| 5 - hydroxytryptamine 4 (5-HT4) receptor agonists are a class of drugs to promote gastric motility via excitatory cholinergic gastrointestinal tract between neurons and myenteric plexus of 5-HT4 receptor, to promote the release of acetylcholine. They coule enhanced gastrointestinal tract movement and improve functional dyspepsia. They are mainly used for functional dyspepsia accompanied by heartburn, belching, nausea, vomiting, early satiety, abdominal distension and other gastrointestinal symptoms, they can also be used for gastro-esophageal reflux disease, diabetic gastroparesis and partial gastrectomy patients stomach dysfunction. The representation drugs are cisapride and mosapride. Cisapride has been withdrown from the market in 2000 due to cardiac toxicity, while mosapride cardiac is still in the wide range of clinical applications. In addition, P & G's newly developed ATI-7505 is in phaseⅡclinical trials.SHR116958 is a new type of gastric motility drug belonging to the benzamide 5 - HT 4 receptor agonist. Its chemical structure (Figure 1) retains the basic activity of benzamide structure, side-chain replacement for its own nuclear quinoline acid ester. The molecular biology and cytology of activity in vitro screening tests about SHR116958 have shown several times more than Mosapride. And the initial in vivo experiment also showed higher bioavilability and faster absorption of the higher excellent pharmacokinetic properties. The safety aspects of the preliminary tests also showed that the toxicity is not obviously.Therefore, the purpose of this study was to elucidate the absorption, distribution, metabolism and excretion of SHR116958 in rats and Beagle dogs Main contents of this study include:1. Development and validation of a LC/MS/MS method for determination of SHR116958 in biological samplesThe establishment of LC / MS / MS method for determination of SHR116958 and its metabolites in biological samples, including specificity, sensitivity, accuracy, precision, liner and recovery. Similarly, for different biological samples, the LC / MS / MS method based on the revision and optimization of drug extraction methods, the establishment of a variety of important tissues and organs of homogenate, feces, urine, bile and other biological samples.2. The concentration-time curves and absorption of SHR116958 and its metabolites following a single oral or intravenous administration in rats and dogs(1) rat pharmacokineticsChose one dose (36 mg/kg) as the single oral dose and the intravenous injection dose. The blood collection time points are 0h after administration, 0.25,0.5,0.75, 1,1.5,2,3,4,6,8,12,36 and 48 h.(2) Beagle pharmacokinetics①D etermination of a single oral administration of 3 doses (10,30,50 mg.kg-1): blood concentration - time curves and related parameters, to determine whether there is proportional to the AUC and Cmax increased dependent on the linear pharmacokinetics, or the non-linear pharmacokinetics. The blood collection time points are 0hafter administration 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24h;②chose one dose for oral administration for 7 days, the comparison between day 1 and day 7 of concentration-time curve to determine whether there is elimination or inhibition.③using self-crossover design, chose the middle-dose group of the oral administration for the same group of the single intravenous injection dose group after the cleaning period of 14 days.3. The distribution of SHR116958 in ratsOne dose and 4 time points (6 rats/group) were selected to extract heart, liver, brain, spleen, lung, kidney, stomach, intestine, muscle, fat, gonads and other important tissues and organs of rats. Then determinate the concentrations of SHR116958 in these various tissues and organs by LC/MS/MS.4. The preliminary identification of the metabolites of SHR116958In invitro studies, SHR116958 was incubated with liver-microsomes to preliminarily identify possible metabolites of SHR116958. Meanwhile study the metabolic stability and metabolic differences between species. The metabolic profile of SHR116958 in ineubation samples were characterized using LC/MS/MS methods.5. Excretion of SHR116958 in ratsChoose one dose of oral administration to feces, urine and bile excretion studies.6. The determination of plasma protein binding rateThree doses of SHR116958 mixed with blank rat, Beagle dog and human plasma were incubated with human serum albumin orα1-acid glycoprotein. The incubation samples were characterized using LC/MS/MS method.7. The toxicokinetics study of SHR116958Three doses of 10, 30, 60 mg·kg-1 were used for oral administration, one time per day for 30 days in beagle dogs. The test was used to study the existence of accumulation, and their dose relationship.The main results of this study include:1. Development and