Construction Of A High-efficiency Delivery Formulation Of TNF-Α Antagonist And Its Application In The Treatment Of Rheumatoid Arthritis

People with rheumatoid arthritis(RA)suffer from joint destruction for a long time.As the disease progresses,the patient will gradually lose mobility and even become disabled.Since RA cannot be cured,clinically,the main goal is to alleviate the pain of patients and control the course of the disease.Serious side effects and drug resistance will occur in long-term clinical treatment,so it is necessary to constantly replace new drugs,which will lead to new side effects.Whether it is the huge cost of long-term treatment or the impact on physical health,it will cause a huge burden on patients.Studies have found that TNF-α is an important cytokine leading to the inflammatory cascade,and blocking the effect of TNF-α can effectively delay the course of RA.TNF-α antagonists(rhTNFR:Fc)can compete with TNF-α to control the course of the disease,but the low stability and low bioavailability of such protein drugs affect the therapeutic effect.Sustainedrelease delivery formulations can effectively improve the bioavailability of drugs,reduce dosing frequency and prolong dosing duration,and have great potential for RA,a chronic disease that requires long-term treatment.Because of its functional shell,the nanocapsule carrier can encapsulate the drug in the shell,so that the drug can be protected from environmental hazards,and the capsule shell layer can be designed to have special functions such as targeting or stimuli response.Polylactic glycolic acid(PLGA)microspheres are often used as drug sustained-release carriers.By loading therapeutic drugs into PLGA microspheres,the release cycle of the drug can be effectively prolonged,bioavailability is enhanced,and the number of administrations can be greatly reduced.To sum up,this paper aims at the problems existing in the current clinical use of rhTNFR:Fc.Taking advantage of the performance advantages of nanocapsules and PLGA sustained-release microsphere drug carrier,two rhTNFR:Fc sustained-release preparations for different administration routes were designed and developed,and the RA efficacy of the preparations was evaluated in detail.The details are as follows:(1)Preparation of rhTNFR:Fc nanomedicine capsules and study on the efficacy of RA.By in-situ polymerization,2-Methacryloyloxyethyl phosphorylcholine(MPC)with good blood compatibility was used as the monomer and polylactic acid-polyethylene glycol-polylactic acid acrylate(AC-PLA-PEG-PLA-AC)with p H-responsive degradability was used as a cross-linking agent,and a polymer capsule layer was in situ coated on the surface of rhTNFR:Fc,to obtain a rhTNFR:Fc nano drug capsule(n(rhTNFR:Fc))with excellent blood circulation stability and p H-responsive drug release.The prepared n(rhTNFR:Fc)was spherical particles with a size of about 10 nm.The in vitro dissolution test proved that the nanomedicine capsules have a sensitive acid-responsive drug release behavior,and the drug release rate in an acidic p H 5.5 environment is about 3 times that in a p H 7.4environment.Further,in vitro experiments proved that n(rhTNFR:Fc)can inhibit the proliferation of human rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS)by binding to extracellular TNF-α,and reduce the expression of TNF-α level.In vivo animal experiments have proved that after a week of tail vein injection of n(rhTNFR:Fc),the mice can still maintain about 50% of the plasma concentration.In addition,in vivo imaging experiments demonstrated that n(rhTNFR:Fc)can be effectively enriched in the leg lesions of the collagen-induced arthritis(CIA)mouse model,with good targeting ability.Finally,the mouse joint index score and the detection of inflammatory factors proved that the therapeutic effect of n(rhTNFR:Fc)on CIA model mice was significantly improved compared with the original drug.Its long-acting sustained-release and high bioavailability properties have great application potential for improving the clinical treatment of RA.(2)Preparation of porous PLGA sustained-release microspheres loaded with rhTNFR:Fc and its efficacy study on subcutaneous injection in the treatment of RA.First,we successfully prepared a nano-hydroxyapatite(nHAP)with ultra-small size and high dispersion stability using hypergravity technology.The nHAP/PLGA microspheres were prepared by double emulsion method with PLGA,and then the surface of the nHAP/PLGA composite microspheres formed a porous structure by means of Na OH etching.Finally,we prepared nHAP/PLGA-PDA composite microspheres by in situ polymerization of dopamine(DA)on the surface and in the pores of the microspheres.In vitro accelerated degradation test proved that nHAP has an effective buffering effect on the acidity of PLGA after degradation.The drug loading and drug release assays proved that the porous microsphere structure and in situ polymerized PDA in the pores can effectively improve the loading efficiency of rhTNFR:Fc,eliminate the drug burst effect,and prolong the drug release period.Further,in vitro cell experiments proved that nHAP/PLGAPDA sustained-release microspheres(nHAP/PLGA-PDA-rhTNFR:Fc)loaded with rhTNFR:Fc could effectively inhibit the proliferation of RA-FLS cells and reduce the expression level of inflammatory factors.After subcutaneous injection of nHAP/PLGA-PDA-rhTNFR:Fc,the effective drug concentration in blood can be maintained for 15 days.Finally,compared with subcutaneous injection of rhTNFR:Fc,nHAP/PLGA-PDA-rhTNFR:Fc exhibited superior arthritis treatment effect.