O-phospho-L-serine, multi-functional excipient for B domain deleted recombinant factor VIII

Factor VIII (FVIII) is an important cofactor in the blood coagulation cascade. A deficiency or dysfunction of FVIII causes hemophilia A, a life-threatening bleeding disorder. FVIII circulates in plasma as a heterodimer comprising 6 domains (heavy chain, A1-A2-B and light chain, A3-C1-C2). Replacement therapy using FVIII is the leading therapy in the management of hemophilia A. However, approximately 15% to 30% of patients develop inhibitory antibodies that neutralize the activity of the protein. Neutralizing antibodies to epitopes in the lipid binding region of FVIII are commonly identified in patients' plasma. In this report, we investigated the effect of O-phospho-L-serine (OPLS), which binds to the lipid binding region, on the immunogenicity of B domain deleted recombinant factor VIII (BDDrFVIII). Sandwich enzyme-linked immunosorbent assay (ELISA) studies showed that OPLS specifically bind to the lipid binding region, localized in the C2 domain of the coagulation factor. Size exclusion chromatography and fluorescence anisotropy studies showed that OPLS interfered with the aggregation of BDDrFVIII. Immunogenicity of free- vs BDDrFVIII-OPLS complex was evaluated in a murine model of hemophilia A. Animals administered subcutaneous (sc) injections of BDDrFVIII-OPLS had lower neutralizing titers compared with animals treated with BDDrFVIII alone. Based on these studies, we hypothesize that specific molecular interactions between OPLS and BDDrFVIII may improve the stability and reduce the immunogenicity of BDDrFVIII formulations.     

Efficacy and safety of a recombinant factor VIII produced from a human cell line (simoctocog alfa)

Introduction: The development of inhibitors against infused factor VIII (FVIII) has a detrimental impact on health and quality of life of patients with hemophilia A. Several observational studies and a recently published randomized trial indicate that the inhibitor risk in previously untreated patients (PUPs) is higher following the use of recombinant FVIII (rFVIII) products compared with plasma-derived FVIII concentrates. There is currently a great interest towards newer rFVIII products that adopt various technological solutions to reduce the inhibitor risk.

Areas covered: This review describes the efficacy and safety of simoctocog alfa (Nuwiq), a human cell-line derived rFVIII developed for the prevention and treatment of bleeding in hemophilia A patients. Particular relevance will be given to the safety aspects of this product, with special emphasis on the rate of inhibitor development, presenting the results of the phase II and III clinical trials on previously treated patients (PTPs) and the interim data from the phase III trial on PUPs.

Expert opinion: The final results from PTP studies and the preliminary data from the PUP study document the high efficacy and safety profile of this rFVIII product, which has the potential to reduce the inhibitor risk in PUPs with severe hemophilia A.     

Nonlinear pharmacokinetics of factor VIII and its phosphatidylinositol lipidic complex in hemophilia A mice

Factor VIII (FVIII) is an important cofactor in the blood coagulation cascade and its deficiency or dysfunction causes hemophilia A (HA), a bleeding disorder. Replacement with recombinant FVIII is limited by a short half‐life and the development of inhibitory antibodies. A phosphatidylinositol (PI) containing lipid nanoparticle was developed that, when associated with FVIII, reduces immunogenicity and prolongs circulation of the therapeutic protein in HA mice. A multiple dose level pharmacokinetic (PK) study of human free FVIII and its FVIII–PI complex over a clinically relevant range of doses (20, 40 and 200 IU/kg) was conducted in HA mice to investigate linearity of the PK and to determine if the reduced catabolism of FVIII following association with PI particles, previously only observed in the terminal phase following 400 IU/kg, could be extendable over a range of doses. The findings suggest that the disposition of FVIII is best characterized by a two‐compartment model with saturable Michaelis‐Menten elimination. Spontaneous complexation of FVIII with PI particles significantly increases plasma survival of the protein at 20 and 40 IU/kg doses. Human simulations at 40 IU/kg project an increase in the terminal half‐life and the time to reach a minimum therapeutic threshold of 0.01 IU/ml of 5.4 h and 40 h, respectively, compared with free FVIII. Formulation with PI containing lipid particles may represent a viable delivery strategy for improving FVIII therapy. Copyright © 2014 John Wiley & Sons, Ltd.     

