布比卡因聚乳酸缓释微球的研究
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摘要
布比卡因于80年代在我国应用于临床，广泛应用于术中及术后疼痛的治疗。布
比卡因产生局部麻醉作用的同时可扩张外周血管，药物经血管吸收加速，可使其效
期缩短，甚至产生中枢神经系统及心血管毒性反应。临床上使用需小剂量频繁给药
以维持其药效，既给患者带来痛苦和不便又因血药浓度积累引起毒副作用。为了延
长布比卡因的作用时效，减少给药次数，降低不良反应。本文用聚乳酸为载体，制
备了布比卡因聚乳酸微球，对微球进行了体内外评价。
本文采用W/O/W乳剂一溶剂挥发法制备了布比卡因聚乳酸微球，通过析因设计
实验，考察了各因素对微球制备及性质的影响。实验结果表明：内相聚合物浓度，
布比卡因浓度、布比卡因与聚乳酸的投料比、有机溶媒的选择、搅拌速度可影响微
球的粒径和分布。微球的含量及包封率受药物与聚合物投料比、分散时间、搅速等
因素的影响。体外释药研究表明：微球无明显的突释现象，释药可用Higuchi方程
Q=5.287+9.525t~(1/2)（r=0.996）和一级动力学方程ln（100-Q）=4.442-0.023t（r=-0.993）
拟合。微球的粒径，含药量及制备过程中的因素均对其释药性能有影响。聚乳酸的
分子量对微球的理化特性及释药有一定影响。
稳定性实验表明：37℃密闭保存，微球产生粘连破坏。4℃和室温下密闭存放六
个月，微球的形态，含药量及体外释药均比较稳定。这与聚乳酸的玻璃化温度有关
（40-55℃）。
本文采用高效液相色谱法测定兔血浆中布比卡因的含量。布比卡因浓度在
0.05-6.0μg/ml范围内线性关系良好（r=0.9998），最低检测限为0.02μg/ml。平
均回收率为98.078％，日内差和日间差分别为2.328％、3.557％。表明用高效液相色
谱法测定血浆中的布比卡因含量方法稳定，结果准确可靠。
本文进行了新西兰兔单剂量布比卡因注射液皮下浸润麻醉和伤口植入布比卡因
微球的药物动力学研究。注射剂组峰浓度高，达峰后血药浓度迅速下降。微球组药
一时曲线相对平缓，Tmax延迟（P＜0.01），MRT明显延长（P＜0.01）。表明微球具有
明显的延缓释药效果，大大提高了使用的安全性。
布比卡因聚乳酸微球的初步药效学实验中，以新西兰兔的局麻直径为指标评价
微球是否具有时效依赖关系。结果表明微球组的作用时效较对照组明显延长。
因此，以聚乳酸作为载体制备布比卡因微球，微球具有明显的缓释作用。达到
延长作用时间及减少给药次数的目的。
Bupivacaine had been widely used for clinical. It is considered a relatively safe and effective method for providing anesthesia and postoperative. Side effects about cardiovascular and nerve center system has been observed, due to the accumulation of bupivacaine plasma concentration. Moreover, the effect that bupivacaine enlarge local vascular will reduce anesthetic time-action. therefore, in order to prolong the anesthetic action, minimize side effects and decrease frequency of administration. The suitable sustained-release microspheres containing bupivacaine was prepared using Polylactic acid(PLA) as carrier and evaluated in vitro and in vivo.
Bupivacaine PLA microspheres were prepared using emulsion-dispersion solvent evaporation method. The effects of different variables on the preparation of PLA microspheres were studied according to a factorial design of experiments. Polymer concentration of internal phase,ratio of bupivacaine:PLA, organic solution and stirring rate could affect microspheres size and size distribution. The drug encapsulation efficiency and drug content of the microspheres was mainly determined by the ratio of bupivacaine:PLA, dispersion time and stirring rate. Studies on the release in vitro showed that nearly no initial burst release could be seen. The in vitro release kinetics of bupivacaine from PLA microspheres can be described by Higuchi equation and First-order equation. It could be expressed by following equation Q=5. 287+9. 525t~(1/2)(r=0. 996): ln(1OO-Q)=4. 442-0. 023t (r=0.993). The release rate was related to rnicrospheres size, drug content and some factors for preparation. To some extent, PLA of different molecular weight could affect the preparation and in vitro release of microspheres.
