**3.1.1 Pinosylvin and methotrexate combination in AA**

PIN [3´,5´-dihydroxystilbene] and PTE [3,5-dimethoxy-4´-hydroxystilbene] used in our experiments were synthesized and purified by Šmidrkal et al. (2010) and Harmatha et al. (2011). PIN and PTE are natural substances from the stilbenoid group, wide-spread in a variety of plants. They are chemically related to resveratrol. Both substances studied inhibited significantly the chemiluminescence (CL) of whole human blood and the CL of isolated human neutrophils (Perečko et al. 2008). The new information on the inhibitory effect of PIN and PTE on HPV, production of ROS and MPO activity suggests that the protective effect of PIN may be beneficial in controlling inflammation in experimental AA (Macickova et al*.*, 2010). PIN was also the most effective in reducing HPV on day 28, when administered in the dose of 30 mg/kg b.w. per os (Fig. 1). According to these findings we chose PIN as a suitable candidate for combination therapy with methotrexate (MTX). In the combination AA-PIN+MTX, arthritic animals were treated twice a week with MTX in the oral dose of 0.4 mg/kg b.w. and daily with PIN in the oral dose of 50 mg/kg b.w. Monotherapy was performed with the same doses. In addition to the routine statistical analysis the combination treatment was compared to individual MTX treatment (#-symbol). In arthritic rats, PIN potentiated the antiarhritic effect of MTX on days 14 and 21, evaluated as decrease of HPV (Table 1). Activity of GGT in spleen homogenate (Table 2), plasma levels of MCP-1 and CRP (Table 3) were not improved by addition of PIN to MTX, due to the prominent effect of MTX alone on these parameters. Arthritic animals showed an increase in OS, evaluated as plasma levels of TBARS. PIN enhanced the antioxidant effect of MTX (Table 2) (Bauerova et al., 2010b).


Table 1. Hind paw volume (HPV) changes in an experiment with methotrexate (MTX) and pinosylvine (PIN) in monotherapy and in combination therapy PIN+MTX measured in time profile. The data were expressed as arithmetic mean ± S.E.M. Statistical significance was evaluated applying Student's t-test for independent variables: \*P < 0.05, \*\*P < 0.01, and \*\*\*P < 0.001 compared to control healthy animals (CO); +P < 0.05, ++P < 0.01, and +++P < 0.001 compared to untreated arthritic animals (AA); #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to methotrexate monotherapy (AA-MTX).

Modern Pharmacological Approaches to Therapies:

**3.2 Natural substances isolated from yeast and mushrooms** 

the results of our studies.

and antioxidative properties.

Substances Tested in Animal Models of Rheumatoid Arthritis 245

apoptosis of T cells, increasing endogenous adenosine release, altering the expression of cellular adhesion molecules, influencing production of cytokines, humoral responses and bone formation (Wessels et al., 2008). The use of MTX has been limited by some of its toxic manifestations, such as abdominal discomfort, alopecia, oral ulcerations, and cytopenia (Alarcon et al*.,* 1989). In this case a lowering of the dose could be beneficial and this could be achieved by combination therapy, for which we could recommend PIN, as indicated by

The control of inflammation in RA patients by natural and synthetic substances with antiinflammatory and/or antioxidant and immunomodulatory effects, which are safe also during long-term administration, could become a relevant part of RA therapy. Modulation of OS accompanying RA can offer a new approach and crucially modify treatment of this disease. The key goal of this proposal will be the investigation of the combination of immunosuppressive therapy of MTX with immunomodulators-antioxidants with the aim to achieve enhancement of its efficacy in RA treatment, which would enable dosage reduction in clinical conditions and, consequently, decrease the frequency of occurrence of its dosedependent side effects. Thus new ways of supplementary or combinatory RA therapy are of great importance. The aim is to find an alternative or additive to classical RA therapy with natural molecules without side effects possessing immunomodulatory, antiinflammatory,

