Research On Methotrexate Thermosensitive Hydrogel For Intra-articular Injection

Articular injection is a kind of topical treatment that directly delivery drugs to the site of inflammation of the rheumatoid arthritis patients.It's mostly based on solutions formulations currently and difficult to achieve therapeutic effect because of the rapid elimination in the intra-articular after injected.So it's often required repeated administration.Now the local injection has been widely studied and used thermosensitive hydrogel formulation as sustained-release preparations of intra-articular injection.This preparation use the polymer copolymer material having a hermosensitive as a carrier.So it occurs phase change and becomes semi-solid after injected in order to achieve the purpose of increasing drug residence time in the joint cavity.It can effectively improve the local drug concentration in the joint cavity,and reduce systemic side effects of oral drugs gastrointestinal tract.Some scholars have used PLLA[1] and PLGA[2] as carriers to prepare methotrexate sustained release microspheres for intra-articular injection Currently,but there is some serious burst release of microsphere preparation and resulting in acute inflammation after early injection.Guilei Ma[3] has used PCL-PEG-PCL as a carrier to prepare methotrexate temperature-sensitive gel and studyed its pharmacokinetic in vivo.The plasma concentration of methotrexate is still higher than the detection limit 24 hours after injection.However this dosage forms used acetonitrile as solvent and the amount of the solvent remained should be strictly controlled under the safe value.This study taking PLGA-PEG-PLGA as gel matrices and mannitol as viscosity modifiers to prepare methotrexate thermosensitive hydrogel for intra-articular injection.The gelation temperature was applied as a target and choose PLGA-PEG-PLGA,methotrexate and mannitol which impact the gelling temperature greater as the investigation factors to select the optimized formulation of methotrexate thermosensitive hydrogel.Gels formulation was finally confirmed as 25% PLGA-PEG-PLGA,0.20% methotrexate and 0.10% mannitol;PLGA-PEG-PLGA25%,0.20% methotrexate and 0.10% mannitol gelation,which the gelation temperature was 35?.The quality control method of methotrexate thermosensitive hydrogel was established.The drug content of methotrexate thermosensitive hydrogel of three batches were determined by HPLC method.This method had good specificity and high precision.The average contents of methotrexate in three batches of samples was more than 90%.The structure of PLGA-PEG-PLGA copolymer was characterized.Then its rheological properties was investigated in order to study the characters of rheology of the gel during phase transition.The impact of the hydrogel concentrations and initial drug loadings on the release rate was investigated by dialysis bag model to study the determination methods of contents and its in vitro release.The data suggested that the methotrexate loaded hydrogel was able to slow down the release rate.And the accumulative drug release was up to 90.58% at 12 days.The release and time were fitted with different release models,and the value of R of the Ritger-peppas model was the largest.So the drug release by the diffusion of drug and the gel dissolution presumably.The healthy SD rats were randomly divided into three groups,injecting penicillin of carboxymethylcellulose sodium and lipopoly saccharide under the skin in order to establish the model of rheumatoid arthritis.At the same time we inject stroke-physiological saline solution,the solution of methotrexate and methotrexate thermsensitive hydrogel respectively.Detecting the level of tumor necrosis factor –?(TNF-?)in the exudate of the rats by ELISA on the third,sixth and ninth day after injected.And then prepare the paraffin section of subcutaneous tissue.The 3d,6d and 9d after injecting drug,the level of TNF-? of the group of the methotrexate solution have lowered 27.31%±4.37%,42.70%±3.92% and 52.28%±4.00% respectively compared to the group of physiological saline solution while the level of TNF-? of the group of methotrexate thermsensitive hydrogel have lowered 24.28%±3.05%,52.45%±3.22% and 68.96%±2.85% respectively.The results of paraffin section shows that the inflammation of the the group of the methotrexate solution and the group of methotrexate thermsensitive hydrogel both ameliorated compared with the group of stroke-physiological saline solution.And the inflammatory cells of the group of methotrexate thermsensitive hydrogel still retain at the lower level on the ninth day after injecting drug,which having a significant difference between the group of stroke-physiological saline solution.Divide SD rats randomly into two groups and inject the physiological saline and methotrexate thermsensitive hydrogel into the subcutaneous airbag to study the biocompatible of the gel.Then prepare the paraffin section of subcutaneous tissue when the 1,3 and 7d after injection.The results showed there was no significant inflammatory cells after administration,which was no significant difference between the two groups when 7d after injection.Divide Kunmin g mice randomly into two groups and inject the solution of methotrexate and methotrexate thermsensitive hydrogel mixed with near infrared absorbing dyes IR-820 through the air-pouch of the rats to study in vivo retention.Observe the fluorescence intensity of the air-pouch and inspect the partial retention of the vector.It was shown that the group of the methotrexate solution have almost eliminated on the day after injected.While the retention quantity of the group of methotrexate thermsensitive hydrogel is still retain 17.11% on the ninth day after injecting drug.Since the temperature-sensitive properties of the formulation,normal injection sterilization method would impact the properties of formulations.So this study using 60 Co radiation sterilization instead of normal method,the appearance,temperature-sensitive properties,drug content and in vitro release behavior of temperature-sensitive gel were evaluated both before and after the sterilization.The results show that the sterilization method is suitable for the preparation.With appearance,temperature-sensitive properties,drug content and in vitro release behavior of temperature-sensitive gel as the indexes,the stability of preparation was evaluated by influencing factor experiment and long-term experiment,The results show that the formulation is unstable under high temperature,light instability,and should be preserved at a low temperature and protected from the light.To sum up,the methotrexate thermosensitive gel conformed with the general quality requirements of injection,having fine injection performance in situ.The thermosensitive hydrogel could phase change quickly after inject into the joint.Morever,the distant treatment outcome of the thermosensitive hydrogel is better than methotrexate solution and have no tissue irritation.In the long-term rheumatoid arthritis medication,the thermosensitive hydrogel can reduce drug side effects and the frequency of administration to avoid joint infection and has obvious advantages and prospects compared with solution preparation.