**3.3.1 Carnosine in monotherapy of AA**

Carnosine (CARN) is a dipeptide consisting of β-alanine and L-histidine. It was shown to be a specific constituent of excitable tissues of all vertebrates accumulating in amounts exceeding that of ATP (Boldyrev & Severin, 1990). The antioxidant capacity of this compound is well documented, as well as its pH buffering, osmoregulating, and metalchelating abilities (Boldyrev, 1990). A potentially useful characteristic of CARN is its ability to act as an anti-glycating agent (Boldyrev, 2002; Boldyrev, 2005; Hipkiss et al., 1998; Hipkiss & Brownson, 2000), to quench superoxide anion and hydroxide radical (Pavlov, et al. 1993; Rubtsov et al., 1991) and to neutralize 4-hydroxy-nonenal (HNE) and other toxic aldehydes (Aldini et al., 2002, 2011, Liu et al., 2003). In order to study the efficiency of carnosine as geroprotector, senescence accelerated mice (SAM), which have increased levels of ROS and deficiency of antioxidant capacity, was used (Boldyrev et al., 2001; Yuneva et al., 2002). CARN decreased the content of protein carbonyls and lipid peroxides in their blood, demonstrating normalization of oxidative metabolism in SAM tissues as a cause of increased life span. Oxygen metabolism has an important role in the pathogenesis of RA. ROS produced in excessive amounts under some pathological states, exceed the physiological ROS buffering capacity and result in OS. Excessive production of ROS can damage proteins, lipids, nucleic acids, and matrix components (Bauerova & Bezek, 1999). With respect to this fact we evaluated CARN in AA. The aim of this study was to assess whether administration of CARN in AA would ameliorate inflammation and disease progression. CARN beneficially affected the clinical parameter HPV in the model of AA measured in time profile (days 14, 21 and 28), significantly on day 14 (Table 9).


Table 9. Hind paw volume (HPV) changes in an experiment with carnosine (CARN) evaluated as monotherapy in time profile. For statistical analysis of data see table 1.

Activity of GGT in joint was significantly reduced by CARN administration (Table 10). Markers of redox imbalance in plasma (TBARS, and protein carbonyls) were significantly decreased (Table 10). Protein carbonyls in brain tissue homogenates were significantly elevated and were decreased by CARN to control values (Table 10). The reduction of immunological markers of inflammation (IL-1α and MCP-1) in plasma by CARN is a result supporting its anti-inflammatory activity (Table 11).

Both antioxidants tended to improve HPV (not significantly) and significantly corrected the parameters of OS. No dose dependency was shown with exception of CoQ influence on GGT in spleen. In the next experiments we evaluated CARN in monotherapy of AA and CoQ in the lower dose for combinatory therapy with MTX in AA. The obtained results are

Carnosine (CARN) is a dipeptide consisting of β-alanine and L-histidine. It was shown to be a specific constituent of excitable tissues of all vertebrates accumulating in amounts exceeding that of ATP (Boldyrev & Severin, 1990). The antioxidant capacity of this compound is well documented, as well as its pH buffering, osmoregulating, and metalchelating abilities (Boldyrev, 1990). A potentially useful characteristic of CARN is its ability to act as an anti-glycating agent (Boldyrev, 2002; Boldyrev, 2005; Hipkiss et al., 1998; Hipkiss & Brownson, 2000), to quench superoxide anion and hydroxide radical (Pavlov, et al. 1993; Rubtsov et al., 1991) and to neutralize 4-hydroxy-nonenal (HNE) and other toxic aldehydes (Aldini et al., 2002, 2011, Liu et al., 2003). In order to study the efficiency of carnosine as geroprotector, senescence accelerated mice (SAM), which have increased levels of ROS and deficiency of antioxidant capacity, was used (Boldyrev et al., 2001; Yuneva et al., 2002). CARN decreased the content of protein carbonyls and lipid peroxides in their blood, demonstrating normalization of oxidative metabolism in SAM tissues as a cause of increased life span. Oxygen metabolism has an important role in the pathogenesis of RA. ROS produced in excessive amounts under some pathological states, exceed the physiological ROS buffering capacity and result in OS. Excessive production of ROS can damage proteins, lipids, nucleic acids, and matrix components (Bauerova & Bezek, 1999). With respect to this fact we evaluated CARN in AA. The aim of this study was to assess whether administration of CARN in AA would ameliorate inflammation and disease progression. CARN beneficially affected the clinical parameter HPV in the model of AA

measured in time profile (days 14, 21 and 28), significantly on day 14 (Table 9).

