Oxidation of recombinant methionyl human granulocyte colony stimulating factor

The oxidation of methionine residues in recombinant methionyl human granulocyte colony stimulating factor with hydrogen peroxide has been investigated. Kinetic data of the oxidation were obtained by using reversed phase-high performance liquid chromatography. The stability-indicating capability of this system was confirmed with micellar electrokinetic capillary chromatography. In the pH range 1.9–7.5, the k_obs value for the oxidation process is constant. Above pH 7.5, k_obs tends to increase with increasing pH. In the pH range 1.9–11.8, four oxidation products were detected in RP-HPLC. Mass spectrometric analysis revealed that one mono-, one di- and two trioxidation products were formed. Using the cyanogen bromide cleavage method the nature of the oxidation products was determined. The mono-oxidation product is the protein with Met¹²¹ oxidized, while the dioxidation product has oxidized Met¹²¹ and Met¹²⁶ residues. The trioxidation products are the proteins with Met¹²¹, Met¹²⁶ and Met¹³⁷ or Met⁰, Met¹²¹ and Met¹²⁶ oxidized.     

重组人粒细胞集落刺激因子宿主蛋白质含量的测定

目的根据重组人粒细胞集落刺激因子 (rhG CSF)的生产工艺 ,建立宿主蛋白质 (HCP)含量的测定方法。方法用不含rhG CSF基因的空质粒转染E .coli宿主 (BL2 1) ,按rhG CSF的生产工艺进行发酵、纯化、制备HCP。常规法免疫家兔 ,制备抗HCP多抗血清 ,经纯化后 ,过碘酸钠法标记辣根过氧化物酶 (HRP) ,建立双抗夹心酶标记免疫吸附测定 (ELISA)法测定HCP在rhG CSF原液中的含量。结果所建HCP测定方法能够测定 7.8～ 2 5 0ng/ml范围内的HCP。结论重组蛋白质药物宿主蛋白质含量测定应依据不同的工艺 ,制定不同的方法。     

重组人粒细胞集落刺激因子致急性肾衰竭

1名54岁男性失代偿期肝硬化患者,皮下注射重组人粒细胞集落刺激因子(rhG-CSF)200μg,1次/d。用药后第2天患者出现尿色变深,第4天出现眼睑水肿,肉眼血尿、少尿等症状。BUN由4.8mmol/L升至7.9mmol/L(最高13.9mmol/L),Cr由113μmol/L升至154μmol/L(最高308μmol/L)。停用rhG-CSF,给予还原型谷胱甘肽、硫普罗宁、呋塞米等对症支持治疗。2周后肾功能恢复正常。     

Pharmacokinetics of recombinant human granulocyte macrophage colony-stimulating factor in Macaca mulata

目的：研究重组人粒细胞巨噬细胞集落刺激因子（ｒｈＧＭＣＳＦ）在恒河猴体内的药物动力学．方法：用酶连接免疫吸附测定法检测血浆中ｒｈＧＭＣＳＦ的含量．结果：ｉｖｒｈＧＭＣＳＦ后血药浓度时间曲线符合三房室模型．第１，２和３相的Ｔ１２分别为００５－００７ｈ，０１４－０５８ｈ和１４－４１ｈ．ＡＵＣ随剂量成比例增加．ｉｖ高剂量和低剂量的Ｃｌ和Ｋ１０都相似．ｓｃｒｈＧＭＣＳＦ后血药浓度的峰值为０９３±０１６μｇ·Ｌ－１，达峰时间为２６５±０１４ｈ，生物利用度为０６１．结论：恒河猴ｒｈＧＭＣＳＦ药物动力学数据为临床试验提供有用参考．     

重组人粒细胞集落刺激因子成品蛋白质含量测定方法的研究

目的研究重组人粒细胞集落刺激因子 (rhG CSF)成品蛋白质含量测定方法。方法用三氯醋酸 Lowry法和高效液相色谱外标法测定rhG CSF成品蛋白质含量。结果两种方法均能有效排除rhG CSF成品中甘露醇的干扰 ,与经典的Lowry法相比 ,蛋白质的回收率均大于 96 % ,RSD均 <5 %。结论三氯醋酸 Lowry法和高效液相色谱外标法均可用于测定rhG CSF成品的蛋白质含量 ,高效液相色谱法操作更简便 ,更准确。     

