A novel synthetic derivative of human erythropoietin designed to bind to glycosaminoglycans

To synthesize long-acting and antiangiogenic erythropoietin to be clinically applied for treatment of patients with solid tumors, we synthesized a hybrid molecule of human erythropoietin added onto the C-terminus with a heparin-binding motif of human PLGF-2 to develop a novel derivative of long-acting and antiangiogenic erythropoietin: heparin-binding erythropoietin (HEPO), and studied the characteristics of this novel erythropoietin derivative. HEPO cDNA was synthesized, expressed in insect cells, and the protein was purified using a heparin-sepharose affinity column. The erythropoietic and angiogenic effects of the partially purified protein were analyzed in vitro and in vivo. The erythropoietic activity of the protein was equivalent to natural EPO in vitro. In vivo administration of the protein to mice revealed its long-acting erythropoietic activity as expected. Administration of the protein inhibited angiogenesis in a mouse limb ischemia model. In conclusion, the heparin-binding motif of PLGF-2 may act as, so to speak, a superendostatin. This novel long-acting erythropoietin derivative may have an advantage to inhibit tumor growth while preserving hematopoietic and tissue-protective effects.     

Pharmacological testing of recombinant human erythropoietin: Implications for other biotechnology products

The development of recombinant DNA and large-scale cell culture technologies has introduced a wide range of biotechnology products or “biologics” including cytokines, vaccines, peptides, hormones, growth factors, and monoclonal antibodies that are being used in the diagnosis, prevention, and treatment of various diseases. As with any new drug development program, pharmacologic evaluation is critical to an understanding of that new drug entity. A well-designed general/safety pharmacology program contribues to the overall safety assessment of that new entity, provides important information to the design of early clinical trials, assists in resolving any issues that arise during preclinical or clinical development, and assists in deciding whether to continue its development. This has been the case for many small molecular weight compounds and is no less important for the new biotechnology products. Because of their complex structural and biological characteristics, biotechnology products present unusual challenges in their development, resulting in pharmacology programs designed on a “case by case” basis. The pharmacologic evaluation of r-HuEPO may well serve as an model for understanding some of the potential issues associated with the development of biologics. With r-HuEPO being marketed worldwide for approximately five years, clinical data are available to assess the predictive value of the pharmacology studies. An analysis of these data serves as a guide to determine which preclinical studies best reflect the clinical data. Appropriate design of a pharmacology program depends on an understanding of the structure of the molecule, the mechanism of action at the cellular level, species specificity, and the clinical plan. On this basis a program can be developed to accurately assess the potential non-pharmacologic effects and ultimately establish a benefit-to-risk ratio. © 1995 Wiley-Liss, Inc.     

多剂量皮下注射重组人促红细胞生成素在健康人体的药代动力学

目的　观察不同剂量国产重组人促红细胞生成素(rhEPO)皮下注射在健康人体的药代动力学。方法　12名健康志愿者随机分为2个剂量组(50和150IU·kg-1 ),每组6例。分别皮下注射rhEPO,共7次。用酶联免疫吸附实验法测定血清rhEPO浓度。结果　给药前12名健康志愿者血清EPO基础水平波动于3. 75~13. 75mU·ml-1。2组谷浓度(Cmin)在给药4~6次后,达稳态水平。高剂量组首次和末次给药后的AUC(0-96h)分别是低剂量组的2. 8倍和3. 0倍,Cmax分别是3. 1倍和3. 6倍。第1次给药后,每个受试者的tmax和t1 /2等药代动力学参数与末次给药后比较无明显差异。结论　剂量在50~150IU·kg-1,多次皮下注射rhEPO,血药浓度水平与给药剂量呈正相关;药物吸收和清除基本上无时间依赖性;在给药7次内,未观察到体内蓄积倾向。     

