A Large-Scale Process to Produce Microencapsulated Proteins

Pharmaceutical Research -     

重组人生长激素缓释微球.Ⅱ.大鼠体内的药物动力学研究

考察重组人生长激素（rhGH）可生物降解微球在动物体内的缓释效果，探讨蛋白稳定剂对微球体内药物动力学行为的影响。大鼠皮下注射rhGH-PLGA微球后，利用放射免疫吸附分析法测定血药浓度。结果表明，rhGH-PLGA微球具有明显的缓释作用，给药后d30，动物体内的rhGH浓度仍维持在正常生长激素水平之上。而大鼠同等剂量给予rhGH溶液24h后，其体内的生长激素浓度即恢复至正常水平。微球内水相中加入泊洛沙姆407（F127），使rhGH的相对生物利用度增加约12．5％，进一步证实了F127对rhGH具有稳定作用。     

The Stability of Recombinant Human Growth Hormone in Poly(lactic-co-glycolic acid) (PLGA) Microspheres

Purpose. The development of a sustained release formulation for recombinant human growth hormone (rhGH) as well as other proteins requires that the protein be stable at physiological conditions during its in vivo lifetime. Poly(lactic-co-glycolic acid) (PLGA) microspheres may provide an excellent sustained release formulation for proteins, if protein stability can be maintained. Methods. rhGH was encapsulated in PLGA microspheres using a double emulsion process. Protein released from the microspheres was assessed by several chromatrographic assays, circular dichroism, and a cell-based bioassay. The rates of aggregation, oxidation, diketopiperazine formation, and deamidation were then determined for rhGH released from PLGA microspheres and rhGH in solution (control) during incubation in isotonic buffer, pH 7.4 and 37°C. Results. rhGH PLGA formulations were produced with a low initial burst (<20%) and a continuous release of rhGH for 30 days. rhGH was released initially from PLGA microspheres in its native form as measured by several assays. In isotonic buffer, pH 7.4 and 37°C, the rates of rhGH oxidation, diketopiperazine formation, and deamidation in the PLGA microspheres were equivalent to the rhGH in solution, but aggregation (dimer formation) occured at a slightly faster rate for protein released from the PLGA microspheres. This difference in aggregation rate was likely due to the high protein concentration used in the encapsulation process. The rhGH released was biologically active throughout the incubation at these conditions which are equivalent to physiological ionic strength and pH. Conclusions. rhGH was successfully encapsulated and released in its fully bioactive form from PLGA microspheres over 30 days. The chemical degradation rates of rhGH were not affected by the PLGA microspheres, indicating that the internal environment of the microspheres was similar to the bulk solution. After administration, the microspheres should become fully hydrated in the subcutaneous space and should experience similar isotonic conditions and pH. Therefore, if a protein formulation provides stability in isotonic buffer, pH 7.4 and 37°C, it should allow for a safe and efficacious sustained release dosage form in PLGA microspheres.     

重组人生长激素缓释微球.Ⅰ.制备与体外释放研究

采用复乳-溶媒蒸发法制备重组人生长激素（rhGH）可生物降解缓释微球，考察蛋白稳定剂泊洛沙姆407（F127）对rhGH体外释放的影响。利用均匀设计优化微球的处方和制备工艺，制得的rhGH-PLGA微球球形圆整，表面光滑致密，粒径均匀且具有较高的包封率和载药量。微球的体外释药行为呈先快后慢的两相释放模式，d30时含有F127的微球累积释药量约79％，较不含稳定剂的微球高22％。表明F127可通过抑制有机溶剂诱导的蛋白聚集而对rhGH起到稳定作用。     

