Effects of 4 Thiopurine Compounds on Nitric Oxide Production and Cell Viability of HIG-82 Synoviocytes and RAW 264.7 Macrophages

1Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia. 2Faculty of Medicine and Health Sciences, UniversitiSains Islam Malaysia, Kuala Lumpur, Malaysia. 3Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Malaysia. 4Laboratory of UPM-MAKNA Cancer Research (CANRES), Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. *Correspondence Author E-mail: nazrulh@upm.edu.my

Worldwide, RA is a serious health problem that affectsup to 1.0% of adults in the developed countries (Parada-Turskaet. al., 2006;Wang et. al., 2006).In the USA alone, the total annual cost of arthritis to society is compensated for by more than 20 million RA patients with severe developmental disabilities on a daily basis and estimated at $100 billion (Scott et. al., 2010). In Malaysia, 5 in 1000 adults are reported to be the RA patient by a year (Shahriret. al., 2008). As consequent, RA has a significant impact on physical, emotional, psychological and social activities of patients in the daily basis; they restricted in most functioning on works as well reducing life expectancy, premature mortality further causing the massive economic burden to the society (Too et. al., 2012).
To date, patients suffering from arthritis such as Crohn's, ulcerative colitis, and chronic arthritis have been prescribed with a long-single potent drug that has experienced serious side effects.As example, the potent drugs like diseasemodifying anti-rheumatic drugs (DMARD's) (e.g: azathioprine, methotrexate and tofacitinib), non-steroidal anti-inflammatory drugs (NSAID's) (e.g; diclofenac) and glucocorticoids (Nielsen et. al., 2001). DMARD drugs are often used with a combination with NSAIDs or glucocorticoids greatly improved the quality of life for many patients after 4-6 months to be fully effective. Unfortunately, DMARDs target the immune system of the patients, which weaken the immune system's ability to fight infections. Subsequently, the chemotherapy to the cells effected disease become resistance toward the drugs. This is a major factor in the failure of many forms of chemotherapy especially to cancer patients. These problems of course reduce the drug's efficacy (Shankeret. al., 2010).
Hyper-proliferation of synovial fluid (SF) and synoviocytes in the synovial membrane lead to the development of pannus which subsequently cause the invasion to the joint and bone that cause damage to the affected area (Smolen et. al., 2014;2007;Firestein, 1996) and SF play a vital role in facilitating inflammatory action, cartilage degradation and joint destruction in RA (Moret. al., 2005). A study using SCID mouse revealed that intrinsic factor with the immune system support contributes to join damage and degradation. Thus, it indicates that the invading products, if synoviocytes may be isolated from inflammatory cells and serve as a key target for progression of arthritis (Muller-Ladner et. al., 2007). In this research, the HIG-82 synoviocyte cell line, which retains both the morphology and differentiation markers of synovial fibroblast, was used as a RA cell model to evaluate the therapeutic action of thiopurine compounds.Phorbol myristate acetate (PMA) is a well-known stimulator to activate necrosis factor-kappa B (NF-êB) in the cell was applied to resemble the inflammatory and arthritic condition in activated synoviocytes (Kim and Ro, 2005;Smith et. al., 1998;Baeuerle and Henkel, 1994;Hasan et. al., 2012). NF-êB regulates the expression of target genes that encode inflammatory cytokines such as interleukin (IL)-1, IL-6, IL-12, tumor necrosis factor-alpha (TNF-á), interferons (IFNs), chemokines and enzymes that cause inflammatory response such as COX-2 and inducible nitric oxide synthase (iNOS) (Piccinini and Midwood, 2010).
