| Mosapride,（±） 4-amino-5-chloro-2-ethoxy-N-[[4-（4-fluorobenzyl）-2-morpholinyl]methyl] benzamide,used as its citrate form,is a potent and selective benzamide gastroprokinetic agent that enhances the gastrointestinal motility by stimulating the 5-hydroxytryptamine 4（5-HT4） receptor.It has been used increasingly in Japan and some other Asian countries.Compared with the comprehensive investigations on its pharmacological activities and therapeutic practices,the studies on the metabolism and pharmacokinetics of mosapride in vivo are limited.Only 4 phaseⅠmetabolites were reported.The aim of this paper was to use the filamentous fungus Cunninghamella（C.） elegans AS 3.156 as a in vitro model to metabolize mosapride,isolate and identify the major metabolites;then,systematically study the metabolism of mosapride in rat and human as well as its pharmacokinetics in rat.1.Metabolism of mosapride in C.elegans AS 3.156.Strain screening and orthogonal tests of the transformation conditions were carried out. The microbial model using C.elegans AS 3.156 to transform mosarpride was developed.The analyses were performed using ultra performance liquid chromatography-photodiode array-electrospray ionization-triple quadrapole tandem mass spectrometry （UPLC-PDA-ES1-MS/MS） and time of flight-mass spectrometry（TOF-MS）.A total of 13 metabolites,including 7 phaseⅠand 6 phaseⅡmetabolites were detected.After isolation and purification,the structures of 4 phaseⅠmetabolites were characterized by TOF-MS and NMR analyses as:mosapride-4’-N-oxide（M4）,4-amino-5-chloro-2-ethoxy-N-[3-（4-fluorobenzyla- mino）-2-hydroxypropyl]benzamide（M5）,4-amino-5-chloro-2-ethoxy-N-（2-morpholinylmeth-yl）benzamide（M6） and 4-amino-5-chloro-2-ethoxy-N-[（4-fonny]-2-morpholinyl）methyl]ben-zamide（M7）.According to drug metabolism rule and the information obtained from ESI-MS, ESI-MS/MS and TOF-MS analyses,the structures of other 9 metabolites were elucidated as: 4-fluorobenzyl-hydroxylated mosapride（Ml）,3-（or 6-）hydroxymosapride（M2）, morpholinyl-hydroxymosapride（M3）,N-[（4-acetyl-2-morpholinyl）methyl]-4-amino-5-chloro-2-ethoxy-benzamide（M8）,4-glucoside-mosapride（M9）,4-amino-5-chloro-N-（2,3-dihydroxy-propyl）-2-ethoxybenzamide（M10）,4-amino-5-chloro-2-ethoxybenzamide（M11）,2-chloro-5-ethoxy-4-[[4-（4-fluorobenzyl）-2-morpholinyl]methylcarbamoyl]phenylsulfamic acid（M12） and 4-amino-5-chloro-2-ethoxy-N-[3-[N-（4-fluorobenzyl）formamido]-2-hydroxypropyl]ben-zamide（M13）.All the metabolites except M6 were new compounds.The fragmentation rules of mosapride and its metabolites under ESI-MS/MS were summarized.2.Metabolism of mosapride in rat and human.The reference standards of M4-M6 were used for the metabolism studies of mosapride in rat and human using UPLC-ESI-MS/MS.In rat and human,18 and 16 metabolites of mosapride were detected,respectively.A total of 19 metabolites were found with at least 16 metabolites reported for the first time in vivo.The structures of 3 new metabolites（M4-M6） were identified by using reference standards.According to drug metabolism rule and the information obtained from ESI-MS and ESI-MS/MS analyses,the structures of other 16 metabolites were elucidated as followed:M2,M9,M11,M12（as mentioned above）, O-glucuronide conjugate of des-p-fluorobenzyl hydroxymosapride（M14）,des-p-fluorobenzyl -3-hydroxymosapride or des-p-fluorobenzyl-6-hydroxymosapride（M15 and M16）, 4-amino-5-chloro-N-（2-hydroxypropylamino）-2-ethoxybenzamide（M17）,4-glucuronide-mo-sapride（M18）,glucuronide conjugate of O-deethyl mosapride（M19）,O-glucuronide conjugate of 3-hydroxymosapride or 6-hydroxymosapride（M20 and M21）,3’-（or 5’-）oxo-des-p-fluorobenzyl mosapride（M22）,O-deethyl mosapride（M23）,3-（or 6-）hydroxymosapride（M24）,4-glucuronide-mosapride（M25,an isomer of M18）.These studies suggested that there was no qualitative species difference in the metabolism of mosapride between rat and human.By comparison of the metabolites in rat bile and feces,it was found that there might be reduction of N-oxide metabolites to parent and hydrolysis of the conjugative metabolites during their passage through the GI tract before excretion in the feces.There were 10 identical metabolism pathways including 7 reported for the first time in rat and human.PhaseⅠmetabolism pathways included:N-dealkylation（N-des-p-fluorobenzyl and iV-dealkylation of the long alkyl chain）,Odeethylation,morpholinyl ring cleavage, hydroxylation,iV-oxidation,and oxidation to form ketone.PhaseⅡmetabolism pathways included:glucose,glucuronide and sulfate conjugation.A metabolite might go through more than one metabolism pathways.There were 25 metabolites found in C.elegans AS 3.156,rat