Study on stability, during stored at 370C, the microspheres fused and aggregated. While stored at 4 0C or room temperature under desiccated
condition for six months , surface morphology, drug content and in vitro release of microspheres did not alter. It related to the glasstransition temperature for PLA.
A high performance liquid chromatographic method was developed to determine bupivacaine plasma concentration. The linear range was 0.05?.0 jig/mi (r~O.9998). The limit was 0. O2ug/ml. The average recovery in plasma was 98. 078%. The within day and between day RSD were 2. 328% and 4.46l%.The accuracy test showed that the determination of bupivacaine in plasma by HPLC method was stable, and the results were reliable.
The pharmacokinetic study was carried out in rabbits. Bupivacaine microspheres was single梚mplant given to animal. Bupivacaine injection was Single梚nject given too. Compared with injection the plasma concentration of bupivacaine was lower, and concentration梩ime curve was gently. It obviously showed that T.ax delay(p(0.0l) and MRT prolong(p(0.01).Prolonged drug action also could be seen.
Study on the beginning pharmacodynamics, a rabbit model for evaluation of regional anesthesia is presented. The regional anesthesia was assessed by direct observation of sensory blockade. It is showed that the regional sensory blockade of microspheres group prolonged compared to the injection group.
In conclusion this biodegradable polymer system provides promising new alternative for the delivery of local anesthetics to produce prolonged acting time and decrease side effects.
比卡因产生局部麻醉作用的同时可扩张外周血管，药物经血管吸收加速，可使其效
期缩短，甚至产生中枢神经系统及心血管毒性反应。临床上使用需小剂量频繁给药
以维持其药效，既给患者带来痛苦和不便又因血药浓度积累引起毒副作用。为了延
长布比卡因的作用时效，减少给药次数，降低不良反应。本文用聚乳酸为载体，制
备了布比卡因聚乳酸微球，对微球进行了体内外评价。
本文采用W/O/W乳剂一溶剂挥发法制备了布比卡因聚乳酸微球，通过析因设计
实验，考察了各因素对微球制备及性质的影响。实验结果表明：内相聚合物浓度，
布比卡因浓度、布比卡因与聚乳酸的投料比、有机溶媒的选择、搅拌速度可影响微
球的粒径和分布。微球的含量及包封率受药物与聚合物投料比、分散时间、搅速等
因素的影响。体外释药研究表明：微球无明显的突释现象，释药可用Higuchi方程
Q=5.287+9.525t~(1/2)（r=0.996）和一级动力学方程ln（100-Q）=4.442-0.023t（r=-0.993）
拟合。微球的粒径，含药量及制备过程中的因素均对其释药性能有影响。聚乳酸的
分子量对微球的理化特性及释药有一定影响。
稳定性实验表明：37℃密闭保存，微球产生粘连破坏。4℃和室温下密闭存放六
个月，微球的形态，含药量及体外释药均比较稳定。这与聚乳酸的玻璃化温度有关
（40-55℃）。
本文采用高效液相色谱法测定兔血浆中布比卡因的含量。布比卡因浓度在
0.05-6.0μg/ml范围内线性关系良好（r=0.9998），最低检测限为0.02μg/ml。平
均回收率为98.078％，日内差和日间差分别为2.328％、3.557％。表明用高效液相色
谱法测定血浆中的布比卡因含量方法稳定，结果准确可靠。
本文进行了新西兰兔单剂量布比卡因注射液皮下浸润麻醉和伤口植入布比卡因
微球的药物动力学研究。注射剂组峰浓度高，达峰后血药浓度迅速下降。微球组药
一时曲线相对平缓，Tmax延迟（P＜0.01），MRT明显延长（P＜0.01）。表明微球具有
明显的延缓释药效果，大大提高了使用的安全性。
布比卡因聚乳酸微球的初步药效学实验中，以新西兰兔的局麻直径为指标评价
微球是否具有时效依赖关系。结果表明微球组的作用时效较对照组明显延长。
因此，以聚乳酸作为载体制备布比卡因微球，微球具有明显的缓释作用。达到
延长作用时间及减少给药次数的目的。
Bupivacaine had been widely used for clinical. It is considered a relatively safe and effective method for providing anesthesia and postoperative. Side effects about cardiovascular and nerve center system has been observed, due to the accumulation of bupivacaine plasma concentration. Moreover, the effect that bupivacaine enlarge local vascular will reduce anesthetic time-action. therefore, in order to prolong the anesthetic action, minimize side effects and decrease frequency of administration. The suitable sustained-release microspheres containing bupivacaine was prepared using Polylactic acid(PLA) as carrier and evaluated in vitro and in vivo.