聚乙二醇化重组人粒细胞集落刺激因子大鼠和Beagle犬的免疫原性

目的：在进行聚乙二醇化重组人粒细胞集落刺激因子（PEG30-rhG-CSF）大鼠和Beagle犬重复给药毒性试验时,观察给药后动物血清中抗PEG30-rhG-CSF抗体的产生和抗体的中和活性,为非临床安全性评价的确切性以及临床给药周期提供依据。方法：大鼠分为赋形剂对照组、PEG30-rhG-CSF0.75、3.0、12mg/kg组,sc给药,隔日1次,连续2周;Beagle犬分为赋形剂对照组、PEG30-rhG-CSF0.25、1.0和4.0mg/kg,sc,每周1次,连续4周。采用ELISA法检测动物血清中抗PEG30-rhG-CSF的抗体,采用NFS-60细胞/MTT比色法检测抗体的中和活性。结果：大鼠在给药期和恢复期各剂量组动物血清中均未检测到抗PEG30-rhG-CSF的结合抗体。Beagle犬在给药的第1周和第2周,未检测到结合抗体,但在第4周时,低、中和高3个剂量组各有5只（5/6）的动物血清中检测到结合抗体,而抗体滴度与剂量无明显相关性。恢复4周后,仅高剂量组1只（1/2）动物出现抗体,且抗体滴度呈下降趋势。另外,在给药期和恢复期,血清中所产生的抗体均无中和PEG30-rhG-CSF的活性。结论：大鼠重复给予PEG30-rhG-CSF的毒性试验中,所有动物血清中均未检测到结合抗体和中和抗体;Beagle犬用药组的绝大部分动物血清中出现抗PEG30-rhG-CSF的抗体,但所产生抗体无中和活性。     

Structural characterization of bovine granulocyte colony stimulating factor: effect of temperature and pH

The protein bovine granulocyte colony stimulating factor (bGCSF) was studied in solution as a function of pH (2-7) and temperature (10 degrees -90 degrees C) using fluorescence, circular dichroism, and Fourier transform infrared spectroscopies, as well as differential scanning calorimetry and optical density as a measurement of aggregation. bGCSF possesses significant conformational lability under the solution conditions examined. Under all pH conditions examined, a major conformational change is observed as a function of temperature at 50 degrees -60 degrees C, although the magnitude and precise temperature at which this occurs varies with pH. Three major conformations are adopted with changing pH. One is observed at pH 2 and 3, a second at pH 4, and a third at pH 5-7. At low pH (2-3), bGCSF adopts a molten globule-like conformation at moderate temperatures (25 degrees -45 degrees C), whereas at pH 4 the protein appears to form a non-molten globule extended conformation. The use of this type of study as complementary data for protein phase diagram development as well as the relationship between the conformational lability demonstrated by bGCSF and that observed for recombinant human granulocyte colony stimulating factor and other similar cytokines is discussed.     

Pharmacokinetic and pharmacodynamic analysis of subcutaneous recombinant human granulocyte colony stimulating factor (lenograstim) administration.

A total of 72 adult healthy volunteers were administered 1 microgram/kg of rhG-CSF. There was no correlation between Cmax and an increase in peripheral neutrophil count, and there was a negative correlation between AUC and this increase. The mechanism of this is probably based on the correlation between the elimination rate constant (ke) and neutrophil increase. The ke probably has a close relationship with uptake by neutrophil and its progenitor via the G-CSF receptor. An individual with higher ke should therefore show a greater increase in neutrophil count. Therefore, AUC is proportional to the rhG-CSF remainder, that is, the proportion that is not consumed in the course of increasing the neutrophil count. In such a situation, the bioavailability calculated from the AUC is unlikely to indicate the absorbed amount. The authors also analyzed the pharmacokinetics using a two-compartment model with zero-order absorption and first-order elimination. This model was sufficient to obtain a good curve fit, and this demonstrates that the absorption process is not a first-order but a zero-order process. Therefore, there might be an upper limit to the rhG-CSF transfer rate from subcutaneous tissue to blood.     

