聚乙二醇化重组人生长激素长效作用及在体生物活性研究

目的:观察聚乙二醇化重组人生长激素(PEG-GH)在去脑垂体大鼠体内的(in vivo)促进生长的生物活性功能和长效药理作用.方法:以摘除脑垂体的大白鼠为动物模型,PEG-GH d 1一次给药;rhGH(国家标准品)作为阳性对照,每天给药一次,连续6 d,使总剂量与PEG-GH一致.同时设模型组(去脑垂体大鼠),只给予 0.1%牛血清白蛋白的生理盐水,d 7 处死大鼠,检测指标为:体重增加、尾长增长、胫骨骨骺板宽度和肝脏重量.结果:实验结果显示,与模型组比,PEG-GH一次给药各剂量组体重显著增加(P＜0.01),尾长也显著增长(P＜0.01),胫骨骨骺板宽度显著增宽(P＜0.01),中、高剂量组肝脏重量增加(P＜0.01),并都具有量效关系;其促进动物生长的生物活性与每天给药的rhGH比没有差别(P>0.05),具有长效的作用,其中 4.3、8.6 IU·kg-1所至生物活性大于多次给药的普通rhGH 8.6 IU·kg-1效果;本批供试品测得生物效价为 5.0 IU·mg-1,比普通rhGH高1倍.结论:本批供试品PEG-GH用药次数少、药效持续时间长,比普通rhGH具有更高的促进动物机体生长的生物活性.     

新型定点修饰的聚乙二醇化重组人生长激素修饰位点研究

目的:建立新型定点修饰的聚乙二醇化重组人生长激素(PEG-rhGH)修饰位点的研究方法,为该类新型修饰技术产物的质量评价提供依据。方法:采用MALDI-TOF质谱分析修饰产物中聚乙二醇(PEG)的修饰个数及修饰肽段的质量数;采用LC-Q-TOF质谱分析修饰位点,分析色谱柱为UPLC色谱柱(Protein ACQUITY BEH C₄ Column, 150 mm×2.1 mm, 1.7μm, 300?),以0.1%甲酸水溶液-0.1%甲酸乙腈溶液为流动相,进行梯度洗脱,柱温为40℃。质谱数据采集条件为MS~E模式,一级质谱能量4 eV,二级碎裂电压30～55 eV。结果:修饰蛋白中PEG的平均相对分子质量为30 000,并且每个PEG-rhGH分子中仅存在1个PEG修饰;原型蛋白重组人生长激素中第134位精氨酸被替换为赖氨酸,且该赖氨酸的ε-氨基与连接子HOOC-O-CH₂-CH₂-N₃中的羧基端共价结合,连接子另一端的叠氮基团与活化后的PEG偶联后生成修饰产物。结论:联合运用多种质谱技术,通过比对...     

单链聚乙二醇化重组人干扰素ω的制备及其性质

采用直链PEG-琥珀酰亚胺琥珀酸酯 (mPEG-SS) 选择性修饰重组人干扰素ω (rhIFNω), 离子交换 色谱和凝胶过滤色谱组合分离纯化单链PEG修饰产物 (PEG-rhIFNω), 基质辅助激光解吸附飞行时间质谱(MALDI-TOF MS) 测定单链PEG-rhIFNω的相对分子量, 并利用RP-HPLC和SDS-PAGE对修饰产物进行分析。在优化的工艺条件下, 分离纯化收集液的单链PEG-rhIFNω, 平均含量达182 μg·mL⁻¹, 分离纯化收率超过22%, 纯度大于98%, SDS-PAGE法测得表观分子质量为60 810, MALDI-TOF MS法测得相对分子质量为43 790; 单链PEG-rhIFNω具有典型PEG修饰蛋白的特性, 抗病毒活性保留率为15.0%, 抗原性降低了64倍, 酸稳定性、抗 胰酶水解能力、血清稳定性和热稳定性均显著提高。单链PEG-rhIFNω的药学性质获得显著改善, 有望开发为 安全、长效的新型干扰素。     