Nitric oxide (NO) is a short-lived, reactive species molecule synthesized by NO synthases (NOS) catalyzed by L-arginine (Nathan, 1991). Many biological responses involved by NO molecule such as inflammation, cell-mediated immune response, relaxation of the blood vessels and neurotransmission (Miyasaka, 1997). There are at least two NOS isoforms; Ca2 + Calmodulindependent constitutive NOS (cNOS), present primarily in endothelial cells and brain, and Ca2 + Calmodulin-independent inducible NOS (iNOS), residing predominantly in macrophages and smooth muscle cells (Nagy et. al., 2007). Nitric oxide (NO) is generally produced in large quantities by activated macrophages as one of the host defence during inflammation. NO can catalyse by distinct three isoforms of nitric oxide synthase. However, the enzyme which plays the most critical role in inflammation is iNOS. After activated by endotoxin or pro-inflammatory cytokines such as IL-1, IFN-gamma (IFN-ã), TNF-á (Miyasaka, 1997), murine macrophage produces large amount of NO (Larouxet. al., 2000). Purinethiol or 6-mercaptopurine (6-MP) ( Figure 1) is a derivative of purine sulphur approved by the Food and Drug Administration ( FDA) as an antitumor drug in 1953 (Pilar et. al., 1996). 6-MP was among the first effective drugs for childhood leukaemia and subsequently refined as an effective anti-cancer and immunosuppressant medication in the previously poor prognosis of the disease (Zaza et. al., 2005;Bell et. al., 2004;Elion, 1989).
Thiopurine compounds are purine antimetabolic, which interferes with biochemical process involving endogenous purines, which are vital components of DNA, RNA and certain co-enzymes (Coulthard, 2012;Cara et. al., 2004;Coulthardet. al., 2002;Lennard, 1992). The thiopurines were initially tested for leukaemia treatment and as immunosuppressant for organ transplantation. The compounds of thiopurine have a fairly small therapeutic index and are capable of life-threatening toxicity (Sahasranamanet. al., 2008). Thiopurines can have a cytotoxic effect in a myriad of areas.Previous reported usesthe thiopurines for inflammatory bowel disease and ulcerative colitis, eventually by controlled clinical trials (Konidari and El-Matary, 2014;Freiet. al., 2013;Mahadevanet. al., 2000;Ricketts, 1998). 6-MP and its derivatives, blocks the bio-activation of NF-êB and its related cytokines (Chang et. al., 2012). They prevent bio-activation by inhibiting de novo purine synthesis and integrate specific cells, including neutrophils, macrophage, lymphocyte, and endothelial cells, into nucleic acids (Ordentlichet. al., 2003; Hussein-Al-Ali et. al., 2012). Possessions are dose-related, low doses of drugs are anti-inflammatory, but higher doses are immunosuppressive and cytotoxic (Polifka and Friedman, 2002).Therefore, effects of thiopurine compounds namely, 6-mercaptopurine (6-MP), 6-MP riboside (6-MPR), 6-thioguanine (6-TG) and 6-thioxanthine (6-TX) on cell viability and inhibitory effects on nitric oxide production were examined on PMA-activated HIG-82 synoviocytes fibroblast and LPS-induced murine macrophage, RAW 264.7 cell lines. The therapeutic effects of thiopurines compounds, particularly on the cell viability with less cytotoxicity is crucial. Therefore, the objectives of the current study: firstly, to examine the effects of immunosuppressive thiopurine anti-metabolites compounds on cell viability of phorbol myristate acetate (PMA)-activated HIG-82 synoviocytes fibroblast and Escherichia coli lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophage cell lines. Secondly, to examine inhibitory effects of immunosuppressive thiopurine anti-metabolites on inducible nitric oxide production of phorbol myristate acetate (PMA)-activated HIG-82 synoviocytes fibroblast and Escherichia coli lipopolysaccharide (LPS)induced RAW 264.7 murine macrophage cell lines and thirdly, to investigate the cytotoxicity effects of immunosuppressive thiopurine anti-metabolic compounds due to nitric oxide production in responding to compound dosage. We hypothesized that these thiopurine compounds will be potentially useful for the treatment of RA.

Induction and stimulation of inflammation phase
Methods previously described by Parada-Turskaet. al. (2008) and Jeounget. al. (2013) were employed with slight modifications. The cells growing at exponential phase with 80-90% confluence were detached from the plastic surface by gently trypsinized with Trypsin-EDTA(0.25%) solution.After the cells had completely detached from the surface of the flask, then the completed media was added to stop the trypsin activity.The cell suspension was relocated into a centrifuge tube of 15 mL and centrifuged at 4 ° C at 190 x g for 10 minutes.After removal of supernatant, the cell pellets were re-suspended with 1 mL of completed growth medium; RAW 264.7 cell in DMEM with 10% fetal bovine serum and HIG-82 cell line in Ham's F12 containing 10% fetal bovine serum and the concentration of cells were adjusted to 1 x 10 6 cells/mL and 1 x 10 4 cells/mL for both cells respectively by adding calculated volume of completed growth medium above. Basically, 10 uL of suspension cells was mixed at ratio 1:1 with 0.4% trypan blue solution and the number of cells was calculated using a haemocytometer. Dead cells absorbed the blue colour of trypan blue due to the membrane permeability, while the viable cells remained unstained. The viability of cells for the assay had to be at least 95%. The cell viability and the volume of completed growth medium needed were calculated.
