Study On The Preparation Of Injectable Nomegestrol Microspheres By Polymer-alloys Method

Emulsion-solvent-evaporation technique is one of the most acceptable methods in drug microencapsulation. So far only one material is usually used as the matrix of microspheres(MS) , the release of three phases pattern usually occurs,e.g. first a initial burst release(often on 1st day) , then a lag time during which little drug is released , and finally a steady release phase until the drug is completely released. The research was conducted to establish a modified polymer-alloys microencapsulation method, in which PLGA and PLA are combined as MS matrix. However, PLGA and PLA vary greatly in physical/chemical characteristics, especially the degradable behavior. Two kinds of polymers having a similar structure e.g. PLGA(poly(lactic-co-glycolic acid)) and PLCG (poly(lactide-co-glycolide)) are used as the matrix in certain mass ratio here, it's hoped that besides the high EE(encapsulation efficiency), the obtained MS can overcome the disadvantages mentioned above, and release drug in a steady rate.Nomegestrol acetate (Nome)——a hydrophobic steroid hormone——is used as a model drug in the thesis, to establish a solid polymer-alloys method for the preparation of 1-month releasing MS..The thesis was divided into 6 parts.1. The characterization study of Nome and polyesterThe research was performed to study the properties of Nome and polyester for the optimization of formulation and technology.The characteristics of polyesters, including thermal behavior, IR spectrum, Mw distribution, crystallinity,?(intrinsic viscosity), were evaluated. The results showed that PLGA and PLCG were amorphous. MS prepared by PLGA(75/25,14kD) were taken as an example for the degradation test in pH7.4 PBS at 37?, the microenviroment pH,?, mass loss of MS were monitored. The results showed that the MS degraded completely in almost 1 month.The characteristics of Nome, including thermal behavior, UV spectrum, crystalline, solubility were evaluated. The results demonstrated that Nome was crystalline, having a Tm at 181.5?, and insoluble in water, only 7.82?g/ml. The HPLC method was used to determine Nome in free state or in MS , which proved to have good accuracy, sensitivity, precision, and specificity.Meanwhile 5% HP-?-CD pH 7.4 PBS which qualified the sink condition was selected as the in vitro release medium of Nome loaded MS . The experiment compared the direct and residue drug determination methods, the data of the latter method was proven to meet the requirement of the results of pharmacodynamics and pharmacokinetics. Therefore it was chosen as the method for the determination of drug release pattern in MS.2. Polymer-alloys methods for the preparation of Nome MSNome MS were prepared by traditional O/W-emulsion- solvent-evaporation method firstly. The criteria to evaluate the MS included the morphology, particle size ,size distribution, EE, the recoverable percent,etc. An analysis of variance (ANOVA) was used to test the effect of various factors on results. Then multiple comparisons were processed with a Turkey's method (T-method) between levels to check the significance of difference further. The principle component analysis was used to find out which factors were the most important to influence the properties of MS so as to guide the further MS preparation .Optimal formulation was chosen and MS were formed with good properties. However either the so-obtained PLGA or PLCG MS showed unsatisfying release patterns in vitro. The in vitro dissolution study of PLGA (50/50,14kD) and PLGA (75/25,14kD) MS displayed no lag time, but the drug released durations were only 5,14d respectively. The duration of MS prepared by PLCG(75/25,?=0.15), PLCG(50/50,?=0.2), PLCG(75/25,?=0.2) were much longer and increased with the rising of?and the L/G ratio, ranging from 28?70d, yet all the MS above had obvious lag time, which increased with the rising of?and the L/G ratio. All the phenomenons above indicated the possibility and scientific proofs of polymer-alloys method.On the basis of the experiments above, the mass ratio 1/3 of PLGA (75/25,14kD) and PLCG (75/25,?