摘要
促红细胞生成素(EPO)是由肾脏分泌的一种活性糖蛋白,能与表达于红细胞表面的EPO受体结合,刺激骨髓中红系造血母细胞的增殖和分化。近年来研究显示,EPO是具有多种活性的生长因子,EPO—RmRNA,EPO—R蛋白,EPO与EPO·R结合广泛存在于大脑,心血管组织,肝脏,胃肠道组织,胰岛等非红细胞生成细胞与器官。自从1989年美国正式批准基因工程药rHuEPO进入市场以来,重组人促红细胞生成素(rHuEPO)已广泛用于临床,治疗各种类型的贫血病。但市场上的各种rHuEPO药物的生物半衰期短(4—8小时),临床使用注射次数多。目前新制剂的发展方向为拉长其半衰期,主要着眼于氨基酸的置换或者氨基酸的修改,增加糖链或者两个独立的EPO片段的连接,这样就延长了rHuEPO生物半衰期,患者由每周两三次注射改变为每周一次或者两周一次,大大减轻了患者的痛苦。由国内研究机构研制的新型长效促红细胞生成素(LL-rHuEPO),目前正拟申报此类新药。研究该药物在大鼠体内的药代动力学规律,阐明药代动力学特点,提供重要的药代动力学参数,为过渡到临床研究提供必要的依据。本研究采用~(125)I同位素示踪法研究LL-rHuEPO不同给药途径及不同给药剂量下在大鼠体内的药代动力学、组织分布以及排泄规律,所得结果如下:
1.LL-rHuEPO在大鼠体内的药代动力学研究:
单次静脉注射1,2,3ug/kg~(125)I—LL-rHuEPO后,通过统计矩的方法计算所得的主要药代动力学参数是:血药浓度—时间曲线下面积(AUC_((0-48h)))分别为:192.137,329.434,641.622(ug/L/h);Cmax分别为:12.1,18.874,29.398(ug/L)。AUC、Cmax与剂量呈线性关系。消除半衰期t1/2z分别为:20.457h,25.053h,20.409h。
单次肌肉注射1,2,3 ug/kg~(125)I—LL-rHuEPO后,通过统计矩的方法计算所得的主要药代动力学参数是:清除率CLz/F从0.001到0.006 L/h/kg;消除半衰期t1/2z从25.258h到287.91h,但多数维持在40—60之间;药物在体内的平均驻留时间MRT(0-t)基本一致,范围从33.664h到34.883h;Cmax与AUC(0-72h)表现与剂量呈明显的线性关系。
2.LL-rHuEPO在大鼠体内的组织分布:
SD大鼠经肌肉注射2ug/kg~(125)I—LL-rHuEPO,测定不同时间不同组织的药物浓度。给药后8h、24h、48h,LL-rHuEPO主要分布在注射部肌肉,血浆,骨髓中,骨髓是其主要作用部位。血流量大的组织:心,肾,肝等次之,脑,小肠,大肠等分布最少。
3.LL-rHuEPO在大鼠体内的排泄:
SD大鼠静脉注射2ug/kg~(125)I—LL-rHuEPO后11天内,粪、尿的总排泄量为22.09%,其中尿累积量占20.7%,而粪只占1.39%。
结论
1.大鼠静脉注射1,2,3ug/kg LL-rHuEPO后,消除半衰期20-25h之间。
2.大鼠肌肉注射1,2,3ug/kg的LL-rHuEPO后,消除半衰期30-60h之间。
3.LL-rHuEPO的消除半衰期明显长于rHuEPO。
4.LL-rHuEPO肌肉注射生物利用度较高(约81%)。
5.大鼠肌肉注射2ug/kg的LL-rHuEPO后组织分布广泛,骨髓是其主要作用部位。
6.LL-rHuEPO主要经肾由尿排泄,在体内经受广泛的代谢转化为代谢物而消除。
Erythropoietin (EPO) is a glycoprotein secreted by the kidneys, which can bind to EPO receptor expressed in the surface of red blood cells, and stimulate the proliferation and differentiation of the red blood cells in the bone marrow. Previous studies show that EPO is one of growth factors with mutil-functions. EPO-RmRNA, EPO-R protein and EPO-EPO·R are widely distubited in the non Erythropoietin cells and organs such as brain, cardiovascular, liver, gastrointestinal tract, insulin. Since the United States formally approved the drug (recombinant Human EPO -rHuEPO) in 1989, rHuEPO has been widely used in clinical treatment of various types of anemia. However, the drug of rHuEPO in current market has short half-life (4-8 hours) and mutil-injections are need in clinical practiacal. Therefore, in order to reduce the suffering of the patients caused by mutil-injection the development of a new rHuEPO with longer biological half-life are needed. Recently, a long-lasting recombinant human erythropoietin (LL-rHuEPO) was newly developed by a research institute of China. The aim of the present study is to study the Preclinical pharmacokinetics, distribution and excretion of the newly developed LL-rHuEPO in rats using ~(125)I isotope tracing techniques. The results are as follows:
1. Pharmacokinetics of LL-rHuEPO in rats:
After single iv injection (1, 2, 3 ug/kg LL-rHuEPO) in rats, the main pharmacokinetic parameters were taken by the method of calculating statistics: AUC (0-48h) was: 192.137, 329.434, 641.622 (ug / L / h); Cmax was: 12.1, 18.874, 29.398 (ug / L). AUC, Cmax are dose related. t_(1/2z) was: 20.457 h, 25.053h, 20.409h.
After single im injection(1,2,3 ug/kg LL-rHuEPO) in rats, the main pharmacokinetics parameters were as follows: CLz / F from 0.001到0.006 L / h / kg; t_(1/2z) from 25.258 to 287.91 hours, but most remain at between 40 to 60hours; MRTs (0-72h) are the similar among 3 different treatments, ranging from 33.664 to 34.883 hours with dose-related and dose-proportional increases of Cmax and AUC ( 0-72h) values.
2. Distribution of LL-rHuEPO in rats:
LL-rHuEPO concentration in different tissue at the certain time after im administration (at the dose of 2ug/kg) was mensurated. After administration of 8h, 24h, 48h: LL-rHuEPO are mainly distributed in the muscle of injection site, plasma, bone marrow. Much less of LL-rHuEPO were found in the tissue of heart, kidney, liver, and .the tissue of brain, small intestine and large intestine with the least LL-rHuEPO. 3. Excretion of LL-rHuEPO in rats:LL-rHuEPO was given to SD rats by iv administration at the dose of 2ug/kg. About 22.09% of the injected amount was excreted in feces and urine within 11das, of which 20.7%, 1.39% was excreted in urine and feces, respectively.
Conclusions:
1. After iv administration of LL-rHuEPO to rats (at the dose of 1, 2, 3 ug / kg, respectively), its elimination half-life was 20h to 25h.
2. After im administration of LL-rHuEPO to rats (at the dose of 1, 2, 3 ug / kg, respectively), its elimination half-life mainly changed from 30h to 60h.
3. The elimination half-life of LL-rHuEPO was obviously longer than rHuEPO.
4. Bioavailability of LL-rHuEPO im injection was high (about 90%).
5. Distribution of LL-rHuEPO following im administration in rats was extensive. Bone marrow was its main functionary poisition.
6. LL-rHuEPO was mainly excreted by urine via kidney. And it could experience widely metabolism and could be eliminated by transforming metabolites.LL-rHuEPO;; isotope tracer;; pharmacokinetics;; distribution;; excretion
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