孕激素缓释微球的研制及其局部应用对子宫内膜异位症的治疗作用
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摘要
研究背景
子宫内膜异位症是一种难以治愈的常见妇科良性疾病,而妊娠是对子宫内膜异位症极为有效的自然疗法,假孕疗法就是用药物模拟妊娠的机体内环境,从而达到治疗目的。然而,全身应用孕激素的副作用多,包括恶心、痤疮、体重增加、水钠潴留、突破性出血、肝功能异常及血栓形成等。患者的依从性较差,常常半途而废,极大地影响了治疗效果。同时常规假孕疗法病灶局部的药物浓度低,对卵巢内膜异位囊肿等病变的治疗效果差。
为了提高疗效同时降低副作用,我们提出诱导局部假孕治疗子宫内膜异位症的假说。即制备一种可长期缓释孕激素的生物降解型微球,注射到子宫内膜异位症病灶局部,在病灶局部形成高浓度孕激素的环境,诱导出高度假孕状态,使异位内膜病灶萎缩、坏死并吸收,从而提高疗效;而血药浓度低,对代谢及卵巢功能影响小,可减少常规假孕疗法的副作用。
研究目的
1.设计一种可注射并长期缓释孕激素的生物降解型微球;
2.通过自体移植子宫内膜建立兔及大鼠子宫内膜异位症动物模型;
3.在兔内异症模型上观察孕激素缓释微球局部应用的疗效与副作用;
4.在大鼠皮下内异症模型上观察单次局部注射孕激素缓释微球疗效的维持时间,并与GnRHa的疗效相比较。
研究方法与结果
第一部分:孕激素缓释微球的制备及特性检测
采用油/水复乳液中蒸发法制备左炔诺孕酮聚乳酸微球,并检测了微球表面性状、粒径分布、注射性、载药率、体外释药及降解。通过优化含药量、蒸发温度和蒸发搅拌速度改善载药微球的表面性状、减少载药微球的突释效应。
含药量严重影响载药微球的表面性状,当分散相含药量在5%及以下时,载药微球表面相对光滑;而含药量在10%及以上时,载药微球的表面粗糙,有药物微晶体黏附,部分微球开裂;蒸发温度也对载药微球的表面性状有一定的影响,在常温下(25℃左右)制备的载药微球表面粗糙有裂纹,而在冰浴条件(0-4℃)下制备的载药微球表面相对光滑;蒸发搅拌速度是微球表面性状的另一影响因素,低速搅拌蒸发制备的载药微球表面光滑,但部分微球出现融合现象,中速搅拌蒸发制备的载药微球表面光滑,形状规则,无融合现象,而高速搅拌蒸发制备的载药微球表面粗糙,大小不一,形状不规则。体外释药曲线显示优化组合条件下制备的载药微球突释效应最小,释药最平稳,最接近零级释放。
扫描电镜显示优化组合条件下制备的载药微球表面非常光滑致密,无孔隙裂纹,粒径分布在45μm左右(45.18±8.96μm),适合18G-23G的针头注射,但对细于26G的针头不具备通针性。含药量在(37.5±1.4) %,经过6个月的体外降解,微球仅仅出现粘连和融合,但仍保持原有形状。
第二部分:子宫内膜异位症动物模型的建立
对45只雌性新西兰大白兔行开腹手术,切除左侧子宫并纵形剖开,剥除肌层及浆膜层,修整成0.5×0.5 cm大小的组织,6–0丝线缝合至腹膜壁层,两个月后,内膜移植物从腹腔凸起并形成血管化的囊肿,动情周期未受影响,囊肿的组织与结构和人内膜异位囊肿相似。
对25只雌性SD大鼠行开腹手术,切除左侧子宫并纵形剖开,剥除肌层及浆膜层,修整成4块0.5×0.5 cm大小的组织,放置在大鼠下腹部皮下,两个月后,皮下内膜移植物变成紫蓝色的囊肿,动情周期未受影响,囊肿的组织与结构和人内膜异位囊肿相似。
第三部分:孕激素缓释微球兔内膜异位囊肿内注射的药理药效学研究
将兔内膜异位模型随机化分为5组,分别接受局部假孕治疗(即将孕激素缓释微球注射到内膜异位囊肿内)、局部空白微球治疗(即将空白微球注射到内膜异位囊肿内)和系统假孕治疗(即反复皮下注射孕激素)。并设去势组作为阳性对照,空白对照组作为阴性对照。与系统假孕组相比,局部假孕组血药曲线平稳,血药浓度远远低于系统假孕组(P < 0.01),而囊肿内药物浓度远远高于系统假孕组(P < 0.01),一次给药可维持6个月。
治疗6个月后,对照组和空白微球组的内膜异位囊肿明显增大(P < 0.01),系统假孕组内膜异位囊肿无显著变化,而局部假孕组和去势组内膜异位囊肿显著减小(P < 0.01)。系统假孕组内膜异位囊肿仅仅发生分泌性改变,而局部假孕组与去势组内膜异位囊肿发生高度萎缩。