rhEPO对光损伤诱导的RPE细胞凋亡的抑制

目的研究重组人促红细胞生成素(rhEPO)在人视网膜色素上皮(RPE)细胞光化学损伤中的保护作用及其作用机制,为年龄相关性黄斑变性等的药物治疗和预防提供理论依据。方法取成人ARPE-19细胞株传代培养的2~5代细胞建立光损伤模型,光照12h后再培养24h终止培养,采用AnnexinV-FITC/PI流式双染法检测不同浓度的rhEPO干预治疗前后RPE细胞凋亡的变化;采用酶联免疫吸附实验(enzyme linked immunosorbant assay,ELISA)及免疫组织化学法检测不同浓度的rhEPO干预治疗前后RPE细胞caspase-3及Bcl-2表达的变化;并添加AG490(Jak2激酶抑制剂),探讨人rhEPO对人RPE细胞光化学损伤的保护性作用机制。结果各rhEPO组均可明显减少光化学损伤诱导的人RPE细胞的凋亡,呈浓度依赖性,以40IU·ml-1EPO组结果最明显,为(4.93±1.45)·ml-1;在40IU·ml-1EPO组caspase-3浓度最低,为(0.125±0.029)μg·L-1;在40IU·ml-1EPO组Bcl-2表达最高(168.21±3.87);在加入AG490组,人RPE细胞凋亡增高为(11.29±2.11)·ml-1;caspase-3浓度增高为(0.362±0.042)μg.L-1;Bcl-2表达降低。rhEPO抑制凋亡作用均被阻止。结论rhEPO可抑制光化学损伤诱导的人RPE细胞的凋亡,抑制caspase-3的浓度,上调Bcl-2的表达,对人RPE细胞的光化学损伤有保护治疗作用;其保护作用机制主要通过EPO与受体结合后激活Jak2激酶途径完成的。     

重组人促红细胞生成素在肿瘤相关性贫血中的应用

贫血是肿瘤患者的常见合并症, 既往治疗肿瘤相关性贫血多用输血,存在诸多缺点及风险.近年来重组人促红细胞生成素在肿瘤相关性贫血中的应用日益受到重视.现简述重组人促红细胞生成素的药物类别、适应证、用法用量、临床应用、缺点、不良反应、使用现状及应用限制等.     

腺相关病毒介导的人红细胞生成素体外表达

目的:研究腺相关病毒载体介导的人红细胞生成素在293细胞的体外表达Ⅰ方法:将人红细胞生成素cDNA克隆至AAV表达载体,采用无辅助病毒共转染法生产重组AAV-EPO病毒,利用RT-PCR和ELISA法检测hEpo的体外表达.结果:成功构建和生产了表达hEpo的重组AAV-EPO病毒.重组AAV-EPO病毒感染293细胞后,RT-PCR结果显示有hEpo基因表达,细胞上清中hEpo的表达水平达500U/L.结论:所制备的重组AAV-EPO病毒可以有效表达hEpo,可以进一步用于动物体内实验.     

肌肉注射裸DNA后人红细胞生成素在小鼠体内的表达

目的研究肌肉注射裸DNA以后 ,人红细胞生成素 (hEPO)在小鼠体内的表达。方法利用PCR技术扩增hEPOcDNA ,构建真核表达质粒pAAV EPO ,小鼠肌肉注射后 ,ELISA和血红细胞压积法测定hEPO的体内表达。结果小鼠血清hEPO浓度和血红细胞压积明显高于对照组。结论裸DNA直接肌肉注射是有效的基因治疗手段之一。     

早产儿注射促红细胞生成素对贫血的防治效果及护理

目的观察重组人类促红细胞生成素（recombinant human erythropoietin,rh—EPO）对早产儿贫血的防治效果。方法将早产后在我科住院的胎龄小于34周,体质量低于2000g的新生儿50例,按入院顺序随机分成两组,治疗组25例,对照组25例。两组患儿入院后常规给予保暖,维持体温、血糖、血压等内环境稳定,营养支持等处理,必要时输血。治疗组于生后第7天开始给予重组人类促红细胞生成素200IU／（kg·d）,皮下注射,每周3次,共4周。对照组仅用常规治疗。分别于生后第1、2、3、4、5周抽取外周静脉血,检测并比较不同时间两组早产儿的血红蛋白（haemoglobin,Hb）、网织红细胞（reticulocyte,Ret）和血细胞比容（hematocrit,HCT）。结果两组早产儿出生后血红蛋白均下降,但治疗组下降缓慢,治疗结束后两组差异非常显著（P〈0．01）;治疗组治疗后网织红细胞计数和血细胞比容较对照组明显升高（P〈0．01）;治疗结束后两组网织红细胞计数差异缩小（P〉0．05）,但血细胞比容差异仍显著（P〈0．01）;治疗组的输血率（8％）较对照组的输血率（32％）明显减少（P〈0．05）。结论重组人类促红细胞生成素可以有效的防治早产儿贫血,减少输血。     

Anti-inflammatory effects of a novel peptide designed to bind with NF-κB p50 subunit

AIM: To explore the anti-inflammatory effects of a novel peptide designed to bind with the NF-kappaB p50 subunit. METHODS: The affinity of the peptide binding with p50 was measured with a biosensor. Levels of tumor necrosis factor-alpha(TNF-alpha) and interleukin 6 (IL-6) from a human acute monocytic leukemia cell line (THP-1) treated with lipopolysaccharide (LPS) were measured using the ELISA method. In vivo anti-inflammatory effects of the peptide were tested with phorbol myristate acetate (PMA)-induced ear edema and zymosan A-induced peritonitis in mice. RESULTS: The peptide has the ability to interact with the NF-kappaB p50 subunit and can effectively inhibit TNF-alpha and IL-6 production in the THP-1 cell line, PMA-induced ear edema and zymosan A-induced peritonitis in mice. CONCLUSION: The peptide may have therapeutic potential for the treatment of local acute inflammation.     

