SHR116958的非临床药代动力学研究
|
摘要
5-羟色胺4(5-HT4)受体激动剂是一类促进胃动力药,通过兴奋胃肠道胆碱能中间神经元及肌间神经丛的5-HT4受体,促进乙酰胆碱的释放,从而增强胃肠道运动,改善功能性消化不良。主要用于功能性消化不良伴有胃灼热、嗳气、恶心、呕吐、早饱、上腹胀等消化道症状,也可用于胃食管返流性疾病、糖尿病性胃轻瘫及部分胃切除患者的胃功能障碍[1-10]。此类药物中的代表性药物有西沙必利和莫沙必利,其中西沙必利1988年上市后由于心脏毒性已于2000年退市,莫沙必利心脏毒性较弱,目前临床应用广泛[11-16]。此外,宝洁公司新开发的药效更强安全性更高的ATI-7505正在Ⅱ期临床试验阶段[17-19]。
SHR116958是新开发的一类胃动力新药,属于苯甲酰胺类的5-羟色胺4受体激动剂,其化学结构(图1)保留了此类药物的苯甲酰胺基本活性结构,侧链替换为己酸喹核酯。此药在分子生物学及细胞学体外筛选活性试验中已显示出比莫沙必利高数十倍的活性,初步体内试验也表现出比莫沙必利吸收速度更快生物利用度更高的优异药代动力学性质,安全性方面的初步试验也表明几乎没有明显毒性反应。
本研究的目的是系统地研究SHR116958在大鼠与比格犬体内的吸收、分布、代谢、排泄过程,阐明SHR116958及其主要代谢物体内药动学特征,为药物的药效、毒理评价及临床新药报批提供资料,也为其他此类化合物的药代动力学研究提供参考和新思路。
本研究的主要内容包括:
一、建立测定生物样品中SHR116958及其代谢物的方法建立LC/MS/MS方法测定血浆中SHR116958及其代谢物(水解代谢物)的浓度,并进行方法学确证,包括方法的特异性、灵敏度、准确度、定量线性范围、日内及日间精密度、回收率等。同样,针对不同生物样本,在LC/MS/MS方法的基础上,修订和优化药物提取方法,建立各种重要组织器官匀浆、粪、尿、胆汁等生物样本中SHR116958药物浓度的测定方法。
二、SHR116958及其代谢物的血药浓度-时间曲线及吸收
1、大鼠体内药代动力学
大鼠:选择一个剂量(36 mg/kg)进行单次口服给药的药物代谢动力学研究,并选择一个剂量进行静脉注射,计算口服给药在大鼠体内的生物利用度。采血时间点初步定为给药前、给药后0.25、0.5、0.75、1、1.5、2、3、4、6、8、12、36、48h[20]。
2、比格犬体内药代动力学
①测定单次口服给药高中低3个剂量(10、30、50 mg/kg)的血药浓度-时间图形和相关参数,确定是否存在AUC和Cmax依赖于成正比增高的线性药代动力学,还是非线性药代动力学采血时间点初步定为给药前、给药后0.25、0.5、0.75、1、1.5、2、3、4、6、8、12、24h;②选择一个剂量进行口服连续7天给药,每天1次,连续7天,比较第1天和第7天时的血药浓度曲线,确定是否存在药物诱导代谢消除速率的加快或抑制,使药代动力学行为随给药次数而变化;③采用自身交叉设计,选择清洗期后的中剂量组比格犬在同等剂量进行单次静脉注射,测定血药浓度-时间图形和相关参数,用此计算口服给药在比格犬体内的生物利用度。
三、SHR116958在大鼠体内的分布
选择1个剂量组,6只/组大鼠进行口服给药,在药物的吸收、血药浓度达峰、消除等3-4个时间点处死,解剖,提取心、肝、脑、脾、肺、肾、胃、肠、肌肉、脂肪、生殖腺等重要组织、器官进行药物提取和LC-MS/MS药物浓度测定,比较SHR116958在各组织器官中的分布,有无靶向性,获取血脑屏障的通过情况等。
四、SHR116958的代谢方式及主要代谢产物
采用SHR116958与空白人血浆、肝微粒体酶系统孵育的体外试验方法,确定此药可能存在的代谢方式及主要代谢产物(N-去烷基化代谢物、水解代谢物)。并考察SHR116958在人、猴、犬、大鼠4种属肝微粒体中的代谢稳定性和代谢种属差异,为体内药动学研究、安全性评价、药效学评价实验选择合适的动物模型提供依据。
五、SHR116958在大鼠体内的排泄
粪尿和胆汁排泄研究:选择1个剂量口服给药进行粪、尿和胆汁排泄研究。
六、血浆蛋白结合试验
选择高中低3个剂量浓度的药物分别与空白大鼠、比格犬、人血浆、人血清白蛋白、α1-酸性糖蛋白等孵育,采用超过滤法,结合LC/MS/MS测定,研究SHR116958与不同种属动物血浆蛋白的结合情况,为临床用药提供理论依据。
七、SHR116958的毒代动力学研究
试验设高、中、低和3个剂量组,剂量分别为10、30、60 mg·kg~(-1),每组6只动物,雌雄各半。采用口服给药,1次/天,连用30天,共给药30次,给药期限为4周半,研究SHR116958在比格犬体内是否存在蓄积,以及其与剂量之间的关系。
本研究的主要结果包括:
一、生物样品中SHR116958及其代谢物的LC/MS/MS方法学建立和确证本研究建立了快速、灵敏、易操作的LC/MS/MS法测定大鼠、犬血浆以及大鼠组织等生物样品中的SHR116958及其代谢物的浓度。用盐酸坦洛新为内标,血浆样品经乙酸乙酯萃取,以甲醇:乙腈: 0.1%甲酸水溶液=50:25:25 (v/v/v)为流动相,Angilent Eclipse C18柱(150nm×2.1nm,5μm)进行分离,以高纯氮气作为Curtain gas, collision gas ESI源正离子扫描,MRM扫描方式进行检测。测定犬血浆中SHR116958及M1的线性范围为1-3000 ng·mL~(-1)。血浆中SHR116958及M1最低定量限为1 ng·mL~(-1);大鼠血浆中SHR116958及M1的线性范围为2.71-5560 ng.mL~(-1),最低定量限为2.71 ng?mL~(-1)。本方法专属性好,准确、快速。适用于SHR116958及其代谢物的临床药代动力学研究。
二、SHR116958及其代谢物的血药浓度—时间曲线及吸收
1、SHR116958及其代谢物在大鼠体内的药物代谢动力学大鼠静脉注射SHR116958后迅速被水解代谢为M1,检测不到其它代谢产物,而且M1的血浆药物浓度明显高于同时间点的原形SHR116958浓度;静脉注射后血浆中SHR116958及代谢物M1药物浓度随时间的变化而迅速下降,呈典型的二项指数型曲线,而且SHR116958及代谢物M1药物浓度变化规律基本一致,大多数个体的血药浓度可检测至24h。
SD大鼠口服SHR116958(36 mg·kg~(-1))给药的血药浓度与静脉给药相比有大幅降低,8倍于静脉注射剂量口服后,SHR116958和代谢物M1血药的峰浓度仅是静脉注射的8.8%和5.4%,而且吸收不规则,多有双峰现象,表明大鼠口服SHR116958后吸收很低。
SD大鼠按4.5 mg·kg~(~(-1))剂量静脉注射给药和按36 mg·kg~(~(-1))剂量口服给药后原形药SHR116958的主要动力学参数如下:静脉给药组和口服给药组的原形药SHR116958半衰期t1/2分别为(10.8±3.8)和(18±8.9) h;清除率CL分别为(9.6±2.7)和(85.5±19.4) L·h~(-1)·kg~(~(-1));表观分布容积Vss分别为(53.1±18.6)和(767.9±198.1)L·kg~(-1);曲线下面积AUC0～∞分别为(505.6±167.8)和(1397.53±1087.04) ng·h·mL~(-1);平均驻留时间MRT分别为(5.5±1.5)和(8.95±1.03)h;峰浓度Cmax分别为(569.8±410.8)和(51.8±23.5)ng·mL~(-1);达峰时间Tmax分别为(0.05和2.17±2.29)h,消除速率常数Kel分别为0.07±0.02和0.04±0.02。采用等方差t检验进行比较,具有显著差异的参数有MRT、CL、Vss、Cmax、Tmax。
SD大鼠按照4.5 mg·kg~(-1)剂量静脉注射给药和按照36 mg·kg~(-1)剂量口服给药后代谢物M1的主要动力学参数如下:静脉给药组和口服给药组的代谢物M1半衰期t1/2分别为(8.4±3.6)和(13.6±2.8)h;清除率CL分别为(4.31±1.64)和(74±18.3 )L·h~(-1)·kg~(-1);表观分布容积Vss分别为(21.5±10.5)和(597.6±193.3) L·kg~(-1);曲线下面积AUC0～∞分别为(1192±491.9)和(513.9±135.9)ng·h·mL~(-1);平均驻留时间MRT分别为(4.8±1.07)和(7.96±0.70 )h;峰浓度Cmax分别为(1472.5±1079.6)和(92.3±49.9)ng·mL~(-1);达峰时间Tmax分别为(0.17±0.29)和(0.79±1.10)h,消除速率常数Kel分别为0.09±0.04和0.05±0.01。采用等方差t检验进行比较,具有显著差异的参数有AUC(0-24)、AUC(24-inf)、MRT、CL、Vss、T1/2、Kel、Cmax、Tmax。
SD大鼠口服SHR116958(4.5 ng·mL~(-1))后SHR116958的生物利用度为9.4%,代谢物M1的生物利用度为4.98%,表明大鼠口服SHR116958后吸收很差。
