A novel in vitro release technique for peptide containing biodegradable microspheres.
A novel in vitro release technique for peptide containing biodegradable microspheres.
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DOI：
10.1208/pt010104
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发表时间：
2000-03-09
影响因子：
3.3
通讯作者：
DeLuca, P P
中科院分区：
文献类型：
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作者：
Kostanski, J W;DeLuca, P P
本研究的目的是开发和评估一种透析体外释放技术，用于含有多肽的聚（d， l-丙交酯-乙醇酸酯）（PLGA）微球（ms），该技术将与体内数据相关。采用促黄体生成素释放激素类似物（LHRH）， Orntide醋酸酯，在0.1 M磷酸盐缓冲液（PB）， pH 7.4和0.1 M醋酸缓冲液（AB）， pH 4.0中，高效液相色谱（HPLC）测定溶解度和稳定性，并测定肽通过透析膜（分子量截断300000）的通透性。采用分散/溶剂萃取/蒸发法制备Orntide质粒，并对其药物含量（HPLC）、粒度分布（激光衍射法）和表面形貌（扫描电镜）进行表征。在37℃孵育条件下，采用传统提取法和新型透析法研究PB中的体外释放。用重量分析法分析PB和AB中聚合物的质量损失和基质水化，以及肽对空白PLGA ms （50:50， M(w) 28 022）的吸附。醋酸奥肽的溶解度受pH的影响；约2.3 mg/mL溶解于PB中，18 mg/mL溶解于AB中。稳定性与pH和温度有关。该肽在pH 4.0, 4℃时非常稳定，但在pH 7.4, 37℃时降解迅速，搅拌加速了肽通过透析膜的渗透性，48小时内达到95%的平衡。透析法总释放率较高。Orntide ms的质量损失在AB组（3周内损失50%，35天内损失95%）比PB组（35天内损失65%）快。相比之下，35 d水化后的PB含量增加了4倍。PB （128 μ g Orntide/10 mg PLGA）对空白ms的非特异性吸附高于AB （< 5 μ g Orntide/10 mg PLGA）。给大鼠服用30天Orntide PLGA ms后，6小时后血清Orntide初始水平为21 ng/mL， 6天后Cmax为87 ng/mL。服用ms （3 mg Orntide /Kg）后，睾酮水平立即被抑制，从5.2 ng/mL降至0.3 ng/mL（24小时后），并持续38天。缓冲体系的pH和质量损失对奥肽醋酸酯的溶解度和降解动力学有显著影响；基质水合作用以及对空白质谱的非特异性吸附与ph值有关。通过透析方法在AB中获得的体外释放谱与体内数据具有良好的相关性，从而提供了更可靠的体内性能预测。
The purpose of this study was to develop and evaluate a dialysis in vitro release technique for peptide-containing poly(d, l-lactide-co-glycolide) (PLGA) microspheres (ms) that would correlate with in vivo data. Using a luteinizing hormone- releasing hormone analogue (LHRH), Orntide acetate, solubility and stability were determined in 0.1 M phosphate buffer (PB), pH 7.4, and in 0.1 M acetate buffer (AB), pH 4.0, with high-performance liquid chromotography (HPLC), and peptide permeability through a dialysis membrane (molecular weight cut-off 300,000) was determined. Orntide ms were prepared by a dispersion/solvent extraction/evaporation method and characterized for drug content (HPLC), particle size distribution (laser diffraction method), and surface morphology (scanning electron microscopy). In vitro release was studied in PB using a conventional extraction method and with a new dialysis method in AB. Gravimetric analyses of polymer mass loss and matrix hydration, and peptide adsorption to blank PLGA ms (50:50, M(w) 28 022) were carried out in PB and AB upon incubation at 37 degrees C. Serum Orntide and testosterone levels in rats after administration of Orntide ms were determined by radioimmunoassay. Orntide acetate solubility was influenced by pH; approximately 2.3 mg/mL dissolved in PB and > 18 mg/mL in AB. Stability was pH- and temperature-dependent. The peptide was very stable at pH 4.0, 4 degrees C, but degraded rapidly at pH 7.4, 37 degrees C. Peptide permeability through the dialysis membrane was accelerated by agitation and >95% equilibrium was reached within 48 hours. The overall release rate was higher with the dialysis method. Mass loss of the Orntide ms was faster in AB (50% loss in 3 weeks; 95% in 35 days) than in PB (65% in 35 days). In contrast, hydration after 35 days was 4-fold higher in PB. The nonspecific adsorption to blank ms was greater in PB (128 microg Orntide/10 mg PLGA) compared with AB (< 5 microg Orntide/ 10 mg PLGA). Administration of 30-day Orntide PLGA ms to rats resulted in an initial serum Orntide level of 21 ng/mL after 6 hours and a Cmax of 87 ng/mL after 6 days. Testosterone levels were suppressed immediately after ms administration (3 mg Orntide /Kg) from 5.2 ng/mL to 0.3 ng/mL (after 24 hours) and remained suppressed for 38 days. Orntide acetate solubility and degradation kinetics were markedly influenced by pH of the buffer systems and mass loss; matrix hydration, as well as the nonspecific adsorption to blank ms, was pH-dependent. The in vitro release profile obtained with the dialysis method in AB correlated well with the in vivo data, thereby providing a more reliable prediction of in vivo performance.