Effect Of Hepatic Impairment On The Pharmacokinetics Of Imrecoxib And Its Mechanism Study

As the largest and most important metabolic organ,the liver plays important roles in drug absorption,distribution,metabolism,and excretion（ADME）.It may lead to various degrees of alteration in drug metabolism and dispositions under hepatic disease states.Drug specifications have shown that the dosages of several medications,such as the non-steroidal anti-inflammatory drugs celecoxib and nimesulide,need to be adjusted for patients living with hepatic impairment.Therefore,it is of great clinical importance to clarify the particular effect and mechanism of insufficient liver function on the pharmacokinetics of drugs for individualized medication.The present study used imrecoxib,a cyclooxygenase-2 inhibitor,to systemically evaluate the effect of moderate hepatic impairment on the pharmacokinetic properties of imrecoxib and its active metabolites M1 and M2,as well as to further elucidate the mechanism which underlies the PK variations in terms of absorption and metabolism,as well as excretion using multiple in vivo and in vitro models.The research provides a theoretical foundation for rational medication under disease states,special populations,and drug-drug interaction.1.The effect of moderate hepatic impairment on the pharmacokinetics of imrecoxib and its major metabolites.The metabolites of imrecoxib in plasma and urine samples were identified via the ultra-performance liquid chromatography/ultraviolet quadrupole time-of-flight mass spectrometry combined with MetabolitePilot software,following an oral administration of 100 mg of imrecoxib to patients with moderate hepatic impairment and matched healthy subjects.Results indicated that the parent drug underwent extensive metabolism in both the hepatic impaired patients and the healthy subjects,including major metabolitc pathways:namely hydroxylation of the benzylic carbon motif（M1）,oxidation of the benzylic carbon motif to form carboxylic acid（M2）,glucuronidation of M1（M10-1）,glucuronidation of M2（M11）.Other minor metabolic reactions included mono-oxidation,N-dealkanation and mono-oxidation,oxidation of the benzylic carbon motif to form carboxylic acid and N-dealkanation,dehydrogenation,mono-oxidation and dehydrogenation,di-oxidation and di-dehydrogenation,di-oxidation,tri-oxidation and dehydrogenation,mono-oxidation and glucuronidation,and di-oxidation and glucuronidation.According to ultraviolet detection,M0,M1,and M2 were the major circulating substances in both healthy subjects and patients with moderate hepatic impairment.A rapid,sensitive,and reliable LC-MS/MS method was established and validated to simultaneously quantify the parent drug（M0）,M1,and M2 in plasma or urine samples,to evaluate the pharmacokinetics of imrecoxib and its major metabolites in both patients with hepatic impairment and healthy subjects.The pharmacokinetic parameters were calculated based on non-compartmental analysis using WinNonlin7.0 software.Results,based on geometric mean ratios,indicated that when compared to matched healthy subjects,AUC_0-∞increased in moderate hepatic impairment patients by 9.2-,3.3-,and 1.5-fold for imrecoxib,M1,and M2 respectively.C_max were also increased by 4.8-and 1.8-fold for M0 and M1,whereas it decreased by 28.1%for M2.The observed T_max were all delayed from 1.0,1.0,and 2.0 h to 3.0,4.0,and 4.0hours for the M0,M1,and M2 respectively.Other PK parameters(such as t_1/2z)were comparable between the moderate patients and healthy groups for the three major circulating components.Results suggested that moderate hepatic dysfunction leads to the prolonged plasma peak time and increased plasma exposure of imrecoxib and its active metabolites M1 and M2.The plasma protein binding of imrecoxib,M1,and M2 in moderate hepatic impaired patients and healthy subjects was evaluated using the in vitro rapid equilibrium dialysis approach.Results demonstrated that the plasma binding of the three circulating substances were all concentration-independent in both the patients and healthy subjects.The mean plasma binding values of M0,M1,and M2 were89.3±0.5,73.7±0.4,and 84.3±0.9%in the moderate hepatic impaired patients respectively,all of which were significantly decreased when compared to the healthy subjects（93.3±1.5,78.4±2.3,and 87.6±0.6%）.Significant