2、SHR116958及其代谢产物在比格犬体内的药代动力学研究比格犬单次口服10 mg·kg~(-1)、30 mg·kg~(-1)、50 mg·kg~(-1) 3个剂量的SHR116958后,其原型药物SHR116958的达峰时间在0.75～1.5 h左右,其达峰浓度分别为(129.8±61.7)、(695.8±87.4)和(1357.5±1338.7)ng.mL~(-1),而SHR116958的活性代谢物M1的达峰时间也在0.75～1.5 h左右,其达峰浓度为(208.1±108.0)、(541.8±226.3)和(413.7±606.7)ng·mL~(-1),以后呈单项指数消除特征,10 mg·kg~(-1)剂量组SHR116958及其代谢产物M1血药浓度只能检测至12h,其它两个计量组可检测至24h,检测不到其它代谢产物如M2。
由于SHR116958原型药物在血浆中迅速水解为其活性代谢产物M1,在静脉给药中测得的血浆药物浓度较低,代谢物M1的全身暴露水平是SHR116958的6倍并且在8h以后几乎测不到其血药浓度,而活性代谢产物的血浆浓度约是SHR116958的10倍,Cmax是原形SHR116958的18.0倍。而口服给药后,原型药物SHR116958与其代谢物M1的血药浓度差异不是很显著;所以生物利用度的计算选择了活性代谢产物M1的相关药代动力学参数。口服SHR116958后代谢物M1生物利用度为(23.7±9.6)%。
比格犬口服SHR116958低、中、高3个剂量组(10 mg·kg~(-1)、30 mg·kg~(-1)和50 mg·kg~(-1),剂量之比为1:3:5)后, AUC增长比值为1:5.6:10.9;达峰时间Tmax分别为1.33h、1.45h和1.33h; Cmax比值为1:4.1:9.46;平均滞留时间MRT分别为(3.22±0.51)、(4.18±0.46)和(4.15±0.74) h;清除率CL分别为(23.05±7.50)、(11.5±1.68)和(14.24±7.95) L·kg~(-1)·h~(-1);低、中、高3个剂量组末端消除相半衰期t1/2为5.88±4.94、4.94±3.18、4.96±3.09。在10～50 mg·kg~(-1)的范围内AUC和峰浓度与给药剂量基本成正相关,但稍高于正比例关系。代谢物M1AUC增长比值为1:2.2:5.3;低、中、高3个剂量组血药浓度达峰时间分别1.17 h、1.05 h和1.42 h;Cmax比值为1:2.1:5.8。平均滞留时间MRT分别为(5.08±0.69)、(4.14±2.15)和(4.53±0.95)h;清除率CL分别为(14.60±4.68)、(18.71±6.97)和(11.81±5.85) L·kg~(-1)·h~(-1);低、中、高3个剂量组末端消除相半衰期t1/2为(3.95±1.53) h(,4.96±2.50) h(,5.07±3.39) h。在10～50 mg·kg~(-1)的范围内AUC和峰浓度与给药剂量成正相关,约为正比例关系,呈明显的线性动力学特征。
比格犬连续多次口服SHR116958后第1次给药的药代参数及与第7次给药的药代参数均无明显差异, SHR116958的蓄积因子AUC(0～∞)d7/ AUC(0～∞)d1为0.94,活性代谢物M1的蓄积因子AUC(0～∞)d7/ AUC(0～∞)d1为1.0,表明多次给药后,SHR116958与其代谢物在比格犬体内基本无明显蓄积情况。
三、SHR116958及其代谢产物在大鼠体内的组织分布
SD大鼠36 mg·kg~(-1)口服给药SHR116958后,SHR116958的含量按AUC排序由大到小依次为:肝、胃、脾、肾、肺、脂肪、胸腺、卵巢、骨髓、心脏、睾丸、肌肉、血浆、脑。其中在肝的含量最高,胃次之,说明SHR116958经由肝脏代谢,主要分布并作用于靶器官胃,符合其作为胃动力药的特征;其次,通过脑中药物浓度极低可以推断此药不易通过血脑屏障。口服给药后血浆中原形药浓度较低,表明大鼠口服给药后存在明显的首过效应;药物作用的靶点胃组织中药物浓度也非常高,可能是SHR116958被胃组织直接吸收相关。SHR116958水解活性产物M1按AUC排序由大到小依次为:胃、肝、肺、肾、卵巢、血浆、脾、脂肪、骨髓、心脏、睾丸、肌肉、胸腺、脑。胃中代谢物浓度最高,说明原形药SHR116958及其水解后的主要活性产物M1大部分分布于主要靶器官胃,分布规律与原形药大体一致,而与莫沙必利不一致。
四、SHR116958的代谢方式及主要代谢产物
体外代谢孵育试验表明,SHR116958的代谢具有CYP依赖性,主要由CYP催化代谢。而SHR116958对CYP2C9, CYP2C19, CYP2D6, CYP2E1没有明显抑制作用,对CYP1A2,CYP3A4的IC50值均大于10μΜ,根据文献资料, IC50>10μΜ属于较弱的抑制;1μΜ< IC50<10μΜ属于中等程度抑制;IC50<1μΜ属于强烈抑制,所以SHR116958对人体各主要CYP的抑制较弱。
SHR116958在各种属肝微粒体孵育体系中均有显著代谢,共鉴定出9种代谢产物,其中羟基化代谢物和酯水解代谢物为主要代谢物。SHR116958在人肝微粒体代谢速率小于各动物。其中犬与人肝微粒体中代谢物种类及相对数量最为接近。五、SHR116958及其代谢产物在大鼠体内的排泄
SHR116958主要以水解活性产物M1的形式通过尿液排出,少量以原形药的形式通过尿和粪排出。48h内尿和粪以代谢物M1的形式的累计排出量分别达到给药量的72.36%和3.78%,尿粪合计排出达到给药量的76.13%。48h内尿和粪以原形药的形式的累计排出量分别达到给药量的13.33%和0.392%,尿粪合计排出达到给药量的13.72%。48h时粪尿中以原形和代谢物M1的形式累及排出的量接近90 %。
SHR116958主要以水解活性产物M1的形式通过胆汁排泄,少量以原形药的形式排泄。24h内胆汁以原形和代谢物M1的形式的累计排出量分别达到给药量的3.2%和0.27 %, 24h时胆汁中以原形和代谢物M1的形式累及排出的量为3.5 %,与最终从粪便排泄量相当。
六、SHR116958及其代谢产物的血浆蛋白结合
SHR116958及其水解活性产物M1与人血浆、大鼠血浆和犬血浆蛋白结合率在97-99%之间,种属之间血浆蛋白结合差别不大,略高于人血清白蛋白(HSA)的结合率,表明SHR116958及其水解活性产物M1除与血浆中的主要结合蛋白白蛋白结合外,还有少部分可能与其它结合蛋白如α1-酸性糖蛋白、脂肪、蛋白结合,同时说明SHR116958及其代谢产物M1主要与血浆中的白蛋白结合。
七、SHR116958及其代谢产物的毒代动力学
比格犬口服SHR116958后SHR116958、代谢物M1血药浓度随剂量的加大而增高,第1次与第30次相同时间点的血药浓度经配对t-检验后,大部分时间点血药浓度的差别无统计学意义(P > 0.05)。第1次给药后比格犬低、中、高剂量组的SHR116958平均AUC(0-t)值为(3884.9±1974.8)、(21731.7±17764.6)和(56985.9±29413)ng·h·mL~(-1),第30次给药后比格犬低、中、高剂量组的SHR116958平均AUC(0-t)值为(18190.3±1786.9)、(52792.7±16381.3)和(77844.2±21821.2)ng·h·mL~(-1)。第1次给药后比格犬低、中、高剂量组的SHR116958的Cmax值为(1974.2±1226.2)、(12561.7±11279.9)和(28733.3±23867)ng·mL~(-1),30次给药后SHR116958低、中、高剂量组的Cmax值为(1799.2±560.8)、(8250.0±2738.4)和(15205±4750.2)ng·mL~(-1)。
第1次给药后比格犬低、中、高剂量组的M1平均AUC(0-t)值为(1310.3±677.8)、(4095.6±1451.7)和(12212.5±4496.5) ng·h·mL~(-1),第30次给药后比格犬低、中、高剂量组的M1平均AUC(0-t)值为( 4039.4±359.2)、(10917.8±1578.6)和(18628.0±1038.6) ng·h·mL~(-1)。第1次给药后比格犬低、中、高剂量组的M1的Cmax值为(466.7±282.3)、(1479.2±495.3)和(3349.2±868.8) ng·mL~(-1),第30次给药后M1的Cmax值为(1195.0±177.1)、(3103.3±521.1)和(7145.0±1026.7) ng·mL~(-1)。
连续30次给药后,SHR116958的蓄积系数低、中、高剂量组分别为4.68、2.43、1.37; M1的蓄积系数低、中、高分别为3.08、2.67、1.52。
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 samples
The 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 pharmacokinetics
Chose 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 pharmacokinetics
①D 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 rats
One 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 SHR116958
In 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 rats
Choose one dose of oral administration to feces, urine and bile excretion studies.