聚乙二醇化重组人生长激素对去垂体幼鼠胰岛分泌的影响及其机制

目的观测长效生长激素聚乙二醇化重组人生长激素(PEG-rhGH)是否引起胰岛素抵抗及其与短效生长激素注射用重组人生长激素(rhGH)的差异。方法 3周龄SD雄性幼鼠106只,随机取88只进行去垂体手术造模,取成模的幼鼠54只分为模型组、rhGH组、PEG-rhGH组,另18只假手术组为对照组。分别给予生理盐水(0.25 mg·kg-1·d-1)、rhGH(0.25mg·kg-1·d-1)、PEG-rhGH(1.4 mg·kg-1·周-1)处理。4周后进行糖耐量实验和胰岛素释放试验,计算胰岛素曲线面积与葡萄糖曲线面积比值,胰岛素稳态模型(HOMA-IR);检测血清生长抑素水平;免疫组织化学法检测胰岛胰岛素(INS)、胰高血糖素(GLU)、生长抑素(SS)、胰多肽(PP)。结果 PEG-rhGH组HOMA-IR较对照组、模型组低(P<0.05),与rhGH组比较无差异。AUCI/AUCG值组间比较显示PEG-rhGH组高于rhGH组,但无统计学差异。PEG-rhGH组GLU蛋白表达与模型组表达无差异。PEG-rhGH组INS表达较模型组表达上升(P<0.05)。PEG-rhGH组SS、PP表达与rhGH无差异。血清SS各组间均无明显差异。结论未观察到PEG-rhGH引起去垂体大鼠胰岛素抵抗和胰岛β细胞分泌能力下降,对胰岛分泌蛋白的表达无明显影响。     

ELISA法研究聚乙二醇化重组人生长激素小鼠体内的药动学

建立了双抗夹心ELISA法检测小鼠血清中聚乙二醇化重组人生长激素（PEG—GH）的浓度。结果表明，PEG-GH和重组人生长激素（rhGH）浓度均在1～150ng/ml范围内呈良好的线性关系。PEG—GH以静脉注射与皮下注射两种途径给药，所得t/2β分别为静注rhGH的34.8倍和39.8倍，清除率明显下降。表明PEG—GH具有长效作用。     

聚乙二醇重组人生长激素治疗儿童特发性矮小症的研究进展

特发性矮小症(ISS)是临床常见病,发病率高且持续上升,可能对患儿造成终身影响,引起严重心理疾患,甚至威胁生命。临床常采用重组人生长激素治疗,能有效促进患儿骨骼生长发育,但因其代谢半衰期较短,需要每日注射,患儿依从性差,从而影响治疗效果。而聚乙二醇重组人生长激素(PEG-rhGH)的研发,成功延长了药物代谢半衰期,可1周仅注射1次,降低了注射频率,提升了患儿的耐受度和依从性,进一步提高了治疗效果和身高增长速度。本文就PEG-rhGH治疗儿童ISS的研究进展进行综述,为该病的临床治疗提供参考。     

聚乙二醇化人重组生长激素对去垂体幼鼠脏器功能的影响

目的 探讨聚乙二醇化重组人生长激素(PEG-rh GH)对去垂体幼鼠主要脏器的影响。方法 SD雄性幼鼠随机取88只进行去垂体手术造模,另18只假手术组为对照组。术后2周,选成模幼鼠54只,分为模型组、重组人生长激素(rh GH)组、PEG-rh GH组。每晚sc生理盐水0.25 mg/kg,并且rh GH组每晚sc注射用重组人生长激素0.25 mg/kg,PEG-rh GH组每周第1天晚上sc注射用重组人生长激素1.40 mg/kg。治疗4周后,检测肝功能ALT、AST、TBIL、DBIL水平,肾功能BUN、CREA水平,心肌酶谱CK、CKMB及肝脏、心脏、肾脏的病理组织和肝脏的转录生长因子β1(TGF-β1)和肾脏C-fos蛋白表达。结果 肝功能中AST、TBIL水平下降;肾功能中CREA水平升高;肝脏、肾脏、心脏病理未见明显异常;肝脏TGF-β1、肾脏C-fos蛋白表达组间无差异。结论 PEG-rh GH对肝脏、肾脏、心脏的组织形态和功能无明显不良影响。     