Solid Surface Chemical and Physical Effects on the Adsorption of Recombinant Factor VIII

The adsorption of a recombinant Factor VIII to silica surfaces coated to present different hydrophilic/hydrophobic, electrostatic and steric characteristics was monitored in situ, with ellipsometry. rFVIII adsorption affinity was high for hydrophobic surfaces and hydrophilic, charged surfaces. A lower affinity was recorded between rFVIII and a neutral hydrophilic surface, with substantial reduction in rFVIII adsorption to electronically neutral surfaces presenting pendant poly[ethylene oxide] chains. We conclude that steric repulsion is a requirement for minimizing rFVIII adsorption at solid surfaces.     

A retrospective survey on the safety of Replenate, a high-purity factor VIII concentrate

PURPOSE: To assess the safety of a highly purified, plasma-derived factor VIII concentrate (Replenate) in routine clinical use. METHODS: Following guidelines entitled safety assessment of marketed medicines (SAMM), safety data were collected in the UK on 194 patients who received an estimated 47.6 million IU of Replenate. This population included 47 patients undergoing 53 surgical operations or dental extractions. RESULTS: The study detected four cases of new factor VIII inhibitor development and twelve other adverse events, five that were unrelated to the product, five whose causality was unknown, one that was possibly product-related and one case due to possible lack of efficacy. Only one of these cases had been notified to the manufacturer through conventional spontaneous reporting procedures. Three patients were switched from Replenate as a result of an adverse event (one case of infusion site irritation and two cases of a rise in titre of an existing inhibitor), but no unexpected adverse reactions were noted and there were no reports of virus transmission. The median factor VIII recovery value was 2.17 IU/dl per IU/kg, but recovery was shown to be dependent on several variables, namely inhibitor status, treatment centre and the patient's body weight. The median factor VIII recovery in inhibitor-free patients was 2.28 IU/dl per IU/kg (range: 1.20-6.62). CONCLUSIONS: The study confirms that Replenate is well tolerated by the majority of patients in routine clinical practice.     

基因重组人粒-巨噬细胞集落刺激因子/白细胞介素-3融合蛋白在猕猴中的药代动力学

目的　研究重组人粒 /巨噬细胞集落刺激因子 白细胞介素 3融合蛋白 (PIXY32 1)在猕猴中的药动学。方法　12 5I 标记结合反相高效液相和酸沉淀法。结果　 12 5I PIXY32 1纯度为 94 5 % ,标记前后PIXY32 1对TF 1细胞增殖的ED50 分别为 0 12 5和 0 119μg·L-1。12 5I PIXY32 1在体内迅速降解。iv和sc后末端T1/ 2 相近 ,为 6 6～ 8 2h。AUC随sc剂量增大 ,全身清除率ClS 相近 ,sc生物利用度6 3 %± 2 1%。泌尿系统浓度最高 ,胆汁其次 ,骨髓和脾脏高于其它组织略低于血清 ,脑内最低。主要经尿排泄 ,少部分在尿中以原型排出。结论　猕猴sc12 5I PIXY32 12 0～ 80 μg·kg-1后为线性药代动力学。肾脏在12 5I PIXY32 1的降解中起一定作用     

Circular dichroism of reduced and oxidized recombinant human epidermal growth factor

To further elucidate the role of the disulfide bonds in determining the protein folding of recombinant human epidermal growth factor (r-HuEGF) we studied the structure of reduced and oxidized r-HuEGF using circular dichroism (CD). The far UV CD spectrum of reduced r-HuEGF in 10 mm sodium phosphate pH 3.0 is very different from that of the oxidized molecule. The spectrum of the reduced molecule consists of a plateau from 225 to 200 nm, consistent with the presence of α-helix, β-sheet, and unordered structure. The addition of the α-helix inducer trifluoroethanol to the reduced molecule resulted in an enhancement of α-helix, at the apparent expense of β-sheet, while the oxidized molecule was unaffected by the presence of this reagent. Secondary structure predictions based on the amino acid sequence of EGF correlate most closely with the structure of the reduced molecule. From these results, it appears that the r-HuEGF has a more regular secondary structure in the absence of the disulfide bonds than in their presence. This suggests that the folding of EGF occurs by destroying the regular secondary structure that was present in the reduced state, and that the structure of the native molecule is dictated largely by disulfide bonding.     

The use of pharmacokinetics in dose individualization of factor VIII in the treatment of hemophilia A

Introduction: Hemophilia A is a bleeding disorder resulting from a lack of clotting factor VIII (FVIII), and treatment typically consists of prophylactic replacement of the deficient factor. However, high between subject variability precludes the development of a ‘one size fits all’ dosing strategy and necessitates an individualized approach. We sought to summarize the data on the pharmacokinetics of FVIII available as a basis for the development of population pharmacokinetic models to be used in dose tailoring.