Sustained Release of Human Growth Hormone from PLGA Solution Depots

Purpose. The effects of altering the dynamics of phase inversion of a polylactic glycolic acid (PLGA) solution depot on the sustained-release delivery profile of human growth hormone (hGH) were evaluated. The impact of adjusting the protein particle composition was also studied in a slow phase-inverting formulation. Methods. Protein release profiles of depots prepared from four model solvents were generated by injecting formulations into the subcutaneous space of normal rats and monitoring hGH serum levels over the course of 1 month. Scanning electron microscopy, Coulometric Karl Fischer titration, size-exclusion liquid chromatography, and reversed-phase liquid chromatography were used to observe depot morphologies, bulk water absorption, PLGA degradation, and protein particle dissolution rates, respectively. Results. An extended-release profile and significantly reduced burst effect resulted when the aqueous affinity of the depot solvent was reduced. As seen earlier in in vitro experiments, lowering the solvent's aqueous affinity slows the phase inversion rate, which in turn produces depot morphologies favorable to prolonged release. Protein burst on injection was entirely eliminated in a slow phase-inverting formulation by densifying the lyophilized protein particles. Unlike the use of metal cations to prolong release of some proteins in PLGA microsphere depots, this technique is more universal, and thus is potentially usable with any protein or highly soluble drug agent. The onset of biodegradation was observed to occur at 14 days for all depot formulations, however the bulk biodegradation rate slowed as the aqueous affinity of the depot solvent decreased. This result supports the hypothesis that, in a slow phase-inverting system, drug release over the first few weeks is governed by the diffusion rate of drug through the polymer solution. Conclusions. By taking advantage of the effects of low aqueous affinity and protein particle densification, a PLGA solution depot was produced with the capability of sustaining hGH levels in normal rats at a serum level of 10 to 200 ng/ml for 28 days.     

Monodisperse Liquid-filled Biodegradable Microcapsules

Purpose Encapsulation of liquids into biodegradable polymer microcapsules has been a challenging task due to production limitations stemming from solution viscosity, phase stabilization, molecular localization, and scalable production. We report an extension of Precision Particle Fabrication (PPF) technology for the production of monodisperse liquid-filled microcapsules containing an oil or aqueous core and contrast these results to double-walled microspheres. Materials and Methods PPF technology utilizes a coaxial nozzle to produce a liquid core jet surrounded by a polymer annular jet, which is further encompassed by a non-solvent carrier stream, typically 0.5% wt/vol polyvinyl alcohol in water. Jet diameters are controlled by the volumetric flow rate of each phase. The compound jet is then disrupted into uniform core/shell droplets via a controllable acoustic wave and shell material is hardened by solvent extraction. Results Monodisperse polymeric microcapsules demonstrated a narrow size distribution and the formation of a continuous shell leading to efficient encapsulation of various liquid cores. The intermingling of core and shell phases and the localization of different molecular probes (fluorescent dyes and fluorescently labeled proteins) to the core or shell phase provided additional evidence of phase separation and molecular partitioning, respectively. We also demonstrate the pulsatile release of bovine serum albumin encapsulated in an aqueous core. Conclusions PPF technology provided exceptional control of the overall size and shell thickness of microcapsules filled with various types of oil or water. This technique may enable advanced delivery profiles of pharmaceuticals or nutraceuticals.     

放射免疫法研究乳糖基重组人生长激素和重组人生长激素的药代动力学

目的 :比较研究乳糖基重组人生长激素 (L -rhGH )和重组人生长激素 (rhGH )在小鼠血和肝中的药代动力学。方法 :用注射免疫法测定L -rhGH和rhGH在小鼠血、肝脏中的药物浓度。结果 :静脉注射后 ,两者的药代动力学过程存在显著性差异 ,L -rhGH在肝中的分布量较rhGH增加 ,且T1/2 仅为rhGH的17 9 % ;L -rhGH在血中亦表现为短维持时间、低分布量。结论 :L -rhGH呈现出更符合人体生理的药代动力学特征     

Stabilization of pH-Induced Degradation of Porcine Insulin in Biodegradable Polyester Microspheres

The purpose of this research project was to stabilize the pH-induced degradation of porcine insulin encapsulated within biodegradable polyester microspheres through the incorporation of a basic additive. Insulin microspheres fabricated using Poly(L-lactide) (L-PLA) and Poly(DL-lactide-co-glycolide) (50:50 DL-PLGA) were subjected to in vitro release studies and the stability of unreleased insulin encapsulated within microspheres was investigated. The intramicrosphere pH was estimated by encapsulating acid-base indicators covering a wide pH transition range within 50:50 DL-PLGA microspheres. Finally, a basic excipient sodium bicarbonate was incorporated in 50:50 DL-PLGA microspheres to minimize acid-induced insulin degradation. The in vitro release was slow and incomplete (<30% in 30 days). Extraction and analyses of the unreleased insulin within the microspheres revealed that an average of ～11% remained intact. The degradation products observed consisted of ～15% of three distinct deamidated hydrolysis products including A-21 Desamido insulin, ～22% Covalent Insulin Dimer and trace amounts of High Molecular Weight Transformation Products. Comparison of the degradation profile of unreleased insulin contained in various microsphere formulations with the in vitro release kinetics indicated that an increase in covalent dimer formation within the microspheres prior to release is associated with a decrease in the cumulative percent insulin released during a 30-day incubation period. In an attempt to correlate insulin degradation with the drop in intra-microsphere pH due to polymer hydrolysis, it was determined that the pH within a degrading microsphere reaches a value of ～1.8 after 4 weeks. The incorporation of a basic excipient, sodium bicarbonate, in 50:50 DL-PLGA microspheres resulted in an improved in vitro release profile (cumulative release ～47.3% in 30 days) as well as a significant reduction in covalent dimerization of the unreleased insulin to barely detectable levels. The low pH microenvironment within a degrading microsphere is one of the major factors leading to protein instability, and the degradation of proteins encapsulated within polyester microspheres can be minimized by the incorporation of a basic excipient.     