ELISA法研究聚乙二醇重组人生长激素注射液单次给药人体药代动力学

目的 建立ELISA方法研究聚乙二醇重组人生长激素(PEG-rhGH)注射液单次给药人体药代动力学.方法 将30名健康受试者随机分成4组(其中两组为自身对照),分别单次皮下注射PEG-rhGH注射液(0.1 mg·kg-1、0.2 mg·kg-1、0.4 mg·kg-1)、注射用重组人生长激素(rhGH)(0.067mg· kg-1).ELISA法测定不同时间点PEG-rhGH、rhGH的血药浓度,并计算药代动力学参数.结果 PEG-rhGH、rhGH血药浓度分别在0.312 5～40.0000 ng·ml-1、0.312 5～10.0000 ng· ml-1范围内线性关系良好,最低检测性均为0.312 5ng· ml-1,批间、批内RSD均＜15％. PEG-rhGH(0.1、0.2、0.4 mg·kg-1)、rhGH(0.067mg· kg-1)的t1/2分别为:(31.70±4.70)h、(32.19±4.58)h、(30.39±5.93)h、(1.95±0.44)h,Tmax为:(22.20±9.82)h、(29.40±10.75)h、(40.80±8.39)h、(3.20±1.10)h,Cmax:(105.24±45.37)ng·ml-1、(379.09±109.61)ng·m1-1、(920.69±293.21)ng·ml-1、(30.17±3.20)ng·m1-1,CL/F:(26.97±13.86) ml· kg-1· h-1、(9.21±4.05) ml· kg-1· h-1、(6.29±2.87) ml· kg-1· h-1、(284.26±43.47)ml· kg-1· h-1,AUC0→∞:(4657.70±2337.30) ng· m1-1·h、(25279.58±9407.63) ng· ml-1·h、(74438.89±29 007.81) ng·ml-1·h、(240.97±39.40) ng·ml-1·h.结论 PEG-rhGH体内过程符合线性动力学特征,与rhGH相比,明显推迟达峰时间、延长半衰期、减慢清除率,具有长效特征.     

重组人生长激素长效制剂的研究进展

重组人生长激素长效制剂能延长药物在体内的滞留时间,有效增加患者用药的顺应性、降低用药相关的医护成本。本文重点综述微球、生物可降解性凝胶、聚乙二醇化等重组人生长激素长效制剂的制备方法及其研究进展。     

半乳糖化重组人生长激素和重组人生长激素在小鼠体内的药物动力学

目的：比较半乳糖化重组人生长激素和重组人生长激素在小鼠体内的药物动力学特征。方法：用＾１２５Ｉ标记Ｇａｌ－ｒｈＧＨ和ｒｈＧＨ、通过小鼠尾静脉ｉｖ，测定血和肝中相对放射性随时间的变化，以３Ｐ８７药物动力学程序进行模型拟合和参数求算。结果：＾１２５Ｉ－ｒｈＧＨ－Ｇａｌ在血中的相对放射性呈现双峰，其体内药行动力学特征与＾１２５Ｉ－ｒｈＣＨ明显不同，进一步证实了Ｇａｌ－ｒｈＧＨ的肝靶向性。     

Clinical evaluation of recombinant human growth hormone injection in children with growth hormone deficiency

Recombinant human growth hormone (rhGH) has been widely used in the clinical treatment of growth hormone deficiency. To simplify the injection process and increase drug compliance, application of the GH injection has become a new treatment plan in recent years. The purpose of the current study was to evaluate the efficacy and safety of rhGH injection for the treatment of growth hormone deficiency (GHD) in children in China. In a nationwide, noncomparative, prospective, randomized, open trial, 31 children with confirmed complete GHD received subcutaneous injection of rhGH at 0.25 mg/kg·wk (0.107 IU/kg·d). The injection was given daily and the total weekly amount was separated into 6–7 injections. The patients were followed up at 3-month intervals and the treatment duration was 12 months. The height (HT), annual growth velocity (GV), mean height standard deviation score (HT SDS), blood serum insulin-like growth factor I (IGF-I), insulin-like growth factor binding protein 3 (IGFBP-3), and bone maturity before and after treatment were compared, and the safety of the treatment was analyzed. The mean HT, GV, and HT SDS were increased from 109.0±14 cm, 2.7±0.9 cm/yr, and −4.62 ±1.46 at baseline to 121.8±13.4 cm, 12.9±3.3 cm/yr, and −2.47±1.86 after 12 months of treatment, respectively (P<0.001). At the same time, blood IGF-I and IGFBP- 3 were increased significantly [41.27±64.43 μg/L vs 159.21±167.92 μg/L and 1540.00±1325.11 mg/L vs 3533.93±1413.82 mg/L, respectively (P<0.001)]. The bone age assessments performed 6 and 12 months after the treatment showed that no advanced bone maturation was noted. No serious adverse events occurred during the treatment, and the drug-related adverse events were mainly decreased thyroid function. We conclude that rhGH injection is a safe and effective drug for treatment of growth hormone deficiency in children.     