Bupivacaine PLA microspheres were prepared using emulsion-dispersion solvent evaporation method. The effects of different variables on the preparation of PLA microspheres were studied according to a factorial design of experiments. Polymer concentration of internal phase,ratio of bupivacaine:PLA, organic solution and stirring rate could affect microspheres size and size distribution. The drug encapsulation efficiency and drug content of the microspheres was mainly determined by the ratio of bupivacaine:PLA, dispersion time and stirring rate. Studies on the release in vitro showed that nearly no initial burst release could be seen. The in vitro release kinetics of bupivacaine from PLA microspheres can be described by Higuchi equation and First-order equation. It could be expressed by following equation Q=5. 287+9. 525t~(1/2)(r=0. 996): ln(1OO-Q)=4. 442-0. 023t (r=0.993). The release rate was related to rnicrospheres size, drug content and some factors for preparation. To some extent, PLA of different molecular weight could affect the preparation and in vitro release of microspheres.
Study on stability, during stored at 370C, the microspheres fused and aggregated. While stored at 4 0C or room temperature under desiccated
condition for six months , surface morphology, drug content and in vitro release of microspheres did not alter. It related to the glasstransition temperature for PLA.
A high performance liquid chromatographic method was developed to determine bupivacaine plasma concentration. The linear range was 0.05?.0 jig/mi (r~O.9998). The limit was 0. O2ug/ml. The average recovery in plasma was 98. 078%. The within day and between day RSD were 2. 328% and 4.46l%.The accuracy test showed that the determination of bupivacaine in plasma by HPLC method was stable, and the results were reliable.
The pharmacokinetic study was carried out in rabbits. Bupivacaine microspheres was single梚mplant given to animal. Bupivacaine injection was Single梚nject given too. Compared with injection the plasma concentration of bupivacaine was lower, and concentration梩ime curve was gently. It obviously showed that T.ax delay(p(0.0l) and MRT prolong(p(0.01).Prolonged drug action also could be seen.
Study on the beginning pharmacodynamics, a rabbit model for evaluation of regional anesthesia is presented. The regional anesthesia was assessed by direct observation of sensory blockade. It is showed that the regional sensory blockade of microspheres group prolonged compared to the injection group.
In conclusion this biodegradable polymer system provides promising new alternative for the delivery of local anesthetics to produce prolonged acting time and decrease side effects.
引文
[1] David B，Charles B；et al．Prolonged regional nerve blockade by controlled release of local anesthetic from a biodegradable polymer matrix．Anesthesiology 1993；79：340-346
[2] Shigehito S,Yauyuki B；et al．Prolongation of epidural anesthesia in the rabbit with the use of a biodegradable copolymer paste．Anesth．Analy．1995；80：97-101
[3] Curley J,Castillo J；et al．Prolonged regional nerve blockade：injectable biodegradable／polyester microspheres．Anesthesiology 1996；84(6) ：1401-1410
[4] Estebe J．P，Malledant Y; et al．Prolongation of spinal anesthesia with bupivacaine-loaded(DL-lactide)microspheres．Anesth．Analg．1995；81(1) ：99-103
[5] 中华人民共和国药典 (二部)，2000版．587
[6] 陆彬主编．药物新剂型与新技术．人民卫生出版社出版 165
[7] 胡一桥等．胰岛素聚乳酸微球处方筛选．中国药学杂志 1999；34(12) ：822-826
[8] Chida T，Yoshida K；et al．Preparation and characterization of polylacfic acid microspheres containing water-soluble dyes using a novel W／O／W emulsion solvent evaporation method．J．Microencapsulation．1996；13(2) ：219-228