Areas covered: We reviewed the pharmacokinetics of FVIII as used for the treatment of hemophilia A, with a focus on the variability observed between patients and the application of pharmacokinetic methods to dose individualization. We also explored the covariates affecting pharmacokinetic parameters, the differences between plasma-derived and recombinant FVIII and the development of extended half-life products.

Expert opinion: The pharmacokinetics of factor VIII in patients with hemophilia shows a high interpatient variability, and is affected by age, weight, level of von Willebrand factor, and blood group. A population approach to estimating individual pharmacokinetics is likely to provide the most successful strategy to tailor factor concentrate dosing to the individual needs and to ensure optimal patient outcomes, while also improving the cost-effectiveness of prophylactic replacement therapy.     

重组人表皮生长因子凝胶剂的研制

目的：研制用于创伤修复的重组人表皮生长因子（recombinant human epidermal growth factor rhEGF)凝胶剂。方法：以卡泊姆为水溶性凝胶基质制备rhEGF的凝胶剂,并以大鼠皮肤急性创伤模型和大鼠皮肤烫伤模型评价了rhEGF凝胶剂在创伤修复中的效果。结果：所得rhEGF凝胶剂制备工艺简单,质量可靠,使用方便,对于大鼠皮肤急性创伤和大鼠皮肤烫伤具有明显的疗效。结论：rhEGF凝胶剂可望应用于临床。     

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对健康供者免疫细胞的影响及其应用安全性

目的:观察重组人粒细胞集落刺激因子(rhG-CSF)在动员造血干细胞时对健康供者外周血T细胞表面活化信号(CD4 CD28 ,CD8 CD28 )、活化T细胞(CD4 CD25low)和调节性T细胞(CD4 CD25high)的影响及其应用的安全性。方法:30例造血干细胞健康供者[男19,女11,中位年龄28(14～56)岁]皮下注射rhG-CSF5μg/(kg.d),连续4～6d。用药前及停药后第1天、第3天及第7天用流式细胞术检测供者外周血CD4 T细胞和CD8 T细胞表面CD28表达的相对荧光强度(RFI),以及CD4 CD25low和CD4 CD25highT细胞分别在CD4 T细胞中的百分比;并在用药前及停药后第1天、第7天及第30天进行外周血白细胞、血小板、肝功能、肾功能以及脾超声检查。结果:CD4 CD28T细胞和CD8 CD28T细胞相对荧光强度表达在用药前分别为14.91±6.10和11.10±3.74,停药后第3天分别降至11.93±5.39和8.53±3.74;差异有统计学意义(P=0.034,P=0.033)。CD4 CD25lowT细胞在CD4 T细胞中百分比,用药前为(21.4±8.87)%,停药后第3天降至(18.23±5.89)%;差异无统计学意义(P>0.05)。CD4 CD25highT细胞在CD4 T细胞中百分比,用药前为(4.16±1.62)%,停药后第1天升至(6.43±2.46)%,差异有统计学意义(P=0.000)。白细胞计数和脾超声检查用药前分别为(5.91±1.02)×109/L和(33.76±2.76)cm2,停药后第1天分别升至(34.13±8.07)×109/L和(46.85±4.53)cm2;血小板计数用药前为(228.07±73.69)×109/L,停药后第7天降至(158.27±40.69)×109/L,差异均有统计学意义(P均=0.000)。ALT和Cr用药前分别为(28.23±7.69)IU/L和(60.70±15.86)μmol/L,停药后第1天分别为(27.17±7.23)IU/L和(61.10±16.38)μmol/L,差异均无统计学意义(P均>0.05)。结论:rhG-CSF能减弱CD28的表达,降低CD4 CD25low和增加CD4 CD25high在CD4 T细胞中百分比,停药后第7天恢复至动员前水平,对健康供者肝肾功能无不利影响。     

重组人酸性成纤维细胞生长因子的稳定性研究

目的研究重组人酸性成纤维细胞生长因子(rhaFGF)在不同温度下的存放稳定性。方法rhaFGF冻干粉和溶媒在4,25和37℃分别保存30个月;rhaFGF液体在4℃保存56d,在25和37℃分别保存35d。采用MTT法测定rhaFGF的生物活性,同时进行外观性状、水分含量、pH值的测量和无菌试验。结果4℃时,rhaFGF冻干粉、液体和溶媒的各项性质在观察期内都无变化。25℃时,rhaFGF冻干粉、液体的生物活性分别在存放18个月和35d有下降趋势。37℃时,rhaFGF冻干粉、液体的生物活性分别在存放12个月和35d有下降趋势。结论rhaFGF冻干粉在4℃时可有效保存2年;rhaFGF冻干粉溶解后于4℃时保存28d内使用有效;溶媒在4℃时保存2年质量稳定。     