基因重组人生长激素治疗青春期前特发性生长激素缺乏症临床试验

目的:评价基因重组人生长激素(rhGH)治疗青春期前特发性生长激素缺乏症(IGHD)患儿6个月的疗效和安全性。方法:对49例IGHD患儿进行rhGH治疗,每晚睡前皮下注射0．5IU·kg~(-1),每周分5次注射,共26周。评价治疗前后身高、生长速度、身高标准差计数(SDS)等指标。结果:在rhGH治疗期间,46例患儿生长速度由每年(2．5±1．0)cm提高到(11．5±2．5)cm(P＜0．005)。身高SDS由治疗前(-4．2±1．8)增为(-3．7±1．8)。同时患儿13和26周血清胰岛素样生长因子(IGF-1)和IGF结合蛋白(IGFBP,)水平均较治疗前明显升高(P＜0．05)。其体重与骨龄无明显变化。治疗后共有15．7%患儿出现甲状腺功能降低,抗hGH抗体阳性率为21．7%,但所有这些现象均未影响患者体格的线性增长。在治疗期间所有患者肝肾功能、血尿常规和代谢性指标(如血糖和血脂水平)等均保持在正常范围。结论:rhGH是治疗IGHD安全有效的药物。     

Efficacy of needle-free administration of recombinant human growth hormone in adults with growth hormone deficiency

AIM: Needle-free administration of recombinant human growth hormone (rhGH) is effective in the treatment of growth hormone deficiency (GHD) in children, but has not been studied in adult patients. Therefore, we evaluated the efficacy of needle-free administration of rhGH in adults with GHD. METHODS: Insulin-like growth factor-I (IGF-I) concentrations were compared in newly diagnosed patients with GHD (n = 21) and in patients previously treated by subcutaneous injection of rhGH (switchers, n = 34), at baseline, 12 months and 24 months. RESULTS: In the new patients, IGF-I standard deviation scores (SDS) increased from - 1.82 +/- 0.46 to + 0.75 +/- 0.33 at 12 months and to + 0.65 +/- 0.41 at 24 months (P < or = 0.001 vs. baseline). In switchers, IGF-I SDS remained unchanged with values of + 0.98 +/- 0.32 at baseline, + 0.87 +/- 0.23 at 12 months and + 0.73 +/- 0.29 at 24 months (P = 0.696 vs. baseline). In new patients, the rhGH dose was 0.46 +/- 0.03 mg day(-1) at 12 months and 0.47 +/- 0.03 mg day(-1) at 24 months. In switchers, the rhGH dose was 0.53 +/- 0.04 mg day(-1) at baseline (s.c. injection), 0.52 +/- 0.03 mg day(-1) at 12 months and 0.48 +/- 0.03 mg day(-1) at 24 months (NS between the different time points). There was no difference in the dose of rhGH at 12 and 24 months between the two groups. Side-effects were generally minor and consisted of local tissue reactions. CONCLUSION: Administration of rhGH by needle-free, transdermal injection is effective in maintaining IGF-I concentrations in the normal range for age in adults with GHD, and is as effective as traditional subcutaneous injection of rhGH.     