\*\*\*

+

Table 9. Hind paw volume (HPV) changes in an experiment with carnosine (CARN) evaluated as monotherapy in time profile. For statistical analysis of data see table 1.

Activity of GGT in joint was significantly reduced by CARN administration (Table 10). Markers of redox imbalance in plasma (TBARS, and protein carbonyls) were significantly decreased (Table 10). Protein carbonyls in brain tissue homogenates were significantly elevated and were decreased by CARN to control values (Table 10). The reduction of immunological markers of inflammation (IL-1α and MCP-1) in plasma by CARN is a result

**AA** 40.84±4.53

**AA-CARN** 23.58±4.88

supporting its anti-inflammatory activity (Table 11).

**(%) Day 14 Day 21 Day 28 CO** 8.81±1.91 11.67±2.54 13.09±2.61

> 91.32±6.00 \*\*\*

72.76±12.91 74.19±9.60

88.42±5.44 \*\*\*

described below.

**3.3.1 Carnosine in monotherapy of AA** 

**HPV** 

In the present experiment, the GGT activity was elevated in peripheral joint tissue. This finding is in good agreement with clinical studies of patients with RA who had increased levels of GGT not only in serum and urine but also in synovial fluid (Rambabu et al., 1990). CARN effectively reduced the activity of GGT in joint. Administration of CARN lowered the level of secondary products of lipid peroxidation in plasma measured as TBARS. Cheng (Cheng et al., 2011) showed that CARN, but not other conventional antioxidants, could protect neurons against MDA-induced injury through decomposition of protein crosslinking and may serve as a novel agent in the treatment of neurodegenerative diseases. The "anti-carbonyl" effect of CARN administration was also evidenced by other authors (Aldini et al., 2010).


† - nmol 4-nitroaniline /min /g tissue

Table 10. Activity of GGT(-glutamyltransferase) in joint, plasmatic TBARS (thiobarbituric acid reactive substances), and protein carbonyls in plasma and brain measured on experimental day 28 in an experiment with carnosine (CARN) evaluated as monotherapy. For statistical analysis of data see table 1.

We found that CARN was effective in decreasing protein carbonyls in plasma as well as in brain tissue homogenate of arthritic rats. These findings might provide, at least partially, an explanation for the antiinflammatory activity of CARN in chronic autoimmune disease, such as RA. The action of CARN resulted in decreased systemic inflammation in AA, monitored by plasmatic level of proinflammatory cytokine IL-1α and chemokine MCP-1. CARN was also effective in reducing the MCP-1 level in plasma in our experiment, suggesting that it may have a good potential in the treatment of chronic inflammatory diseases including RA where IL-1 and MCP-1 are involved.

Modern Pharmacological Approaches to Therapies:

effective than the individual substances (Table 13).

**AA** 42.411±7.411

**AA-MTX** 13.307±2.673

**AA-CoQ+MTX** 4.965±1.026

time profile. For statistical analysis of data see table 1.

\*\*\*

++

+++ / #

**HPV** 

not directly coupled.

