中国医药生物技术
中國醫藥生物技術
중국의약생물기술
CHINESE MEDICINAL BIOTECHNOLOGY
2012年
6期
401-411
,共11页
富岩*%马金玲*%孙丽%杜杰%张延林%徐楠楠%孙云霞%于在林
富巖*%馬金玲*%孫麗%杜傑%張延林%徐楠楠%孫雲霞%于在林
부암*%마금령*%손려%두걸%장연림%서남남%손운하%우재림
白蛋白类%干扰素 α%重组蛋白质类%药代动力学%毒理学
白蛋白類%榦擾素 α%重組蛋白質類%藥代動力學%毒理學
백단백류%간우소 α%중조단백질류%약대동역학%독이학
Albumins%Interferon-alpha%Recombinant proteins%Pharmacokinetics%Toxicology
目的对具有独立自主知识产权的注射用重组人血清白蛋白/干扰素α2a 融合蛋白新药的临床前药效学、药代动力学和安全性评价进行研究.
方法通过重组人血清白蛋白/干扰素α2a 融合蛋白(rHSA/IFNα2a)对 HepG22.2.15分泌乙型肝炎病毒(HBV)细胞作用产生的抗病毒进行体外药效学评价;以给药后的食蟹猴体内2'-5'寡腺苷酸合成酶活性作为干扰素药效学标志基因表达考察药效动力学指标;药代动力学观察了不同剂量的125I-rHSA/IFNα2a 单次皮下注射给予小鼠、大鼠后,各组织器官的分布、排泄情况及 rHSA/IFNα2a 单次和多次皮下注射给予食蟹猴后其体内的代谢情况;安全性评价中,安全药理学研究观察了 rHSA/IFNα2a 对小鼠中枢神经系统及对狗呼吸和心血管系统功能的影响;毒理学研究观察了rHSA/IFNα2a 单次注射给予小鼠(皮下和静脉)、食蟹猴(皮下)的毒性反应,及反复皮下注射给予大鼠和食蟹猴的毒性反应,食蟹猴反复给药的毒性试验中还伴行了毒代动力学的研究.妊娠 Wistar 大鼠皮下注射给予 rHSA/IFNα2a 对孕鼠生殖能力及胎仔的影响.
结果试验药物 rHSA/IFNα2a 和阳性对照品(PEG-rhIFNα2a)的最大无毒剂量为3.13×103 IU/ml,当以低于最大无毒剂量的 rHSA/IFNα2a 和阳性对照品处理 HepG22.2.15细胞,显示细胞分泌 HBV 病毒的能力受到抑制.细胞上清液中的 HBV 核酸量随时间而减少,HBV 的 s 抗原和 e 抗原表达也同步受到抑制,与 PEG-rhIFNα2a 对HBV 病毒分泌的相对抑制率检测结果相同.药代动力学研究结果显示静脉 rHSA/IFNα2a 给药组和对照组的 t1/2分别为(88.88±9.65)h 和(35.81±4.91)h;食蟹猴皮下注射末端半衰期较长为61.20~83.57 h.安全药理学研究表明单次皮下注射重组人血清白蛋白/干扰素α2a 融合蛋白在100、300、600μg/kg 剂量范围内对小鼠中枢神经系统无明显影响,与戊巴比妥钠无协同作用;在50、200μg/kg 剂量范围内对狗呼吸和心血管系统无明显影响.rHSA/IFNα2a单次皮下和静脉给予小鼠的最大耐受量≥50 mg/kg,单次皮下注射给予食蟹猴的耐受剂量≥10 mg/kg.以100、300和1000μg/kg 剂量反复皮下注射 rHSA/IFNα2a 给予大鼠,以50、150和500μg/kg 反复皮下注射给予食蟹猴,每周给药1次,连续13周,基本安全剂量分别为1000μg/kg和500μg/kg,注射局部仅见可逆的轻度刺激性反应;多次给药后,动物体内产生抗 rHSA/IFNα2a 和 IFNα2a 的抗体,且抗体具有中和 rHSA/IFNα2a 活性的作用,且停药4周后,抗体滴度明显降低.多次给药后由于中和抗体的产生,血浆中的抗原量低于检测限.单次给药食蟹猴各代谢动力学参数呈现线性动力学特征.当给药剂量高达1000μg/kg时未见孕鼠母体毒性和胎仔毒性,提示 rHSA/IFNα2a 无致畸作用.