Eph受体酪氨酸激酶A2和金属蛋白酶2在大肠癌组织的表达及其与大肠癌发生发展的关系

目的 探讨Eph受体酪氨酸激酶A2(EphA2)和金属蛋白酶2(MMP-2)在大肠癌组织中的表达及其与临床病理特征和预后关系.方法 采用免疫组织化学法检测76例大肠癌患者手术标本中癌组织和癌旁组织的EphA2蛋白和MMP-2蛋白表达情况.结果 EphA2蛋白在大肠癌组织中的表达概率明显高于癌旁组织(92.10%比26.31%,P<0.05),MMP-2蛋白在大肠癌组织中的表达概率同样明显高于癌旁组织(80.20%比15.90%,P<0.05);EphA2蛋白表达水平与组织分化程度大肠癌、Dukes分期、淋巴结转移、年龄密切关联;MMP-2蛋白表达水平与大肠癌组织学分化程度、Dukes分期、淋巴结转移、年龄密切关联.结论 EphA2蛋白和MMP-2蛋白在大肠癌组织中的高表达可能在大肠癌的发生、发展过程中发挥重要作用.     

Stabilization and encapsulation of recombinant human erythropoietin into PLGA microspheres using human serum albumin as a stabilizer

The aim of this study was to prepare recombinant human erythropoietin (rhEPO) loaded poly(lactic-co-glycolic acid) (PLGA) microspheres using human serum albumin (HSA) as a stabilizer. Prior to encapsulation, the rhEPO-HSA mixture microparticles were fabricated using a modified freezing-induced phase separation method. The microparticles were subsequently encapsulated into PLGA microspheres. Process optimization revealed that the polymer concentration in the organic phase and the sodium chloride (NaCl) concentration in the outer water phase of the s/o/w emulsion played critical roles in determining the properties of the resultant microspheres. An in vitro release test showed that rhEPO was released from PLGA microspheres in a sustained manner up to 30 days. A single injection of rhEPO-loaded PLGA microspheres in Sprague-Dawley rats resulted in elevated hemoglobin and red blood cell concentrations for about 33 days. The stability of the rhEPO within the PLGA microspheres was systematically investigated by size-exclusion high-performance liquid chromatography (SEC-HPLC), SDS-PAGE, western blot and in vivo biological activity assay. The stability of rhEPO released from rhEPO-loaded microspheres was also examined by western blot. The results suggested that the integrity of rhEPO was successfully protected during the encapsulation process and the release period from polymeric matrices.     

维生素A联合重组人促红细胞生成素预防早产儿贫血的疗效

目的在给予重组人促红细胞生成素的基础上,观察口服维生素A预防早产儿贫血的效果。方法选取120例早产儿,分成3组,对照组给予常规治疗,观察A组给予重组人促红细胞生成素(rHu-EPO)及维生素D滴剂,观察B组给予重组人促红细胞生成素及维生素AD滴剂。3组患儿均在出生后第14天开始给予维生素E、赖氨肌醇维生素B12及依地酸铁钠口服溶液,疗程共4周;对比3组早产儿治疗前后体质量、血红蛋白(Hb)、红细胞比容(Hct)、血清铁蛋白(SF)及网织红细胞(Ret)的变化情况,统计其输血例数。结果治疗后,患儿Hb、Hct、Ret水平及输血例数各组间均有显著差异(P<0.05);其SF水平观察B组高于观察A组及对照组(P<0.05),观察A组与对照组无显著差异;观察组W水平显著高于对照组(P<0.05),但观察A组及B组间差异不明显。结论在给予重组人促红细胞生成素的基础上加用维生素A能有效地预防早产儿贫血。     