重组人表皮细胞生长因子治疗烧伤残余创面的研究

目的　观察重组表皮生长因子 (rhEGF)对大面积烧伤患者晚期迁延不愈的治疗作用。方法　应用rhEGF对 6 7例烧伤残余创面进行局部治疗 ,采用自身对照法。 (对照药物 :0 9%生理盐水 ) ,以创面愈合为指标 ,与对照组平行比较。结果　rhEGF用于烧伤残余创面的治疗 ,明显缩短创面愈合时间 ,较对照组缩短约 15d ,差异具有显著性 (P <0 0 1)。结论　rhEGF能促进烧伤残余创面的愈合     

Immunogenicity and pharmacokinetic studies of recombinant Factor VIII containing lipid cochleates

Replacement therapy using recombinant factor VIII (rFVIII) is currently the most common therapy for hemophilia A, a bleeding disorder caused by the deficiency of FVIII. However, 15–30% of patients develop inhibitory antibodies against administered rFVIII, which complicates the therapy. Encapsulation or association of protein with lipidic structures can reduce this immune response. Previous studies developed and characterized rFVIII-containing phosphatidylserine (PS) cochleate cylinders using biophysical techniques. It was hypothesized that these structures may provide a reduction in immunogenicity while avoiding the rapid clearance by the reticuloendothelial system (RES) previously observed with liposomal vesicles of similar composition. This study investigated in vivo behavior of the cochleates containing rFVIII including immunogenicity and pharmacokinetics in hemophilia A mice. The rFVIII-cochleate complex significantly reduced the level of inhibitory antibody developed against rFVIII following intravenous (i.v.) administration. Pharmacokinetic modeling allowed assessment of in vivo release kinetics. Cochleates acted as a delayed release delivery vehicle with an input peak of cochleates showed limited RES uptake and associated rFVIII displayed a similar disposition to the free protein upon release from the structure. Incomplete disassociation from the complex limits systemic availability of the protein. Further formulation efforts are warranted to regulate the rate and extent of release of rFVIII from cochleate complexes.     

Factor VIII associated with lipidic nanoparticles retains efficacy in the presence of anti‐factor VIII antibodies in hemophilia A mice

The development of inhibitory antibodies against factor VIII (FVIII) is a major challenge in hemophilia A (HA) therapy. Such antibodies develop in nearly 30% of patients receiving replacement FVIII, abrogating therapeutic efficacy. This work evaluated whether B‐domain deleted FVIII encapsulated in phosphatidylinositol containing lipid nanoparticles (PI‐BDD FVIII) could serve as an efficacious FVIII replacement therapy in the presence of inhibitors. The HA mice were given clinically relevant doses of FVIII to develop inhibitors. The efficacy of free and PI‐BDD FVIII was studied in inhibitor‐positive HA mice using a tail clip assay. Mathematical modeling of these data was conducted to evaluate the hypothesis that lipid association sterically shields the protein from inhibitor binding. The immunization protocol resulted in a mean inhibitory titer level of 198 ± 52 BU/ml. Free BDD FVIII was ineffective at controlling blood loss in inhibitor‐positive HA mice as early as 2 h post dose. In contrast, PI‐BDD FVIII treated animals retained partial hemostatic efficacy as long as 18 h post dose. Mathematical modeling supports the hypotheses that a greater fraction of lipid‐associated FVIII remains unbound to inhibitors and that PI‐BDD FVIII has lower binding affinity to inhibitors than does the free protein. In addition, the modeling approaches extend current efforts to model the impact of immunogenicity on PK and the therapeutically meaningful endpoint of efficacy, thereby addressing an important knowledge gap, particularly in the FVIII scientific literature. Clinical translation of these findings could result in a significant improvement in the quality of care of inhibitor‐positive HA patients. Copyright © 2016 John Wiley & Sons, Ltd.     