围术期应用粒细胞集落刺激因子对大鼠心脏术后心功能的影响

目的 探讨围术期应用粒细胞集落刺激因子(G-CSF)对大鼠心脏术后心功能的影响.方法 将Wistar雄性大鼠随机分为3组,每组10只,暴露心脏后于左心室沿冠状动脉左前降支附近作一切口后立即缝合切口并关胸.对照组不使用G-CSF,仅完成手术过程;G-CSF预处理组术前通过鼠尾静脉注射G-CSF 5 d;G-CSF预处理+心内注射组除术前通过鼠尾静脉注射G-CSF 5 d外,手术过程中将G-CSF直接注射在心室切口周围.术后第7、28天行超声心动图检查评价心脏结构和心功能.结果 第7大时,对照组左心室射血分数(LVEF)和缩短分数(FS)分别为(52.8±2.9)％、(22.2±1.4)％,G-CSF预处理组和G-CSF预处理+心内注射组LVEF[分别为(56.5±4.3)％、(61.2±3.6)％]、FS[分别为(25.2±2.5)％、(29.5±2.3)％]均明显高于对照组,差异有统计学意义(均P＜0.05);G-CSF预处理组和G-CSF预处理+心内注射组左心室收缩末内径(LVESD)[分别为(6.25±0.92)、(5.51±0.85)mm]和左心室收缩末容量(LVESV)[分别为(0.57±0.18)、(0.44±0.14)ml]低于对照组[分别为(6.60±1.10)mm、(0.67±0.31)ml],G-CSF预处理+心内注射组LVESD和LVESV低于G-CSF预处理组,差异均有统计学意义(均P＜0.05).第28大时G-CSF预处理组和G-CSF预处理+心内注射组的LVEF[分别为(59.6±4.7)％、(63.8±1.9)％]和FS[分别为(26.2±2.8)％、(30.9±1.6)％]均明显高于对照组[分别为(53.4±3.6)％、(23.6±1.7)％],LV ESD[分别为(5.92±0.72)、(5.35±0.54)mm]、左心室舒张末内径(LVEDD)[分别为(7.48±0.61)、(7.26 ±0.55)mm] 、LVESV [分别为(0.49±0.14)、(0.41±0.08)m1]、左心室舒张末容量(LVEDV)[分别为(1.05±0.25)、(1.02±0.27)ml]低于对照组[分别为(6.98±0.37)mm、(8.04±0.42)mm、(0.76±0.15)ml、(1.37±0.18)ml],差异均有统计学意义(均P＜0.05);G-CSF预处理+心内注射组LVEF、FS明显高于G-CSF预处理组,LVESD、LVEDD、LVESV、LVEDV明显低于G-CSF预处理组,差异均有统计学意义(均P＜0.05).结论 围术期应用G-CSF可明显改善大鼠心脏术后心脏收缩及舒张功能.     

基因重组人粒细胞集落刺激因子防治肺癌化疗反应

目的 :探讨国产基因重组人粒细胞集落刺激因子 (rHuG CSF)对肺癌病人化疗所致白细胞减少的防治作用及不良反应。方法 :采用随机分组、自身交叉对比的方法 ,将 6 0例肺癌VIP方案化疗病人分成A ,B两组 (各 30例 ) ,A组第 1周期化疗结束 4 8h开始皮下注射rHuG CSF 3μg·kg- 1,qd ,7～14d ,第 2周期单用化疗 ;B组第 1周期单用化疗 ,第 2周期化疗结束 4 8h开始皮下注射rHuG CSF 3μg·kg- 1,qd ,7～ 14d。自化疗开始隔日查血常规 1次 ,观察白细胞及中性粒细胞的变化。结果 :治疗周期白细胞总数及中性粒细胞最低值均高于对照周期 ,最低值的持续时间和化疗开始至细胞恢复到正常值以上的时间均少于对照周期。骨痛、肌痛和乏力发生率分别为 10 %和 7% ,偶有发热。结论 :rHuG CSF对化疗引起的白细胞减少具有预防和治疗作用 ,不良反应轻微 ,安全可靠。     

Outline of clinical studies on recombinant human granulocyte colony stimulating factor (KRN 8601) in Japan.

In phase I studies of KRN 8601, single administration and six-day consecutive administration studies were conducted in healthy male adults using intravenous drip infusion and subcutaneous administration. Safety and tolerance to KRN 8601 were confirmed and a dose-related increase of neutrophil counts by KRN 8601 was observed. In an early phase II study, the safety and tolerance to KRN 8601 in cases of neutropenia following cancer chemotherapy were shown at doses of 25-800 micrograms/m2 and an improvement of neutropenia was seen at doses of more than 50 micrograms/m2. In a phase II study, the optimal dose was investigated for subcutaneous administration and intravenous drip infusion in cases of neutropenia induced by chemotherapy for malignant lymphomas. The optimal dose was 75 micrograms/body (about 50 micrograms/m2) for subcutaneous administration and 100-200 micrograms/m2 for intravenous drip infusion. In a phase III study, a double-blind prospective randomized trial comparing KRN 8601 with an inactive placebo against malignant lymphomas was performed and the inhibitory, improvement and recovery-promoting effects of KRN 8601 (75 micrograms/body, sc) on neutropenia were demonstrated.     

