Can curcumin improve the methotrexate based treatment for rheumatoid arthritis?

Darekar, Ashish Sahadev (2016) Can curcumin improve the methotrexate based treatment for rheumatoid arthritis? Doctoral thesis, University of Central Lancashire.

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Rheumatoid arthritis (RA) is an autoimmune disorder characterised by its varied and unpredictable origin, eventual destruction of cartilage and bone and the perpetual length of treatment involved. Even though significant developments have taken place over the past couple of decades in the treatment, the efficacy of drugs for RA is a major concern due to the side effects involved. Methotrexate (MTX) is currently used as first line treatment due to its ability to modify the rheumatic conditions so that disease progression can be prevented. However, at the same time prolonged exposure to MTX can lead to severe side effects such as lung fibrosis and hepatotoxicity. Recent research has focused on developing an alternative to MTX with similar efficacy but with reduced adverse effects. One such promising option is curcumin, an active compound extracted from Indian spice Turmeric. Various studies have indicated the synergistic properties exhibited by curcumin and its ability to modulate the underlying inflammatory pathways involved in RA. However, no previous studies have been reported with regards to using a combination of MTX and curcumin for the treatment of RA. A novel RP-HPLC stability indicating method was developed in order to establish the compatibility of the two compounds. The method was developed using Waters Reverse Phase (XBridgeTM Shield RP18 4.6x250 mm, 5 µM) column. A gradient system, consisting of two mobile phases with acetonitrile concentrations of 35% and 60% respectively, was designed to optimise the separation of the two compounds while taking into consideration the difference in hydrophobicity. The wavelengths for detection were 305 nm and 430 nm for MTX and curcumin, respectively. The retention time for MTX and curcumin was 4.8 ± 0.10 min and 12.3 ± 0.10 min, respectively. The Page 4 of 192 total run time of analysis was 25 minutes. The developed method was validated for parameters such as accuracy, precision, linearity, limit of detection and lower limit of quantification. The system was tested through intraday and interday repeatability and reproducibility. The method was used to analyse the MTX and curcumin under different stress conditions such as pH, UV radiation, temperature and humidity to establish their compatibility. The degradation products are successfully separated and therefore, the method can be effectively used as stability indicating method. Gene expression profiling was performed using HFLS-RA cells treated with MTX and curcumin separately and concurrently. The DNA microarray data identified 53 genes that were downregulated and 21 genes that were upregulated in all the treated samples using both stringent and non-stringent filtering. The total of 13 genes were selected based on the fold change obtained in the microarray data and their potential as therapeutic biomarkers based on previous research. The gene validation using qRTPCR confirmed the higher efficacy of curcumin in the inhibition of the proinflammatory genes such as ANGPTL7, CD248, CH25H, COL14A1, CXCL12, CYTL1, IFITM1 and IL7. Curcumin was found to also increase the expression levels of genes associated with anti-inflammatory roles, namely BCAR4, CD274, HSPA6, OTP and RELT. The increased gene expression in samples treated with both MTX and curcumin confirmed the possible synergistic activity which is encouraging, taking in to account the fact that these compounds are compatible according to the stability studies carried out and thus could be used in combination for the treatment of RA. This could improve the current treatment by reducing the severity of side-effects attributed to MTX while maintaining the efficacy of the treatment due to the ability of curcumin to modulate specific therapeutic biomarkers involved in the RA pathogenesis.

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