**1. Introduction**

232 Rheumatoid Arthritis – Treatment

Yue, D., W. Brintnell, et al. (2010). "CTLA-4Ig blocks the development and progression of

Zeuner, R. A., K. J. Ishii, et al. (2002). "Reduction of CpG-induced arthritis by suppressive

oligodeoxynucleotides." *Arthritis Rheum* 46(8): 2219-2224.

62(10): 2941-2952.

citrullinated fibrinogen-induced arthritis in DR4-transgenic mice." *Arthritis Rheum*

Preclinical research on animal models of rheumatoid arthritis (RA) is very important for alerting the healthcare and scientific community and pharmaceutical companies of the existence of new or "forgotten" molecules. Most antirheumatics have side-effects when used in higher doses and/or within long-term dosage. Combinatory therapy is expected to have a higher efficacy without increased toxicity. Methotrexate (MTX) has become the main immunosuppressive substance used in the treatment of patients with RA. However, the use of MTX has to be limited due to its toxic manifestations, e.g. abdominal disorder, alopecia, oral ulcers, and cytopenia (Alarcon et al., 1989). Ineffectiveness of treatment can be also observed. In the survey of McKendry and Dale (McKendry & Dale, 1993), due to the risk of treatment, termination was substantiated in 75% of patients with RA taking MTX for 60 months. An adverse drug effect proved to be a more common reason for treatment termination (53%) compared to loss/lack of beneficial effect (22%), other reasons (16%), or lost of follow-up (9%). On the other hand, the therapeutic efficacy of MTX can be increased by combination with other synthetic drugs or inhibitors of TNF-α (Smolen et al., 2010). Application of biological therapy (antibodies or soluble receptors of TNF-α, IL-1 and IL-6) represents a great progress in the therapy of RA. Yet biological treatment is also frequently combined with MTX (Maini et al., 1998; Weinblatt et al., 1999). There are countless possibilities for combinations with MTX. Many substances were neglected when they failed to show good efficacy in monotherapy compared to standard antirheumatics. They would not get a second chance if the expected reduction of clinical parameters (mainly edema of joints) did not materialize, despite the fact that they improved many biochemical disease markers. Our research in the last years was focused on evaluation of new therapeutics for the combinations of the classical immunosuppressive treatment with immunomodulators and compounds affecting reduction/oxidation homeostasis.

Development of drugs for the therapy of RA has been very intensive in recent years. Biological therapy targeted on neutralizing the effect of antiinflammatory cytokines, particularly TNF-α, IL-1 and IL-6, by using antibodies or soluble receptors provided a great progress in RA therapy. However, not even these expensive drugs are able to cure RA

Modern Pharmacological Approaches to Therapies:

to the disease process (Joe et al., 1999).

**2.1 Markers applied in adjuvant arthritis** 

significant (p < 0.05); not significant (p > 0.05).

**2.1.1 Clinical markers** 

**2.1.2 Biochemical markers** 

al., 2007).

Substances Tested in Animal Models of Rheumatoid Arthritis 235

cells play major roles in the disease. Humoral immune mechanisms appear not to contribute

AA has been extensively used for pharmaceutical testing, and therefore much data exists for comparison in humans. While this model does not mimic perfectly the condition of human arthritis, it is easily reproducible, well defined and has proven useful for the development of new therapies for arthritis, as exemplified also by cytokine blockade therapies (Bendele et al., 1999). AA has been used in the evaluation of nonsteroidal inflammatory drugs, such as phenylbutazone and aspirin during the early 1960s, and later COX-2 inhibitors such as celecoxib were studied. AA in rats shares many features with human arthritis, including genetic linkage, synovial CD4+ cells and T cell dependence. On the contrary, one of the major differences between the AA model and human arthritis is simply that the inciting agent is known in the model, though the need for any specific antigen is controversial.

The parameters characterizing efficacy of the substances tested on the immunological, oxidative and inflammatory processes will be reported in this chapter. The spectrum of these parameters is suitable for the description of AA development and provides optimal possibilities for studying pharmacological influence of the substances tested as well as for the elucidation of the mechanisms of their action. In our laboratory, plasma and blood samples were collected regularly from animals in light anesthesia. Tissue samples were collected at the end of the experiment after sacrificing the anesthesised animals. The end of the experiment was mostly day 28 after injection of *Mycobacterium butyricum*. All characteristics monitored could be divided into three groups: clinical, biochemical and immunological parameters. For data interpretation and statistical analysis the data were expressed in terms of arithmetic mean ± S.E.M. In all cases the untreated arthritis group was compared with healthy control animals (\*-symbol), the treated arthritis groups were compared with the untreated arthritis animals (+-symbol). For significance calculations the unpaired Student's t-test (two samples unequal variance) was used with the following significance designations extremely significant (p < 0.001), highly significant (p < 0.01),

We monitored one basic clinical parameter: the hind paw volume (HPV). The HPV increase was calculated as the percentage increase in the HPV on a given experimental day relative to the HPV at the beginning of the experiment. The hind paw volume was recorded on days 1, 14, 21, and 28 with the use of an electronic water plethysmometer (UGO BASILE, Comerio-Varese, Italy). In one of our previous experiments, we confirmed that this clinical parameter became significantly modified starting around day 14 and its significant increase in comparison with healthy controls is maintained until the end of the experiment (Bauerova et

Numerous studies have suggested an important role of oxidative stress (OS) in the pathogenesis of RA (Bauerova & Bezek, 1999; Bohanec et al., 2009). Several clinical studies as well as preclinical studies using animal models of RA have documented an imbalance in the body's redox homeostasis to a more pro-oxidative environment, suggesting that therapies

definitely, although they remarkably inhibit the development of arthritis and improve the life quality of patients. Following treatment interruption, a fast development of RA occurs. Biological therapy also has adverse effects, such as development of resistance and secondary infections. For these reasons, the search for new drugs which could avoid these infections or suppress them is still an up-to-date problem. As already mentioned, the most frequently applied conventional drug for RA has been MTX. Its application is usually additional to the biological therapy or it is used in combination with other conventional drugs. Intensive immunosuppressive treatment with MTX or biological therapy adversely affects immunological homeostasis of the organism and increases the risk of infections. For these reasons, there is a search for alternative immunomodulatory approaches, which could minimize side effects of immunosuppressive therapy on cellular and humoral immunity. One possibility is represented by the combination of immunosuppressive and immunostimulatory substances or compounds regulating redox balance of the organism. Their application can establish immunological and redox homeostasis and increase resistance of the organism to infections.

The focus of this chapter is mostly on substances of natural origin possessing antiinflammatory, antioxidative or immunomodulating properties along with minimal side effects when administered to animals. The safety of long-term therapy of RA is very important, because patients with RA are usually treated for two or more decades. We describe our results obtained in adjuvant arthritis (AA) with endogenous antioxidants as carnosine and coenzyme Q10, glucomannan and Imunoglukan®, as well as selected extracts and compounds from plants. These results will be confronted with results of other authors from preclinical and clinical studies. The aim is to present an overview of the potential of new compounds for the therapy of RA with the focus on approving their ability for combination therapy with methotrexate.