differences in the geometric mean ratios of AUC_0-∞for the moderate hepatic impaired patients versus the healthy subjects were computed using total drug or free drug concentrations.These values were 9.2 and 14.7 respectively,suggesting that medication safety should be evaluated in the clinical use of imrecoxib for hepatic impairment patients and that a reduced dose should be considered.The urinary accumulative excretion percentage-time curves were plotted after M0,M1 and M2 were determined in urine samples of patients with hepatic impairment and healthy subjects.Results suggested that urine imrecoxib concentrations account for no more than 1%of the dose.The urinary accumulative excretion of M1 and M2 were8.25±2.4 and 27.4±6.7%,which were higher for patients than for healthy subjects（3.30±1.53 and 23.1±7.8%）.However,renal elimination parameters,which were measured based on the urinary excretion and plasma exposure of the metabolites suggested that the renal clearance of both M1 and M2 were significantly decreased in patients,which were 50.5 and 60.6%those in healthy subjects,respectively.2.Investigation of the metabolic mechanism of imrecoxib.Investigation on the metabolism of imrecoxib in humans found that the benzylic carbon motif is its major site of oxidative metabolism,producing the major metabolites M1 and M2.The plasma exposure of M2 is four times higher than those of both M0 and M1 in humans.However,this metabolite is far less formed than M1 in both in vitro human liver microsomes（HLMs）and human hepatocytes.Therefore,this study set out to investigate the formation mechanism of M2 as well as to further explore the reason for discrepancies between in vitro and in vivo metabolic data.In this study,M0,M1,and chemically synthesized aldehyde imrecoxib metabolic intermediate（M-CHO）were selected as substrates,while human hepatocytes,HLMs,human liver cytosols（HLCs）,recombinant enzymes,and selective enzyme inhibitors were employed as in vitro metabolic models.The metabolic map of imrecoxib in humans was elaborated as follow:the parent drug was initially hydroxylated and mainly mediated by CYP3A4 and CYP2D6（with relative contribution ratios of 68%and 32%）to form M1.M1 was then oxidized to M-CHO in HLMs and HLCs,a process which was the rate-limiting step in the generation for the end-product M2.In further M-CHO metabolism,two opposing reactions occurred:namely rapid oxidation catalyzed by HLM enzymes（CYP3A4 and CYP2D6）and HLC enzyme（aldehyde oxidase）to form M2 versus reduction to regenerate M1,mediated by NADPH-dependent reductases（such as cytochrome P450 reductase）.The two metabolic pathways were competitive.Therefore,the amount of M2 in static in vitro incubations was considerably underestimated.These findings give one explanation for the discrepancy between in vitro and in vivo metabolic data,and suggest that the effect of competitive reduction on static oxidation metabolism should be considered in in vitro metabolic experiments.3.Mechanism study on the effect of hepatic impairment on the pharmacokinetics of imrecoxib and its major metabolites.Investigation into the metabolic fate of imrecoxib showed that the major metabolic enzymes involved were CYP3A4,CYP2D6,and aldehyde oxidase,whose protein contents or the activities were reported to be variably reduced in patients with chronic liver disease.This may result in a higher exposure of the parent drug and lower exposures of M1 and M2 for those with moderate hepatic impairment subjects.In contrast,the AUC of the two active metabolites were increased by 3.3 and 1.5 folds in patients in the present study.Additionally,significant differences in the renal excretion of the two metabolites M1 and M2 were observed between patients with moderate hepatic impairment and the healthy subjects,represented by the finding that the urinary accumulative excretions of M1 in hepatic impaired patients were higher in hepatic impaired patients,while the renal clearance of both M1 and M2 were significantly decreased when compared to the healthy groups.Therefore,this research uncovers the mechanism which underlies the effect of hepatic impairment on the pharmacokinetic variations of imrecoxib and its major metabolites from the perspectives of absorption and metabolism as well as excretion.In the mechanism study associated with metabolism and absorption,we