重组人类促红细胞生成素防治早产儿贫血的临床研究

目的　应用不同剂量国产重组人类促红细胞生成素 (rhu EPO)防治早产儿贫血 ,探讨其最适剂量和疗效。方法　 83例孕周 <36周 ,出生体重 <2 5 0 0克早产儿 ,以入院顺序按rhu EPO剂量随机分为第 1组 2 5例 (rhu EPO 75 0IU·kg-1·w-1) ,第 2组 18例 (45 0IU·kg-1·w-1) ,第 3组 2 0例(30 0IU·kg-1·w-1) ,分为每周 3次 ,静脉注射 ,疗程 4周。另设第 4组 2 0例为对照组。结果　 4组早产儿生后血红蛋白 (Hb)、红细胞压积 (Hct)渐行下降。第 1组下降最轻 ,第 2组次之 ,对照组最明显 ,经方差分析有显著性差异 (P <0 0 1) ;网织红细胞 (Ret)明显增高 ,且Ret升高程度和rhu EPO剂量有关 ;治疗后血清EPO浓度以第 1组最高 ,对照组最低 ,有显著性差异 (P <0 0 1) ,但第 3组与对照组比较无显著性差异 (P >0 0 5 ) ;血清铁 (Fe)生后均逐渐下降 ,治疗各组略低于对照组 ,无显著性差异。结论　早期rhu EPO治疗可提高Hb、Hct、Ret,并且疗效和剂量有关 ,体内充足的铁储备是确保rhu EPO疗效的重要因素。     

重组人红细胞生成素糖基化与生物活性关系的研究

目的　研究重组人红细胞生成素糖基化与生物活性的关系。方法　采用间苯二酚显色法测定rhEPO的唾液酸含量 ;网织红细胞法测定rhEPO的生物活性 ;并对二者进行相关性分析。结果　当唾液酸 EPO小于 8.2 8时 ,其生物比活性低于12 0 0 0 0IU mg。结论　重组人红细胞生成素的唾液酸含量高低与其生物比活性具有显著正相关性 (r=0 .64 92 79)。     

Mixed-effects modelling of the interspecies pharmacokinetic scaling of pegylated human erythropoietin

The aim of this study was to develop a population pharmacokinetic model for interspecies allometric scaling of pegylated r-HuEPO (PEG-EPO) pharmacokinetics to man. A total of 927 serum concentrations from 193 rats, 6 rabbits, 34 monkeys, and 9 dogs obtained after a single dose of PEG-EPO, administered by the i.v. (dose range: 12.5–550 μg/kg) and s.c. (dose range: 12.5–500 μg/kg) routes, were pooled in this analysis. An open two-compartment model with first-order absorption and lag time (Tlag) and linear elimination from the central compartment was fitted to the data using the NONMEM V software. Body weight (WT) was used as a scaling factor and the effect of brain weight (BW), sex, and pregnancy status on the pharmacokinetic parameters was investigated. The final model was evaluated by means of a non-parametric bootstrap analysis and used to predict the PEG-EPO pharmacokinetic parameters in healthy male subjects. The systemic clearance (CL) in males was estimated to be 4.08WT1.030 × BW−0.345 ml/h. In females, the CL was 90.7% of the CL in males. The volumes of the central (Vc) and the peripheral (Vp) compartment were characterized as 57.8WT0.959 ml, and 48.1WT1.150 ml, respectively. Intercompartmental flow was estimated at 2.32WT0.930 ml/h. Absorption rate constant (Ka) was estimated at 0.0538WT−0.149. The absolute s.c. bioavailability F was calculated at 52.5, 80.2, and 49.4% in rat, monkey, and dog, respectively. The interindividual variability in the population pharmacokinetic parameters was fairly low (<35%). Non-parametric bootstrap confirmed the accuracy of the NONMEM estimates. The mean model predicted pharmacokinetic parameters in healthy male subjects of 70 kg were estimated at: CL: 26.2 ml/h; Vc: 3.6 l; Q: 286 l/h; Vp: 6.9 l, and Ka: 0.031 h⁻¹. The population pharmacokinetic model developed was appropriate to describe the time course of PEG-EPO serum concentrations and their variability in different species. The model predicted pharmacokinetics of PEG-EPO in humans suggest a less frequent dosing regimen relative to erythropoietin and darbepoetin, potentially leading to a simplification of anemia management.     

