive first-pass metabolism.The concomitant therapy with CYP3 A inhibitors or inducers is likely to change TPN729 plasma concentration in vivo.Hence,attention should be paid to the high potential of TPN729 as a victim for drug-drug interactions with CYP3 A.On the contrary,TPN729 exhibits potent inhibition on P-gp-mediated efflux transport.Clinically relevant dose of TPN729 significantly inhibited P-gp mediated gastrointestinal efflux of digoxin and increased its systemic exposure.Therefore,the potential of TPN729 as a perpetrator in drug-drug interactions associated with P-gp substrates should also be considered in clinical practice.Moreover,TPN729 inhibits the efflux of overexpressed P-gp in resistant tumor cells in a concentration-dependent manner and enhances the intracellular accumulation of chemotherapeutic drugs,thus providing effective MDR reversal and tumor growth retardation.The sufficient MDR reversal effect supports the development of TPN729 as a chemo-resensitizer in combination with conventional P-gp substrate chemotherapeutic drugs to overcome multidrug resistance.