For cell stimulation and treatment,50 µL of the diluted cell suspension (1 x 10 6 cells/ mL) were dispensed into a tissue culture grade 96-well flat bottom plate (Becton Dickinson, NJ, USA) except for blank. The plate was incubated for 3 hours for RAW 264.7 cell line and 24 hours for HIG-82 cell line at 37 °C, 5% CO 2 to allow the cells to attach to the surface. While waiting the cell attachment, the tested compound stock was serial diluted to a decreasing concentration with completed growth medium. For preliminary screening, six concentration of tested compound were prepared (100, 50, 25, 12.5, 6.25 and 3.125 µM). After incubation hours, the media was gently removed to discard unattached cells and the attached cells were then induced with 50 mL of 10 µg/mL (final concentration) of Escherichia coli lipopolysaccharide (LPS) from serotype 055:B5 (Sigma, USA) and 10 nM (final concentration) phorbol myristate acetate (PMA) for the cells respectively. Then, 50 µL from each of serially diluted samples were transferred into each well of the prepared tissue culture plate except the control groups. The five controls were set up and the control group were prepared in the last row of the tissue culture plate. The plate was then incubated for another 24 hours overnight at 37 °C, 5% CO 2 . After incubation, the culture cells were prepared for MTT cytotoxicity assay.

-( 4 , 5 -d i m e t h y l t h i a z o l e -2 -y l ) -2 , 5-diphenyltetrazolium bromide (MTT) cell viability assay
The cell viability was determined by assaying the reduction of MTT reagents to formazan salts. MTT is a water soluble tetrazolium salt which yellow in colour. Metabolically active cells are able to convert MTT molecule into a water-insoluble dark blue formazan salt by reductive cleavage of the tetrazolium ring (Pozzoliniet. al., 2003). After treatment, the supernatant of 96-wells plate containing cells were replaced with the fresh media and 20 µL of MTT reagents (5 g/mL) were added into each well. After 4 hours, the spent media was removed completely and the formazan salts were dissolved with 100 µL 100% DMSO. The absorbance was then measured at 570 nm using an ELISA microplate reader (Infinite M200 Tecan Microplate Reader; Tecan Inc. Durham, North Carolina, USA). The percentage of cell viability was calculated.

Griess assay
The production of nitric oxide (NO) was determined by measuring the increase of nitrite in the supernatant of the samples. The inhibitory activity of compounds on NO production assayed using Griess reaction (Ahmad et. al., 2006). Briefly, 50 µL of supernatant from each well transferred into corresponding well of clean non-sterile 96-well plate. The exact volume 50 µL (1:1) of Griess reagent [1% sulpanilamide, 0.1% N-(1naphtyl)-ethylene diamine dihydrochloride in 2.5% phosphoric acid] and mix well with the cell culture supernatant. The absorbance was read at 550 nm with microplate reader (Tecan Infinite 200 PRO, Switzerland). The amount of nitrite in a sample was calculated from sodium nitrite standard curve Value were mean ± SD (n=3 independent test) (*) Significant different (P < 0.05) when compared to normal freshly prepared in deionised water (0-100 µM). Then, the percentage on nitrite inhibition of each sample was calculated using formula below: Statistical analysis Data is expressed as mean ± SEM of duplicates using GraphPad Prism 5 software, using different experiments of triplicate sets of each template. Using variance analysis, statistical significance was established at p Â0.05. Significant treatment means were further subjected to Bonferroni post-tests. Table 1 and 2 showed a dose-dependent suppression effects to dose increment compounds on HIG-82 synovicytes and murine macrophage RAW 264.7 cell lines due to    Interestingly, 6-MP and diclofenac possess a similar effect on RAW 264.7 macrophages cell same trend effects to HIG-82 synoviocyte cell line. However, from the both findings, HIG-82 synoviocyte cell was seem more sensitive and less viability to thiopurine compounds when compared to RAW 264.7 cell within the dosage range of the compounds but the HIG-82 cell was more consistent pattern effects to thiopurine compounds. The result findings from cell viability MTT assay showed that anti-metabolic thiopurine compounds namely 6-MP, 6-MPR, 6-TG and 6-TX were not cytotoxicity to HIG-82 synoviocyte cell and RAW 264.7 murine macrophage cell line of the selected dosages range. This is because the