结论获得的大量药效学、药代动力学、毒理学试验数据显示 rHSA/IFNα2a 具有较好的安全性、成药性和长效性,研究结果可供正在开展的临床试验研究参考.
目的對具有獨立自主知識產權的註射用重組人血清白蛋白/榦擾素α2a 融閤蛋白新藥的臨床前藥效學、藥代動力學和安全性評價進行研究.
方法通過重組人血清白蛋白/榦擾素α2a 融閤蛋白(rHSA/IFNα2a)對 HepG22.2.15分泌乙型肝炎病毒(HBV)細胞作用產生的抗病毒進行體外藥效學評價;以給藥後的食蟹猴體內2'-5'寡腺苷痠閤成酶活性作為榦擾素藥效學標誌基因錶達攷察藥效動力學指標;藥代動力學觀察瞭不同劑量的125I-rHSA/IFNα2a 單次皮下註射給予小鼠、大鼠後,各組織器官的分佈、排洩情況及 rHSA/IFNα2a 單次和多次皮下註射給予食蟹猴後其體內的代謝情況;安全性評價中,安全藥理學研究觀察瞭 rHSA/IFNα2a 對小鼠中樞神經繫統及對狗呼吸和心血管繫統功能的影響;毒理學研究觀察瞭rHSA/IFNα2a 單次註射給予小鼠(皮下和靜脈)、食蟹猴(皮下)的毒性反應,及反複皮下註射給予大鼠和食蟹猴的毒性反應,食蟹猴反複給藥的毒性試驗中還伴行瞭毒代動力學的研究.妊娠 Wistar 大鼠皮下註射給予 rHSA/IFNα2a 對孕鼠生殖能力及胎仔的影響.
結果試驗藥物 rHSA/IFNα2a 和暘性對照品(PEG-rhIFNα2a)的最大無毒劑量為3.13×103 IU/ml,噹以低于最大無毒劑量的 rHSA/IFNα2a 和暘性對照品處理 HepG22.2.15細胞,顯示細胞分泌 HBV 病毒的能力受到抑製.細胞上清液中的 HBV 覈痠量隨時間而減少,HBV 的 s 抗原和 e 抗原錶達也同步受到抑製,與 PEG-rhIFNα2a 對HBV 病毒分泌的相對抑製率檢測結果相同.藥代動力學研究結果顯示靜脈 rHSA/IFNα2a 給藥組和對照組的 t1/2分彆為(88.88±9.65)h 和(35.81±4.91)h;食蟹猴皮下註射末耑半衰期較長為61.20~83.57 h.安全藥理學研究錶明單次皮下註射重組人血清白蛋白/榦擾素α2a 融閤蛋白在100、300、600μg/kg 劑量範圍內對小鼠中樞神經繫統無明顯影響,與戊巴比妥鈉無協同作用;在50、200μg/kg 劑量範圍內對狗呼吸和心血管繫統無明顯影響.rHSA/IFNα2a單次皮下和靜脈給予小鼠的最大耐受量≥50 mg/kg,單次皮下註射給予食蟹猴的耐受劑量≥10 mg/kg.以100、300和1000μg/kg 劑量反複皮下註射 rHSA/IFNα2a 給予大鼠,以50、150和500μg/kg 反複皮下註射給予食蟹猴,每週給藥1次,連續13週,基本安全劑量分彆為1000μg/kg和500μg/kg,註射跼部僅見可逆的輕度刺激性反應;多次給藥後,動物體內產生抗 rHSA/IFNα2a 和 IFNα2a 的抗體,且抗體具有中和 rHSA/IFNα2a 活性的作用,且停藥4週後,抗體滴度明顯降低.多次給藥後由于中和抗體的產生,血漿中的抗原量低于檢測限.單次給藥食蟹猴各代謝動力學參數呈現線性動力學特徵.噹給藥劑量高達1000μg/kg時未見孕鼠母體毒性和胎仔毒性,提示 rHSA/IFNα2a 無緻畸作用.