In recent decades, polysaccharides isolated from botanical sources (mushrooms, algae, lichens, and higher plants) have attracted a great deal of attention in the biomedical arena because of their broad spectrum of therapeutic properties and relatively low toxicity (Tzianabos, 2000). Plant and mushroom polysaccharides reveal immunomodulatory effects that depend on polysaccharide structure and molecular weight (low molecular weight – inhibition, high molecular weight – activation) (Schepetkin & Quinn, 2006). Prokopova (Prokopova et al., 1993) were the first to describe a therapeutic effect of simple carbohydrates on AA. We were first to report on the protective antioxidant and antiinflammatory activities of carboxylated (1-3)-beta-D-glucan isolated from *Saccharomyces cerevisiae* in Lewis rats with AA (Kogan et al., 2005). Glucomannans (GM) from *Candida utilis* were evaluated in the same model. The antiarthritic activity for cell-wall GM was associated with antioxidant activity *in vivo* (Bauerova et al., 2006; Mihalova et al., 2007). In the following experiment, the beneficial action of GM was revealed mainly in HPV decrease. Further a decrease of the activity of GGT in the spleen, hind paw joint and muscle tissue homogenates, decrease of the plasmatic activity of N-acetyl-beta-D-glucosaminidase (NAGA), and finally suppression of lysozyme and peroxidase activity assessed in peritoneal macrophages were observed in arthritic animals treated with GM. All these findings speak in favor of the antiinflammatory activity of GM. Moreover, a significant improvement of the arthritis induced suppression of total antioxidant status and decrease of the level of the arthritis-associated protein carbonyls in plasma were detected. In this experiment two doses of GM – 5 and 7.5 mg/kg b.w. were evaluated successfully. Peroral and intraperitoneal ways of administration were also compared (Bauerova et al., 2008b). In the following study, we tested the effect of GM in a higher dose of 15 mg/kg b.w. administered per os. On day 28 after *Mycobacterium butyricum* induced AA, GM was found to reduce HPV. Neutrophil count in whole blood was significantly increased on day 28 after induction of AA, yet GM in the dose of 15 mg/kg b.w. did not change it significantly. The spontaneous and PMA-induced CL was significantly increased in whole blood of rats with AA in comparison with healthy


† - nmol 4-nitroaniline /min /g tissue

Table 2. Plasmatic level of TBARS (thiobarbituric acid reactive substances) and activity of GGT (-glutamyltransferase) in spleen measured on experimental day 28 in an experiment with methotrexate (MTX) and pinosylvine (PIN) in monotherapy and in combination therapy PIN+MTX. For statistical analysis of data see table 1.


Table 3. Plasmatic level of MCP-1 (monocyte chemotactic protein-1) and CRP (C-reactive protein) measured on experimental day 14 in an experiment with methotrexate (MTX) and pinosylvine (PIN) in monotherapy and in combination therapy PIN+MTX. For statistical analysis of data see table 1.

Effect of PIN and MTX, applied separately or in combination, was further studied on spontaneous and stimulated chemiluminescence and neutrophil count in blood of arthritic rats. In rats treated with MTX, all the arthritis-induced changes were significantly reduced and this inhibition became more pronounced when MTX was applied along with PIN. MTX alone decreased neutrophil count, spontaneous and stimulated chemiluminescence by 28%, 41% and 43%, respectively, whereas in combination with PIN, it inhibited these parameters by 59%, 69% and 63%, respectively. Monotherapy with PIN failed to induce any detectable changes either in the number of neutrophils or in oxidant concentration (Jancinova et al., 2010).