Substances Tested in Animal Models of Rheumatoid Arthritis 253

study, the decreasing effect of MTX monotherapy on hind paw swelling was evident on all monitored days (Table 12). The significance of this effect was a confirmation of its well known antiarthritic effect, which we proved also previously on the AA model (Jurcovicova et al., 2009; Nosal et al., 2007; Rovensky et al., 2009). As shown in Table 12, the combination therapy was the most effective in decreasing the HPV of arthritic animals on all experimental days selected. Moreover, for day 14, we found a statistically significant difference between MTX monotherapy and its combination with CoQ10. These promising clinical results were further completed by measurements of HNE- and MDAprotein adducts and protein carbonyls in plasma (Table 13). Changes in all groups with arthritis were calculated with respect to the control value assessed for healthy control animals on experimental day 28. The dashed line represents the value of control as 100%. We obtained a good agreement of HPV with the parameters of OS: the effect was increasing in the order CoQ10 alone, MTX alone, combination of CoQ10 and MTX. The most pronounced effect found for the combination of MTX and CoQ10 was significant for all OS parameters compared with non-treated arthritic animals. Moreover, the combination decreased all parameters close to the control group values, being more

> **(%) Day 14 Day 21 Day 28 CO** 6.228±0.942 13.64±1.891 14.874±1.744

**AA-CoQ** 28.094±4.515 77.635±3.599 59.949±6.039

Table 12. Hind paw volume (HPV) changes in an experiment with coenzyme Q10 (CoQ) and methotrexate (MTX) evaluated as monotherapy and combination therapy (CoQ+MTX) in

As shown in Table 14, the arthritis process increases significantly the level of CoQ9 in comparison with healthy controls. The effect of therapy on this phenomenon unveils a picture comparable to that found for other OS parameters (Table 13). The combination therapy was again the most effective and significant in comparison to the untreated arthritis group and the improvement was on the level of CO (Table 14). Table 14 shows also that the effects of the given treatments on the AA-increased IL-1α levels are very close to the effects illustrated in table 13. The improving effect on the increased cytokine plasmatic levels is raising in the order CoQ10, MTX and CoQ10+MTX. Furthermore, a statistically significant difference was found between MTX monotherapy and its combination with CoQ10. For the local inflammatory parameter — the activity of GGT in joint homogenate — an approximately double increase was recorded on comparing arthritic animals with CO (Table 14). The treated groups presented similar results as already described for IL-1α. All these findings suggest that the cycles of GGT and CoQ are

81.083±7.901 \*\*\*

23.793±4.946 +++

12.552±2.328 +++

68.629±8.952 \*\*\*

26.996±8.201 ++

19.571±2.426 ++


Table 11. IL-1α (interleukin-1α) and MCP-1 (monocyte chemotactic protein-1) measured on experimental day 28 in an experiment with carnosine (CARN) evaluated as monotherapy. For statistical analysis of data see table 1.

#### **3.3.2 Coenzyme Q10 in combination with MTX in AA**

Based on our results with mitochondrial energetics modification and the observed antiinflammatory and antioxidant effects (Bauerova et al., 2005a, 2008a; Gvozdjakova et al., 2004; Ponist et al., 2007), we chose CoQ10 as a candidate for combinatory therapy of RA. Patients with RA often suffer muscle weakness and atrophy. It is assumed that progressive muscle atrophy in RA patients is caused by damaged myofibrils and impaired mitochondria (De Palma et al., 2000). Disruption of mitochondrial bioenergetics caused by free radicals is involved in the development of myopathies. OS-caused alteration of mitochondrial functions can manifest in different manners (Cardoso et al., 1999). Leakage of free radicals from the respiratory chain leads to damaged mitochondrial membrane, proteins, DNA and inhibits oxidative phosphorylation (Luft, 1995; Miesel et al., 1996). Maneiro et al. (2003) found inhibition of functions of complex II and III of the respiratory chain and higher frequency of energetically "exhausted" mitochondria in chondrocytes of patients with osteoarthritis compared to healthy donors. In light of these findings, we decided to support the impaired mitochondrial functions by CoQ10 supplementation and thus to reduce the increased OS in AA. Some evidence from the literature showed that antirheumatic therapies which increased the level of CoQ10 were able to slow down RA progression (Comstock et al*.,* 1997; Knekt et al., 2000; Kucharska et al., 2005). The hind-paw muscle of arthritic animals lies very close to the inflamed joint and could be also sensitive to joint inflammation (Ponist et al., 2007). Moreover, AA is a systemic inflammatory disease and we might also expect impairment in myocardial mitochondrial functions. We found that the reactions of skeletal muscle and myocardium muscle on CoQ supplementation in AA were different, which was not so surprising in view of their different structure and functions in the organism (Gvozdjakova et al., 2007). The results with solubilized CoQ10 (water-soluble form) indicated its therapeutic effect in the experimental model of AA (Bauerova et al., 2005a, 2008a; Gvozdjakova et al., 2004; Ponist et al., 2007). These findings are of potential significance in the treatment of patients with RA.