6. The determination of plasma protein binding rate
Three 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 SHR116958
Three 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 samples
This 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 rats
After 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 dogs
After 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 0.75~1h, 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 rats
After 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 SHR116958
The 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 rats
SHR116958 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 rate
The plasma protein binding rate between SHR116958 and M1 and human, rat and dog plasma is in 97~99%. 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 SHR116958
After 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.
SHR116958是新开发的一类胃动力新药,属于苯甲酰胺类的5-羟色胺4受体激动剂,其化学结构(图1)保留了此类药物的苯甲酰胺基本活性结构,侧链替换为己酸喹核酯。此药在分子生物学及细胞学体外筛选活性试验中已显示出比莫沙必利高数十倍的活性,初步体内试验也表现出比莫沙必利吸收速度更快生物利用度更高的优异药代动力学性质,安全性方面的初步试验也表明几乎没有明显毒性反应。
本研究的目的是系统地研究SHR116958在大鼠与比格犬体内的吸收、分布、代谢、排泄过程,阐明SHR116958及其主要代谢物体内药动学特征,为药物的药效、毒理评价及临床新药报批提供资料,也为其他此类化合物的药代动力学研究提供参考和新思路。
本研究的主要内容包括:
一、建立测定生物样品中SHR116958及其代谢物的方法建立LC/MS/MS方法测定血浆中SHR116958及其代谢物(水解代谢物)的浓度,并进行方法学确证,包括方法的特异性、灵敏度、准确度、定量线性范围、日内及日间精密度、回收率等。同样,针对不同生物样本,在LC/MS/MS方法的基础上,修订和优化药物提取方法,建立各种重要组织器官匀浆、粪、尿、胆汁等生物样本中SHR116958药物浓度的测定方法。
二、SHR116958及其代谢物的血药浓度-时间曲线及吸收
1、大鼠体内药代动力学
大鼠:选择一个剂量(36 mg/kg)进行单次口服给药的药物代谢动力学研究,并选择一个剂量进行静脉注射,计算口服给药在大鼠体内的生物利用度。采血时间点初步定为给药前、给药后0.25、0.5、0.75、1、1.5、2、3、4、6、8、12、36、48h[20]。
2、比格犬体内药代动力学
①测定单次口服给药高中低3个剂量(10、30、50 mg/kg)的血药浓度-时间图形和相关参数,确定是否存在AUC和Cmax依赖于成正比增高的线性药代动力学,还是非线性药代动力学采血时间点初步定为给药前、给药后0.25、0.5、0.75、1、1.5、2、3、4、6、8、12、24h;②选择一个剂量进行口服连续7天给药,每天1次,连续7天,比较第1天和第7天时的血药浓度曲线,确定是否存在药物诱导代谢消除速率的加快或抑制,使药代动力学行为随给药次数而变化;③采用自身交叉设计,选择清洗期后的中剂量组比格犬在同等剂量进行单次静脉注射,测定血药浓度-时间图形和相关参数,用此计算口服给药在比格犬体内的生物利用度。
三、SHR116958在大鼠体内的分布
选择1个剂量组,6只/组大鼠进行口服给药,在药物的吸收、血药浓度达峰、消除等3-4个时间点处死,解剖,提取心、肝、脑、脾、肺、肾、胃、肠、肌肉、脂肪、生殖腺等重要组织、器官进行药物提取和LC-MS/MS药物浓度测定,比较SHR116958在各组织器官中的分布,有无靶向性,获取血脑屏障的通过情况等。
四、SHR116958的代谢方式及主要代谢产物
采用SHR116958与空白人血浆、肝微粒体酶系统孵育的体外试验方法,确定此药可能存在的代谢方式及主要代谢产物(N-去烷基化代谢物、水解代谢物)。并考察SHR116958在人、猴、犬、大鼠4种属肝微粒体中的代谢稳定性和代谢种属差异,为体内药动学研究、安全性评价、药效学评价实验选择合适的动物模型提供依据。
五、SHR116958在大鼠体内的排泄
粪尿和胆汁排泄研究:选择1个剂量口服给药进行粪、尿和胆汁排泄研究。
六、血浆蛋白结合试验
选择高中低3个剂量浓度的药物分别与空白大鼠、比格犬、人血浆、人血清白蛋白、α1-酸性糖蛋白等孵育,采用超过滤法,结合LC/MS/MS测定,研究SHR116958与不同种属动物血浆蛋白的结合情况,为临床用药提供理论依据。
七、SHR116958的毒代动力学研究
试验设高、中、低和3个剂量组,剂量分别为10、30、60 mg·kg~(-1),每组6只动物,雌雄各半。采用口服给药,1次/天,连用30天,共给药30次,给药期限为4周半,研究SHR116958在比格犬体内是否存在蓄积,以及其与剂量之间的关系。
本研究的主要结果包括:
一、生物样品中SHR116958及其代谢物的LC/MS/MS方法学建立和确证本研究建立了快速、灵敏、易操作的LC/MS/MS法测定大鼠、犬血浆以及大鼠组织等生物样品中的SHR116958及其代谢物的浓度。用盐酸坦洛新为内标,血浆样品经乙酸乙酯萃取,以甲醇:乙腈: 0.1%甲酸水溶液=50:25:25 (v/v/v)为流动相,Angilent Eclipse C18柱(150nm×2.1nm,5μm)进行分离,以高纯氮气作为Curtain gas, collision gas ESI源正离子扫描,MRM扫描方式进行检测。测定犬血浆中SHR116958及M1的线性范围为1-3000 ng·mL~(-1)。血浆中SHR116958及M1最低定量限为1 ng·mL~(-1);大鼠血浆中SHR116958及M1的线性范围为2.71-5560 ng.mL~(-1),最低定量限为2.71 ng?mL~(-1)。本方法专属性好,准确、快速。适用于SHR116958及其代谢物的临床药代动力学研究。
二、SHR116958及其代谢物的血药浓度—时间曲线及吸收
1、SHR116958及其代谢物在大鼠体内的药物代谢动力学大鼠静脉注射SHR116958后迅速被水解代谢为M1,检测不到其它代谢产物,而且M1的血浆药物浓度明显高于同时间点的原形SHR116958浓度;静脉注射后血浆中SHR116958及代谢物M1药物浓度随时间的变化而迅速下降,呈典型的二项指数型曲线,而且SHR116958及代谢物M1药物浓度变化规律基本一致,大多数个体的血药浓度可检测至24h。
SD大鼠口服SHR116958(36 mg·kg~(-1))给药的血药浓度与静脉给药相比有大幅降低,8倍于静脉注射剂量口服后,SHR116958和代谢物M1血药的峰浓度仅是静脉注射的8.8%和5.4%,而且吸收不规则,多有双峰现象,表明大鼠口服SHR116958后吸收很低。
SD大鼠按4.5 mg·kg~(~(-1))剂量静脉注射给药和按36 mg·kg~(~(-1))剂量口服给药后原形药SHR116958的主要动力学参数如下:静脉给药组和口服给药组的原形药SHR116958半衰期t1/2分别为(10.8±3.8)和(18±8.9) h;清除率CL分别为(9.6±2.7)和(85.5±19.4) L·h~(-1)·kg~(~(-1));表观分布容积Vss分别为(53.1±18.6)和(767.9±198.1)L·kg~(-1);曲线下面积AUC0～∞分别为(505.6±167.8)和(1397.53±1087.04) ng·h·mL~(-1);平均驻留时间MRT分别为(5.5±1.5)和(8.95±1.03)h;峰浓度Cmax分别为(569.8±410.8)和(51.8±23.5)ng·mL~(-1);达峰时间Tmax分别为(0.05和2.17±2.29)h,消除速率常数Kel分别为0.07±0.02和0.04±0.02。采用等方差t检验进行比较,具有显著差异的参数有MRT、CL、Vss、Cmax、Tmax。
SD大鼠按照4.5 mg·kg~(-1)剂量静脉注射给药和按照36 mg·kg~(-1)剂量口服给药后代谢物M1的主要动力学参数如下:静脉给药组和口服给药组的代谢物M1半衰期t1/2分别为(8.4±3.6)和(13.6±2.8)h;清除率CL分别为(4.31±1.64)和(74±18.3 )L·h~(-1)·kg~(-1);表观分布容积Vss分别为(21.5±10.5)和(597.6±193.3) L·kg~(-1);曲线下面积AUC0～∞分别为(1192±491.9)和(513.9±135.9)ng·h·mL~(-1);平均驻留时间MRT分别为(4.8±1.07)和(7.96±0.70 )h;峰浓度Cmax分别为(1472.5±1079.6)和(92.3±49.9)ng·mL~(-1);达峰时间Tmax分别为(0.17±0.29)和(0.79±1.10)h,消除速率常数Kel分别为0.09±0.04和0.05±0.01。采用等方差t检验进行比较,具有显著差异的参数有AUC(0-24)、AUC(24-inf)、MRT、CL、Vss、T1/2、Kel、Cmax、Tmax。