percentage of cell viability for all four compounds were showed more than 70% viability cell at all concentrations tested. Table 2 demonstrated the inhibitory effects of thiopurine compounds on nitric oxide upon PMA-activated HIG-82 synoviocytes cell line.Among the thiopurine compound tested, there are no inhibitory action of NO showed on PMA-activated HIG-82 cells. This is due to the PMA probably does not cause the production of NO oxide. Thus, the inhibitory assessment of thiopurine compound cannot be determined. This same goes to control drug, diclofenac and the iNOS inhibitor L-NAME action in suppressing NO cannot be determined. The dosages selected were same with dosages range with cytotoxicity effects. The evaluation of NO inhibitory effects of thiopurine compounds and its derivatives is based on the dose response activities effect at six different concentrations (3.125-100µM; 2-folds). Among four thiopurine compounds and diclofenac were tested, the inhibitory effects of NO were not determined. This is probably due to very less or undetectable concentration of NO was produced by the PMA-stimulated HIG-82 cell line. Positive control (L-NAME) also was demonstrated same respond like the compounds due to mechanism of PMA to the type of cell.

Effects of thiopurine compounds on nitrite oxide (NO) production
The concentration used for screening NO production of RAW 264.7 murine macrophage cell was covered a wide range (3.125-100 µM) to determine the effective working concentration for   Table 2 showed the effects of 6-MP, which dose dependent effects decreasing the level of nitrite concentration. The decreasing level of NO production pattern also demonstrated by other thiopurine compounds. Among four thiopurine compounds used, 6-mercaptopurine showed most potent NO inhibitory effect on LPS-induced RAW 264.7 murine macrophage cells with 11.61±10.85% NO inhibition activity at 100 µM compared to the rest compounds. The potency thiopurine compounds as NO inhibitor from strongest to weakest compounds as following in order; 6-mercaptopurine > 6-mercaptopurine riboside > 6-thioxanthine > 6-thioguanine. Interestingly, the inhibitory effects of NO production of all thiopurine compounds were showed at very strong activity of immunosuppressive when compared to L-NAME, a standard iNOS inhibitor as a positive drug control, which significantly inhibited NO production at the concentration of 250 µM. The efficacy of the thiopurine immunosuppressive compounds on nitrite production of RAW 264.7 murine macrophage cell line is shown in Table 2. Table 3 and Table 4 demonstrated the percentage of cell viability reduction and inhibitory effects on NO production at 100 µM respectively compared to Normal after 24 hrs incubation.

DISCUSSIONS
Results of the present study demonstrated the cytotoxicity effects and NO production by HIG-82 synociocyte and RAW 264.7 cell lines after treated with four immunosuppressive thiopurine anti-metabolic compounds, namely the 6-mercaptopurine (6-MP), 6-mercaptopurine riboside (6-MPR), 6-thioguanine (6-TG) and 6-thioxanthine (6-TX). As mentioned these compounds exhibits dose-dependent cytotoxicity effects on HIG-82 synoviocytes and murine macrophage RAW 264.7 cell lines after incubation 24 hours. In general, the cytotoxicity effect on PMA-activated HIG-82 and LPS-induced RAW 264.7 cell lines after treated with thiopurine compounds were demonstrated a dose-dependent manner with higher proliferation activity on HIG-82 synoviocytes cells compared to murine macrophage RAW 264.7 cells approximately 70% (30.77% reduction) and 60% (39.28% reduction) proliferation activity after 24 hrs incubation (Table  3). Furthermore, all thiopurine compounds except 6-MP riboside were showed more toxicity effects on LPS-induced RAW 264.7 murine macrophage cell line especially after treated with 6-thioguanine. In other side, only 6-MP riboside was demonstrated less toxicity to RAW 264.7 murine macrophage cell, thus promoting more proliferation activity (Mawatariet. al., 2001). According to our finding in this study, 6-thioguanine was demonstrated a very potent thiopurine as well at 100 µM on RAW 264.7 murine macrophage cell (Table 3) was believed involved in HGPRT up-regulation in inflammation mechanisms. Endless, the toxic accumulation of 6-TG in cells has activated an apoptosis or cell death programme which therefore provokes tumour regenericity in tissues and organs (Yatscoff and Aspeslet, 1998).
