Studies On The Intra-Articular Injectable Tetramethylpyrazine Loaded Sustained-Release PLGA Microspheres

Osteoarthritis (OA) is one of the common arthritis with high attack rate and it usually needs multiple treatment cycles to cure it. Intra-articular injection is one of the most common treatment methods for OA. However, drug solution injection always leaks into the systemic circulation after the intra-articular injection. Thus, the local drug concentration falls quickly and the therapy effect will be low. The other problems meet with this treatment including frequent administration frequency, serious side effects and relative low patient compliance. This article innovationally designed sustained-release microspheres encapsulating tetramethylpyrazine (ligustrazine, TMPZ) for intra-articular injection with the purpose of improving the therapeutic effects and avoiding the existing problems of the intra-articular injection of drug solution mentioned above.As one of the widely accepted sustained release drug delivery systems, microspheres have been deeply investigated. It exerts sustained-release effects through the specific skeleton material, poly(lactic-co-glycolic acid, PLGA), which is biodegradable with harmless products H2O and CO2. PLGA was used as adjuvant which had been approved by the Food and Drug Administration (FDA), USA. The intra-articular injectable sustained-release microspheres preparation to heal OA can reduce administration frequency, extent therapy duration, reduce the patient pain and enhance the patient compliance.TMPZ was selected as the model drug and PLGA used as the carrier material in this experiment. The TMPZ loaded sustained-release microspheres were prepared by O/W emulsion solvent evaporation method. This article was composed of the following seven parts:the preformulation study, the optimization of the formulation and technological parameters, the characteristics of the preparations and in vitro drug release study, the sterilization and stability study, the leakage and local tissue distribution study in vivo, and the in vivo biocompatibility and pharmacodynamic study.1. The preformulation study The equilibrium solubility and oil-water partition coefficient of TMPZ were investigated firstly, in order to determine the evaluating indexes and the preparation method of the TMPZ loaded sustained-release microspheres. The results indicated that TMPZ was well dissolved in some organic solvents such as methylene chloride and ethyl acetate, and the coefficient of distribution in propyl butyl-carbinol-water was 12.33. Encapsulation efficiency and loading efficiency were evaluated by ultraviolet-visible spectroscopy and related material was determined by high performance liquid chromatography (HPLC) according to the results of the methodology investigation. The in vitro release of the TMPZ from the sustained-release microspheres were evaluated by dynamic membrane dialysis method using pH 7.4 phosphate buffer solution (with 1% lauryl sodium sulfate and 0.02% nitrine sodium) as release media.2. The optimization of the formulation and the technological parameters of the TMPZ loaded sustained-release microspheresThe TMPZ loaded sustained-release microspheres were prepared using modified O/W emulsion solvent evaporation method under the consideration of the drug properties and the requirements of the clinical application of the intra-articular injectable sustained-Release microspheres. The evaluating indexes included diameter, encapsulation efficiency, drug loading and initial burst release. The formulation and technological parameters were optimized based on the results of the single factor study and the orthogonal design. The obtained microspheres were dried using freeze-drying method. The results showed that the modified O/W emulsion solvent evaporation method was suitable for the preparation of the TMPZ loaded sustained-release microspheres with the advantages of easy to operation, the temperate preparation conditions and perfect reproducibility. The mean diameters of the 3 batch TMPZ loaded sustained-release microspheres prepared under the optimized formulation and the technological parameters was (10.0±2.11)μm, the encapsulation efficiency was (81.36±1.15)% and the drug loading was (8.2±0.25)%.The results of the in vitro drug release study showed that (16.57±2.94)% of total loaded drug released from the sustained-release microspheres in 24 hours and (82.24±0.51)% released within 32 days. These results indicated that the encapsulation efficiency achieved the requirements of pharmacopoeia and in vitro drug release met the predetermined requirements of the project.3. The establishment of in vitro drug release method and the research on drug release mechanismThe formation of the TMPZ loaded sustained-release microspheres was validated by different scanning calorimetry (DSC). The different in vitro drug released conditions (including the release medium and the bacteriostatic agent) was compared; meanwhile, an accelerated release method was established to shorten the experimental time of in vitro release study. The linear relationship between the data obtained from the conventional release method and the data from the novel accelerated release method was confirmed by point-to-point regression equation and the result showed that it's available to anticipate the conventional release property by the data from the novel accelerated release method. Thus, the novel accelerated release method is expected to be popularized and applicated widely.The in vitro drug release mechanism of the TMPZ loaded sustained-release microspheres was analyzed by determining the weight loss and by comparing the scanning electron microscope (SEM) photographs. The results indicated that after releasing for 20 days, the microspheres were adhered and fused; demarcation line disappeared and pores of the microspheres increased. However, no obvious shape changes were