SD大鼠口服SHR116958(4.5 ng·mL~(-1))后SHR116958的生物利用度为9.4%,代谢物M1的生物利用度为4.98%,表明大鼠口服SHR116958后吸收很差。
2、SHR116958及其代谢产物在比格犬体内的药代动力学研究比格犬单次口服10 mg·kg~(-1)、30 mg·kg~(-1)、50 mg·kg~(-1) 3个剂量的SHR116958后,其原型药物SHR116958的达峰时间在0.75～1.5 h左右,其达峰浓度分别为(129.8±61.7)、(695.8±87.4)和(1357.5±1338.7)ng.mL~(-1),而SHR116958的活性代谢物M1的达峰时间也在0.75～1.5 h左右,其达峰浓度为(208.1±108.0)、(541.8±226.3)和(413.7±606.7)ng·mL~(-1),以后呈单项指数消除特征,10 mg·kg~(-1)剂量组SHR116958及其代谢产物M1血药浓度只能检测至12h,其它两个计量组可检测至24h,检测不到其它代谢产物如M2。
由于SHR116958原型药物在血浆中迅速水解为其活性代谢产物M1,在静脉给药中测得的血浆药物浓度较低,代谢物M1的全身暴露水平是SHR116958的6倍并且在8h以后几乎测不到其血药浓度,而活性代谢产物的血浆浓度约是SHR116958的10倍,Cmax是原形SHR116958的18.0倍。而口服给药后,原型药物SHR116958与其代谢物M1的血药浓度差异不是很显著;所以生物利用度的计算选择了活性代谢产物M1的相关药代动力学参数。口服SHR116958后代谢物M1生物利用度为(23.7±9.6)%。
比格犬口服SHR116958低、中、高3个剂量组(10 mg·kg~(-1)、30 mg·kg~(-1)和50 mg·kg~(-1),剂量之比为1:3:5)后, AUC增长比值为1:5.6:10.9;达峰时间Tmax分别为1.33h、1.45h和1.33h; Cmax比值为1:4.1:9.46;平均滞留时间MRT分别为(3.22±0.51)、(4.18±0.46)和(4.15±0.74) h;清除率CL分别为(23.05±7.50)、(11.5±1.68)和(14.24±7.95) L·kg~(-1)·h~(-1);低、中、高3个剂量组末端消除相半衰期t1/2为5.88±4.94、4.94±3.18、4.96±3.09。在10～50 mg·kg~(-1)的范围内AUC和峰浓度与给药剂量基本成正相关,但稍高于正比例关系。代谢物M1AUC增长比值为1:2.2:5.3;低、中、高3个剂量组血药浓度达峰时间分别1.17 h、1.05 h和1.42 h;Cmax比值为1:2.1:5.8。平均滞留时间MRT分别为(5.08±0.69)、(4.14±2.15)和(4.53±0.95)h;清除率CL分别为(14.60±4.68)、(18.71±6.97)和(11.81±5.85) L·kg~(-1)·h~(-1);低、中、高3个剂量组末端消除相半衰期t1/2为(3.95±1.53) h(,4.96±2.50) h(,5.07±3.39) h。在10～50 mg·kg~(-1)的范围内AUC和峰浓度与给药剂量成正相关,约为正比例关系,呈明显的线性动力学特征。
比格犬连续多次口服SHR116958后第1次给药的药代参数及与第7次给药的药代参数均无明显差异, SHR116958的蓄积因子AUC(0～∞)d7/ AUC(0～∞)d1为0.94,活性代谢物M1的蓄积因子AUC(0～∞)d7/ AUC(0～∞)d1为1.0,表明多次给药后,SHR116958与其代谢物在比格犬体内基本无明显蓄积情况。
三、SHR116958及其代谢产物在大鼠体内的组织分布
SD大鼠36 mg·kg~(-1)口服给药SHR116958后,SHR116958的含量按AUC排序由大到小依次为:肝、胃、脾、肾、肺、脂肪、胸腺、卵巢、骨髓、心脏、睾丸、肌肉、血浆、脑。其中在肝的含量最高,胃次之,说明SHR116958经由肝脏代谢,主要分布并作用于靶器官胃,符合其作为胃动力药的特征;其次,通过脑中药物浓度极低可以推断此药不易通过血脑屏障。口服给药后血浆中原形药浓度较低,表明大鼠口服给药后存在明显的首过效应;药物作用的靶点胃组织中药物浓度也非常高,可能是SHR116958被胃组织直接吸收相关。SHR116958水解活性产物M1按AUC排序由大到小依次为:胃、肝、肺、肾、卵巢、血浆、脾、脂肪、骨髓、心脏、睾丸、肌肉、胸腺、脑。胃中代谢物浓度最高,说明原形药SHR116958及其水解后的主要活性产物M1大部分分布于主要靶器官胃,分布规律与原形药大体一致,而与莫沙必利不一致。
四、SHR116958的代谢方式及主要代谢产物
体外代谢孵育试验表明,SHR116958的代谢具有CYP依赖性,主要由CYP催化代谢。而SHR116958对CYP2C9, CYP2C19, CYP2D6, CYP2E1没有明显抑制作用,对CYP1A2,CYP3A4的IC50值均大于10μΜ,根据文献资料, IC50>10μΜ属于较弱的抑制;1μΜ< IC50<10μΜ属于中等程度抑制;IC50<1μΜ属于强烈抑制,所以SHR116958对人体各主要CYP的抑制较弱。
SHR116958在各种属肝微粒体孵育体系中均有显著代谢,共鉴定出9种代谢产物,其中羟基化代谢物和酯水解代谢物为主要代谢物。SHR116958在人肝微粒体代谢速率小于各动物。其中犬与人肝微粒体中代谢物种类及相对数量最为接近。五、SHR116958及其代谢产物在大鼠体内的排泄
SHR116958主要以水解活性产物M1的形式通过尿液排出,少量以原形药的形式通过尿和粪排出。48h内尿和粪以代谢物M1的形式的累计排出量分别达到给药量的72.36%和3.78%,尿粪合计排出达到给药量的76.13%。48h内尿和粪以原形药的形式的累计排出量分别达到给药量的13.33%和0.392%,尿粪合计排出达到给药量的13.72%。48h时粪尿中以原形和代谢物M1的形式累及排出的量接近90 %。
SHR116958主要以水解活性产物M1的形式通过胆汁排泄,少量以原形药的形式排泄。24h内胆汁以原形和代谢物M1的形式的累计排出量分别达到给药量的3.2%和0.27 %, 24h时胆汁中以原形和代谢物M1的形式累及排出的量为3.5 %,与最终从粪便排泄量相当。
六、SHR116958及其代谢产物的血浆蛋白结合
SHR116958及其水解活性产物M1与人血浆、大鼠血浆和犬血浆蛋白结合率在97-99%之间,种属之间血浆蛋白结合差别不大,略高于人血清白蛋白(HSA)的结合率,表明SHR116958及其水解活性产物M1除与血浆中的主要结合蛋白白蛋白结合外,还有少部分可能与其它结合蛋白如α1-酸性糖蛋白、脂肪、蛋白结合,同时说明SHR116958及其代谢产物M1主要与血浆中的白蛋白结合。
七、SHR116958及其代谢产物的毒代动力学
比格犬口服SHR116958后SHR116958、代谢物M1血药浓度随剂量的加大而增高,第1次与第30次相同时间点的血药浓度经配对t-检验后,大部分时间点血药浓度的差别无统计学意义(P > 0.05)。第1次给药后比格犬低、中、高剂量组的SHR116958平均AUC(0-t)值为(3884.9±1974.8)、(21731.7±17764.6)和(56985.9±29413)ng·h·mL~(-1),第30次给药后比格犬低、中、高剂量组的SHR116958平均AUC(0-t)值为(18190.3±1786.9)、(52792.7±16381.3)和(77844.2±21821.2)ng·h·mL~(-1)。第1次给药后比格犬低、中、高剂量组的SHR116958的Cmax值为(1974.2±1226.2)、(12561.7±11279.9)和(28733.3±23867)ng·mL~(-1),30次给药后SHR116958低、中、高剂量组的Cmax值为(1799.2±560.8)、(8250.0±2738.4)和(15205±4750.2)ng·mL~(-1)。
第1次给药后比格犬低、中、高剂量组的M1平均AUC(0-t)值为(1310.3±677.8)、(4095.6±1451.7)和(12212.5±4496.5) ng·h·mL~(-1),第30次给药后比格犬低、中、高剂量组的M1平均AUC(0-t)值为( 4039.4±359.2)、(10917.8±1578.6)和(18628.0±1038.6) ng·h·mL~(-1)。第1次给药后比格犬低、中、高剂量组的M1的Cmax值为(466.7±282.3)、(1479.2±495.3)和(3349.2±868.8) ng·mL~(-1),第30次给药后M1的Cmax值为(1195.0±177.1)、(3103.3±521.1)和(7145.0±1026.7) ng·mL~(-1)。
连续30次给药后,SHR116958的蓄积系数低、中、高剂量组分别为4.68、2.43、1.37; M1的蓄积系数低、中、高分别为3.08、2.67、1.52。
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 samples
The 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 pharmacokinetics
Chose 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 pharmacokinetics
①D 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 rats
One 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 SHR116958
In 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 rats