[9] Claudia W，Eric D；et al．Influence of the microencapsulation method and peptide loading on poly(lactic acid) and poly(lactic-co-glycolic acid) degradation during in vitro testing．J．Controlled．Release．1998；51：327-341
[10] Laurence B，Dan B；et al．Slow-release effect of pH-adjusted bupivacaine． Int．J．Pharm．1992；84：33-37
[11] Ogana Y，Yamamoto M；et al．Controlled-release of leuprolide acetate from polylactic acid or copoly(lactic／glycolic) acid microcapsules：influence of molecular weight and copolymer ratio of polymer．Chem．Pharm．Bull． 1988；36：1095-1103
[12] Spenlehauer G，Veillar M；et al．Formation and characterization of cisplatin loaded poly(D，L-lactide) microspheres for chemoembolization．J．Pharm．Sci． 1986；75：750-755
[13] Lin S．Y，Chen K．S；et al．Functionality of protective colloids affecting the formation，size uniformity and morphology of drug-free polylactic acid microspheres．J．Microencapsulation．1998；15(3) ：383-390
[14] 王正容，陆彬等．左炔诺孕酮-聚 3-羟基丁酸酯缓释微球的研究．药学学报 1999：34 (1) ：54-57
[15] 钟延强等．关节腔内注射用氟比洛芬明胶微球的制备及体外释药研究．药学实践杂志 1998；16 (6) ：341-345
[16] 杨俊杰等．醋酸亮丙瑞林微球．国外医学药学分册 1998；25 (5) ：298-301
[17] 中华人民共和国药典 (二部)，2000 版．附录75
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[19] Corre L，Rytting J；et al．In vitro controlled release kinetics of local anaesthetics from poly(D，L-lactide) and poly(lactide-co-glycolide) microspheres． J．Microencapsulation．1997；14(2) ：243-255
[20] Wada R，Hyon S；et al．Lactic acid oligomer microspheres containing hydrophilic drugs．J．Pharm．Sci．1990; 79(10) ：919-924
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[22] Gorner T，Gref R；et al．Lidocaine-loaded biodegradable nanopheres．I Optimization of the drug incorporation into the polymer matrix． J．Controlled．Release. 1999；57：259-268
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[24] Mhando J．R，Po L．W；et al．Controlled release of local anaesthetic agents from liquid-solid emulsion gels．Int．J．Pharm．1990；62：249-258
[25] 朱颐申，胡一桥等．聚乳酸及乳酸／羟基乙酸共聚物微球的研究．中国药科 大学学报．1999；30 (1) ：73-77
[26] Benita S,Benoit J．P；et al．Characterization of drug loaded poly(D，L-lactide) microspheres．J．Pharm．Sci．1984；73(12) ：1721-1724
[27] Wakiyana N，Juni K；et al．Preparation and evalution in vitro of polylactic acid microspheres containing local anesthetics．Pharm．Bull．1981；29(11) ：3363-3368
[28] Wakiyama N，Juni K；et al. Influences of physicochemical properties of polylactic acid on the characteristics and in vitro release：patterns of polylactic acid microspheres containing local anesthetics．Chem．Pharm．Bull．1982；30(70) ：2621-2628
[29] Vert M，Chabot F；et al．Biodegradable cisplatin microspheres prepared by the solvent evaporation method：morphology and release characteristics． J．Controlled．Release．1988；7：217-229
[30] Bodmeier R，Mcginity J．W；et al．Poly(lactic acid) microspheres containing quinidine base and quinidine sulphate prepared by the solvent evaporation technique．J．Microencapsulation．1987；4(4) ：279-288
[31] Corre L，Guevello L；et al．Preparmion and characterization of bupivacaine-loaded polylactide and polylactide-co-glycolide microspheres．Int．J．Pharm． 1994；107：41-49
[32] Juni K,Nakano M；et al．Preparation and evaluation in vitro and in vivo of poly(lactic acid) microspheres containing doxorubicin．Chem．Pharm．Bull． 1985；33(1) ：313-318
[33] Benoit J．P，Thies C；et al．A physiochemical study of the morphology of progesterone loaded poly(d，l-lactide) microspheres．Int．J．Pharm．1986；29：95-102
[34] Fong J．W，Pearson J；et al．Evaluation of biodegradable microsapsules prepared by solvent evaporation process using sodium oleate as emulsifier． J．Controlled．Release．1986；3：119-130