NO is an important modulator of the inflammatory cascade (Korhonen et. al., 2005).
However, due to the instable and volatile properties of NO if long air exposure, it is difficult to quantify the level of NO production in biological samples. Griess assay is a commonly used, simple and rapid spectrophotometric method to detect the presence of organic nitrite compounds in biological samples, which is also one of the short stable end product(s) of NO formation (Moshage, 2009). Nitric oxide (NO) is a free radical that commonly involved in biological process (Stadleret. al., 1991). However, overproduction of NO can cause cytotoxic and cytostatic effect (Choudhariet. al., 2013;Connelly et. al., 2001;Palmer et. al., 1988). Few years back, several study in RA patients had revealed with strong evidences that overproduction of NO may be important in the pathogenesis of RA suggested a predominant M1 macrophage phenotype (McInnes and Schett, 2011) and the inflammation joints in RA are the main source of NO (Korhonen et. al., 2005;Miyasaka, 1997). In human, level of nitrite in synovial fluid was highly elevated compared to serum in RA patient thus suggesting that inflamed synovial joint and synoviocytes were contributed to RA (Moshage, 2009;Jovanovic et. al., 2002).
NO producing cells can be varied inflammatory synoviums. Several specialized cells are capable of providing NO in the inflamed synovium, including osteoblasts, osteoclasts, macrophages, fibroblasts, neutrophils, and endothelial cells. (Otero and Goldring, 2007). Articular chondrocytes synthesise NO in response to IL-1 and lipopolysaccharide (LPS) reactions (Otero and Goldring, 2007;Stadler et. al., 1991). Nevertheless, the number of chondrocytes existing in the synovium may not be sufficient to compensate for the level of NO produced in the synovium. Synovial fibroblasts develop NO in response to IL-1 and TNF-á, while its development is inhibited by the transforming growth factor-â (TGF-â) (Stichtenothet. al., 1995). In comparison, it also notes the localization of iNOS immunoreactivity in synovial macrophages (Miyasaka, 1997). The NOS inhibitor (L-NAME) has been reported to reduce disease activity in experimental RA (McCartney-franciset. al., 1993). However, in this study, the level of nitric oxide produce cannot be determined due to no or too little of NO production being induced by the PMA-activated HIG-82 synoviocyte cell. It seems PMA suppresses its production as same reaction demonstrated by TGF-â (Stichtenothet. al., 1995). This is because PMA was not an appropriate activator for the HIG-82 synoviocyte cell to produce NO instead of activating protein kinase C (PKC) signalling pathways for modulating diverse cellular responses such as gene transcription, cell proliferation and differentiation, induction of apoptosis, immune response, and receptor desensitisation (Chang et. al., 2005). PMA has a minimal effect on NO synthesis (Hulkoweret. al., 1992;Stadler et. al., 1991;Georgescuet. al. 1988;Zakaria et. al., 2006). On the other hand, PMA is a protein kinase C (PKC) activator alone had no effects, whereas PMA with recombinant interferon (rIFN)-gamma synergistically increased NO synthesis (Yoon et. al., 1994). PMA-sensitive PKC isoforms (a and e) function in such a negative regulatory role of NOS and PMA-sensitive isoforms (a, b or e) may play a role in the inhibition of NOS induction (Paul et. al., 1997). These findings revealed that the dosages of thiopurine compounds were not toxic to synoviocytes HIG-82 cells.