Choose one dose of oral administration to feces, urine and bile excretion studies.
6. The determination of plasma protein binding rate
Three 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 SHR116958
Three 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 samples
This 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 rats
After 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 dogs
After 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 0.75~1h, 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 rats
After 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 SHR116958
The 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 rats
SHR116958 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 rate
The plasma protein binding rate between SHR116958 and M1 and human, rat and dog plasma is in 97~99%. 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 SHR116958
After 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.
引文
[1] Rahman SK, Piper DC, King FD. 5-HT3 receptor antagonists: Central and peripheral nervous system. Expert Opinion on Therapeutic Patents , 1994,4:6(669-680)
[2] Eglen RM, Hegde SS. 5-Hydroxytryptamine (5-HT)4 receptors: Physiology, pharmacology and therapeutic potential. Expert Opinion on Investigational Drugs , 1996,5:4(373-388)
[3] Graul A, Silvestre J, Castaner J. Tegaserod Maleate. 5-HT4 agonist, prokinetic, treatment of irritable bowel syndrome. Drugs of the Future , 1999,24:1(38-44)
[4] DePonti F, Tonini M. Irritable bowel syndrome: New agents targeting serotonin receptor subtypes. Drugs , 2001,61:3(317-332)
[5] Tonini M. Recent advances in the pharmacology of gastrointestinal prokinetics. Pharmacological Research , 1996,33:4-5(217-226)
[6] Kato S, Fujiwara I, Yoshida N. Nitrogen-containing heteroalicycles with serotonin receptor binding affinity: development of gastroprokinetic and antiemetic agents. Medicinal Research Reviews , 1999,19:1(25-73)
[7] Uchiyama Tsuyuki Y, Saitoh M, Muramatsu M. Identification and characterization of the 5-HT4 receptor in the intestinal tract and striatum of the guinea pig. Life Sciences, 1996,59:25-26(2129-2137)
[8] Yoshida N, Omoya H, Furukawa K, Oka M, Ito T. Pharmacological studies on mosapride citrate (AS-4370), a gastroprokinetic agent (1) - Comparison with cisapride and metoclopramide. Japanese Pharmacology and Therapeutics , 1993,21:9(249-264)
[9] Kii Y, Ito T. Effects of 5-HT4-receptor agonists, cisapride, mosapride citrate, and zacopride, on cardiac action potentials in guinea pig isolated papillary muscles. Journal of Cardiovascular Pharmacology , 1997,29:5(670-675)
[10] Kim HS, Choi EJ, Park H. The effect of mosapride citrate on proximal and distal colonic motor function in the guinea-pig in vitro. Neurogastroenterology and Motility, 2008,20:2(169-176)
[11] Inui A, Yoshikawa T, Nagai R, Yoshida N, Ito. Effects of mosapride citrate, a 5-HT4 receptor agonists, on colonic motility in conscious guinea-pigs. Japanese Journal of Pharmacology, 2000,82:1(P-541) .
[12] Yoshikawa T, Yoshida N, Mine Y, Hosoki K.Affinity of mosapride citrate, a new gastroprokinetic agent, for 5-HT4 receptors in guinea pig ileum. Japanese Journal of Pharmacology, 1998, 77:1(53-59)
[13] Katayama K, Morio Y, Haga K, Fukuda T. Cisapride, a gastroprokinetic agent, binds to 5-HT4 receptors. Folia Pharmacologica Japonica , 1995,105:6(461-468)
[14] Matsui Y, Satomura K, Nishiwaki T, Matsuoka N, Nakamura H. Antigenicity study of mosapride citrate. Japanese Pharmacology and Therapeutics , a1993,21:10(159-167)
[15] Sanger GJ. Translating 5-HT receptor pharmacology. Neurogastroenterol Motil. 2009 Dec;21(12):1235-8.
[16] Ruth M, Hamelin B, Rohss K, Lundell L. The effect of mosapride, a novel prokinetic, on acid reflux variables in patients with gastro-oesophageal reflux disease. Alimentary Pharmacology and Therapeutics , 1998,12:1(35-40)
[17] Camilleri M, Vazquez-Roque MI, Burton D, Ford T, McKinzie S, Zinsmeister AR, Druzgala P. Pharmacodynamic effects of a novel prokinetic 5-HT receptor agonist, ATI-7505, in humans. Neurogastroenterol Motil. 2007 Jan;19(1):30-8.
[18] Bayés M, Rabasseda X, Prous JR. Gateways to clinical trials. Methods Find Exp Clin Pharmacol. 2007 Jun, 29(5):359-73.
[19] Sanger GJ. Translating 5-HT receptor pharmacology. Neurogastroenterol Motil. 2009 Dec;21(12):1235-8
[20]国家食品药品监督管理局.化学药物非临床药代动力学研究技术指导原则.2005.