同时,系统假孕组转氨酶、血脂、血糖和体重增加均显著升高,而局部假孕组对各代谢指标无明显影响。系统假孕组排卵受到抑制,无动情周期,而局部假孕组动情周期未受影响。
第四部分:孕激素缓释微球囊内单次注射对大鼠皮下内膜异位囊肿疗效的长期观察
将大鼠皮下内膜异位模型随机化分为3组,分别接受局部假孕治疗(即将孕激素缓释微球注射到内膜异位囊肿内)和6个月的GnRHa治疗,并设空白对照组。每月测量囊肿最大径变化,连续阴道涂片观察性周期变化。治疗后第6、12月取血样和内膜异位囊肿,观察代谢指标变化、囊肿组织学变化、雌/孕激素受体的表达变化。
治疗6个月后,局部假孕治疗和GnRHa治疗均引起内膜异位囊肿显著萎缩(P < 0.01)。局部假孕组雌孕激素受体表达普遍减弱(P < 0.01);而GnRHa组雌激素受体表达普遍增强(P < 0.01),孕激素受体在上皮的表达也显著增强(P < 0.01)。之后,局部假孕组囊肿仍保持萎缩状态,而GnRHa组囊肿在停药后快速增长,于第9月达到治疗前的水平并保持到第12月。局部假孕组雌/孕激素受体的表达在第12月仍低于对照组水平,而GnRHa组雌/孕激素受体的表达在第12月与对照组无显著差异。局部假孕治疗和GnRHa治疗对各代谢指标均无显著影响。局部假孕组动情周期正常,而GnRHa组在治疗后6至7个月无动情周期。
结论
1.优化组合条件下制备的孕激素缓释微球表面性状最好,突释效应最小,最接近零级释放,在体外条件下一次给药可释药长达7个月,适合常规注射。
2.自体移植子宫内膜组织可成功建立兔及大鼠内异症动物模型,兔模型适合研究孕激素缓释微球局部应用的疗效和药物代谢特点;大鼠皮下模型适合研究孕激素缓释微球单次应用疗效的维持时间和局部作用的机制。
3.孕激素缓释微球局部应用后,病变局部药物浓度高,使内膜异位囊肿发生高度萎缩,治疗效果与去势相当,显著优于全身用药;而血药浓度低,无明显的毒副作用。
4.单次局部注射孕激素缓释微球对大鼠皮下内膜异位囊肿的治疗效果可维持近一年,同时无明显的毒副作用,因而可通过重复局部注射孕激素缓释微球预防子宫内膜异位症的复发。
Background
Endometriosis is a chronic disease that responds to systemic pseudo-pregnancy therapy. However, side effects limit their long-term use, and recurrence often occurs after cessation of medication. Reducing side effects whilst improving therapeutic efficacy of pseudo-pregnancy therapy seems contradictory, but appealing. In order to address this dilemma, the efficacy and side effects of local pseudo-pregnancy therapy via progestogen-loaded microspheres were investigated for the first time in endometriosis animal model.
Objective
1. To devise an injectable progestogen controlled release system with stable drug release profile.
2. To establish endometriosis animal model through autotransplantation of endometrium to ectopic sites in rabbits and rats.
3. To study the efficacy and side effects of local pseudo-pregnancy therapy via local application of levonorgestrel-loaded polylactic acid microspheres (LNG-microspheres) in an endometriosis rabbit model.