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。     

重组人表皮生长因子对宫颈糜烂物理治疗后创面愈合的影响

目的 :探讨宫颈糜烂物理治疗后局部喷洒重组人表皮生长因子 (rhEGF)对宫颈创面愈合的影响及安全性。方法 :将5 14例中、重度宫颈糜烂的患者分为冷冻 +rhEGF组 (A组 ,n =118)和冷冻对照组 (B组 ,n =10 4) ,电熨 +rhEGF组 (C组 ,n =15 2 )和电熨对照组 (D组 ,n =140 )。治疗前做宫颈的细胞学检查 ,排除宫颈上皮的异常变异。A与C组在宫颈糜烂物理治疗后 ,使用rhEGF作宫颈的局部喷洒 ,每天一次 ,在宫颈物理治疗后的 3mo,作疗效的判断 ,并在 3 ,6,12mo时随访宫颈刮片检查。对照组给NS局部喷洒。结果 :A组和B组的治愈率分别为 92 4%和 81 7% ;宫颈创面愈合时间分别为 5 8d和 79d ;阴道排液时间分别为 2 5d和 3 9d ;生殖系统感染率分别为 1 69%和 8 65 % ;阴道排液量A组比B组有明显减少 ;以上 2组比较 ,P均 <0 0 5。C组和D组治愈率分别为 96 1% ,85 6% ;宫颈创面愈合时间分别为 45d ,63d ;阴道排液时间分别为 17d ,3 0d ;生殖系统感染率分别为 1 3 4% ,7 86% ;以上 2组比较 ,Ρ均 <0 0 5。C组阴道排液量与D组无明显差别 (Ρ >0 0 5 )。宫颈细胞学检查 ,所有患者未发现宫颈的上皮细胞有异常增生的改变。未发现有过敏或其他的药物不良反应。结论 :rhEGF能促进宫颈糜烂物理治疗后创面愈合 ,提高治愈率 ;未发     

外用重组人表皮生长因子治疗慢性溃疡的效果观察

目的对比观察外用重组人表皮生长因子（rhEGF）啧雾荆治疗慢性浅表性溃疡的疗效。方法在经门诊诊断为慢性下肢溃疡的63例患者中随机分为治疗组和对照组。治疗组采用rhEGF喷湿创面，再敷盖庆大霉素浸湿纱布和凡士林纱布包扎；对照组只用庆大霉素浸湿纱布包扎。观察2组患者创面上皮、肉芽组织生长情况，创面愈合时间。结果治疗组47例患者的创面上皮、肉芽组织生长明显快于对照组，愈合时间缩短平均25d。结论rhEGF用于治疗慢性下肢溃疡有明显促进愈合作用，具有潜在的临床应用价值。     

重组人粒细胞集落刺激因子壳聚糖胶囊的制备和体外释药特性

目的 :制备人重组粒细胞集落刺激因子 (rhG CSF)冻干粉剂壳聚糖胶囊 ,并对其体外释药性能进行评价。方法 :将rhG CSF冻干粉剂装入壳聚糖胶囊中 ,再以邻苯二甲酸羟丙基甲基纤维素 (HPMCP)包裹胶囊 ,用氮兰四唑蓝 (MTT)比色法测定其在人工胃液及小肠液中的体外释放性能。将荧光素钠 (FS)作为模型化合物在相同条件下进行实验 ,以激发波长 470nm、发射波长 5 13nm荧光检测FS壳聚糖胶囊在人工大肠液中的体外释放性能。用扫描电镜法评价壳聚糖胶囊在大肠内容物中的降解作用。结果 :在人工大肠液中壳聚糖具有明显降解作用。rhG CSF壳聚糖胶囊在人工胃液 (2h)和人工小肠液 (6h)内累积释药量为 (15 .5± 6 .5 ) % ,n =6。而在人工大肠液中 ,FS壳聚糖胶囊 4h释药基本完全。结论 :用HPMCP包膜的rhG CSF壳聚糖胶囊具有潜在的结肠靶向释药特性。     

重组粒细胞集落刺激因子对大鼠急性缺血性脑梗死后损伤修复的研究

目的观察人重组粒细胞集落刺激因子(rhG-CSF)对大鼠急性局灶性脑梗死轴突、血管再生的影响。方法健康雄性Wistar大鼠建立局灶性脑缺血模型,随机分为脑缺血组及相应时间点的假手术组、药物组。免疫组织化学法检测脑内血管内皮细胞生长因子(VEGF)变化及反转录-聚合酶链反应(RT-PCR)法检测神经生长相关蛋白(GAP)-43表达。结果①缺血组及药物组1～10d GAP-43均呈增高趋势,2组各时间表达量有区别;②缺血组VEGF表达1～10d内缓慢增高;药物组上调VEGF表达,1～4d迅速增高,4d时达到高峰,4～10d有降低趋势,10d与假手术组相比仍有差别。结论G-CSF可促进轴突再生、血管增殖,延长缺血治疗窗,发挥脑保护作用。