[21] Yokoyama I, Mizuki Y, Yamaguchi T, Fujii T. Simultaneous enantiomeric determination of a gastroprokinetic agent mosapride citrate and its metabolite in plasma using .alpha.1-acid glycoprotein HPLC column. Journal of Pharmaceutical and Biomedical Analysis, 1997,15:9-10(1527-1535)
[22] Aoki Y, Hakamata H, Igarashi Y, Uchida K, Kobayashi H, Hirayama N, Kotani A, Kusu F. Liquid chromatography-tandem mass spectrometric method for determination of mosapride citrate in equine tissues. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences , 2007,858:1-2(135-142)
[23] Qin F, Chen LY, Ma YY, Wang D, Liu J, Lu XM, Li FM. Determination of mosapride in human plasma by high performance liquid chromatography tandem massspectrometry. Yao Xue Xue Bao. 2007 Aug; 42 (8):882-5
[24] Matsumoto S, Tagawa M, Hatoyama T, Fujii T, Miyazaki H, Sekine Y.Absorption, distribution, metabolism and excretion of [carbonyl-14C]mosapride citrate after repeated oral administration in rats. Arzneimittel-Forschung, 1993, 43:10(1103-1108)
[25] Matsumoto S, Tagawa M, Amejima H, Nakao M, Kagemoto A, Fujii T, Miyazaki H, Sekine Y. Absorption, distribution and excretion of [carbonyl-14C]mosapride citrate after a single oral administration in rats, dogs and monkeys. Arzneimittel-Forschung, 1993, 43:10(1084-1094)
[26] Sakashita M, Mizuki Y, Ashizume T, Yamaguchi T, Miyazaki H, Sekine Y. Pharmacokinetics of the gastrokinetic agent mosapride citrate after intravenous and oral administrations in rats. Arzneimittel-Forschung, 1993, 43:8(859-863)
[27] Sakashita M, Mizuki Y, Yamaguchi T, Miyazaki H, Sekine Y. Pharmacokinetics of the gastrokinetic agent mosapride citrate after intravenous and oral administrations in dogs and monkeys. Arzneimittel-Forschung, 1993, 43:8(864-866)
[28] Sakashita M, Mizuki Y, Yamaguchi T, Miyazaki H, Sekine Y. Pharmacokinetics of the gastrokinetic agent mosapride citrate after single and multiple oral administrations in healthy subjects. Arzneimittel-Forschung, 1993,43:8(867-872)
[29]周怀梧,沈佳庆,何雁.双部位吸收和肝肠循环并存的药动学模型及生物利用度.数理医药学杂志,1994,7: 1
[30] Ezzet F, Krishna G, Wexler DB, Statkevich P, Kosoglou T, Batra VK. A population pharmacokinetic model that describes multiple peaks due to enterohepatic recirulation of ezetimibe. Clinical Therapeutics, 2001,23:871.
[31] Lee SH, Lee MG. Pharmacokinetics and pharmacodynamics of azosemide after intravenous and oral administration to rats: absorption from various GI segments. Journal of pharmacokineics and Biopharmaceutics, 1996, 24:551.
[32] Suttle AB, Pollack GM, Brouwer KL, et al. Use of a pharmacokinetic model in corporating discontinuous gastrointestinal absorption to examine the occurrence of double peaks in oral concentration-time profiles. Pharmaceutical Research, 1992,9:350.
[33] Witcher JW, Boudinot FD. Application and simulations of a discontinuous oral absorption pharmacokinetic model. Pharmaceutical Research, 1996,13:1720.
[34]王广基,刘晓东,刘晓泉.药物代谢动力学.化学工业出版社,2005.
[35]刘晓东,谢林,王进,周云曙,王真,刘国卿.双部位吸收模型拟合吉非贝齐在人体中药代动力学数据.药学学报,1996,31:737-741。
[36]吴笑春,靳桂明,李馨。苯妥英吸收的多峰现象。中国医药学杂志,1995,15:299-300。
[37]郑萍,胡敏燕,刘世霆,杨凌。浅析卡马西平血药浓度的多峰现象。中国药房,1999,10:45。
[38]马越鸣,程能能,孙瑞元。安定代谢物去甲安定血浆浓度的双峰现象。中国药理学通报,1996,12:96。
[39]路通,宋钰,谢林,王广基,刘晓东。HPLC法同时测定灌胃黄芩水煎剂后大鼠血浆中黄芩苷和汉黄苷的质量浓度及药动学研究。中草药,2005,36:870-873。
[40]江波,印春华。水飞蓟素磷脂复合物家兔体内生物利用度的研究。中国医药工业杂志,2002,33:598-600。
[41]欧卫平,宓穗卿,黄天来,叶少梅。补骨脂内HPLC测定及以内动力学研究。中药新药与临床药理,2000,11:107-109。
[42]任平。张莉,黄熙。王跃民,王骊丽。麻醉犬心房内注射复方川芎汤后血清中川芎嗪药时曲线双峰与血流动力学效应的关系。第四军医大学学报,2000,21:157-160。
[43]陈淑娟,张斌,杨毅梅,代宗顺,曾繁典。蝙蝠葛碱犬体内药代动力学研究。中国临床药理学与治疗学,2000,5:213-217。
[44]陈淑娟,杨毅梅,刘弈明,张斌,庞雪冰,曾繁典。蝙蝠葛碱大鼠体内药物代谢动力学研究。中国药理学通报,2001,17:225-229。
[45]孟德胜,胡友梅。粉防已碱在小鼠药时曲线中的双峰现象。中国临床药学杂志,1999,8:303-305。
[46]李洪燕,张福荣,吴玲娟,徐潘生,籍秀娟。蒿苯酯在大鼠的药物代谢动力学。药学学报,1995,30:422-427。
[47]马越鸣。药物C-T曲线的双峰现象。国外医学《药学分册》,1987,3:168-173。
[48]卢海侨。药-时曲线上的双峰现象。青海医药杂志,1995,25:31。
[49] Garg DC. Ranitidine bioavailability and kinetics in normal male subjects. Clinical pharmacology and Therapeutics, 1983, 33:445-452.
[50]行杰。黄芩苷在动物体内的呼吸和代谢研究。沈阳药科大学博士学位论文。沈阳:沈阳药科大学。2005,5。
[51] Chan OH, Stewart BH. Pysicochemical and drug delivery consideratuons for oral drug bioavailability. Drug Discovery Today, 1996,1:461-473.
[52]魏树礼.生物药剂学与药物动力学.第一版.北京:北京医科大学出版社,1997.
[53] Rowland M, Tozer TN著,彭彬译。临床药动学。长沙:湖南科学技术出版社,1999。
[54] Matsumoto S, Yoshida K, Itogawa A, Tagawa M, Fujii T, Miyazaki H, Sekine Y Metabolism of [carbonyl-14C] mosapride citrate after a single oral administration in rats, dogs and monkeys. Arzneimittel-Forschung, 1993,43:10(1095-1102)
[55] Sun X, Niu L, Li X, Lu X, Li F. Characterization of metabolic profile of mosapride citrate in rat and identification of two new metabolites: Mosapride N-oxide and morpholine ring-opened mosapride by UPLC-ESI-MS/MS. J Pharm Biomed Anal. 2009 Aug 15;50(1):27-34.
[56] Yuan R, Madani S, Wei XX, Reynolds K, Huang SM. Evaluation of cytochrome P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions. Drug Metab Dispos. 2002, 30:1311-1319.
[57] Hickman D, Wang JP, Wang Y, Unadkat JD. Evaluation of the selectivity of In vitro probes and suitability of organic solvents for the measurement of human cytochrome P450 monooxygenase activities. Drug Metab Dispos. 1998, 26:207-215.
[58] Yan Z, Caldwell GW. Metabolism profiling, and cytochrome P450 inhibition & induction in drug discovery. Curr Top Med Chem. 2001 Nov;1(5):403-25.