Novel Long-Acting Crystal Formulation of Human Growth Hormone

PURPOSE: The aim of the study is to solve a significant challenge of extending the half-life of therapeutic proteins using crystalline biopharmaceuticals and without redesigning the molecules. METHODS: Crystals of recombinant human growth hormone were coated with a monomolecular layer of positively charged poly(arginine). The pharmacokinetics and pharmacodynamics of this poly(arginine)-coated human growth hormone crystalline formulation were determined in hypophysectomized rats and monkeys. RESULTS: Here we have demonstrated for the first time that crystals of human growth hormone coated with positively charged poly(arginine) allowed for in vivo pharmacokinetic release profiles of over several days in animal models. The efficacy of this crystalline formulation injected subcutaneously once a week was found to be equivalent to seven daily soluble injections in the standard weight gain assay using the hypophysectomized rat model and in measurement of serum insulin-like growth factor in monkeys. The nonviscous nature of the suspension facilitated easy administration through a fine, 30-gauge needle and should provide for improved patient convenience and compliance. CONCLUSIONS: The approach described here offers an exciting possibility of being broadly applicable to other therapeutic proteins.     

Effect of Freezing on Aggregation of Human Growth Hormone

The effect of freezing on formation of soluble and insoluble aggregates of human growth hormone (hGH) was studied. The amount of soluble aggregates was affected very little by freezing regardless of the cooling rate. In contrast, the formation of insoluble aggregates (particulates), as determined by light scattering in the 340- to 360-nm range, was found to increase sharply with increasing cooling rates. The amount of these particulates was also dependent on the pH of the solution. Freezing hGH solutions formulated at pH 7.4 resulted in highly scattering solutions, whereas pH 7.8 formulations showed significantly less scattering. These results emphasize the importance of understanding the freezing phenomenon for protein solutions and suggest that the formation of soluble aggregates and insoluble particulates may have different mechanisms.     

One-Step Surface Modification of Poly(lactide-<Emphasis Type="BoldItalic">co</Emphasis>-glycolide) Microparticles with Heparin

Purpose The aim of this study was to modify the surface of poly(lactide-co-glycolide) (PLGA) microparticles with heparin. The heparin-coated PLGA may enhance blood and tissue compatibility of PLGA devices and provide a novel approach to deliver growth factors. Materials and Methods A one-step method using heparin to replace traditional emulsifiers (e.g., PVA) during emulsion-solvent evaporation process was employed to surface-entrap heparin in PLGA microspheres. The emulsifying activity of heparin was modified via varying counter ion form, including univalent (Na⁺, K⁺, Li⁺, and ) and divalent (Ca²⁺, Mg²⁺, Ba²⁺, and Zn²⁺) cations, and complexation with amino acids (Arg, Lys, Leu, Val, Gly and Glu). Surface accessible and total heparin loading were determined by a modified toluidine blue assay and elemental analysis, respectively. Results Heparin bound with univalent counter ions and amino acids exhibited emulsifying activity to varying degrees, whereas divalent heparin salts tended to cause complete aggregation of the PLGA o/w emulsion. Increasing pH (≥7.4) of hardening medium enhanced heparin adsorption and significantly stabilized the PLGA o/w emulsion. The initial surface density of heparin on the PLGA microspheres prepared using univalent heparin salts was around 8–33 mg/m². Surface associated heparin desorbed quickly; potassium heparin showed the best retention, with ~0.2 and 0.1 mg/m² detected on PLGA microsphere surface following 1- and 14-day incubation in PBST at 37°C, respectively. Conclusions PLGA microparticles were successfully surface-modified with heparin. Univalent salts and amino acid complexes of heparin, as effective emulsifiers, can become surface-immobilized in PLGA microspheres.     