=0.15) was applied as matrix for 20%-Nome-loaded MS. The in vitro dissolution test displayed a zero-order kinetics release pattern for 1 month . When sceening the apoporiate mass ratio for 1-month-Nome MS by the means of polymer-alloys method, a linear correlation between the polymer mass ratio and rate constant(k)/drug duration was found, which may give a guideline for other polymer-alloys products, yet whether it could be applied to all products or not remained to be testified.MS with drug loading ranging from 10% to 40% were obtained by the polymer-alloys method, and the burst release on 1st day was only 6.22% for 10%-Nome-loaded MS, 7.07% for 20%-Nome-loaded MS, 18.48% for 40%-Nome-loaded MS.The formulation of the optimal polymer-alloys MS were processed triplicately and the thermal characteristics, morphology, in vitro dissolution behavior were evaluated. The mechanisms of polymer-alloys were studied using the means of partial solubility parameters to guide the development of other products in the future.3. The accelerated test methodThe study was to try to set up an accelerated release method in which drug release follow the same mechanism as a real- time release in vitro with a 1:1 correlation. The addition of organic solvent and increase in temperature methods were used. The results showed that the former changed the release mechanism to diffusion controlled kinetics, this change resulted in lack of correlation between accelerated and real-time release profiles. When 5% HP-?-CD pH7.4 PBS (50?) was used, the data was proven to correlate well with the real time release profiles. All the work above layed a solid foundation for the quality control of MS.4. The study of pharmacokineticsRefering to the determination of LHRH analogue MS release pattern in animals, the in vivo release of the Nome MS was evaluated indirectly by measuring the quantities of residual drug in the injection sites at different times in rats. The method had good accuracy, sensitivity and specificity. The results displayed a good correlation with the profile in vitro release.The in vitro-in vivo correlation equation was y=0.9923x-1.263, with a coefficient 0.9986.5. The study of pharmacodynamicsThe research was performed to test the therapeutic effect of Nome MS on contraception and endometriosis(EM). The inhibition on sexual cycle and oulation on SD rat for single administration of Nome MS was observed to verify the drug effect sustained period. Results showed that anestrum of SD rat maintained for 35.2±0.83 days after injecting MS prepared by polymer-alloys method, indicating that the MS had reliable sustained effects.Two EM models were established, one made by surgically autotransplanting endometrium to the peritoneum in rats, the other by hypodermic injection in mice. When the dose was 0.5mg/kg*d, no significant suppression effects on EM were observed in SD rats, yet significant for 2, 4mg/kg*d, especially significant for 4mg/kg*d, which had no difference with the positive control given Gestrinone (R2323) MS (P=0.118). The Nome doses of 0.5 and 2mg/kg.d decreased the height of endometrium epithelium and serum E2 level significantly. Nome had no effect on serum P level.The Nome doses of 6,12mg/kg*d decreased serum FSH and LH level in mice significantly, while the doses of 3, 24mg/kg*d could decrease serum FSH and LH level, yet the effect was insignificant stastically.6. Study of rbST (recombinant bovine somatropin) MSThe research was conducted to develop a optimized method for rbST microencapsulation. It's of great importance to assure protein not denatured during protein microencapsulating process. In this chapter, the influence of microencapsulation processes, organic solvents, protectants to the stability of rbST were evaluated. The results showed that the oil/water interface had a great influence on the stability of rbST, MS with expected properties couldn't be obtained either by W/O/W or by S/O/W emulsion-solvent-evaporation method. MS with high drug loading(?10%), high protein activity could be retained by S/O/O method in the presence of suitable additives.The innovations of this paper were listed as below:1. It hasn't been mentioned before using PLGA and PLCG which have similar properties as matrix in polymer-alloys method.The in vitro dissolution study revealed that the release of Nome in MS obtained by the method was in zero-order kinetics, lasted 4weeks without initial burst and lag time compared with the MS prepared by common method.2. The acceleration tests using the methods by adding organic solvent and elevating temperature revealed that there was a good correlation between accelerated and real time release profiles when elevating the temperature. It's an objective experimental method, and meaningful for pharmaceutical industry.3. Nomegestrol acetate is mainly used for contraception as a new kind of progestin overseas, and hasn't appeared in demostic market. The only sustained-release dosage is silicon-rubber implants, which requires surgery to place in and move out, thus causing compliance problems.Yet the microspheres developed in the study can be injected by normal injectors, and needn't to be moved out after the drug is released completely. The MS can release the drug for 1month in almost zero-order. It's also discovered that Nome has a good effect in treating EMs, which hasn't been reported before.This can be a guidance for further development.