and human.The corresponding metabolism positions,types and pathways were summarized.3.Pharmacokinetic study of mosapride in rat.It was found that M4 might be reduced to parent mosapride,a so-called interconversion process,which might further influence the efficacy and toxicity of mosapride.In order to study the systemic exposure of M4,a rapid and sensitive UPLC-MS/MS method was developed for the simultaneous determination of mosapride,M4 and M6 in rat after p.o. administration for the first time.The ratios of A UCs of mosapride,M4 and M6 in male and female rat plasma were 1:0.024:9.7 and 1:0.013:0.40,respectively,while those of Cmax were 1:0.025:10 and 1:0.019: 0.38,respectively.In male rat plasma,mosapride was of low concentration and M6 was of much higher concentration;while in female rat plasma,the situation was different.In both sexes of rat,the AUCs of M4 were less than 3.2%of that of mosapride,indicating a low systemic exposure of M4.There was more obvious first-pass effect of mosapride metabolized to M6 in male rat than in female rat.Obvious sex differences in the absorption of mosapride, M4 and M6 were observed.The absorption and elimination of them were faster in male rat than in female in male rat.There was no obvious sex difference of mosapride in tissue distribution.Its concentration was higher in duodenum,caecum,liver and stomach wall.There were higher tissue concentrations and slower elimination of mosapride in female rat than in male rat.The tissue distribution of M4 was more rapid in male rat than in female rat.The concentration of M4 was higher in duodenum,caecum and small intestine wall.In female rat,the tissue concentrations of M4 were higher than those in male rat.This was in consistence with the result that the A UC of M4 in female rat was larger than that in male rat.There was no sex difference of mosapride in excretion.The excretion amounts of mosapride,M4 and M6 accounted for more than 2/3 of the total dose.The main excretion route was urine,then feces and bile,the ratio of their cumulative excretion amount was about 3:2:1 in the form of mosapirde,M4 and M6.M6 was excreted in largest dose percentage,then mosapride and M4,their cumulative excretion amounts,expressed as dose percentage,were about 37%,28%and 12%in male rat,while 24%,26%and 16%in female rat,respectively. Mosaprdie was excreted unchanged mainly in feces,then bile,there was little parent mosapride in urine.M4 and M6 were excreted most in urine,then bile.The cumulative excretion amount of M6 in male rat was larger than that in female,while M4 was the opposite case.The results indicated for the first time that,in both sexes of rat,the urinary cumulative excretion amount of M4 accounted for 10 14%of the dose,which was much higher than that of mosapride.M4 was an important excretion way of mosapride.4.Stability of mosapride and mosapride-4’-N-oxide in simulated gastric and intestinal fluids.The concentrations of mosapride and mosapride-4’-N-oxide after incubation in simulated gastric and intestinal fluids at different time intervals were determine by UPLC-MS/MS method.The results showed that they were stable and there was no interconversion between mosapride and mosapride-4’-N-oxide,indicating that the low bioavailability of mosapride was not due to its degradation in gastrointestinal tract.5.The enhancement effects of mosapride and its 3 new metabolites,M4,M5 and M7,on 5-HT4 receptor-mediated contraction of isolated guinea pig ileum（GPI）.The effects of mosapride and its 3 new metabolites on the contraction of isolated GPI were studied.Compared with the enhancement effect of parent mosapride on isolated GPI,the effect of M4 was stronger at higher accumulative concentrations,the effect of M5 was stronger,while the effect of M7 was weaker.There was no obvious difference them in the enhancement effects of contraction of isolated GPI,but M5 had a slightly better 5-HT4 receptor agonizing effect than mosapride.The results were helpful for further research and development of M5.In all,the metabolism of mosapride in rat and human were systematically studied with the comprehensive metabolic pathways proposed for the first time.The pharmacokinetics of mosapride in rat was fully studied with an emphasis on its new metabolite M4, mosaprdie-4’-N-oxide.Both mosaprdie and mosaprdie-4’-N-oxide were stable in simulated gastric and intestinal fluids.The 5-HT4 receptor agonizing effects of 3 new metabolites, M4-M6,were studied.All those studies are essential and important for fully understanding the efficacy and safety of mosapride. |
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