RA is a common severe joint disease affecting all age groups. It is thus of great importance to develop new strategies for its treatment. As disease modifying anti-rheumatic drugs (DMARDs) often have side effects at high doses and/or during long-term administration, increased efficacy without increased toxicity is expected for combination therapy of RA. MTX, a folic acid antagonist, has become the predominant immunosuppressive agent used in the treatment of patients with RA (Williams et al., 1985). MTX acts mainly on actively proliferating cells during the S-phase of proliferation, suppresses macrophage function, modulates interleukin-1 (IL-1) and superoxide anion production, and inhibits neutrophil chemotaxis (Moreland et al., 1997). Furthermore, MTX treatment was shown to decrease synovial collagenase gene expression in patients with RA (Genestier et al., 2000). The effects of MTX in vivo may be mediated by reducing cell proliferation, increasing the rate of

**Parameters CO AA AA-PIN AA-MTX AA-PIN-**

17.89±0.47 14.33±0.95

\*\*\* 85.90±4.07 37.38±2.95

9.470±0.603 ++

+++

+++

8.446±0.616 ++

+++

662.7±19.6 385.4±27.4

\*\*\*

Table 2. Plasmatic level of TBARS (thiobarbituric acid reactive substances) and activity of GGT (-glutamyltransferase) in spleen measured on experimental day 28 in an experiment with methotrexate (MTX) and pinosylvine (PIN) in monotherapy and in combination

**Parameters CO AA AA-PIN AA-MTX AA-PIN-**

Table 3. Plasmatic level of MCP-1 (monocyte chemotactic protein-1) and CRP (C-reactive protein) measured on experimental day 14 in an experiment with methotrexate (MTX) and pinosylvine (PIN) in monotherapy and in combination therapy PIN+MTX. For statistical

Effect of PIN and MTX, applied separately or in combination, was further studied on spontaneous and stimulated chemiluminescence and neutrophil count in blood of arthritic rats. In rats treated with MTX, all the arthritis-induced changes were significantly reduced and this inhibition became more pronounced when MTX was applied along with PIN. MTX alone decreased neutrophil count, spontaneous and stimulated chemiluminescence by 28%, 41% and 43%, respectively, whereas in combination with PIN, it inhibited these parameters by 59%, 69% and 63%, respectively. Monotherapy with PIN failed to induce any detectable changes either in the number of neutrophils or in oxidant concentration (Jancinova et al.,

RA is a common severe joint disease affecting all age groups. It is thus of great importance to develop new strategies for its treatment. As disease modifying anti-rheumatic drugs (DMARDs) often have side effects at high doses and/or during long-term administration, increased efficacy without increased toxicity is expected for combination therapy of RA. MTX, a folic acid antagonist, has become the predominant immunosuppressive agent used in the treatment of patients with RA (Williams et al., 1985). MTX acts mainly on actively proliferating cells during the S-phase of proliferation, suppresses macrophage function, modulates interleukin-1 (IL-1) and superoxide anion production, and inhibits neutrophil chemotaxis (Moreland et al., 1997). Furthermore, MTX treatment was shown to decrease synovial collagenase gene expression in patients with RA (Genestier et al., 2000). The effects of MTX in vivo may be mediated by reducing cell proliferation, increasing the rate of

\*\*\*

\*\*\*

**TBARS** 

**Activity of GGT in spleen (†)** 

**MCP-1 (µg/ml)** 

**CRP** 

analysis of data see table 1.

2010).

† - nmol 4-nitroaniline /min /g tissue

**(nmol/ml)** 10.59±0.23 19.10±0.53

19.977±1.843 90.45±4.52

therapy PIN+MTX. For statistical analysis of data see table 1.

6.896±0.438 14.089±1.159

**(µg/ml)** 337.4±14.8 722.6±49.3

**MTX** 

11.78±0.47 +++/#

35.47±1.89 +++

**MTX** 

8.316±0.257 ++

366.2±8.8 +++

apoptosis of T cells, increasing endogenous adenosine release, altering the expression of cellular adhesion molecules, influencing production of cytokines, humoral responses and bone formation (Wessels et al., 2008). The use of MTX has been limited by some of its toxic manifestations, such as abdominal discomfort, alopecia, oral ulcerations, and cytopenia (Alarcon et al*.,* 1989). In this case a lowering of the dose could be beneficial and this could be achieved by combination therapy, for which we could recommend PIN, as indicated by the results of our studies.