[59]和凡。细胞色素P450酶活性评价的体外“cocktail”高通量筛选模型的建立和应用。中山大学博士学位论文。广州:中山大学临床药理研究所。2007,5。
[60]曾苏主编。药物代谢学。浙江大学出版社。2004。
[61]宋振玉主编。药物代谢研究意义、方法、应用。北京:人民卫生出版社。1990.3。
[62]郝琨、柳晓泉、王广基。临床前毒代动力学研究进展。中国药科大学学报,2004,35:195-199
[63] Funabashi H, Terada Y, Nakamura H, Shimazu H, Sugisawa K. Reproductive and developmental toxicity studies of mosapride citrate. (3) Perinnatal and postnatal study in rats. Japanese Pharmacology and Therapeutics, 1993, 21:10(3447-3468)
[64] Funabashi H, Terada Y, Nakamura H, Matsuoka T, Takigami H. Reproductive and developmental toxicity studies of mosapride citrate (4) - Teratogenicity study in rabbits. Japanese Pharmacology and Therapeutics, 1993, 21:10(147-157)
[65] Okimoto K, Toyosawa K, Matsuoka N, Ueda Y, Utsumi K, Iida M, Nakamura H, Umemura T. Toxicity study of mosapride citrate (II) - Thirteen week repeated oral dose toxicity study in rats. Japanese Pharmacology and Therapeutics, 1993, 21:10(29-56)
[66] Yatera S, Okimoto K, Koujitani T, Toyosawa K, Utsumi K, Iida M, Nakamura H. Toxicity study of mosapride citrate (I) - Single dose toxicity study in mice, rats and dogs.Japanese Pharmacology and Therapeutics, 1993, 21:10(21-28) ,
[67] Yatera S, Koujitani T, Yasuba M, Matsuoka N, Ueda Y, Utsumi K, Iida M, Nakamura H、Toxicity study of mosapride citrate (III) - Thirteen-week repeated dose oral toxicity study in beagle dogs..Japanese Pharmacology and Therapeutics, 1993, 21:10(57-73)
[68] Funabashi H, Nishimura K, Terada Y, Shigematsu K, Tateishi Y, Nakamura H. Reproductive and developmental toxicity studies of mosapride citrate (2) - Teratogenicity study in rats. Japanese Pharmacology and Therapeutics. 1993, 21:10(101-123)
[69] Funabashi H, Terada Y, Nakamura H, Shimazu H, Sugisawa K. Reproductive and developmental toxicity studies of mosapride citrate (3) - Perinatal and postnatal study in rats. Japanese Pharmacology and Therapeutics . 1993, 21:10(125-146)
[70] Okimoto K, Matsuoka N, Nakamura H, Mochizuki M, Okazaki S. Toxicity study of mosapride citrate (IV) - Twenty-six-week repeated oral dose toxicity study in rats. Japanese Pharmacology and Therapeutics, 1993, 21:10(75-87)
[2] Eglen RM, Hegde SS. 5-Hydroxytryptamine (5-HT)4 receptors: Physiology, pharmacology and therapeutic potential. Expert Opinion on Investigational Drugs , 1996,5:4(373-388)
[3] Graul A, Silvestre J, Castaner J. Tegaserod Maleate. 5-HT4 agonist, prokinetic, treatment of irritable bowel syndrome. Drugs of the Future , 1999,24:1(38-44)
[4] DePonti F, Tonini M. Irritable bowel syndrome: New agents targeting serotonin receptor subtypes. Drugs , 2001,61:3(317-332)
[5] Tonini M. Recent advances in the pharmacology of gastrointestinal prokinetics. Pharmacological Research , 1996,33:4-5(217-226)
[6] Kato S, Fujiwara I, Yoshida N. Nitrogen-containing heteroalicycles with serotonin receptor binding affinity: development of gastroprokinetic and antiemetic agents. Medicinal Research Reviews , 1999,19:1(25-73)
[7] Uchiyama Tsuyuki Y, Saitoh M, Muramatsu M. Identification and characterization of the 5-HT4 receptor in the intestinal tract and striatum of the guinea pig. Life Sciences, 1996,59:25-26(2129-2137)
[8] Yoshida N, Omoya H, Furukawa K, Oka M, Ito T. Pharmacological studies on mosapride citrate (AS-4370), a gastroprokinetic agent (1) - Comparison with cisapride and metoclopramide. Japanese Pharmacology and Therapeutics , 1993,21:9(249-264)
[9] Kii Y, Ito T. Effects of 5-HT4-receptor agonists, cisapride, mosapride citrate, and zacopride, on cardiac action potentials in guinea pig isolated papillary muscles. Journal of Cardiovascular Pharmacology , 1997,29:5(670-675)
[10] Kim HS, Choi EJ, Park H. The effect of mosapride citrate on proximal and distal colonic motor function in the guinea-pig in vitro. Neurogastroenterology and Motility, 2008,20:2(169-176)
[11] Inui A, Yoshikawa T, Nagai R, Yoshida N, Ito. Effects of mosapride citrate, a 5-HT4 receptor agonists, on colonic motility in conscious guinea-pigs. Japanese Journal of Pharmacology, 2000,82:1(P-541) .
[12] Yoshikawa T, Yoshida N, Mine Y, Hosoki K.Affinity of mosapride citrate, a new gastroprokinetic agent, for 5-HT4 receptors in guinea pig ileum. Japanese Journal of Pharmacology, 1998, 77:1(53-59)
[13] Katayama K, Morio Y, Haga K, Fukuda T. Cisapride, a gastroprokinetic agent, binds to 5-HT4 receptors. Folia Pharmacologica Japonica , 1995,105:6(461-468)
[14] Matsui Y, Satomura K, Nishiwaki T, Matsuoka N, Nakamura H. Antigenicity study of mosapride citrate. Japanese Pharmacology and Therapeutics , a1993,21:10(159-167)
[15] Sanger GJ. Translating 5-HT receptor pharmacology. Neurogastroenterol Motil. 2009 Dec;21(12):1235-8.
[16] Ruth M, Hamelin B, Rohss K, Lundell L. The effect of mosapride, a novel prokinetic, on acid reflux variables in patients with gastro-oesophageal reflux disease. Alimentary Pharmacology and Therapeutics , 1998,12:1(35-40)
[17] Camilleri M, Vazquez-Roque MI, Burton D, Ford T, McKinzie S, Zinsmeister AR, Druzgala P. Pharmacodynamic effects of a novel prokinetic 5-HT receptor agonist, ATI-7505, in humans. Neurogastroenterol Motil. 2007 Jan;19(1):30-8.
[18] Bayés M, Rabasseda X, Prous JR. Gateways to clinical trials. Methods Find Exp Clin Pharmacol. 2007 Jun, 29(5):359-73.
[19] Sanger GJ. Translating 5-HT receptor pharmacology. Neurogastroenterol Motil. 2009 Dec;21(12):1235-8
[20]国家食品药品监督管理局.化学药物非临床药代动力学研究技术指导原则.2005.
[21] Yokoyama I, Mizuki Y, Yamaguchi T, Fujii T. Simultaneous enantiomeric determination of a gastroprokinetic agent mosapride citrate and its metabolite in plasma using .alpha.1-acid glycoprotein HPLC column. Journal of Pharmaceutical and Biomedical Analysis, 1997,15:9-10(1527-1535)
[22] Aoki Y, Hakamata H, Igarashi Y, Uchida K, Kobayashi H, Hirayama N, Kotani A, Kusu F. Liquid chromatography-tandem mass spectrometric method for determination of mosapride citrate in equine tissues. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences , 2007,858:1-2(135-142)
[23] Qin F, Chen LY, Ma YY, Wang D, Liu J, Lu XM, Li FM. Determination of mosapride in human plasma by high performance liquid chromatography tandem massspectrometry. Yao Xue Xue Bao. 2007 Aug; 42 (8):882-5
[24] Matsumoto S, Tagawa M, Hatoyama T, Fujii T, Miyazaki H, Sekine Y.Absorption, distribution, metabolism and excretion of [carbonyl-14C]mosapride citrate after repeated oral administration in rats. Arzneimittel-Forschung, 1993, 43:10(1103-1108)
[25] Matsumoto S, Tagawa M, Amejima H, Nakao M, Kagemoto A, Fujii T, Miyazaki H, Sekine Y. Absorption, distribution and excretion of [carbonyl-14C]mosapride citrate after a single oral administration in rats, dogs and monkeys. Arzneimittel-Forschung, 1993, 43:10(1084-1094)
[26] Sakashita M, Mizuki Y, Ashizume T, Yamaguchi T, Miyazaki H, Sekine Y. Pharmacokinetics of the gastrokinetic agent mosapride citrate after intravenous and oral administrations in rats. Arzneimittel-Forschung, 1993, 43:8(859-863)
[27] Sakashita M, Mizuki Y, Yamaguchi T, Miyazaki H, Sekine Y. Pharmacokinetics of the gastrokinetic agent mosapride citrate after intravenous and oral administrations in dogs and monkeys. Arzneimittel-Forschung, 1993, 43:8(864-866)
[28] Sakashita M, Mizuki Y, Yamaguchi T, Miyazaki H, Sekine Y. Pharmacokinetics of the gastrokinetic agent mosapride citrate after single and multiple oral administrations in healthy subjects. Arzneimittel-Forschung, 1993,43:8(867-872)
[29]周怀梧,沈佳庆,何雁.双部位吸收和肝肠循环并存的药动学模型及生物利用度.数理医药学杂志,1994,7: 1
[30] Ezzet F, Krishna G, Wexler DB, Statkevich P, Kosoglou T, Batra VK. A population pharmacokinetic model that describes multiple peaks due to enterohepatic recirulation of ezetimibe. Clinical Therapeutics, 2001,23:871.