4. To determine how long the suppressive effect of single intra-cystic injection of LNG-microspheres lasts and to compare the results with those obtained with six-month GnRH agonist (GnRHa) treatment. Methods and Results
Part One: Preparation and characteristics of progestogen-loaded microspheres
The levonogestrel-loaded polylactic acid microspheres (LNG-microspheres) were prepared by using an o/w emulsification-solvent evaporation method. Characteristics of LNG-microspheres which including surface morphology, particle size distribution, injectability, drug loading efficiency, in vitro drug release profiles and degradation were studied. In order to reduce the initial burst from polylactide acid (PLA) microspheres enclosing levonorgestrel, we prepared the microspheres with a smooth surface by varying drug content, solvent evaporation conditions such as operating temperature and stirring speed.
It was found that the surface morphology of the PLA microspheres strongly depended on the drug content. When the drug content in the dispersed phase is less than 5%, LNG-microspheres had smooth surfaces. On the other hand, the degree of unevenness in the surface morphology was remarkable for microspheres with the drug content greater than 10%. Meanwhile, the surface of the LNG-microspheres was rough and full of crackles when evaporation process was conducted under normal temperature (25℃). However, the surface of the LNG-microspheres was relatively smooth when it was conducted under ice-cold temperature (0-4℃). Evaporation stirring speed also has influence on the surface morphology of LNG-microspheres. The surface was smooth under low evaporation stirring speed but coalescence was found. The LNG-microspheres was rough and irregular under high evaporation stirring speed. While smooth and spherical LNG-microspheres without coalescence were produced under moderate evaporation stirring speed. In vitro release experiments show that initial burst of microspheres with smooth surface was less than that of those with rough surface.
The surface morphology of the LNG-microspheres was perfect when they were produced by optimization of these factors. The microspheres were approximately spherical with a very smooth, non-porous surface under scanning electron microscope (SEM). Virtually no crystalline drug or fragment of polymer was found adhered to the surface. Under 1500x magnification, the microspheres appeared to be homogeneous and parenchymatous. The particle size of LNG-microspheres was mainly distributed around 45μm with the average value of 45.18±8.96μm, which is injectable using 18-gauge to 23-gauge needles, but not with needles finer than 26-gauge. The drug loading efficiency determined from three batches was estimated to be (37.5±1.4) %. After incubating in PBS at 37°C for six months, the drug-loaded microsphere developed adherence and coalesce, but still kept their shapes.
Part Two: Establishment and evaluation of two endometriosis animal models.
45 female New Zealand White rabbits were anesthetized by pentobarbital sodium and laparotomized. The left uterus horn was cut down and incised longitudinally, and the outer layer of myometrium was peeled away. Then 0.5×0.5 cm pieces taken by microscissors were implanted with 6–0 suture onto the abdominal peritoneum. Two months later, the endometrial implants protruded from the abdominal peritoneum and became vascularized cysts. Estrous cycle was not disturbed in the process of establishment. The histological appearance of endometrial cysts resembled those of human endometrioma with a layer of cuboidal epithelium, glands and stromal tissue.
25 female 12-week-old Sprague-Dawley rats were anesthetized by using pentobarbital sodium and laparotomized. The left uterus was cut down and incised longitudinally, and the outer layer of myometrium was peeled away. The endometrium was sectioned into four pieces (5×5mm) and implanted subcutaneously between the abdominal muscle and skin on both sides of the midline incision. The implants grew into dark bloody fluid-filled, ovoid cysts by 2 months. Estrous cycle was not disturbed in the process of establishment. Histologically, endometrial cysts resembled those of human endometrioma with a layer of endometrial epithelium and stromal tissue inside the cyst.
Part Three: Local application of LNG-microspheres in rabbit endometriosis model
Rabbits with experimental endometriosis were randomized to treatment with local pseudo-pregnancy therapy, local blank microspheres, systemic pseudo-pregnancy therapy, ovariectomy or the control. Local pseudo-pregnancy was induced by injection of LNG-microspheres directly into endometrial cysts. Compared with the systemic pseudo-pregnancy group, significantly higher intra-cystic drug levels were maintained for at least six months with much lower serum levels in the local pseudo-pregnancy group (P < 0.01). Meanwhile, the profile in plasma was rather stable and the concentration in plasma was much lower in the local pseudo-pregnancy group (P < 0.01).
