Metabolism And Brain Distribution Of [¹⁴C] Mefuparib In Rats

Objectives:The objective of this study is to investigate the radiopharmacokinetics（PK）,mass balance（MB）and tissue distribution（TD）of the innovative drug[¹⁴C]Mefuparib（CVL218）in rats using ¹⁴C-labeled radioisotope tracer technology,to analyze the radioactive metabolite profiles in rats and to identify relevant metabolites.To elucidate and validate the types of transporters affecting CVL218 in the brain,a screening strategy for blood-brain barrier-crossing drug transporters was developed based on simulated the unbound brain-to-plasma drug partition coeffcient(K_p,uu,brain)values in Gastro Plus software.The study’s findings are used to evaluate the potential of CVL218 as a chemotherapy for the treatment of cerebral metastases.Methods:The PK,MB,and TD studies of[¹⁴C]CVL218 in rats were followed by a liquid scintillation counter and oxidative combustion.Metabolic profiles and metabolites were identified using HPLC-β-RAM/HPLC-Fraction Collector and HPLC-Q Exactive Plus MS.Quantitative whole-body autoradiography（QWBA）was used to quantitatively analyze areas of interest（AOI）in brain tissue.Two strategies were used to evaluate K_p,uu,brainvalues:one based on Gastro Plus software,and the other based on classical brain homogenization studies in experimental animals to calculate K_p,uu,brainvalues.The reliability of the software simulation strategy was investigated by comparing the results of the two strategies.To confirm the type of transporter associated with K_p,uu,brainvalues,CVL218 was evaluated in overexpressing HEK293cells as a substrate for the uptake transporters OATP1B1,OATP1B3,OAT1,OAT3and OCT2.CVL218 and metabolites were evaluated in rats as the substrate for the efflux transporter P-glycoprotein（P-gp）and breast cancer drug-resistant protein（BCRP/Bcrp）.Results:Radiopharmacokinetics:the maximum plasma concentration(C_max)of[¹⁴C]CVL218（20.0 mg/kg,100.0μCi/kg）was 5863.37±2436.23 ng eq./m L after single oral administration in Sprague-Dawley rats（SD rats）.The time to reach C_max(T_max)was 3.67±2.25 h.The following parameters were calculated by Win Nonlin Software:the area under the curve to infinity(AUC_0-∞)was 52985.69±16534.52 ng eq./m L·h,and the half-life(t_1/2)was 2.69±0.32 h.After intravenous（i.v.）administration of[¹⁴C]CVL218（5.0 mg/kg,25.0μCi/kg）,the plasma C_0.083hwas1700.29±592.62 ng eq./m L.The following parameters were calculated by Win Nonlin Software:AUC_0-∞was 9768.97±1083.77 ng eq./m L·h,and the t_1/2was 4.38±0.97 h.Mass balance:the total cumulative excretion of drug-related radioactive substances in urine and feces of rats within 168 h accounted for 97.15±1.26%of the dose.The excretion was predominantly fecal,with 79.18±2.47%.The main excretion time was 48 hours.The amount of radioactive material excreted in the bile of SD rats within 72 hours of bile duct cannulation（BDC）administration was 41.87±11.77%.Tissue distribution:CVL218 was widely distributed in 23 tissues of Long-Evans rats（LE rats）.Radioactive substances were detected in brain tissue.QWBA study in AOI brain:drug-related substances were evenly distributed in AOI brain.AUC_0-48hof AOI brain was 20255.25±3686.98 ng eq./m L·h.Metabolic profiles and major metabolites:CVL218 and 11 metabolites（7 of which were novel metabolites）were detected in plasma,urine,feces,bile,and brain tissue,with the major metabolic pathways being N-demethylation,amide hydrolysis,and glucuronic acid binding.CVL218 accounted for 0.89%and 2.07%of the total plasma radioactivity in male and female rats,respectively.Metabolite M8（4-（7-carbamoyl-5-fluorobenzofuran-2-yl）benzoic acid）,the parent drug deaminated by oxidation to carboxylic acid,was the major metabolite in plasma（male,59.37%;female,77.42%）.In the urine samples of male and female rats,CVL218 accounted for0.82%（male rats）and 8.13%（female rats）of the dose.The most abundant metabolite in urine was M8.CVL218 accounted for 7.28%of the dose in the feces of the male rats and was not detected in feces samples of the female rats.In feces,the most abundant metabolite was M8.In the rat brain,the predominant component was CVL218.K_p,uu,brainprediction value:after obtaining the PK parameters for i.v.administration,the physicochemical parameters（e.g.,p Ka and log P）by ACD Software and in vivo parameters(e.g.,_,)were input into Gastro Plus software to obtain the prediction of K_p,uu,brainvalue of brain tissue.The PK parameters(t_1/2,AUC,and MRT_0-∞)related to clearance rate（clearance,CL）after i.v.administration given by Win Nonlin and the Poulin-Theil model of Gastro Plus software were fitted.The simulated value of K_p,uu,brain（8.4）was the highest among all simulated tissues.K_p,uu,brainmeasured value:According to the metabolite profiles,CVL218accounted for 2.16%（1054.10 ng eq./g·h）of the total plasma radioactivity.CVL218accounted for 100%（16448.36 ng eq./g·h）of the brain total radioactivity.Equilibrium dialysis experiments showed that[¹⁴C]CVL218 exhibited the low plasma protein binding rate and the high brain protein binding rate.The measured K_p,uu,brainvalue of CVL218 was 7.0,which was similar to that of the simulated value by Gastro Plus software.Transporter studies:（1）Uptake transporter study:in vitro studies of HEK293cells overexpressing the uptake transporters OATP1B1,OATP1B3,OAT1,OAT3,and OCT2 proteins showed that the uptake rate ratios of CVL218 were all less than 2.0,and the concentrations did not decrease by 50%after the addition of inhibitors.CVL218 is not a substrate for the uptake transporters（OATP1B1,OATP1B3,OAT1,OAT3,and OCT2）.（2）Efflux transporter study:inhibition PK studies in rats revealed no difference in PK results between the inhibitor group and the intravenous CVL218group.Conclusion:CVL218 is a fast-metabolizing drug and excreted primarily in feces.After a single oral administration,CVL218 can penetrate the BBB and enter the brain tissue.The drug-related substances were evenly distributed in the AOI brain.A total of 11metabolites（7 of which were novel metabolites）were detected.M8 was the major metabolite.Brain tissue distribution was dominated by CVL218.The K_p,uu,brainvalue simulated by the Gastro Plus software strategy（8.4）was found to be similar to that of the classical brain homogenization studies（7.0）.K_p,uu,brainvalues suggest that the entry of CVL218 into the brain might be mediated by the uptake transporters.CVL218 is not a substrate for the uptake transporters（OATP1B1,OATP1B3,OAT1,OAT3,and OCT2）.CVL218 and its metabolites are not substrates for the efflux transporters P-gp and BCRP.CVL218 might be a potential chemotherapy for the treatment of cerebral metastases.