特发性生长激素缺乏性矮小症在重组人生长激素治疗1年后身体构成的变化

目的:探讨青春期前特发性人生长激素缺乏症(IGHD)用基因重组人生长激素(rhGH)治疗后其身体构成的改变。方法:17例年龄为(14．6±4．5)岁,骨龄(12．1±0．8)岁的IGHD青少年(男14例,女3例),在rhGH治疗前生长速度每年(2．9±0．7)cm,患者每周0．5 u·kg-1,分5次于临睡前皮下注射rhGH,共1年。测定患者治疗前和治疗后1,3,6,9和12个月身高、体重、体内脂肪百分含量(F％)、去脂肪量(FFM)、左右手握力、血胰岛素样生长因子-1(IGF-1)和血胰岛素样生长因子结合蛋白-3(IGFBP-3)水平,并以2002年中国北方177例正常青少年值作为对照。结果:治疗前IGHD患者的身高、体重、FFM及血IGF-1和IGFBP-3水平均显著低于正常青少年(P<0．05),F％明显高于正常青少年(P<0．05),但体重指数(BMI)值无明显差异。用rhGH治疗1个月及随后治疗中,患者F％均显著低于治疗前(P<0．05),与正常青少年无统计学差异。经rhGH治疗6个月,17例IGHD患者的身高、生长速度、FFM及血IGF-1和IGFBP-3水平均明显增加(均P<0．05),其改变值均与F％的下降值相关(P均<0．01)。治疗1年后,所有患者的握力均明显增加。结论:IGHD青少年在使用rhGH后,身高、FFM及握力均显著增加,F％减少,提示患者骨骼和肌内组织的增加及脂肪含量的降低,即身体的构成发生了明显的改变。     

荧光光度法测定重组人生长激素注射液中聚山梨醇酯20的含量

目的:建立荧光光度法测定重组人生长激素注射液中聚山梨醇酯20的含量。方法:利用 N-苯基-1-萘胺(NPN)能摄取溶液中的聚山梨醇酯20并产生荧光,以其荧光强度与浓度正相关的特性来测定聚山梨醇酯20的含量。同时考察该方法的影响因素,确定了最佳实验条件。结果:根据影响因素与荧光强度的关系确定了35℃、0.05%NPN 浓度反应5 min 的试验条件,该法的平均仪器精密度、方法精密度和板间精密度分别为2.0%,4.0%,5.0%,平均加样回收率为102.6%。本法测定的2批生长激素注射液结果,与企业采用的方法结果一致。结论:本法快速、简便、准确,可用于测定重组人生长激素注射液中聚山梨醇酯20的含量。     

Properties of a cleaved two-chain form of recombinant human growth hormone

Escherichia coli cells transformed with plasmids engineered for the expression of recombinant human growth hormone as a secreted product also produced a proteolytically cleaved form of rhGH. This variant is isolated at a high resolution anion exchange chromatography stage during the manufacturing process. The higher isoelectric point of this form is demonstrated by isoelectric focusing and chromatofocusing and the two-chain nature by tryptic mapping, N- and C-terminal sequence analyses, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. These data indicate that the single site of cleavage is between Thr-142 and Tyr-143, in contrast to the two-chain variant isolated from human pituitary glands, which has a clip after residue Phe-139. The recombinant two-chain form was further characterized by reversed-phase high performance liquid chromatography at both acidic and basic pHs. The assay utilizing bicarbonate-containing mobile phases was determined to be the most efficient and sensitive method. The bioactivity of this two-chain form was measured by the in vivo rat weight gain assay and by the in vitro Nb2 cell bioassay. Its immunological similarity to intact one-chain rhGH was demonstrated with an enzyme-linked immunosorbent assay.     

Biophysical and biological characterization of PEGylated recombinant human endostatin

Recombinant human endostatin (MES), showing potent inhibition on angiogenesis and tumour growth, has great potential as a therapeutic agent for tumours. The aim of this study was to evaluate the biophysical and biological characterization of PEGylated recombinant human endostatin (M₂ES). Recombinant human endostatin was mono‐PEGylated by conjugation with methoxy polyethylene glycol aldehyde (mPEG‐ALD), and the modification site was identified by digested peptide mapping and matrix assisted laser desorption/ionization–time of flight–mass spectrometry (MALDI–TOF–MS). The purity was assessed by SDS‐PAGE, high‐performance liquid chromatography (HPLC), and capillary zone electrophoresis. The physicochemical property was analyzed through fluorescence spectroscopy, and circular dichroism. The bioactivity and anti‐tumour efficacy of M₂ES were evaluated using an in vitro endothelial cell migration model and a null‐mouse xenograft model of a prostatic cancer, respectively. M₂ES molecules contain a single 20 kDa mPEG‐ALD molecule conjugated at the N‐terminal portion of MES. The purity of M₂ES was greater than 98%. The physicochemical analysis demonstrated that PEGylation does not change the secondary and tertiary structure of MES. Notably, M₂ES retards endothelial cell migration and tumour growth when compared to control group. These biophysical and biological characterization study data contribute to the initiation of the ongoing clinical study.