The high intra-cystic levonorgestrel simulated a state of potent pregnancy, which induced size reductions and endometrial atrophy comparable to those of ovariectomy. In contrast, rabbits treated with systemic pseudo-pregnancy therapy showed a slight non-significant increase in the cyst volume with respect to the initial value although the cysts were still significantly smaller than in the control group (P < 0.01). Systemic pseudo-pregnancy therapy only induced secretory changes with intracellular vacuoles in the epithelium and gland however typical decidulization was not observed in the stroma.
Major metabolic parameters and ovarian function were not disturbed by local pseudo-pregnancy therapy. However, in the systemic pseudo-pregnancy group, the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (CHO), triglycerides (TG), plasma glucose (GLU) and weight gain were significantly (P < 0.01) elevated, estrous cycle was also arrested.
Part Four: Long term efficacy of local application of LNG-microspheres in rat endometriosis model
Rats with subcutaneous endometrial cysts were randomized to treatment with local pseudo-pregnancy therapy, GnRHa or the control. Local pseudo-pregnancy was induced by injection of LNG-microspheres directly into the endometrial cysts. Major diameter was measured monthly to evaluate size changes of the cysts. Sequential vaginal smears were examined daily for evaluation of estrous stage. Blood samples and endometriotic cysts were obtained at month 6 and month 12 to study metabolic side effects, histology, expression of estrogen receptor (ER) and progesterone receptor (PR).
Both local pseudo-pregnancy therapy and GnRHa treatment caused significant regression of endometriotic cysts in the former six months (P < 0.01). For local pseudo-pregnancy group, size reduction peaked at month 6 and sustained almost to month 12. For GnRHa group, however, the cysts grew up immediately after the cessation of treatment and reached its pre-treatment level at month 9. Expression of ER and PR were significantly reduced in local pseudo-pregnancy group at month 6 (P < 0.01) and were not fully recovered at month 12. For GnRHa group, the expression of ER was significantly augmented in both epithelium and stroma (P < 0.01), and the staining of PR in epithelium was also enhanced at month 6 (P < 0.01). However, at month 12, the expression
and distribution of ER and PR in GnRHa group was similar with the control. Neither local pseudo-pregnancy therapy nor GnRHa treatment had any significant influence on body weight, liver function, serum lipids and glucose. There is no significant difference among these groups. GnRHa treatment induced arrest of estrous cycle during the former 6 to 7 months, while no cycle disturbances were noted in the local pseudo-pregnancy group.
Conclusions:
1. The lowest initial burst and almost zero order release were obtained with optimization method. The stable release of LNG could maintain for about seven months for LNG-microspheres prepared under optimization conditions. Therefore, LNG-microspheres prepared in this study were appropriate for routine injection.
2. Autotransplantation of endometrium to ectopic sites has been succeesfully established in both rabbits and rats. The implants developed into fluid-filled, ovoid, cystic structures composed of endometrial tissue by two months. Histological characteristics of these endometrial cysts resemble those of human disease. Rabbits with peritoneal endometrial cysts are of high value for the study of in vivo drug release profile and the efficacy of local application of progestogen-loaded microspheres. Rats with subcutaneous endometrial cysts are good animal model for studying the recurrence of ectopic endometrial tissue after regression and the mechanisms underlying local application of progestogen-loaded microspheres.
3. By local application of LNG-microspheres directly into endometrial cysts, exceptional high concentrations of LNG were maintained in the lesions for at least six months with much lower levels in plasma. Local pseudo-pregnancy induced by LNG-microspheres could achieve therapeutic efficacy comparable to that of ovariectomy without provoking any marked side effects in rabbit endometriosis model. Thus it may be a preferable option for patients with endometriosis.
4. The suppressive effect of single intra-cystic injection of progestogen-loaded microspheres could maintain for almost one year in a rat endometriosis model. Therefore, sustained local pseudo-pregnancy via repeated administration of LNG-microspheres is a potential therapy for preventing recurrence of endometriosis.