[35] Burkhard W，Peter R；et al．A new method for the preparation of drug containing polylactic acid microparticles without using oranic solvent．J．Controlled．Release． 1990；14：269-283
[36] Juni K,Nakano M；et al．Preparation and evaluation in vitro and vivo of poly(lactic acid) microspheres containing dibucaine．Chem．Pharm．Bull． 1982；30(10) ：3719-3727
[37] Guevello L，Corre L；et al．High performance liquid chromatographic determination of bupivacaine in plasma samples for biopharmaceutical studies in application to seven other local anesthetics．J．Chromatography．1993；622：284-290
[38] 杨树德，薛延等．人血中利多卡因、布比卡因及丁卡因的高效液相色谱法 测定．药学学报 1984：19 (8) ：611-615
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[40] 董英海等．盐酸利多卡因缓释胶丸对术后切口镇痛作用的实验研究．中国 疼痛医学杂志 1999；3：177-181
[41] Malinovsky J．M，Bernard J．M；et al．Motor and blood pressure effects of epidural sustained-release bupivacaine from polymer microspheres．Anesth．Analg． 1995；81：519-24
[42] Corre L，Estebe J．P；et al．Spinal controlled delivery of bupivacaine from D，L-lactic acid oligomer microspheres．J．Pharm．Sci．1995；84(1) ：75-80
[43] David B，Charles B；et al．Sustained local anesthetic release from bioerodible polymer matrices：A potential method for prolonged regional anesthesia． Pharm．Res．1993；10(10) ：1527-1532
[44] Blanco M．D，Bernardo M．D；et al．Bupivacaine-loaded comatrix formed by albumin microspheres included in a poly(1actide-co-glycolide) film．
Biomaterials 1999;20:1919-1925
[45] Curley J,Castillo J;et al. Prolonged regional nerve blockade injectable biodegradable bupivacaine polyester microspheres. Anesthesiology 1996;84(6) :1401-1410
[46] Castillo J,Curley J;et al. Glucorticoids prolong rat sciatic nerve blockade in vivo from bupivacaine microspheres. Anesthesiology 1996;85(5) :1157-1166
[2] Shigehito S,Yauyuki B；et al．Prolongation of epidural anesthesia in the rabbit with the use of a biodegradable copolymer paste．Anesth．Analy．1995；80：97-101
[3] Curley J,Castillo J；et al．Prolonged regional nerve blockade：injectable biodegradable／polyester microspheres．Anesthesiology 1996；84(6) ：1401-1410
[4] Estebe J．P，Malledant Y; et al．Prolongation of spinal anesthesia with bupivacaine-loaded(DL-lactide)microspheres．Anesth．Analg．1995；81(1) ：99-103
[5] 中华人民共和国药典 (二部)，2000版．587
[6] 陆彬主编．药物新剂型与新技术．人民卫生出版社出版 165
[7] 胡一桥等．胰岛素聚乳酸微球处方筛选．中国药学杂志 1999；34(12) ：822-826
[8] Chida T，Yoshida K；et al．Preparation and characterization of polylacfic acid microspheres containing water-soluble dyes using a novel W／O／W emulsion solvent evaporation method．J．Microencapsulation．1996；13(2) ：219-228
[9] Claudia W，Eric D；et al．Influence of the microencapsulation method and peptide loading on poly(lactic acid) and poly(lactic-co-glycolic acid) degradation during in vitro testing．J．Controlled．Release．1998；51：327-341
[10] Laurence B，Dan B；et al．Slow-release effect of pH-adjusted bupivacaine． Int．J．Pharm．1992；84：33-37
[11] Ogana Y，Yamamoto M；et al．Controlled-release of leuprolide acetate from polylactic acid or copoly(lactic／glycolic) acid microcapsules：influence of molecular weight and copolymer ratio of polymer．Chem．Pharm．Bull． 1988；36：1095-1103
[12] Spenlehauer G，Veillar M；et al．Formation and characterization of cisplatin loaded poly(D，L-lactide) microspheres for chemoembolization．J．Pharm．Sci． 1986；75：750-755
[13] Lin S．Y，Chen K．S；et al．Functionality of protective colloids affecting the formation，size uniformity and morphology of drug-free polylactic acid microspheres．J．Microencapsulation．1998；15(3) ：383-390
[14] 王正容，陆彬等．左炔诺孕酮-聚 3-羟基丁酸酯缓释微球的研究．药学学报 1999：34 (1) ：54-57
[15] 钟延强等．关节腔内注射用氟比洛芬明胶微球的制备及体外释药研究．药学实践杂志 1998；16 (6) ：341-345
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