Basic Study On Danazol Alginate Microspheres Used For Uterine Fibroid Embolization

Basic Study on Danazol Alginate Microspheres Used for Uterine Fibroid EmbolizationBACKROUNDUterine fibroids are the commonest tumor in women during the reproductive years. Surgery in the form of abdominal hysterectomy or myomectomy has been the traditional treatment for symptomatic fibroids. Although hysterectomy combines surety of symptom relief with minimal risk of fibroid recurrence, but it results in loss of reproductive function and high complication rates. Seeking conservative alternatives to hysterectomy is important for the patients desiring future pregnancy. Recent years, a number of uterus- sparing and minimally invasive treatments of fibroids have emerged. Among them, Uterine fibroid embolization has attracted public attention with a highly effectiveness.Uterine fibroid embolization has become so rapidly accepted as a part of fibroid therapy and now widely used in the world. At present in the procedure nonspherical polyvinyl alcohol (PVA) particles, gelatin sponge and alginate microspheres have been extensively used as embolization agent. Microspheres containing medicine have yet to be used for embolic agent to treat fibroids, while it appeared that means a new direction of development for technique of uterine fibroid embolization. Microspheres containing medicine has dual effect of a ingenious combination of medication and embolization, which characters are targeting and sustained-release drug.Alginate microsphere is natural and degradable as a new embolic agent, it has been widely accepted for uterine fibroid embolization. Danazol are considered treatment drugs for fibroids. By microcapsule technology, Danazol can be packed with alginate microsphere and prepared for Donazol-loaded alginate microspheres. As a new embolic agent for uterine fibroid embolization, it might be more effective than alginate micro- spheres because of sustained-release drug locally. At present, Danazol-loaded alginate microspheres have not yet entered clinical trials and application. Basic study on Danazol Alginate Microspheres is very important, it can provide a basis for clinical application in the future.OBJECTIVES1. To verify the main characteristics of danazol alginate microspheres such as the drug loading, sustained-release drug in vitro/vivo.2. To investigate therapeutic effects, optimal embolic size and the mechanism of danazol alginate microspheres used for uterine fibroid embolization.3. To investigate the impact of danazol alginate microspheres used for uterine fibroid embolization on ovarian function and fertility.METHODS1. The shape, particle diameter and structure of danazol alginate microspheres were observed by light and transmission electron microscopic examination; The drug loading were determined by high performance liquid chromatography (HPLC), Sustained-release drug in vitro were observed in liquid mediator for 1-28 days; Femoral artery of rabbits were embolized with danazol alginate microspheres and were tested releasing drug in serum and tissue fluid locally for 1-14 days after embolization.2. Animal myoma model were induced by estrogen and progesterone with female guinea pigs and beagle dogs. The model were verified through anatomy, histology and imaging.3. To simulate clinical UAE-operation process with animal myoma model and evaluate effects of danazol alginate microspheres and alginate microspheres. To investigate the mechanism of danazol alginate microspheres used for uterine fibroid embolization by anatomy and histology. The expressions of Bcl-2 were evaluated by IHC. Apoptotic cells were assessed by TUNEL assay.4. To compare the effects on myometrium of size 100-200μm and 300-500μm danazol alginate microspheres in embolization of the uterine arteries in beagle dogs. Evaluation based on histopathologic study of the scores of necrosis.5. To simulate clinical UAE-operation process with female rabbits and determined the level of serum E₂, FSH, T, LH by ELISA for 1-3 months after embolization. These female rabbits mated in breeding field. Estrus, pregnancy rate were observed by Veterinary follow-up evaluationRESULTS1. In vitro danazol alginate microspheres have a rapid drug-release in 1-7 days and stable release in 14-28 days. It released a cumulative rate about 40% for 28 days. In vivo there are very little drug-release (＜0.145μg/ml) in serum and tissue fluid locally for 1-14 days. 2. For guinea pigs myoma model, uterine weight and maximum diameter increased. Smooth muscle cells proliferate and disarray which consistent with the changes of leiomyoma in histology. For beagle dogs myoma model, new enhanced scan masses in uterine were observed by CT examination. The imaging features are similar to uterine myoma.3. After UAE-operation process with danazol alginate microspheres, uterine weight, maximum diameter and fibroids' number were decreased obviously, for myoma model of guinea pigs, myometrium thinningz and endometrial gland shrink. Apoptosis rate and Bcl-2 expression increased.4. Size of 100-200μm danazol alginate microspheres had a higher score of uterine necrosis than did size of 300-500μm.5. After UAE with danazol alginate microspheres, there were no significant changes from baseline in the mean FSH, LH, E₂, T levels measured at 1, 2, and 3 months. The total pregnancy rate is lower than control groups within 2-10 months, especially in 2-4 months no successful pregnancy occurred.CONCLUSIONDanazol alginate microspheres have the characteristics of sustained-release drug for microspheres containing medicine. There are quick release and plateau in vitro and cumulative release rate increased over time. For very little drug-release in vivo, at present, the therapy based embolization treatment of myoma secondary medication.Animal myoma model can be induced by estrogen and progesterone with female guinea pigs successfully. Danazol alginate microspheres will be expected to become new type embolic agent used for UAE, according to the effect in our test. The therapeutic mechanism include myoma avascular necrosis after UAE and apoptotic cells increase with danazol releasing locally and with ischemia-reperfusion caused by microsphere degradation.Danazol alginate microspheres are associate with intense uterine necrosis and extensive arterial occlusion regard of size.There is no impact obviously of danazol alginate microspheres used for uterine fibroid embolization on animal ovarian function. After UAE animals can become pregnant, while harmful effects will be with pregnancy in short term.