子宫内膜异位症是一种难以治愈的常见妇科良性疾病,而妊娠是对子宫内膜异位症极为有效的自然疗法,假孕疗法就是用药物模拟妊娠的机体内环境,从而达到治疗目的。然而,全身应用孕激素的副作用多,包括恶心、痤疮、体重增加、水钠潴留、突破性出血、肝功能异常及血栓形成等。患者的依从性较差,常常半途而废,极大地影响了治疗效果。同时常规假孕疗法病灶局部的药物浓度低,对卵巢内膜异位囊肿等病变的治疗效果差。
为了提高疗效同时降低副作用,我们提出诱导局部假孕治疗子宫内膜异位症的假说。即制备一种可长期缓释孕激素的生物降解型微球,注射到子宫内膜异位症病灶局部,在病灶局部形成高浓度孕激素的环境,诱导出高度假孕状态,使异位内膜病灶萎缩、坏死并吸收,从而提高疗效;而血药浓度低,对代谢及卵巢功能影响小,可减少常规假孕疗法的副作用。
研究目的
1.设计一种可注射并长期缓释孕激素的生物降解型微球;
2.通过自体移植子宫内膜建立兔及大鼠子宫内膜异位症动物模型;
3.在兔内异症模型上观察孕激素缓释微球局部应用的疗效与副作用;
4.在大鼠皮下内异症模型上观察单次局部注射孕激素缓释微球疗效的维持时间,并与GnRHa的疗效相比较。
研究方法与结果
第一部分:孕激素缓释微球的制备及特性检测
采用油/水复乳液中蒸发法制备左炔诺孕酮聚乳酸微球,并检测了微球表面性状、粒径分布、注射性、载药率、体外释药及降解。通过优化含药量、蒸发温度和蒸发搅拌速度改善载药微球的表面性状、减少载药微球的突释效应。
含药量严重影响载药微球的表面性状,当分散相含药量在5%及以下时,载药微球表面相对光滑;而含药量在10%及以上时,载药微球的表面粗糙,有药物微晶体黏附,部分微球开裂;蒸发温度也对载药微球的表面性状有一定的影响,在常温下(25℃左右)制备的载药微球表面粗糙有裂纹,而在冰浴条件(0-4℃)下制备的载药微球表面相对光滑;蒸发搅拌速度是微球表面性状的另一影响因素,低速搅拌蒸发制备的载药微球表面光滑,但部分微球出现融合现象,中速搅拌蒸发制备的载药微球表面光滑,形状规则,无融合现象,而高速搅拌蒸发制备的载药微球表面粗糙,大小不一,形状不规则。体外释药曲线显示优化组合条件下制备的载药微球突释效应最小,释药最平稳,最接近零级释放。
扫描电镜显示优化组合条件下制备的载药微球表面非常光滑致密,无孔隙裂纹,粒径分布在45μm左右(45.18±8.96μm),适合18G-23G的针头注射,但对细于26G的针头不具备通针性。含药量在(37.5±1.4) %,经过6个月的体外降解,微球仅仅出现粘连和融合,但仍保持原有形状。
第二部分:子宫内膜异位症动物模型的建立
对45只雌性新西兰大白兔行开腹手术,切除左侧子宫并纵形剖开,剥除肌层及浆膜层,修整成0.5×0.5 cm大小的组织,6–0丝线缝合至腹膜壁层,两个月后,内膜移植物从腹腔凸起并形成血管化的囊肿,动情周期未受影响,囊肿的组织与结构和人内膜异位囊肿相似。
对25只雌性SD大鼠行开腹手术,切除左侧子宫并纵形剖开,剥除肌层及浆膜层,修整成4块0.5×0.5 cm大小的组织,放置在大鼠下腹部皮下,两个月后,皮下内膜移植物变成紫蓝色的囊肿,动情周期未受影响,囊肿的组织与结构和人内膜异位囊肿相似。
第三部分:孕激素缓释微球兔内膜异位囊肿内注射的药理药效学研究
将兔内膜异位模型随机化分为5组,分别接受局部假孕治疗(即将孕激素缓释微球注射到内膜异位囊肿内)、局部空白微球治疗(即将空白微球注射到内膜异位囊肿内)和系统假孕治疗(即反复皮下注射孕激素)。并设去势组作为阳性对照,空白对照组作为阴性对照。与系统假孕组相比,局部假孕组血药曲线平稳,血药浓度远远低于系统假孕组(P < 0.01),而囊肿内药物浓度远远高于系统假孕组(P < 0.01),一次给药可维持6个月。
治疗6个月后,对照组和空白微球组的内膜异位囊肿明显增大(P < 0.01),系统假孕组内膜异位囊肿无显著变化,而局部假孕组和去势组内膜异位囊肿显著减小(P < 0.01)。系统假孕组内膜异位囊肿仅仅发生分泌性改变,而局部假孕组与去势组内膜异位囊肿发生高度萎缩。
同时,系统假孕组转氨酶、血脂、血糖和体重增加均显著升高,而局部假孕组对各代谢指标无明显影响。系统假孕组排卵受到抑制,无动情周期,而局部假孕组动情周期未受影响。