[31] Lee SH, Lee MG. Pharmacokinetics and pharmacodynamics of azosemide after intravenous and oral administration to rats: absorption from various GI segments. Journal of pharmacokineics and Biopharmaceutics, 1996, 24:551.
[32] Suttle AB, Pollack GM, Brouwer KL, et al. Use of a pharmacokinetic model in corporating discontinuous gastrointestinal absorption to examine the occurrence of double peaks in oral concentration-time profiles. Pharmaceutical Research, 1992,9:350.
[33] Witcher JW, Boudinot FD. Application and simulations of a discontinuous oral absorption pharmacokinetic model. Pharmaceutical Research, 1996,13:1720.
[34]王广基,刘晓东,刘晓泉.药物代谢动力学.化学工业出版社,2005.
[35]刘晓东,谢林,王进,周云曙,王真,刘国卿.双部位吸收模型拟合吉非贝齐在人体中药代动力学数据.药学学报,1996,31:737-741。
[36]吴笑春,靳桂明,李馨。苯妥英吸收的多峰现象。中国医药学杂志,1995,15:299-300。
[37]郑萍,胡敏燕,刘世霆,杨凌。浅析卡马西平血药浓度的多峰现象。中国药房,1999,10:45。
[38]马越鸣,程能能,孙瑞元。安定代谢物去甲安定血浆浓度的双峰现象。中国药理学通报,1996,12:96。
[39]路通,宋钰,谢林,王广基,刘晓东。HPLC法同时测定灌胃黄芩水煎剂后大鼠血浆中黄芩苷和汉黄苷的质量浓度及药动学研究。中草药,2005,36:870-873。
[40]江波,印春华。水飞蓟素磷脂复合物家兔体内生物利用度的研究。中国医药工业杂志,2002,33:598-600。
[41]欧卫平,宓穗卿,黄天来,叶少梅。补骨脂内HPLC测定及以内动力学研究。中药新药与临床药理,2000,11:107-109。
[42]任平。张莉,黄熙。王跃民,王骊丽。麻醉犬心房内注射复方川芎汤后血清中川芎嗪药时曲线双峰与血流动力学效应的关系。第四军医大学学报,2000,21:157-160。
[43]陈淑娟,张斌,杨毅梅,代宗顺,曾繁典。蝙蝠葛碱犬体内药代动力学研究。中国临床药理学与治疗学,2000,5:213-217。
[44]陈淑娟,杨毅梅,刘弈明,张斌,庞雪冰,曾繁典。蝙蝠葛碱大鼠体内药物代谢动力学研究。中国药理学通报,2001,17:225-229。
[45]孟德胜,胡友梅。粉防已碱在小鼠药时曲线中的双峰现象。中国临床药学杂志,1999,8:303-305。
[46]李洪燕,张福荣,吴玲娟,徐潘生,籍秀娟。蒿苯酯在大鼠的药物代谢动力学。药学学报,1995,30:422-427。
[47]马越鸣。药物C-T曲线的双峰现象。国外医学《药学分册》,1987,3:168-173。
[48]卢海侨。药-时曲线上的双峰现象。青海医药杂志,1995,25:31。
[49] Garg DC. Ranitidine bioavailability and kinetics in normal male subjects. Clinical pharmacology and Therapeutics, 1983, 33:445-452.
[50]行杰。黄芩苷在动物体内的呼吸和代谢研究。沈阳药科大学博士学位论文。沈阳:沈阳药科大学。2005,5。
[51] Chan OH, Stewart BH. Pysicochemical and drug delivery consideratuons for oral drug bioavailability. Drug Discovery Today, 1996,1:461-473.
[52]魏树礼.生物药剂学与药物动力学.第一版.北京:北京医科大学出版社,1997.
[53] Rowland M, Tozer TN著,彭彬译。临床药动学。长沙:湖南科学技术出版社,1999。
[54] Matsumoto S, Yoshida K, Itogawa A, Tagawa M, Fujii T, Miyazaki H, Sekine Y Metabolism of [carbonyl-14C] mosapride citrate after a single oral administration in rats, dogs and monkeys. Arzneimittel-Forschung, 1993,43:10(1095-1102)
[55] Sun X, Niu L, Li X, Lu X, Li F. Characterization of metabolic profile of mosapride citrate in rat and identification of two new metabolites: Mosapride N-oxide and morpholine ring-opened mosapride by UPLC-ESI-MS/MS. J Pharm Biomed Anal. 2009 Aug 15;50(1):27-34.
[56] Yuan R, Madani S, Wei XX, Reynolds K, Huang SM. Evaluation of cytochrome P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions. Drug Metab Dispos. 2002, 30:1311-1319.
[57] Hickman D, Wang JP, Wang Y, Unadkat JD. Evaluation of the selectivity of In vitro probes and suitability of organic solvents for the measurement of human cytochrome P450 monooxygenase activities. Drug Metab Dispos. 1998, 26:207-215.
[58] Yan Z, Caldwell GW. Metabolism profiling, and cytochrome P450 inhibition & induction in drug discovery. Curr Top Med Chem. 2001 Nov;1(5):403-25.
[59]和凡。细胞色素P450酶活性评价的体外“cocktail”高通量筛选模型的建立和应用。中山大学博士学位论文。广州:中山大学临床药理研究所。2007,5。
[60]曾苏主编。药物代谢学。浙江大学出版社。2004。
[61]宋振玉主编。药物代谢研究意义、方法、应用。北京:人民卫生出版社。1990.3。
[62]郝琨、柳晓泉、王广基。临床前毒代动力学研究进展。中国药科大学学报,2004,35:195-199
[63] Funabashi H, Terada Y, Nakamura H, Shimazu H, Sugisawa K. Reproductive and developmental toxicity studies of mosapride citrate. (3) Perinnatal and postnatal study in rats. Japanese Pharmacology and Therapeutics, 1993, 21:10(3447-3468)
[64] Funabashi H, Terada Y, Nakamura H, Matsuoka T, Takigami H. Reproductive and developmental toxicity studies of mosapride citrate (4) - Teratogenicity study in rabbits. Japanese Pharmacology and Therapeutics, 1993, 21:10(147-157)
[65] Okimoto K, Toyosawa K, Matsuoka N, Ueda Y, Utsumi K, Iida M, Nakamura H, Umemura T. Toxicity study of mosapride citrate (II) - Thirteen week repeated oral dose toxicity study in rats. Japanese Pharmacology and Therapeutics, 1993, 21:10(29-56)
[66] Yatera S, Okimoto K, Koujitani T, Toyosawa K, Utsumi K, Iida M, Nakamura H. Toxicity study of mosapride citrate (I) - Single dose toxicity study in mice, rats and dogs.Japanese Pharmacology and Therapeutics, 1993, 21:10(21-28) ,
[67] Yatera S, Koujitani T, Yasuba M, Matsuoka N, Ueda Y, Utsumi K, Iida M, Nakamura H、Toxicity study of mosapride citrate (III) - Thirteen-week repeated dose oral toxicity study in beagle dogs..Japanese Pharmacology and Therapeutics, 1993, 21:10(57-73)
[68] Funabashi H, Nishimura K, Terada Y, Shigematsu K, Tateishi Y, Nakamura H. Reproductive and developmental toxicity studies of mosapride citrate (2) - Teratogenicity study in rats. Japanese Pharmacology and Therapeutics. 1993, 21:10(101-123)
[69] Funabashi H, Terada Y, Nakamura H, Shimazu H, Sugisawa K. Reproductive and developmental toxicity studies of mosapride citrate (3) - Perinatal and postnatal study in rats. Japanese Pharmacology and Therapeutics . 1993, 21:10(125-146)
[70] Okimoto K, Matsuoka N, Nakamura H, Mochizuki M, Okazaki S. Toxicity study of mosapride citrate (IV) - Twenty-six-week repeated oral dose toxicity study in rats. Japanese Pharmacology and Therapeutics, 1993, 21:10(75-87)