尼索地平微球的制备

采用溶剂挥发法制备了尼索地平微球 ,考察了制备工艺中影响微球质量的 5个主要因素 ,筛选出较理想的处方和工艺。所得微球形态圆整 ,表面光滑 ,粒径 (18.2± 3.8) μm,载药量 2 1.2 % ,包封率 85 .4 %。     

Nasal Absorption Enhancers for Biosynthetic Human Growth Hormone in Rats

The effects of several prospective absorption enhancers were assessed on the nasal absorption of biosynthetic human growth hormone (hGH) in the rat. These enhancers function by alternative mechanisms that include enzyme inhibition, reduction in mucus viscosity, and enhancement of membrane fluidity. The levels of plasma hGH achieved were determined by an enzyme-linked immunosorbent assay. The increase in peak height was calculated relative to nasal administration of hGH alone without any enhancers and the relative bioavailability was calculated with reference to subcutaneous injection data. A lysophospholipid, lysophosphatidylcholine, gave the highest peak concentration, with an increase in peak height of 450% and a relative bioavailability of 25.8%. However, the greatest increase in AUC (291%) was achieved with the aminopeptidase inhibitor, amastatin, which gave a relative bioavailability of 28.9%. A mucolytic agent, N-acetyl-L-cysteine, and a transmembrane fatty acid transporter, palmitoyl-DL-carnitine, were also found to promote the nasal absorption of hGH in this model, with relative bioavailabilities of 12.2 and 22.1%, respectively. Bestatin, an enzyme inhibitor, was not an effective absorption enhancer for hGH in this model.     

重组人生长激素治疗儿童生长激素缺乏症的临床观察

目的：探讨重组人生长激素（rhGH）对生长激素缺乏症（GHD）患儿的治疗效果及对甲状腺功能、血清胰岛素生长因子-1（IGF-1）、25羟维生素D（25（OH）D）水平的影响。方法：选取我院2013年1月至2017年12月我院门诊治疗的70例GHD患儿作为试验组,另外选取70名同龄健康儿童作为对照组,试验组采用rhGH治疗,分析试验组在治疗6个月、治疗12个月后的身高、身高标准差积分、生长速率、骨龄/实际年龄,检测试验组治疗前及治疗12个月后的血清游离三碘甲状腺原氨酸（FT3）、游离甲状腺激素（FT4）、促甲状腺激素（TSH）、IGF-1、25（OH）D水平,并与对照组进行比较。结果：在治疗6个月、12个月后,试验组患儿的身高、身高标准差积分、生长速率、骨龄/实际年龄测定值较治疗前均显著的增加,差异有统计学意义（P<0.05）;在治疗12个月后,试验组患儿的血清FT3、IGF-1、25（OH）D水平升高,差异具有统计学意义（P<0.05）,对照组的血清FT3高于试验组治疗前水平（P<0.05）,对照组的血清IGF-1、25（OH）D高于试验组治疗前及治疗后的水平（P<0.05）。结论：rhGH对GHD患儿的治疗效果显著,能明显改变患儿FT3、血清IGF-1、25（OH）D水平。     

国产与进口重组人生长激素治疗儿童原发性生长激素缺乏症的药物经济学分析

目的:比较国产与进口重组人生长激素治疗儿童原发性生长激素缺乏症的疗效和经济学效果,为临床用药提供依据。方法:采用二次文献研究方法,检索出符合纳入标准的国内文献共4篇,对国产重组人生长激素(A组)与进口重组人生长激素(B组)治疗儿童原发性生长激素缺乏症进行成本-效果分析,并对结果进行敏感度分析。结果:A、B组均有显著促身高增长作用,且无明显不良反应。治疗时间为6个月时,A、B组的单位效果成本分别为2660.5元/cm和13011.6元/cm;治疗时间为12个月时,A、B组的单位效果成本分别为3040.6元/cm和12775.0元/cm。敏感度分析结果表明,药品价格浮动不会影响分析结果。结论:国产与进口重组人生长激素在治疗儿童原发性生长激素缺乏症上效果相似,国产重组人生长激素更具价格优势。     

GM-1PLGA肌注微球的制备及其体外释药评价

目的 研究以聚乳酸羟基乙酸共聚物为囊材,制备长效缓释的单唾液酸四己糖神经节苷脂微球制剂.方法运用复乳溶剂挥发法,考虑多个条件对制备工艺的影响,并采用正交设计对处方进行优化.测定微球的载药量、包封率和释放曲线.结果制备得到的GM-1微球粒径大小均匀,球形致密圆整,微球粒径为(8.2±6.0)μm,载药量和包封率分别为18...     