validation of a LC/MS/MS method for determination of SHR116958 in biological samplesThis study established a rapid and sensitive LC/MS/MS method for the determination of SHR116958 and M1 in plasma and other biological samples. Tamsulosin hydrochloride as internal standard, biological samples extracted with ethyl acetate, methanol: acetonitrile: water (containing 0.1% formic acid) = 50:25:25 (v / v / v) as the mobile phase. Chromatographic separation was performed on a Angilent Eclipse C18 column (150nm×2.1nm, 5μm). High-purity nitrogen as the Curtain gas, collision gas ESI source of positive ion scan, MRM scanning mode for testing. The linearity of the calibration curves was good. The lower limit of quantitation (LLOQ) of the method was 1.0 ng·mL-1 for SHR116958 and M1 in dog plasma and 2.71 ng·mL-1 in rat plasma. The method is good, specificity and fast.2. The concentration-time curves and absorption of SHR116958 and its metabolites following a single oral or intravenous administration in rats and dogs(1)In ratsAfter intravenous injection, SHR116958 WAS rapidly metabolized into M1, and other metabolites was not detected. The concentration of M1 was significantly higher than SHR116958 at the same time point. The concentration of SHR116958 and M1 decreased rapidly with time, most of the concentration can be detected until 24 h. Comparing with intravenous administration, the plasma concentration of rats after orally administration has a significant reduction. The peak plasma concentration of SHR116958 and M1 after oral administration is only 8.8% and 5.4% of intravenous injection with irregular absorption, indicating a very low absorption in rats after oral administration of SHR116958.The main parameters of SHR116958 are as follows: eliminated half-life (t1/2) of intravenous and oral administration group were 10.8±3.8 and 18±8.9 h; clearance (CL) were 9.6±2.7 and 85.5±19.4 L·h - 1·kg-1; apparent volume of distribution (Vss) were 53.1±18.6 and 767.9±198.1 L·kg-1; area under the curve (AUC0∞) were 505.6±167.8 and 1397.53±1087.04 ng·h·mL-1; mean residence time (MRT) were 5.5±1.5 and 8.95±1.03 h; peak concentration (Cmax) were 569.8±410.8 and 51.8±23.5 ng·mL-1; peak time (Tmax) were 0.05 and 2.17±2.29h, elimination rate coefficient (Kel) were 0.07±0.02 and 0.04±0.02. There were significant differences in parameters MRT, CL, Vss, Cmax, T max. The main parameters of M1 are as follows: eliminated half-life (t1/2) of intravenous and oral administration group were 8.4±3.6 and 13.6±2.8 h; clearance (CL) were 4.31±1.64 and 74±18.3 L·h-1·kg-1; apparent volume of distribution (Vss) were 21.5±10.5 and 597.6±193.3 L·kg-1; area under the curve (AUC0∞) were 1192±491.9, and 513.9±135.9 ng·h·mL-1; mean residence time (MRT) were 4.8±1.07 and 7.96±0.70 h; peak concentration (Cmax) 1472.5±1079.6, respectively, and 92.3±49.9 ng·mL-1; peak time (Tmax) were 0.17±0.29 and 0.79±1.10h, elimination rate coefficient (Kel) were 0.09±0.04 and 0.05±0.01. There were significant differences in parameters MRT, CL, Vss, Cmax, T max. The bioavailability of SHR116958 and M1 were 9.4% and 4.98%, indicating poor absorption after oral administration of SHR116958 in rats.(2) In beagle dogsAfter three doses (10 mg·kg-1, 30 mg·kg-1, 50 mg·kg-1) of oral administration of SHR116958, the peak time of SHR116958 and M1 were all about 0.751h, with the Cmax of 129.8±61.7,695.8±87.4 and 1357.5±1338.7 ng.mL-1 for SHR116958 and 208.1±108.0,541.8±226.3, and 413.7±606.7 ng·mL-1 for M1. The 10 mg·kg-1 dose group of SHR116958 and M1 can only be detected until 12 h, the other two groups can be detected to 24 h. Other metabolites, such as M2, were not detected. After intravenous administration, the plasma concentration of M1 is about 10 times of SHR116958 and the Cmax was 18 times of SHR116958. While after oral administration, the difference between the concentrations of SHR116958 and M1 is not very significant. The bioavailability of M1 was 23.7±9.6%.Three dose group of oral administration were 10 mg·kg-1, 30 mg·kg-1 and 50 mg·kg-1, dose ratio of 1:3:5. The increase ratio of AUC was 1:5.6:10.9; Tmax of three doses were 1.33h, 1.45h and 1.33h; Cmax ratio was 1:4.1:9.46; MRT were 3.22±0.51,4.18±0.46 and 4.15±0.74 h; CL were 23.05±7.50,11.5±1.68 and 14.24±7.95 L·kg-1·h-1; t1/2 were 5.88±4.94,4.94±3.18, 4.96±3.09. Following oral administration, the increase in plasma AUC and Cmax was approximately dose-proportional.The increase ratio of AUC for M1 was 1:2.2:5.3; low, Tmax of three doses were 1.17 h, 1.05 h and 1.42 h; The ratio of Cmax was 1:2.1:5.8. MRT were 5.08±0.69,4.14±2.15 and 4.53±0.95 h; CL were 14.60±4.68,18.71±6.97 and 11.81±5.85 L·kg-1·h-1; t1/2 were 3.95±1.53 h, 4.96±2.50 h, 5.07±3.39 h. Following oral administration, the increase in plasma AUC and Cmax was approximately dose-proportional.After