focused on the effect of hepatic first-pass on the bioavailability of imrecoxib.The in vivo investigations were conducted in mice whose metabolisms were similar to humans.The PK profiles of M0 were characterized after intravenous（i.v.）and hepatic portal vein（p.v.）route infusion with the same dose of imrecoxib to C57 mice.Results found that the plasma exposures of M0 were 23979±2852 and 6449±695 ng/mL·h after i.v.and p.v.administration,respectively,which suggests that the parent drug was subjected to extensive first-pass metabolism with a calculated value of 73%.Concentration of imrecoxib in the portal vein plasma,where the parent drug was located prior to entry into liver,was much higher than for those in the systemic plasma,which was the main site for the compounds following hepatic metabolism and disposition.Results show that imrecoxib passing through the liver was rapidly extracted into hepatocytes,with a high liver extraction ratio of 0.72.Additionally,hepatic fibrosis mouse models were successfully constructed utilizing the CCl₄induction approach,which needs continuous intraperitoneal injection for six weeks（two times per week）.Absolute bioavailability values of the parent drug were calculated as 7.6%and 2.5%respectively,while the degrees of first-pass metabolism were calculated as 55%and74%respectively for the CCl₄ induced mice and control mice following intravenous,oral and hepatic portal vein infusion administration of 10 mg/kg imrecoxib.This further confirms that the hepatic first-pass effect could be reduced under liver disease states.The in vitro investigations were then performed using Caco-2 cells,human primary hepatocytes and hepatic transporter overexpressed HEK293 cell lines,in order to evaluate absorption in intestinal epithelial cells,the transmembrane pattern,and the metabolism intensity.Results demonstrated that the apparent permeability coefficients from(P_app,a-b)values were 16.3±1.2×10^-6,15.4±1.6×10^-6,and 19.1±1.1×10^-6 cm/s at the imrecoxib concentration of 2,10,and 50μM respectively,all of which were greater than 10×10^-6 cm/s,while the ratios of P_app,b-a to P_app,a-b were 1.40,1.41,and 1.02 respectively.This indicated that imrecoxib has excellent permeability,little efflux transport of P-gp.Effects of the temperature or the substrate concentration on the imrecoxib uptake was studied for the attached hepatocytes.Results indicated that the accumulated amount of imrecoxib was comparable between the 4°C and 37°C groups,while the uptake of the parent drug linearly increased with the elevated substrate concentration.The effects of hepatic uptake transporters on the imrecoxib disposition were assessed,and the results showed that the uptake ratio of imrecoxib in OATP1A1-,OATP1A3-,or OATP2B1-HEK293 cells to Mock cells were all less than two.This confirms that the uptake of imrecoxib into hepatocytes was mainly dependent on passive diffusion.Mouse heptaocytes and human liver S9 were chosen to evaluate the in vitro metabolic stability of imrecoxib.We found that the in vitro hepatic clearance of imrecoxib in mice and humans were 54.3 and 11.3 mL/（min·kg）,respectively,which suggests that imrecoxib is a medication which results in intermediate hepatic clearance.This further indicated that imrecoxib was eliminated in the forms of metabolites.It also suggested that imrecoxib might undergo extensive first-pass metabolism in humans as same as the mice did.These studies explain why the exposures of imrecoxib,M1 and M2 were overall increased in patients.This is because the first-pass effect of imrecoxib was attenuated in the presence of liver disease,which leads to an increased bioavailability and enhanced absorption of the parent drug,while concomitantly generating more of metabolites M1 and M2.The result was in accordance with the calculated systemic exposure ratio of parent drug/M1/M2 for healthy subjects of approximately 1:1:4,while this was approximately 2:1:2 for the patient group.It is suggested that for drugs undergoing strong first-pass metabolism,hepatic dysfunction leads to an increase not only for the parent drug but also for metabolites.In the mechanism study associated with drug excretion,the efflux transporter transfected MDCK cell lines and renal uptake transporter overexpressed HEK293 cell lines were performed as in vitro models to evaluate participation