Animal And Clinical Study For The Effect Of Pranoprofen In The Treatment Of Dry Eye

Dry eye is a multifactorial disease of the tears and the ocular surface that results in symptoms such as ocular discomfort,visual disturbance,and tear film instability with potential damage to the ocular surface.The core mechanisms of dry eye are driven by tear hyperosmolarity and tear film instability.Any factor that stimulate or damage the ocular surface microenvironment can result in tear film instability decline and noninfectious inflammatory response of ocular surface epithelial cells.In fact,inflammation can happen at any level of dry eye,they can not only promote the progression of dry eye through causing chronic damages of ocular surface and lacrimal gland,but also can be the result of dry eye and further aggravate patients'symptoms and signs.Both the 2007 report of the international dry eye workshop and the 2013 Chinese expert consensus of dry eye clinical diagnosis/treatment are clear about the important role of inflammation in the development of dry eye disease.In the U.S.and European,Cyclosporin A was widely used in the treatment of dry eye inflammation as an important immunosuppressant,its mechanism and clinical effect has been deeply investigated.In recent years,foreign scholars also proved that corticosteroids could effectively improve the symptoms and signs in moderate to severe dry eye through clinical research.However,the value of corticosteroids and cyclosporine was limited in mild to moderate dry eye patients due to its relatively excessive efficacy and potential side effects.Therefore,new management and treatment with fewer side effects,less irritation,and/or moderate anti-inflammation effect for mild to moderate dry eye patients is desirable and required for clinical practice.Non-steroidal anti-inflammatory drugs(NSAIDs)are a class of drugs that provides analgesic(pain-killing)and antipyretic(fever-reducing)effects,and,in higher doses,anti-inflammatory effects.NSAIDs inhibit the activity of both cyclooxygenase-1(COX-1)and cyclooxygenase-2(COX-2),and thereby,the synthesis of prostaglandins and its related inflammation response.In ocular surface,NSAIDs were often used as post-surgical antiinflammation drug in cataract surgery,refractive surgery.Interestingly,these surgeries will lead to the occurrence of dry eye themself,and these kinds of inflammations are non-infectious inflammation,which were similar to the dry eye inflammation.Moreover,the latest research suggests that patients with dry eye has higher concentration of prostaglandin E2 in tears compared with normal person,and the expression level of prostaglandin E2 is positively correlated with dry eye symptoms,which further indicated that NSAIDs is a potential dry eye anti-inflammatory drugs.The core of our research is to explore the effect of the NSAID drug,pranoprofen,in the treatment of dry eye.Research is divided into two parts,animal experiments and clinical trial.In the animal study,we built the dry eye mouse model by 0.2%benzalkonium chloride,and detected the key factor of prostaglandin synthesis pathway and dry eye inflammation factors,explored their relationship with the occurrence and development of dry eye.In the clinic trial,we investigated the efficacy of 0.1%pranoprofen in the treatment of dry eye through an multi-center,randomized,controlled,parallel group study study,used Conjunctival impression cytology and real-time polymerase chain reaction(RT-PCR)to detect the change of Human Leukocyte Antigen DR(HLA-DR)and Intercellular Adhesion Molecule 1(ICAM-1)during the treatment of 0.1%Pranoprofen.Therefore provide basic and clinical evidence for the efficacy of 0.1%pranoprofen in the treatment of dry eye.PART I Prostaglandin synthesis pathway and inflammatory changes in benzalkonium chloride induced dry eye mouse model Purpose To evaluate prostaglandin synthesis pathways and inflammatory changes in 0.2%benzalkonium chloride induced dry eye mouse model.Methods 30 normal male BALB/c mice were randomly divided into 2 groups:normal group and benzalkonium chloride group 15 in each group and right eye for study.Mice in normal group did not make any drug processing,benzalkonium chloride group mice were treated by 0.2%benzalkonium chloride,twice daily for 14 days to build the dry eye model.HE-staining,K10 immunofluorescence staining and TUNEL kit were performed to detect the morphology and function changes of cornealepithelial cells.Western Blot and Enzyme-linked immuno sorbent assay(ELISA)were performed to detect the