multiple administrations, the accumulation factor for SHR116958 and M1 were 0.94 and 1.0, indicating that SHR116958 and M1 has no significant accumulation in beagle dogs.3. The distribution of SHR116958 in ratsAfter oral administration of 36mg.kg-1 the SHR116958 by AUC in descending order were: liver, stomach, spleen, kidney, lung, fat, thymus, ovary, bone marrow, heart, testis, muscle, plasma, brain. It shows that SHR116958 metabolism by the liver, mainly acting on the stomach, consistent with its characteristics as a gastro-kinetic agent; secondly, through the very low drug concentration in the brain, it may infer that this drug is not easy to get through the blood-brain barrier. After oral administration the low concentration of SHR116958 indicating that there is an obvious first-pass effect. The concentration in gastric tissue is in a high condition, shows that SHR116958 be absorbed directly to gastric tissue. M1 by AUC in descending order were: stomach, liver, lung, kidney, ovary, plasma, spleen, fat, bone marrow, heart, testis, muscle, thymus, brain. The highest concentration was in stomach, indicating SHR116958 and M1 was mostly distributed in the major target organ of stomach.4. The preliminary identification of the metabolites of SHR116958The result of incubation experiment showed that, SHR116958 is mainly catalyzed by CYP metabolism. The inhibition of SHR116958 to CYP2C9, CYP2C19, CYP2D6, CYP2E1 were not significant. The IC50 values of CYP1A2, CYP3A4 are greater than 10μΜ. The inhibition of SHR116958 to the most of the human CYP is weak.SHR116958 could be significantly metabolism in a variety of incubation system in liver microsomes. Nine metabolisms were identified, including hydroxylation metabolites and ester hydrolysis metabolites as the major metabolite. The metabolic rate of SHR116958 in human liver microsomes is less than the animals. The amount of metabolites in dogs is mostly closest to human.5. Excretion of SHR116958 in ratsSHR116958 excrete mainly in the form of M1 through the urine, a small amount in the form of SHR116958 through the urine and feces. In 48h, the accumulative output of urine and feces in the form of M1 reach to 72.36% and 3.78%, respectively, totally up to 76.13%. In 48h, the accumulative output of urine and feces in the form of SHR116958 reach to 13.33% and 0.392%, respectively, up to 13.72%. In 48h, the accumulative output of urine and faeces in the form of SHR116958 and M1 is up to 90%.SHR116958 excrete mainly in the form of M1 through the bile, a small amount in the form of SHR116958. In 24h, the accumulative output of bile in the form of M1 reaches to 3.2% and 0.27%, respectively. In 24h, the accumulative output of bible in the form of SHR116958 and M1 reach to 3.5% which is consistent with the excretion of feces.6. The determination of plasma protein binding rateThe plasma protein binding rate between SHR116958 and M1 and human, rat and dog plasma is in 9799%. The difference between the species was not significant, slightly higher than the rate of human serum of human serum albumin (HSA). It shows that SHR116958 and M1 mainly binding to the major binding protein albumin. A small amount may bind to other binding protein.7. The toxicokinetics study of SHR116958After oral administration of SHR116958, the concentrations of SHR116958 and M1 increased with the dose increasing. The difference between the 1st and 30th times was not statistically significant (P> 0.05). After the 1st administration of low, middle and high-dose group, mean AUC(0-t) value of SHR116958 were 3884.9±1974.8,21731.7±17764.6 and 56985.9±29413 ng·h·mL-1. After the 30th administration, the mean AUC (0-t) value of SHR116958 were 18190.3±1786.9, 52792.7±16381.3 and 77844.2±21821.2 ng·h·mL-1. And after the 1st administration of low, middle and high-dose group, the Cmax value were 1974.2±1226.2,12561.7±11279.9 and 28733.3±23867 ng·mL-1, and after the 30th administration, the Cmax value were 1799.2±560.8,8250.0± 2738.4 and 15205±4750.2 ng·mL-1.After 1st administration of low, middle and high-dose group, the mean AUC(0-t) value were 1310.3±677.8,4095.6±1451.7 and 12212.5±4496.5 ng·h·mL-1. After the 30th administration, the mean AUC (0-t) value of M1 were 4039.4±359.2, 10917.8±1578.6 and 18628.0±1038.6 ng·h·mL-1. And after 1st administration of low, middle and high-dose group, the Cmax value of M1 were 466.7±282.3,1479.2±495.3 and 3349.2±868.8 ng·mL-1. And after the 30th administration of three dose group, the Cmax value of M1 were 1195.0±177.1,3103.3±521.1 and 7145.0±1026.7 ng·mL-1. After 30 consecutive administrations, the accumulation coefficient of low, medium and high-dose group for SHR116958 were 4.68, 2.43 and 1.37, respectively, for M1 were 3.08, 2.67 and 1.52, respectively. |
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