第四部分:孕激素缓释微球囊内单次注射对大鼠皮下内膜异位囊肿疗效的长期观察
将大鼠皮下内膜异位模型随机化分为3组,分别接受局部假孕治疗(即将孕激素缓释微球注射到内膜异位囊肿内)和6个月的GnRHa治疗,并设空白对照组。每月测量囊肿最大径变化,连续阴道涂片观察性周期变化。治疗后第6、12月取血样和内膜异位囊肿,观察代谢指标变化、囊肿组织学变化、雌/孕激素受体的表达变化。
治疗6个月后,局部假孕治疗和GnRHa治疗均引起内膜异位囊肿显著萎缩(P < 0.01)。局部假孕组雌孕激素受体表达普遍减弱(P < 0.01);而GnRHa组雌激素受体表达普遍增强(P < 0.01),孕激素受体在上皮的表达也显著增强(P < 0.01)。之后,局部假孕组囊肿仍保持萎缩状态,而GnRHa组囊肿在停药后快速增长,于第9月达到治疗前的水平并保持到第12月。局部假孕组雌/孕激素受体的表达在第12月仍低于对照组水平,而GnRHa组雌/孕激素受体的表达在第12月与对照组无显著差异。局部假孕治疗和GnRHa治疗对各代谢指标均无显著影响。局部假孕组动情周期正常,而GnRHa组在治疗后6至7个月无动情周期。
结论
1.优化组合条件下制备的孕激素缓释微球表面性状最好,突释效应最小,最接近零级释放,在体外条件下一次给药可释药长达7个月,适合常规注射。
2.自体移植子宫内膜组织可成功建立兔及大鼠内异症动物模型,兔模型适合研究孕激素缓释微球局部应用的疗效和药物代谢特点;大鼠皮下模型适合研究孕激素缓释微球单次应用疗效的维持时间和局部作用的机制。
3.孕激素缓释微球局部应用后,病变局部药物浓度高,使内膜异位囊肿发生高度萎缩,治疗效果与去势相当,显著优于全身用药;而血药浓度低,无明显的毒副作用。
4.单次局部注射孕激素缓释微球对大鼠皮下内膜异位囊肿的治疗效果可维持近一年,同时无明显的毒副作用,因而可通过重复局部注射孕激素缓释微球预防子宫内膜异位症的复发。
Background
Endometriosis is a chronic disease that responds to systemic pseudo-pregnancy therapy. However, side effects limit their long-term use, and recurrence often occurs after cessation of medication. Reducing side effects whilst improving therapeutic efficacy of pseudo-pregnancy therapy seems contradictory, but appealing. In order to address this dilemma, the efficacy and side effects of local pseudo-pregnancy therapy via progestogen-loaded microspheres were investigated for the first time in endometriosis animal model.
Objective
1. To devise an injectable progestogen controlled release system with stable drug release profile.
2. To establish endometriosis animal model through autotransplantation of endometrium to ectopic sites in rabbits and rats.
3. To study the efficacy and side effects of local pseudo-pregnancy therapy via local application of levonorgestrel-loaded polylactic acid microspheres (LNG-microspheres) in an endometriosis rabbit model.