The aim of the present study was to examine the combined effect of CoQ10 and MTX on the progression of AA. For this purpose, we used monitoring of HPV along with evaluation of OS and inflammation markers assessed in plasma and tissues. The experiments included healthy animals (CO), arthritic animals not treated (AA), arthritic animals treated with coenzyme Q10 (AA-CoQ), arthritic animals treated with methotrexate (AA-MTX), and arthritic animals treated with the combination of CoQ10 and methotrexate (AA-MTX+CoQ). The two latter groups received a daily oral dose of 20 mg/kg b.w. of CoQ10 either alone or with methotrexate in the oral dose of 0.3 mg/kg b.w. twice a week. AA-MTX was performed as a reference treatment. CoQ10 supplementation to arthritis animals slightly decreased the HPV on all experimental days (Table 12). In the present

**Parameters CO AA AA-CARN** 

Table 11. IL-1α (interleukin-1α) and MCP-1 (monocyte chemotactic protein-1) measured on experimental day 28 in an experiment with carnosine (CARN) evaluated as monotherapy.

Based on our results with mitochondrial energetics modification and the observed antiinflammatory and antioxidant effects (Bauerova et al., 2005a, 2008a; Gvozdjakova et al., 2004; Ponist et al., 2007), we chose CoQ10 as a candidate for combinatory therapy of RA. Patients with RA often suffer muscle weakness and atrophy. It is assumed that progressive muscle atrophy in RA patients is caused by damaged myofibrils and impaired mitochondria (De Palma et al., 2000). Disruption of mitochondrial bioenergetics caused by free radicals is involved in the development of myopathies. OS-caused alteration of mitochondrial functions can manifest in different manners (Cardoso et al., 1999). Leakage of free radicals from the respiratory chain leads to damaged mitochondrial membrane, proteins, DNA and inhibits oxidative phosphorylation (Luft, 1995; Miesel et al., 1996). Maneiro et al. (2003) found inhibition of functions of complex II and III of the respiratory chain and higher frequency of energetically "exhausted" mitochondria in chondrocytes of patients with osteoarthritis compared to healthy donors. In light of these findings, we decided to support the impaired mitochondrial functions by CoQ10 supplementation and thus to reduce the increased OS in AA. Some evidence from the literature showed that antirheumatic therapies which increased the level of CoQ10 were able to slow down RA progression (Comstock et al*.,* 1997; Knekt et al., 2000; Kucharska et al., 2005). The hind-paw muscle of arthritic animals lies very close to the inflamed joint and could be also sensitive to joint inflammation (Ponist et al., 2007). Moreover, AA is a systemic inflammatory disease and we might also expect impairment in myocardial mitochondrial functions. We found that the reactions of skeletal muscle and myocardium muscle on CoQ supplementation in AA were different, which was not so surprising in view of their different structure and functions in the organism (Gvozdjakova et al., 2007). The results with solubilized CoQ10 (water-soluble form) indicated its therapeutic effect in the experimental model of AA (Bauerova et al., 2005a, 2008a; Gvozdjakova et al., 2004; Ponist et al., 2007). These findings are of potential significance in