重组人粒细胞巨噬细胞集落刺激因子凝胶剂治疗溃疡药效学研究

目的观察重组人粒细胞巨噬细胞集落刺激因子(gm-csf)凝胶剂治疗溃疡的药效。方法在豚鼠背部造溃疡模型,将凝胶剂外涂于豚鼠背部的溃疡处,不同时间内考察gm-csf凝胶剂对溃疡的药效。结果gm-csf凝胶剂组及gm-csf原液组同盐水组、基质组比较,溃疡面积明显缩小,有显著差异。结论gm-csf凝胶剂对溃疡有明显的治疗作用。     

The effects of granulocyte colony stimulating factor on chemiluminescence and lipid peroxidation of blood platelets treated with cisplatin

The effects of granulocyte colony stimulation factor (G-CSF) at concentrations of 0.08, 0.8 and 8 microg/ml on reactive oxygen species (ROS) generation and lipid peroxidation induced by cisplatin in pig blood platelets were investigated. The level of reactive oxygen species (O2, H2O2, singlet oxygen and organic radicals) generated in platelets was measured by the chemiluminescence method. Lipid peroxidation was determined by the thiobarbituric acid technique and was expressed as thiobarbituric acid reactive substances. G-CSF at the concentration of 0.08 microg/ml had a strong inhibitory effect (about 60% inhibition) on the production of ROS in the isolated pig platelets. This cytokine also significantly reduced lipid peroxidation in control platelets and platelets treated with cisplatin (p<0.05). In the presence of G-CSF in the incubation medium (0.8 microg/ml) cisplatin-induced generation of ROS was also reduced (p<0.05). This study demonstrates that G-CSF has a protective effect against the oxidative stress in blood platelets caused by cisplatin.     

Formulation of sustained-release microspheres of granulocyte macrophage colony stimulating factor by freezing-induced phase separation with dextran and encapsulation with blended polymers

This study aimed to assess the potential merits of formulating sustained-release microspheres of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) via freezing-induced phase separation (FIPS) of the protein with dextran followed by encapsulation with binary mixture of poly(lactic-co-glycolic acid) (PLGA) 2A (MW～12K) and 3A (MW～47K) or of PLGA2A and polylactic acid (PLA; MW～83K). The formulated dextran particles and microspheres were characterized in?vitro for loading, aggregation, bioactivity and release behavior of the protein where appropriate. rhGM-CSF retained about 60% of bioactivity with no significant aggregation after each formulation step. Encapsulation of protein-loaded dextran particles attained only 80% with the PLGA2A and PLGA3A blend, but 100% with the PLGA2A and PLA mixture. The former formulation exhibited a triphasic in-vitro release profile typical of PLGA microspheres while the latter revealed a much lower initial burst followed by a steady and complete release of rhGM-CSF with preserved bioactivity over a 15-day period.     

重组人粒细胞集落刺激因子注射液动员外周血干细胞10例

目的 :探讨重组人粒细胞集落刺激因子(rhG CSF)注射液动员外周血造血干细胞 (PBSC)的效果。方法 :对 6例恶性血液病病人 ,1d内静脉注射长春新碱 1～ 2mg·m- 2 及环磷酰胺 4～ 7g·m- 2 (分 4次 ,间隔 4h) ,外周血白细胞降至 1×10 9·L- 1以下时 ,加rhG CSF 6～ 8μg·kg- 1,皮下注射 ,qd× 5～ 7d ;对 4例健康供者在采集PBSC前 5d予rhG CSF 6～ 8μg·kg- 1,皮下注射 ,qd× 5d。白细胞升至 10× 10 9·L- 1以上时采集PSBC ,并进行CD+ 34 及粒 巨噬细胞集落形成单位 (CFU GM )检测。结果 :一次收集单个核细胞计数 (3.9±s 1.7)×10 8·kg- 1;CD+ 34 (4 .8± 2 .3)× 10 8·kg- 1;粒 巨噬细胞集落形成单位 (5± 3)× 10 4 ·kg- 1。未出现严重不良反应。结论 :rhG CSF无论对病人自体还是对健康供者均能安全、高效地动员PBSC ,满足移植所需要。     

Sustained in vivo activity of recombinant bovine granulocyte colony stimulating factor (rbG-CSF) using HEPES buffer

