注射用重组人血清白蛋白/促红素融合蛋白的药效学、药代动力学及安全性评价研究

摘要

目的 对拥有独立自主知识产权的世界原创原研注射用重组人血清白蛋白/促红素融合蛋白(rHSA/EPO)创新药开展临床前药效学、药代动力学和安全性评价研究.方法 通过对动物体内外试验研究,观察rHSA/EPO升红细胞(网织红细胞)作用.药效学研究观察rHSA/EPO对小鼠、食蟹猴以及不同的大鼠肾性贫血模型的红细胞生成影响.药代动力学研究则是对rHSA/EPO不同剂量、单次、多次给药大鼠与食蟹猴后的药代/毒代进行研究.毒理学研究通过rHSA/EPO给予小鼠、大鼠和食蟹猴后,观察其一般毒性以及生殖毒性等反应,从而对安全性做出评价.结果 药效学研究结果显示rHSA/EPO可刺激小鼠、大鼠和食蟹猴生成红细胞,升高网织红细胞计数(Ret%),同时对药物或肾切除造成损伤所致的肾性贫血大鼠模型,rHSA/EPO每周或者每2周给药一次,与阳性对照药rhEPO每周给药3次均显示出显著的治疗作用,且较rhEPO具有明显的长效性.药代动力学研究结果显示rHSA/EPO在食蟹猴体内的半衰期平均值约为53.34 h,比rhEPO的平均半衰期7.16 h要长,rHSA/EPO不与血浆蛋白结合,不易透过血脑屏障,主要排泄器官为肾,在动物体内的吸收和消除均慢于rhEPO.毒理学研究结果显示rHSA/EPO以100、300、1000μg/kg单次皮下注射给予ICR小鼠,对其中枢神经系统功能无影响,以25、100、400μg/kg单次皮下注射给予食蟹猴,对其呼吸与心血管系统无明显影响,单次皮下注射给予SD大鼠和食蟹猴未见明显毒性反应,最大耐受剂量分别为10 mg/kg和4 mg/kg,rHSA/EPO未见胚胎毒性和致畸性.食蟹猴及大鼠重复给药毒性试验可见与药理学作用相关的即红细胞增多引起的血液生化、骨髓造血和(或)胃肠、肾脏的一些变化,停药后可见恢复趋势,给药后3周可产生抗rHSA/EPO的结合抗体IgG.大鼠体内产生的抗体较食蟹猴的强,与受试物中含有HSA相关,因而在大鼠重复给药毒性试验中也就出现由于抗体的产生中和了体内的EPO,进而可见严重贫血现象.结论 rHSA/EPO具有显著的升红作用,表现出长效特征,总体安全性好,毒性表现与常规rhEPO产品相似,没有出现新的不良反应.研究结果可为rHSA/EPO临床试验研究提供参考,并具有重要的指导意义.%Objective To evaluate and study in animals for the efficacy, pharmacokinetics and safety of recombinant human serum albumin/erythropoietin fusion protein as a bio-better innovation long-acting drug in pre-clinic. Methods The increase of red blood cells for rHSA/EPO was observed byin vitro andin vivo animal experinments. Effects of rHSA/EPO on the erythropoiesis in mice, cynomolgus monkeys and in different renal anemia rat models were studied from pharmacodynamics. Pharmacokinetics studies were conducted to study the kinetics of rHSA/EPO in rats and cynomolgus monkeys after different doses, single dose, multiple drug administration. The general toxicity and reproductive toxicity of rHSA/EPO in mice, rats and cynomolgus monkeys were studied, and the safety evaluation was made. Results The results from pharmacodynamic studies showed that rHSA/EPO stimulatee mice, rats and monkeys to erythropoiesis, increased reticulocyte count Ret%. At the same time, rHSA/EPO and rhEPO (the positive contro) both exerted obvious curative effect and rHSA/EPO showed more long-acting than rhEPO did in anemia rat model caused by drugs or nephrectomy at rHSA/EPO administered every 1 weeks or once every 2 weeks and rhEPO administered 3 times weekly. From pharmacokinetic studies, the average half-life of rHSA/EPO in the cynomolgus monkeys was about 53.34 hours, much longer than the average half-life of rhEPO at 7.16 hours. rHSA/EPO did not bind to plasma proteins, not easily penetrated the blood-brain barrier, and the main excretory organ was kidney. Furthermore, its body absorption and elimination in animals were slower than the rhEPO. The results from toxicological studies displayed that there was no effects on the central nervous system function after rHSA/EPO was given subcutaneously at the single dose of 100 μg/kg, 300 μg/kg, 1000 μg/kg in ICR mice and no obvious effect on the respiratory and cardiovascular system after cynomolgus monkeys subcutaneously administered with a single dose of 25 μg/kg and 100 μg/kg, 400 μg/kg. No obvious toxicity was observed in SD rats and cynomolgus monkeys subcutaneously given a single injection and then the maximum tolerated dose were 10 mg/kg and 4 mg/kg, respectively. rHSA/EPO showed no embryo toxicity and teratogenicity. The changes of the blood biochemistry, bone marrow and/or gastrointestine, kidney in cynomolgus monkeys and rats with repeated dose toxicity test were associated with pharmacological effects on erythropoiesis and showed recovery trend after drug withdrawal. Anti-rHSA/EPO antibody IgG was produced for 3 weeks after the drug given. Antibodies in rats were produced more easily than that in monkeys because rHSA/EPO contained HSA, for which severe anemia was also occurred due to antibody neutralizing EPO in repeated dose toxicity study. Conclusions rHSA/EPO increases the reticulocytes with the long-acting characteristics of the overall good safety. Furthermore, its toxicity is similar with conventional rhEPO products without new adverse reactions. The results of the studies can provide references and important guiding significances for the clinical trial of rHSA/EPO.