In contrary, the thiopurine compounds have pronounced NO-suppressing activity on RAW 264.7 cell line. Off these, 6-MP was most potent in inhibiting the production of nitrite oxide compared to 6-MPR, 6-TG and 6-TX. Previous study suggested that thiopurines did not act as inhibitor on iNOS system, but they were significantly influenced many respective pharmacological proteins functions which responsible expressing the iNOS especially necrosis factor-êappa B (NF-êB) (Chang et. al., 2012). The central transcriptional mediator in the pathogenesis of several diseases, including stress response, cerebral ischemia and various immune reactions, which have been activated in abundance of cells and stimulated by oxidative stress-related cytokines, is considered to be a vital role of NF-êB. (Chang et. al., 2012). A common response to both cytokines and bacterial lipopolysaccharide (LPS) is an increase in apparent nitric oxide synthase (NOS) activity. This corresponds to the induction of the 130 kDa isoform of the enzyme and has been described in macrophages, smooth muscle cells, renal mesangial cells and hepatocytes (Förstermann and Sessa, 2012;Kunz et. al., 1994;Hortelanoet. al., 1993;Förstermannet. al., 1991).
Our results revealed that 6-MP and 6-TG were decrease more cell viability compared to 6-MPR and 6-TX. This might be due to metabolism of 6-MP to S-methyl-thioinosine 5'-monophosphate (6-Me-Thio-IMP), which is strong inhibitor of purine de novo synthesis (Sahasranamanet. al., 2008). Purine de novo synthesis inhibition is well known to achieve the impact of immunosuppression and block the proliferation of different forms of lymphocyte lines and contribute to the cytotoxic behaviour (Erbet. al., 1998). Thus, 6-TG metabolism produces 5 "triphosphate (dGS) deoxy-6-thioguanosine. It has been shown that integrating dGS into DNA causes cell-cycle arrest and apoptosis via a mechanism involving the mismatch repair pathway (Swan et. al., 1996). It is commonly assumed that cell damage caused mainly by the incorporation of the thioguanine nucleotides (TGN) into DNA is of decisive importance for the cytotoxic effect Previous studies have also shown that DNA has been impaired by the introduction of 6-TG into p388 murine leukemic cells and human cells, which has shown morphological alterations size enlargement and multinucleated nuclei (Almosailleakh and Schwaller, 2019; Daehnet. al., 2011).It's terribly difficult to synthesise, and since its mode of action emerged to be close to that of 6-MP, its metabolic fate and clinical behaviour was addressed a little subsequently (Kowalskaet. al., 2015; Mathews, 2012;Elion, 1989). Those were possibly the key reasons behind 6-TG that have never been used in the maintenance treatment of acute lymphoblastic leukaemia, however, unlike 6-MP, it is more active and less harmful than 6-TG that is further converted to cytotoxic TGN and can be degraded only after deamination by xanthine oxidase (Erbet. al., 1998). In other hands, 6-MPR is a derivative of 6-MP, while 6-TX is major products of metabolism of 6-MP by enzyme xanthine oxidase (Polifka and Friedman, 2002). From the results, 6-MPR decrease more cell viability compare to 6-TX. Although 6-MPR is a minor metabolite, it may be a clinical importance compare the known metabolite, 6-TX which is has less anti-neoplastic activity (Ono et. al., 1997). In contrast, 6-MPR has known has anti-neoplastic activity (Mironet. al., 2009;Solomon et. al., 1984).