結論穫得的大量藥效學、藥代動力學、毒理學試驗數據顯示 rHSA/IFNα2a 具有較好的安全性、成藥性和長效性,研究結果可供正在開展的臨床試驗研究參攷.
목적대구유독립자주지식산권적주사용중조인혈청백단백/간우소α2a 융합단백신약적림상전약효학、약대동역학화안전성평개진행연구.
방법통과중조인혈청백단백/간우소α2a 융합단백(rHSA/IFNα2a)대 HepG22.2.15분비을형간염병독(HBV)세포작용산생적항병독진행체외약효학평개;이급약후적식해후체내2'-5'과선감산합성매활성작위간우소약효학표지기인표체고찰약효동역학지표;약대동역학관찰료불동제량적125I-rHSA/IFNα2a 단차피하주사급여소서、대서후,각조직기관적분포、배설정황급 rHSA/IFNα2a 단차화다차피하주사급여식해후후기체내적대사정황;안전성평개중,안전약이학연구관찰료 rHSA/IFNα2a 대소서중추신경계통급대구호흡화심혈관계통공능적영향;독이학연구관찰료rHSA/IFNα2a 단차주사급여소서(피하화정맥)、식해후(피하)적독성반응,급반복피하주사급여대서화식해후적독성반응,식해후반복급약적독성시험중환반행료독대동역학적연구.임신 Wistar 대서피하주사급여 rHSA/IFNα2a 대잉서생식능력급태자적영향.
결과시험약물 rHSA/IFNα2a 화양성대조품(PEG-rhIFNα2a)적최대무독제량위3.13×103 IU/ml,당이저우최대무독제량적 rHSA/IFNα2a 화양성대조품처리 HepG22.2.15세포,현시세포분비 HBV 병독적능력수도억제.세포상청액중적 HBV 핵산량수시간이감소,HBV 적 s 항원화 e 항원표체야동보수도억제,여 PEG-rhIFNα2a 대HBV 병독분비적상대억제솔검측결과상동.약대동역학연구결과현시정맥 rHSA/IFNα2a 급약조화대조조적 t1/2분별위(88.88±9.65)h 화(35.81±4.91)h;식해후피하주사말단반쇠기교장위61.20~83.57 h.안전약이학연구표명단차피하주사중조인혈청백단백/간우소α2a 융합단백재100、300、600μg/kg 제량범위내대소서중추신경계통무명현영향,여무파비타납무협동작용;재50、200μg/kg 제량범위내대구호흡화심혈관계통무명현영향.rHSA/IFNα2a단차피하화정맥급여소서적최대내수량≥50 mg/kg,단차피하주사급여식해후적내수제량≥10 mg/kg.이100、300화1000μg/kg 제량반복피하주사 rHSA/IFNα2a 급여대서,이50、150화500μg/kg 반복피하주사급여식해후,매주급약1차,련속13주,기본안전제량분별위1000μg/kg화500μg/kg,주사국부부견가역적경도자격성반응;다차급약후,동물체내산생항 rHSA/IFNα2a 화 IFNα2a 적항체,차항체구유중화 rHSA/IFNα2a 활성적작용,차정약4주후,항체적도명현강저.다차급약후유우중화항체적산생,혈장중적항원량저우검측한.단차급약식해후각대사동역학삼수정현선성동역학특정.당급약제량고체1000μg/kg시미견잉서모체독성화태자독성,제시 rHSA/IFNα2a 무치기작용.