4. To determine how long the suppressive effect of single intra-cystic injection of LNG-microspheres lasts and to compare the results with those obtained with six-month GnRH agonist (GnRHa) treatment. Methods and Results
Part One: Preparation and characteristics of progestogen-loaded microspheres
The levonogestrel-loaded polylactic acid microspheres (LNG-microspheres) were prepared by using an o/w emulsification-solvent evaporation method. Characteristics of LNG-microspheres which including surface morphology, particle size distribution, injectability, drug loading efficiency, in vitro drug release profiles and degradation were studied. In order to reduce the initial burst from polylactide acid (PLA) microspheres enclosing levonorgestrel, we prepared the microspheres with a smooth surface by varying drug content, solvent evaporation conditions such as operating temperature and stirring speed.
It was found that the surface morphology of the PLA microspheres strongly depended on the drug content. When the drug content in the dispersed phase is less than 5%, LNG-microspheres had smooth surfaces. On the other hand, the degree of unevenness in the surface morphology was remarkable for microspheres with the drug content greater than 10%. Meanwhile, the surface of the LNG-microspheres was rough and full of crackles when evaporation process was conducted under normal temperature (25℃). However, the surface of the LNG-microspheres was relatively smooth when it was conducted under ice-cold temperature (0-4℃). Evaporation stirring speed also has influence on the surface morphology of LNG-microspheres. The surface was smooth under low evaporation stirring speed but coalescence was found. The LNG-microspheres was rough and irregular under high evaporation stirring speed. While smooth and spherical LNG-microspheres without coalescence were produced under moderate evaporation stirring speed. In vitro release experiments show that initial burst of microspheres with smooth surface was less than that of those with rough surface.
The surface morphology of the LNG-microspheres was perfect when they were produced by optimization of these factors. The microspheres were approximately spherical with a very smooth, non-porous surface under scanning electron microscope (SEM). Virtually no crystalline drug or fragment of polymer was found adhered to the surface. Under 1500x magnification, the microspheres appeared to be homogeneous and parenchymatous. The particle size of LNG-microspheres was mainly distributed around 45μm with the average value of 45.18±8.96μm, which is injectable using 18-gauge to 23-gauge needles, but not with needles finer than 26-gauge. The drug loading efficiency determined from three batches was estimated to be (37.5±1.4) %. After incubating in PBS at 37°C for six months, the drug-loaded microsphere developed adherence and coalesce, but still kept their shapes.
Part Two: Establishment and evaluation of two endometriosis animal models.
45 female New Zealand White rabbits were anesthetized by pentobarbital sodium and laparotomized. The left uterus horn was cut down and incised longitudinally, and the outer layer of myometrium was peeled away. Then 0.5×0.5 cm pieces taken by microscissors were implanted with 6–0 suture onto the abdominal peritoneum. Two months later, the endometrial implants protruded from the abdominal peritoneum and became vascularized cysts. Estrous cycle was not disturbed in the process of establishment. The histological appearance of endometrial cysts resembled those of human endometrioma with a layer of cuboidal epithelium, glands and stromal tissue.
25 female 12-week-old Sprague-Dawley rats were anesthetized by using pentobarbital sodium and laparotomized. The left uterus was cut down and incised longitudinally, and the outer layer of myometrium was peeled away. The endometrium was sectioned into four pieces (5×5mm) and implanted subcutaneously between the abdominal muscle and skin on both sides of the midline incision. The implants grew into dark bloody fluid-filled, ovoid cysts by 2 months. Estrous cycle was not disturbed in the process of establishment. Histologically, endometrial cysts resembled those of human endometrioma with a layer of endometrial epithelium and stromal tissue inside the cyst.
Part Three: Local application of LNG-microspheres in rabbit endometriosis model
Rabbits with experimental endometriosis were randomized to treatment with local pseudo-pregnancy therapy, local blank microspheres, systemic pseudo-pregnancy therapy, ovariectomy or the control. Local pseudo-pregnancy was induced by injection of LNG-microspheres directly into endometrial cysts. Compared with the systemic pseudo-pregnancy group, significantly higher intra-cystic drug levels were maintained for at least six months with much lower serum levels in the local pseudo-pregnancy group (P < 0.01). Meanwhile, the profile in plasma was rather stable and the concentration in plasma was much lower in the local pseudo-pregnancy group (P < 0.01).