Interestingly, our results showed that 6-MP and derivatives exhibits a very good therapeutic ranges in term of less cell dead and high cell lives than diclofenac, a common NSAID used for treating inflammation diseases. Diclofenac is a type NSAID that commonly used to relieve the join swelling but only have minor effect on the disease progression. Diclofenac was expected to inhibit COX synthesis of prostaglandin and help regulate symptoms but fail to prolong even the healing of RA-related joint damage (Wang et. al., 2011;Andreas et. al., 2009). Diclofenac therapy has been shown to have mild impacts on gene expression in SFs (Andreas et. al., 2009).All thiopurine compounds tested in this study were effective in promoting proliferation activity with low toxicity towards HIG-82 synoviocytes and RAW 264.7 murine macrophage cell lines after 24 hrs treatment. However, critical adverse drug reactions (ADR's) in the human body such as fever , rash, diarrhoea, hypertension, hepatitis, pancreatitis, bacterial liver abscesses, cytomegalovirus infections, life-threatening myelosuppression, bone marrow suppression, gastrointestinal symptoms, hypersensitivity reactions, and tumorigenicity may be caused by long-term use of potent drug such as 6-MP in high dose (Gaya et. al., 1995). In addition, the introduction of 6-MP pro-drug; azathioprine may effect the second generations of animal infant in pregnancy's mother (Ramsey-Goldman and Schilling, 1997) and birth defects after first trimester exposure to azathioprine, but the issue is still under debates (Rubinstein and Weinberg, 2012). Intracellular absorption of 6-thioguanine nucleotides in infants is also considered to be responsible for the cytotoxic effects of these drugs by blocking purine synthesis involving DNA synthesis and replication of RNA to new infant tissues and organ (Lennard, 1992).
In regard to these side effects, this present research was performed in observations of very beneficial therapeutic doses as a novel approach to reduce the ADR caused by 6-MP inflammatory arthritis treatment. The usage of glucocorticoids with sufficient thiopurine derivative dosages is another approach to reduce ADRs.Glucocorticoids are often the most successful remission-inducing medications, however regrettably, for patients that neglect to react, and for others that experience side effects or need long-term glucocorticoid treatment, may still show severe side effects, therefore immunomodulatory or immunosuppressive medications combined with an adequate dosage are necessary treatments or substitutes (Nielsen et. al., 2001).Immunomodulatory medication such as 6-MP pro-drug, azathioprine is able to mitigate and reduce the ADR caused by the usage of 6-MP. However, the replacement of 6-MP by azathioprine still facing with immediate and long-term adverse drug reactions of 6-MP via 6-MP metabolism where involved by three routes with superior enzymes in liver and gut; (a) thiopurine-S-methyltransferase (TPMT), added the methyl group to 6-MP through methylation to form 6-methyl-MP; (b) xanthine oxidase, which catalyses 6-MP to thiourate; and (c) hypoxanthine-guanine-phosphoribosyltransferase (HGPRT), which converts 6-MP to 6-thioguanine nucleotides (Karran and Attard, 2008).
A more approach beyond the usage of glucocorticoids and pro-drug replacement is the use of a nano-encapsulated drug delivery system. Recently study was demonstrated, cell proliferation levels in the presence of anti-cancer drugs provided by gold nanoparticles conjugated have been shown to be significantly smaller than those in cells subjected to cytostatic drugs alone (Cuinet. al., 2011), indicating that the transmission of nanoparticipants allowed an improved sensitivity of cancer cells to drugs evaluated with ribavirin. (Tomuleasaet. al., 2012). Nano-particles of diseasemodifying anti-rheumatic nano (DMARNs)medicines medication would have increased effectiveness at a lower dosage of the product concentration in the intended tissues, resulting in a decrease of adverse drug reactions of patients (Rubinstein and Weinberg, 2012). Treatment with DMARD azathioprine/purine derivatives, gold sodium thiomalate and methotrexate successfully restored RA-related chondrocyte gene expression to 'healthy' levels (Andreas et. al., 2009). Antimetabolic thiopurins are thought to be a potential pharmacological agent for the nano-encapsulated drug delivery mechanism for the treatment of multiple pathological disorders with intrinsic inflammatory pathways and components.It often inhibits systemic inflammation by inhibiting several forms of inflammatory cell activation, including antioxidants of free radical molecules in the body system, such as superoxide anion generation (Ordentlichet. al., 2003), cytokine development (Chang et. al., 2005), and molecular adhesion expression (Chang et. al., 2012).As a consequence, by retarding the trans-nuclear transcription of NF-kâ and associated cytokines, thiopurine effectively reduced the synthesis of nitric oxide synthase (Chang et. al., 2011).

CONCLUSION
This research demonstrated the possible use of 6-MP for the in-vitro model of HIG-82 cell culture in treating rheumatoid arthritis disease. Drug candidates are promising usefulness to be identified as a new drug compound used in the treatment of inflammatory arthritis in the future, and further studies need to be conducted with a view to reducing the adverse drug reactions in patients using a nano-encapsulated drug delivery system.