重组人促红素治疗1例肿瘤相关性贫血分析

1例原发性支气管肺癌患者,入院行第4次化疗。入院后血常规提示存在中度贫血,基于前3次化疗后血红蛋白持续下降的情况,医生认为该患者目前不宜化疗。为确定贫血的治疗方案,医生咨询了临床药师,药师查阅文献后建议选用重组人促红素（recombinant human erythropoietin,rhEPO）治疗贫血,待血红蛋白升到80 g/L后,再开始化疗。患者在rhEPO联合铁剂治疗一周后,血红蛋白值升至81 g/L,顺利完成化疗,病情平稳出院。     

铁剂加促红细胞生成素治疗幼儿缺铁性贫血临床效果评价

目的观察铁剂加重组促红细胞生成素治疗幼儿缺铁性贫血的临床疗效。方法 20例患儿（治疗组）接受了重组人红细胞生成素;同期20例患儿作为对照组治疗,两组患儿同时服用铁剂。结果治疗后两组血红蛋白量均高于治疗前,治疗8周后治疗组血红蛋白量高于对照组,两组比较差别有显著性（P〈0.05）。同时治疗后两组红细胞压积均高于治疗前,治疗8周后治疗组红细胞压积明显高于对照组,两组比较有显著性差异（P〈0.05）。结论应用铁剂治疗时加用重组人红细胞生成素能更为有效治疗缺铁性贫血,快速提高血红蛋白等各项指标,值得临床推广。     

重组人促红细胞生成素治疗新生儿缺氧缺血性脑病临床意义

目的探讨重组人促红细胞生成素（r Hu EPO）治疗中、重度新生儿缺氧缺血性脑病（HIE）的疗效。方法 32例中、重度新生儿HIE患者,随机分为治疗组（16例）和对照组（16例）。对照组给予常规治疗,治疗组除常规治疗外,给予皮下注射r Hu EPO 400 IU/（kg·d）,3次/周,总疗程4周。所有患儿28日龄时进行NBNA评分,3月龄时进行智能发育测评（CDCC）。结果治疗组28日龄NBNA评分正常者比例高于对照组,3月龄CDCC评分（包括MDI、PDI评分）治疗组高于对照组,差异有统计学意义（P〈0.05）。结论 r Hu EPO在新生儿缺氧缺血性脑病的损伤中有神经保护作用,可促进红细胞恢复及再生,提高患儿的智力和运动发育。     

Fenoterol stimulates human erythropoietin production via activation of the renin angiotensin system

AIMS: The present study assessed the hypothesis that the beta2 sympathomimetic fenoterol influences the production of erythropoietin (EPO) by activation of the renin angiotensin system (RAS), i.e. angiotensin II. METHODS: In an open, parallel, randomized study healthy volunteers received i.v. either placebo (electrolyte solution), fenoterol or fenoterol in combination with an oral dose of the AT1-receptor antagonist losartan. RESULTS: Compared with placebo treatment AUCEPO(0,24 h) was significantly increased after fenoterol application by 48% whereas no increase in the group receiving fenoterol and losartan could be detected. The rise of PRA was statistically significant under fenoterol and fenoterol plus lorsartan. CONCLUSIONS: Stimulation of EPO production during fenoterol infusion appears to be angiotensin II-mediated. Thus, angiotensin II may be considered as one important physiological modulator of EPO production in humans.     

国产与进口重组人红细胞生成素治疗血透病人贫血的比较

目的：研究国产重组人红细胞生成素（ｒｈ Ｅ Ｐ Ｏ） 对慢性肾功能衰竭伴肾性贫血的临床疗效。方法：将６８ 例慢性肾功能衰竭伴肾性贫血的血液透析（ 血透） 病人随机分成国产ｒｈ Ｅ Ｐ Ｏ 组３８ 例（ 男性２２例,女性１６ 例;年龄４５ ａ ±ｓ ４ ａ） 予国产ｒｈ Ｅ Ｐ Ｏ ８０～１６０ Ｕ／ｋｇ ,ｉｖ , 进口ｒｈ Ｅ Ｐ Ｏ 组３０ 例（ 男性１８ 例,女性１２ 例;年龄４１ ａ ±３ ａ） 予进口ｒｈ Ｅ Ｐ Ｏ ３０ ～８０ Ｕ／ｋｇ ,ｉｖ , 均为每周２ ～３ 次,连续用药治疗２４ ｗｋ ,观察血红蛋白（ Ｈｂ） 、血细胞比容（ Ｈｃｔ） 、血清铁和血清铁蛋白的动态变化。结果：国产ｒｈ Ｅ Ｐ Ｏ 组总有效率为９５ ％ 。进口ｒｈ Ｅ Ｐ Ｏ 组总有效率为１００ ％ （ Ｐ＞０ ．０５） ,２ 组各有２ 例因出现血压升高而被迫停药。结论：国产ｒｈ Ｅ Ｐ Ｏ（８０ ～１６０ Ｕ／ｋｇ） 的疗效基本上与进口ｒｈ Ｅ Ｐ Ｏ 组（３０ ～８０ Ｕ／ｋｇ） 一致。