of the transporters in the excretion of imrecoxib metabolites M1 and M2.Results demonstrated that efflux ratio values of M1 in P-gp,BCRP,and MRP2 overexpressed MDCK cell lines were26.2,8.8,and 3.4 respectively,while the ratios were all considerably decreased（efflux ratio values were 1.4,1.8,and 1.1,respectively）in the presence of positive inhibitors of the individual transporters.This indicated that M1 is the substrate of P-gp,BCRP,and MRP2.In contrast,efflux ratio values of M2 in P-gp,BCRP,and MRP2 overexpressed MDCK cell lines were 1.0,1.2,and 1.0 respectively,indicating that M2 is not a substrate of P-gp,BCRP,and MRP2.The urinary excretion of M1recovered more for the hepatic impaired patients,which may be attributed to the compensatory urinary elimination to overcome impaired hepatobiliary elimination under liver disease states.Additionally,ratios of transporter-mediated uptake to Mock of M1 and M2 for OAT3 were 2.03 and 162 respectively,indicating that the two metabolites were the substrates of renal uptake transporter OAT3.Potential inhibition of the expression or the activity of OAT3 as a result of the secondary renal injury induced by the hepatic impairment was one of the factors for decreased renal clearance of both M1 and M2.4.ConclusionImrecoxib and its two major metabolites,M1 and M2,were significantly affected by hepatic impairment in several processes,including the alteration of pharmacokinetic parameters（such as the plasma exposure）and a plasma binding decrease for the three circulatory substances,as well as urinary excretion increase and renal clearance decrease for the metabolites M1 and M2.During further mechanism studies on the above-mentioned phenomenon,our research systematically described in vivo process of imreocxib and its two major metabolites M1 and M2 in humans as follows:after oral administration of imrecoxib,the parent drug is passively diffused into intestinal epithelial cells with a high permeability characteristic.Following arrival at the liver through the hepatic portal vein,around 70%of the parent drug is eliminated by the first-pass metabolism,while fractions escaping the hepatic elimination enter into the systemic circulation.In hepatocytes,imrecoxib undergoes an extensive metabolism under catalysis by metabolic enzymes such as P450s.The benzylic carbon motif is mainly oxidized to its corresponding benzylic alcohol metabolite M1 mediated by CYP3A4 and CYP2D6,and further to the carboxylic acid metabolite M2 catalyzed by CYP3A4,CYP2D6,and aldehyde oxidases after oxidizing to the metabolic intermediate M-CHO.Previous animal studies have shown that both the bile and the kidney are involved in the clearance of M1 and M2.M1 produced in hepatocytes partially enters the circulation and is excreted through the kidney,where the uptake transporter OAT3 locates and participates in elimination of M1.The rest were excreted through the bile secretion mediated by P-gp,BCRP,and MRP2 expressed on hepatocyte canalicular membrane.Due to its highly polar carboxylic acid group,only a small proportion of M2 produced in hepatocytes returns to circulation and is excreted through the kidney,where OAT3also participates in M2 elimination.A large percentage of M2 is eliminated through the bile secretion.However,this process is not mediated by the general efflux transporter on hepatocyte canalicular membrane.Liver dysfunction in hepatic impairment patients may reduce protein contents or activities of CYP3A4,CYP2D6,and aldehyde oxidase,which attenuate the hepatic first-pass metabolic capacity for imrecoxib.More of the parent drug enters the circulation and is continuously taken up by liver to be metabolized,resulting in a plasma exposure increase,as well as peak time procrastination of M0,M1,and M2.This research has established a relationship between the pharmacokinetic parameters of imrecoxib and its major metabolites,as well as their variations under the hepatic impairment states.It is recognized that the first-pass effect,metabolic enzymes（CYP3A4,CYP2D6,and aldehyde oxidases）and transporters（P-gp,BCRP,MRP2,and OAT3）play key roles in the pharmacokinetics of imrecoxib,M1,and M2.The research also suggested that medication safety should be valued in the clinical use of imrecoxib and that reasonable dose adjustments should be conducted following further clinical evaluation.