The purpose of this study was to develop a long-acting injectable formulation of bG-CSF for veterinary use. However, in order to achieve sustained in vivo activity it was first necessary to stabilize the protein at the injection site. Preformulation studies, as well as literature, suggest that bG-CSF aggregates at neutral pH ranges (i.e., pH 6-8) and at temperatures of approximately 40 degrees C. Therefore, bG-CSF will not retain its activity for an extended period of time at the injection site. During this study we determined that HEPES buffer has a very significant impact on protein stability as well as on biological performance. Recombinant bovine granulocyte colony stimulating factor (rbG-CSF) was formulated in 1 M HEPES buffer for subcutaneous injection into cows. bG-CSF formulated in 1 M HEPES buffer resulted in sustained in vivo activity of bG-CSF compared to the "control" formulation (control formulation: 5% mannitol, 10 mM acetate buffer, 0.004% tween-80, pH 4). White blood cell (WBC) count was used as a marker to evaluate in vivo activity of the formulation. WBC numbers remained above a threshold value for only 24-30 h for the control formula. However, when bG-CSF was formulated in 1 M HEPES, the WBC remained above threshold for 3 days or 72 h. Formulating bG-CSF in 1 M HEPES at pH 7.5 also resulted in greater solution stability. This was surprising since bG-CSF is intrinsically not stable at neutral pH. The effect of 1 M HEPES on the T(M) (temperature at maximum heat flow on calorimetry scan) of bG-CSF was determined by microcalorimetry. In the absence of 1 M HEPES buffer the T(M) was 48 degrees C (onset approximately 40 degrees C), while bG-CSF formulated in 1 M HEPES buffer has a T(M) of 59 degrees C (onset approximately 50 degrees C). Similar organic buffers, such as MOPS, HEPPS, TES, and tricine, also resulted in improved solution stability as well as in sustained in vivo activity. The dramatic effect of these buffers on stability and biological performance of bG-CSF is not well understood. One hypothesis is that the electrostatic interaction between the zwitterionic form of these buffers and bG-CSF provides stabilization against denaturation.     

Selective domain stabilization as a strategy to reduce human serum albumin-human granulocyte colony stimulating factor aggregation rate

Therapeutic proteins must be generally formulated to reduce unwanted aggregation. Fusion proteins, which comprise domains assembled from separate proteins, may require unique formulation strategies in order to maximize their stability. A fusion protein of human serum albumin (HSA) and human granulocyte colony stimulating factor (GCSF; HSA-GCSF) was used as a model to test the hypothesis that formulations that increase the thermodynamic conformational stability of the least stable domain of a fusion protein will stabilize the entire fusion protein against aggregation. Conformational stability of HSA-GCSF was modulated by addition of octanoic acid, which was previously shown to increase the conformational stability of HSA, the least stable domain. Contrary to our hypothesis, increased conformational stability of the HSA domain did not result in increased resistance to aggregation of HSA-GCSF. These results for HSA-GCSF were also compared with similar studies conducted previously on a therapeutic protein formed by the fusion of HSA and human growth hormone (hGH; HSA-hGH).     

脱氧核苷酸注射液治疗急性脑梗死的多中心随机、单盲、安慰剂对照临床试验

目的评价脱氧核苷酸注射液治疗急性脑梗死的有效性和安全性。方法本研究采用目的评价脱氧核苷酸注射液治疗急性脑梗死的有效性和安全性。方法本研究采用多中心、随机单盲对照的方法,于2013年5月至2014年5月共入选144例脑梗死患者,随机分为对照组和治疗组,对照组（n=72煸人给予溶栓、抗凝、抗血小板、活血化瘀等方法进行治疗,治疗组（n=72）在对照组基础上给予脱氧核苷酸注射液（200mg／d）,连续21d,评价患者的神经功能缺损状况并检测血清蛋白含量,记录医院感染发生情况。结果治疗后,两组美国国立卫生院神经功能缺损评分（national institutes of health neurological function,NIHSS）均显著下降、日常生活活动评分（Baahel指数）均明显提高,但治疗组NIHSS评分下降以及Barthel指数提高更显著（P〈0．05）。治疗组与对照组在基本痊愈率（26．0％vs10．3％）、总有效率（91．3％vs73．5％）方面比较差异有统计学意义（P〈0．05）。血清蛋白检测显示,两组总蛋白（total protein,TP）、白蛋白（albumin,ALB）水平均显著下降,但治疗组TP、ALB水平下降程度显著低于对照组（P〈0．05）。治疗21d后,治疗组CD^3＋、CD^4＋、自然杀伤（naturalkiller,NK）细胞水平均显著高于对照组,而医院感染发生率显著低于对照组（P〈0．05）。治疗期间两组除发生低热、皮疹、头痛、轻微恶心呕吐外,均未见其他严重不良反应。结论脱氧核苷酸注射液可改善急性脑梗死患者神经功能缺损状况,增加血清蛋白水平,增强机体免疫力,减少医院感染发生率,治疗过程基本无不良反应。