Induction Of A Local Pseudo-pregnancy Via Levonorgestrel-loaded Microspheres For The Treatment Of Endometriosis In Animal Models

BackgroundEndometriosis 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.Objective1. 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 ResultsPart One: Preparation and characteristics of progestogen-loaded microspheresThe 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 modelRabbits 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 modelRats 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 expressionand 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.