Poly (lactide-co-glycolide)-Polymethacrylate Nanoparticles for Intramuscular Delivery of Plasmid Encoding Interleukin-10 to Prevent Autoimmune Diabetes in Mice

Purpose

Determine the efficiency of cationic nanoparticles prepared by blending poly (lactide-co-glycolide; PLGA) and methacrylate copolymer (Eudragit® E100) to deliver a therapeutic gene encoding mouse interleukin-10, in vitro and in vivo.

Methods

Nanoparticles prepared with PLGA and E100 were evaluated for delivery of plasmid DNA encoding mouse interleukin-10 in vitro and in vivo in mice upon intramuscular injection. Blood-glucose, serum interferon-gamma levels and histology of pancreas were studied to determine therapeutic efficacy. Histological evaluation of skeletal muscle from the injection site was performed to assess the biocompatibility of nanoparticles.

Results

PLGA/E100 nanoparticles showed endosomal escape evidenced by confocal microscopy and buffering ability. Transfecting HEK293 cells with plasmid-loaded PLGA/E100 nanoparticles resulted in significantly (p?Conclusions Nanoparticles prepared by blending PLGA with methacrylate can efficiently and safely deliver plasmid DNA encoding mouse interleukin-10 leading to prevention of autoimmune diabetes.     

重组人生长激素治疗特发性身材矮小患儿的疗效评价

目的:重组人生长激素(rhGH)为基因工程大肠杆菌或哺乳动物细胞产生的人生长激素,其结构和氨基酸排序与人垂体激素雷同,本文评价其治疗特发性身体矮小患者的疗效与安全性.方法:查阅国内外近期相关文献,并结合笔者临床经验进行综述.结果与结论:尽管疗效研究组为重组人生长激素可改善特发性身材矮小患儿的最终身高,但临床上尚有争议,尤其是诊断内源性生长激素缺乏仍是尚待突破的课题.     

The Effect of Sodium Tauro-24,25-Dihydrofusidate on the Nasal Absorption of Human Growth Hormone in Three Animal Models

The ability of a novel permeation enhancer, sodium tauro-24,25-dihydrofusidate (STDHF), to increase the systemic delivery of human growth hormone (hGH); after intranasal administration was investigated in rat, rabbit, and sheep. Formulations of hGH with STDHF exhibited greatly enhanced nasal absorption at concentrations of STDHF above its critical micelle concentration. The increase in bioavailability was 11-fold in rats and in rabbits and 21-fold in sheep for formulations containing 0.5% STDHF as compared to those without STDHF. Glycocholate or taurocholate at 0.5% was three to five times less effective than STDHF at enhancing hGH absorption in rats. Additionally, the pulsatile absorption kinetics observed after intranasal delivery more closely resemble the endogenous secretory pattern of hGH than those obtained following subcutaneous administration.     

Increased Intestinal Delivery of Viable Saccharomyces boulardii by Encapsulation in Microspheres

Purpose Although probiotics are of a major potential therapeutic interest, their efficacy is usually limited by poor bioavailability of viable microorganisms on site. The aim of this study was to protect the probiotic Saccharomyces boulardii from degradation in order to ensure a greater number of viable yeast in the colon. Methods Alginate microspheres coated with or not with chitosan were used to encapsulate the yeast by an extrusion method. The efficiency of encapsulation was assessed both in vitro and in vivo. Results In vitro, less than 1% of the non-encapsulated probiotic survived after 120 min at pH 1.1, whereas the majority of encapsulated yeast cells remained entrapped within both types of microspheres. Further exposure to a pH 6.8 allowed the release of about 35% of viable yeasts. In vivo, the percentage of viable yeast excreted over 96 h after a single oral dose of 2 × 10⁸ cfu/100 g in rats was 2.5% for non-encapsulated yeast and reached 13.3 and 9.0% of the dose administered for the uncoated and chitosan-coated microspheres, respectively. Conclusions Given the dose-dependent efficacy of S. boulardii and the efficiency of microencapsulation in protecting the yeast from degradation, alginate microspheres could be of great interest in therapeutic applications of the yeast.