observed. After releasing about 30 days, the microspheres were porous and the shape changed thoroughly. These results suggested that the release process of the TMPZ loaded sustained-release microspheres was not mainly depended on the polymer degradation. Swelling degree was evaluated by determination of the microspheres particle size in release medium and calculation of the size changes. These results showed that the in vitro releases curve of the TMPZ loaded sustained-release microspheres was depicted by Higuchi equation. The results of the equation fitting and the drug release mechanism research showed that the in vitro release of the drug from the TMPZ loaded sustained-release microspheres was mainly by diffusion mechanism.4. The sterilization and stability studiesThe stability of the TMPZ loaded sustained-release microspheres was evaluated through changes including appearance, drug contents and in vitro release behavior. The results showed that high temperature and high moisture would affect the properties of the TMPZ loaded sustained-release microspheres. The TMPZ loaded sustained-release microspheres were sterilized under lOkGy and 15kGy of 60Co exposure. lOkGy of 60Co exposure was selected to sterilize the TMPZ loaded sustained-release microspheres and the result of the microorganism test indicated that treatment with lOkGy of 60Co was acceptable.5. In vivo leakage and local tissue distribution studiesThe in vivo leakage and the local tissue distribution of the TMPZ loaded sustained-release microspheres after intra-articular injection in rats were investigated and TMPZ injection was used as control. After intra-articular injection of the TMPZ loaded sustained-release microspheres, the plasma drug concentration and the local drug concentration of the joint cavity (joint tissue and synovial fluid) of two kind of preparation were compared. The results indicated that the AUC0-∞of TMPZ injection group was 5.02 times more than that of the TMPZ loaded sustained-release microspheres group. Pharmacokinetic parameters were calculated by statistical moment method. The t1/2 of microspheres group and injection group are 130.75h and 16.93h, respectively, showing of 7.72 folds increasing. MRT0-∞were 26.09h and 223.37h, respectively, showing of 8.56 folds enhancement. AUC0-∞of the TMPZ loaded sustained-release microspheres group was 14.4 times more than that of TMPZ injection group (52586.0 mg/L*h and 3644.5 mg/L*h, respectively). After encapsulated in the microspheres, the drug leakage into the body circulation reduced and the local drug concentration increased obviously, thus the therapy effect was expected to be enhanced obviously.6, The biocompatibility studyThe biocompatibility of the TMPZ loaded sustained-release microspheres and the blank PLGA microspheres in rats was evaluated by pathological examination using normal saline as control. The results showed the TMPZ loaded sustained-release microspheres induced slight inflammation response, but infiltration liquid accumulation, blood vessels and fibrous proliferation were not detected. Therefore, the results showed good compatibility of both the TMPZ loaded sustained-release microspheres and the blank PLGA microspheres.7. The pharmacodynamic studyRat OA model was induced by intra-articular injection of papain and aminothiopropionic acid. The successful modeled rats were injected with TMPZ injection (2.1mg/0.1ml, once a week,6 times in total) and the TMPZ loaded sustained-release microspheres suspensions (4.2mg/0.1ml, injected once), respectively. The rats were sacrificed after injection of different preparation for 6 weeks. Therapy effect was evaluated via (1) the Freemont indexing under the optical microscope for the examination of the articular cartilage changes; (2) the Mankin arthrosis histology classification for the characterization of the cartilage and synovial membrane; (3) knee joint diameter and the knee joint activity; (4) examination of joint synovial fluid cell factor IL-βand PGE2 content for the discussion and comparison of the therapy effect of TMPZ injection and the TMPZ loaded sustained-release microspheres. The results indicated that the administration frequency and total dose can be reduced by TMPZ loaded sustained-release microspheres (from 6 times to 1 time and 12.6 mg to 4.2 mg, respectively). Furthermore, the effect of inflammation treatment of-the TMPZ loaded sustained-release microspheres was better than that of the TMPZ injection, the cartilage repair of the TMPZ loaded sustained-release microspheres was equal with that of the TMPZ injection. The results proved the better therapy effect and the sustained activity of the TMPZ loaded sustained-release microspheres.ConclusionThe TMPZ loaded sustained-release microspheres overcame the disadvantages of the TMPZ injection, including decreasing drug leakage and the frequent administration. The application of the novel dosage form is expected to extend the therapeutic duration, reduce the pain of patients, enhance the patient compliance and access to good social and economic benefits. The TMPZ loaded sustained-release microspheres was prepared using modified emulsion solvent evaporation method with reduced burst release property and increased encapsulation efficiency compared with the conventional preparation method. The accelerated release method was established to shorten the experiment time. It was available to expect the conventional in vitro release characteristics using the novel accelerated release method.In conclusion, the preparation, in vitro and in vivo property studies and pharmacodynamic study of the TMPZ loaded sustained-release microspheres was completed. A novel idea and method were explored for the treatment of joint diseases. This project possessed scientific and technological value and clinical prospect, and the research area of traditional Chinese medicine and long-acting preparations were enriched by this study.