The high intra-cystic levonorgestrel simulated a state of potent pregnancy, which induced size reductions and endometrial atrophy comparable to those of ovariectomy. In contrast, rabbits treated with systemic pseudo-pregnancy therapy showed a slight non-significant increase in the cyst volume with respect to the initial value although the cysts were still significantly smaller than in the control group (P < 0.01). Systemic pseudo-pregnancy therapy only induced secretory changes with intracellular vacuoles in the epithelium and gland however typical decidulization was not observed in the stroma.
Major metabolic parameters and ovarian function were not disturbed by local pseudo-pregnancy therapy. However, in the systemic pseudo-pregnancy group, the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (CHO), triglycerides (TG), plasma glucose (GLU) and weight gain were significantly (P < 0.01) elevated, estrous cycle was also arrested.
Part Four: Long term efficacy of local application of LNG-microspheres in rat endometriosis model
Rats with subcutaneous endometrial cysts were randomized to treatment with local pseudo-pregnancy therapy, GnRHa or the control. Local pseudo-pregnancy was induced by injection of LNG-microspheres directly into the endometrial cysts. Major diameter was measured monthly to evaluate size changes of the cysts. Sequential vaginal smears were examined daily for evaluation of estrous stage. Blood samples and endometriotic cysts were obtained at month 6 and month 12 to study metabolic side effects, histology, expression of estrogen receptor (ER) and progesterone receptor (PR).
Both local pseudo-pregnancy therapy and GnRHa treatment caused significant regression of endometriotic cysts in the former six months (P < 0.01). For local pseudo-pregnancy group, size reduction peaked at month 6 and sustained almost to month 12. For GnRHa group, however, the cysts grew up immediately after the cessation of treatment and reached its pre-treatment level at month 9. Expression of ER and PR were significantly reduced in local pseudo-pregnancy group at month 6 (P < 0.01) and were not fully recovered at month 12. For GnRHa group, the expression of ER was significantly augmented in both epithelium and stroma (P < 0.01), and the staining of PR in epithelium was also enhanced at month 6 (P < 0.01). However, at month 12, the expression
and distribution of ER and PR in GnRHa group was similar with the control. Neither local pseudo-pregnancy therapy nor GnRHa treatment had any significant influence on body weight, liver function, serum lipids and glucose. There is no significant difference among these groups. GnRHa treatment induced arrest of estrous cycle during the former 6 to 7 months, while no cycle disturbances were noted in the local pseudo-pregnancy group.
Conclusions:
1. The lowest initial burst and almost zero order release were obtained with optimization method. The stable release of LNG could maintain for about seven months for LNG-microspheres prepared under optimization conditions. Therefore, LNG-microspheres prepared in this study were appropriate for routine injection.
2. Autotransplantation of endometrium to ectopic sites has been succeesfully established in both rabbits and rats. The implants developed into fluid-filled, ovoid, cystic structures composed of endometrial tissue by two months. Histological characteristics of these endometrial cysts resemble those of human disease. Rabbits with peritoneal endometrial cysts are of high value for the study of in vivo drug release profile and the efficacy of local application of progestogen-loaded microspheres. Rats with subcutaneous endometrial cysts are good animal model for studying the recurrence of ectopic endometrial tissue after regression and the mechanisms underlying local application of progestogen-loaded microspheres.
3. By local application of LNG-microspheres directly into endometrial cysts, exceptional high concentrations of LNG were maintained in the lesions for at least six months with much lower levels in plasma. Local pseudo-pregnancy induced by LNG-microspheres could achieve therapeutic efficacy comparable to that of ovariectomy without provoking any marked side effects in rabbit endometriosis model. Thus it may be a preferable option for patients with endometriosis.
4. The suppressive effect of single intra-cystic injection of progestogen-loaded microspheres could maintain for almost one year in a rat endometriosis model. Therefore, sustained local pseudo-pregnancy via repeated administration of LNG-microspheres is a potential therapy for preventing recurrence of endometriosis.
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