expression level of COX-2 and PGE2.RT-PCR was performed to detect the gene expression level of tumor necrosis factor alpha,macrophage inflammatory protein 2,class II major histocompatibility complex and intercellular adhesion molecule 1.Results Treatment of 0.2%benzalkonium chloride for 14 days aggravated ocular surface inflammation index,corneal fluorescein and tear film stability;Expression level of COX-2,PGE2,tumor necrosis factor alpha,macrophage inflammatory protein 2,class II major histocompatibility complex and intercellular adhesion molecule 1 were all significantly increased.Conclusion 0.2%benzalkonium chloride induced dry eye model is an mixed dry eye model with inflammatory/immune response and activated prostaglandin synthesis pathways.PART ? Efficacy of pranoprofen in the treatment of benzalkonium chloride induced dry eye mouse modelPurpose To evaluate the Efficacy of 0.1%pranoprofen in the treatment of benzalkonium chloride induced dry eye mouse modelMethods 60 normal male BALB/c mice were randomly divided into 4 groups:normal group,benzalkonium chloride group,sodium hyaluronate group and pranoprofen group,15 in each group and right eye for study.Mice in normal group did not make any drug processing,the rest of 3 groups of mice were treated by 0.2%benzalkonium chloride eye drop,twice daily for 14 days to build the dry eye model.Then sodium hyaluronate group and pranoprofen group mice were treated with 0.1%sodium hyaluronate and 0.1%pranoprofen respectively,twice daily for 10 days.All mice were sacrificed for sample collections.Ocular surface inflammation index,corneal fluorescein staining,tear break up time and schirmer test were evaluated before and after the dry eye model built,as well as the 2,4,6,8,10 treatment days.Tear sample were collected on day 1,3,5,7,9.HE-staining,K10 immunofluorescence staining and TUNEL kit were performed to detect the morphology and function changes of corneal epithelial cells.Western Blot and Enzyme-linked immuno sorbent assay(ELISA)were performed to detect the expression level of COX-2 and PGE2.RT-PCR was performed to detect the gene expression level of tumor necrosis factor alpha,macrophage inflammatory protein 2,class ? major histocompatibility complex and intercellular adhesion molecule 1.Results Pranoprofen have better effect in the recovery of ocular surface inflammation index,corneal fluorescein staining tear film stability compared with 0.1%sodium hyaluronate.pranoprofen have better inhibition effect for MHC-2,ICAM-1,TNF-a,MIP-2,COX-2 and PGE2 when compared with 0.1%sodium hyaluronate.Conclusion 0.1%pranoprofen can effectively treat benzalkonium chloride induced dry eye model through its inhibition effect for the COX-2 and dry eye related inflammatory factors.PART ? Clinical efficacy of Pranoprofen in treatment of dry eye patients:a multicenter,random,controlled clinical trial Purpose To investigate the clinical efficacy of 0.1%pranoprofen in the treatment of dry eye.Methods It is a prospective,multi-center,randomized,controlled,parallel group study.One hundred and fifteen patients with mild to moderate dry eye disease(55-60 in each treatment group)participated in this multi-center study.Patients were randomly administered with eyedrops containing 0.1%pranoprofen(PRA)plus 0.1%sodium hyaluronate(SH)or 0.1%sodium hyaluronate(SH)only,three times daily for 28 days,followed by a 1-week post treatment observation.Dry eye symptom score(DESS),fluorescein corneal staining(FLCS),tear break-up time(TBUT),and Shirmer 1 tear test(ST1,without anesthesia)were evaluated or conducted before treatment and at each study visit.Conjunctival impression cytology was taken from the patients treated with PRA plus SH before and after treatment and real-time polymerase chain reaction(RT-PCR)was performed to detect the changes of Human Leukocyte Antigen DR(HLA-DR)and Intercellular Adhesion Molecule 1(ICAM-1).Results Patients treated with PRA plus SH showed gradual improvements of DESS,FLCS,and TBUT.Between-group comparisons of FLCS and TBUT have statistically significant differences from day 14.Good tolerance with no severe adverse events was found in both groups.Patients treated with PRA plus SH had a reduced expression level of HLA-DR and were statistically different after 28 days of therapy.Conclusion The application of PRA at a dose of 0.1%was well tolerated and benefited to the patients with mild to moderate dry eye disease.The underlying mechanism of its efficacy may be associated with the reduction of inflammatory factors of conjunctival epithelial cells.