결론획득적대량약효학、약대동역학、독이학시험수거현시 rHSA/IFNα2a 구유교호적안전성、성약성화장효성,연구결과가공정재개전적림상시험연구삼고.
Objective The evaluation of pharmacodynamics, pharmacokinetics and safety in animals of recombinant human serum albumin/interferon-α2a fusion protein (rHSA/IFNα2a) with independent intellectual property rights.
@@@@Methods For pharmacodynamics study, in vitro antiviral effect of rHSA/IFNα2a on inhibiting the secretion of hepatitis B virus (HBV) from HepG2 2.2.15 cells was evaluated. Our study also examined the bioactivity of interferon-specific pharmacodynamic marker enzyme (2-5oas) after injection of rHSA/IFNα2a in cynomolgus monkeys. For pharmacokinetics study, the distribution and excretion of 125I-rHSA/IFNα2a in different tissues and organs was examined by single subcutaneous injection with different doses in mice and rats. Metabolism in vivo was further performed in cynomolgus monkeys with single and multiple doses. For safety study, effects of rHSA/IFNα2a on the central nervous system in mice as well as the respiratory and cardiovascular system in Beagle dogs were observed. Through acute and chronic toxicity study, the safety evaluation of rHSA/IFNa2a was performed in mice, rats and cynomolgus monkeys with single and repeated doses. The pharmacokinetics/toxicokinetics study was performed to examine the PK parameters. The reproductive toxicity of rHSA/IFNα2a was also evaluated in pregnant Wistar rats and fetuses.
@@@@Results The maximum non-toxic dose for rHSA/IFNα2a and the positive control, PEG-rhIFNα2a (Pegasys), was 3.13 × 103 IU/ml. The secretion of HBV particles was inhibited when the HepG2 2.2.15 cells were treated with a dose lower than the maximum non-toxic amount of rHSA/IFNα2a, and the level of HBV nucleic acid was reduced through the different time periods tested. The expression of HBsAg and HBeAg from HepG2 2.2.15 cells were also inhibited. All the results in vitro were consistent with the relative suppression rate of PEG-rhIFNα2a. Pharmacokinetic study in cynomolgus monkeys through intravenous administration showed the t1/2 was (88.88 ± 9.65) h and (35.81 ± 4.91) h in the rHSA/IFNα2a group and the PEG-rhIFNα2a control group, respectively. When administered through subcutaneous dosing of rHSA/IFNα2a, the t1/2 was 61.20~83.57 h. Safety pharmacology study showed that no apparent effects of rHSA/IFNα2a on the central nervous system of mice were observed at 100, 300 and 600μg/kg doses, and no synergy effects were found when combined with pentobarbital sodium. Also, no apparent effects of the drug were found on the respiratory and cardiovascular systems of Beagle dogs by single subcutaneous injection at 50μg/kg and 200μg/kg. In the single-dose toxicity study, the maximum tolerated dose (MTD) in mice was≥50 mg/kg (i.v. or s.c.) and the MTD in monkeys was ≥10 mg/kg (s.c.). The basic safe doses were 1000μg/kg and 500μg/kg in rats and in cynomolgus monkeys, respectively, after treated subcutaneously with rHSA/IFNα2a once a week for 13 consecutive weeks. A reversible mild irritant reaction was observed in the injection site and anti-IFNα antibodies were produced after repeated administration. These antibodies neutralized the rHSA/IFNα2a activity in vitro. Linear metabolic kinetics characteristics in cynomolgus monkeys were found after a repeat-dose study. When the monkeys were repeatedly treated with rHSA/IFNα2a, the antigen was not detected due to neutralization of the antibodies induced after administration. No toxicity was observed in pregnant rats and fetuses when administered dose increased up to 1000μg/kg, indicating no teratogenic effects of rHSA/IFNα2a.
@@@@Conclusion The data obtained from the studies on pharmacodynamics, pharmacokinetics and toxicity showed that rHSA/IFNα2a is safe and druggabe with long-acting effects. This work provided information for the ongoing human clinical trials.