The aim of the present study was to examine the combined effect of CoQ10 and MTX on the progression of AA. For this purpose, we used monitoring of HPV along with evaluation of OS and inflammation markers assessed in plasma and tissues. The experiments included healthy animals (CO), arthritic animals not treated (AA), arthritic animals treated with coenzyme Q10 (AA-CoQ), arthritic animals treated with methotrexate (AA-MTX), and arthritic animals treated with the combination of CoQ10 and methotrexate (AA-MTX+CoQ). The two latter groups received a daily oral dose of 20 mg/kg b.w. of CoQ10 either alone or with methotrexate in the oral dose of 0.3 mg/kg b.w. twice a week. AA-MTX was performed as a reference treatment. CoQ10 supplementation to arthritis animals slightly decreased the HPV on all experimental days (Table 12). In the present

\*

\*

49.87±4.13 +

5422.00±658.50 +

**IL-1α (pg/ml)** 47.55±3.63 70.5±5.94

**(pg/ml)** 5345.70±734.30 7946.50±878.70

**MCP-1** 

the treatment of patients with RA.

For statistical analysis of data see table 1.

**3.3.2 Coenzyme Q10 in combination with MTX in AA** 

study, the decreasing effect of MTX monotherapy on hind paw swelling was evident on all monitored days (Table 12). The significance of this effect was a confirmation of its well known antiarthritic effect, which we proved also previously on the AA model (Jurcovicova et al., 2009; Nosal et al., 2007; Rovensky et al., 2009). As shown in Table 12, the combination therapy was the most effective in decreasing the HPV of arthritic animals on all experimental days selected. Moreover, for day 14, we found a statistically significant difference between MTX monotherapy and its combination with CoQ10. These promising clinical results were further completed by measurements of HNE- and MDAprotein adducts and protein carbonyls in plasma (Table 13). Changes in all groups with arthritis were calculated with respect to the control value assessed for healthy control animals on experimental day 28. The dashed line represents the value of control as 100%. We obtained a good agreement of HPV with the parameters of OS: the effect was increasing in the order CoQ10 alone, MTX alone, combination of CoQ10 and MTX. The most pronounced effect found for the combination of MTX and CoQ10 was significant for all OS parameters compared with non-treated arthritic animals. Moreover, the combination decreased all parameters close to the control group values, being more effective than the individual substances (Table 13).


Table 12. Hind paw volume (HPV) changes in an experiment with coenzyme Q10 (CoQ) and methotrexate (MTX) evaluated as monotherapy and combination therapy (CoQ+MTX) in time profile. For statistical analysis of data see table 1.

As shown in Table 14, the arthritis process increases significantly the level of CoQ9 in comparison with healthy controls. The effect of therapy on this phenomenon unveils a picture comparable to that found for other OS parameters (Table 13). The combination therapy was again the most effective and significant in comparison to the untreated arthritis group and the improvement was on the level of CO (Table 14). Table 14 shows also that the effects of the given treatments on the AA-increased IL-1α levels are very close to the effects illustrated in table 13. The improving effect on the increased cytokine plasmatic levels is raising in the order CoQ10, MTX and CoQ10+MTX. Furthermore, a statistically significant difference was found between MTX monotherapy and its combination with CoQ10. For the local inflammatory parameter — the activity of GGT in joint homogenate — an approximately double increase was recorded on comparing arthritic animals with CO (Table 14). The treated groups presented similar results as already described for IL-1α. All these findings suggest that the cycles of GGT and CoQ are not directly coupled.

Modern Pharmacological Approaches to Therapies:

**(%)** 41.90±3.56 51.28±3.51

**burst (%)** 27.36±3.44 43.70±3.97

**activity (%)** 40.38±2.04 30.56±5.10

**Phagocytosis** 

**Oxidative** 

**Metabolic** 

**4. Conclusion** 

*Zingiber officinale*).

conditions.

**5. Acknowledgment** 

Substances Tested in Animal Models of Rheumatoid Arthritis 255

**Parameters CO AA AA-CoQ AA-MTX AA-CoQ** 

55.48±4.07 30.07±1.44

42.95±6.29 19.97±0.47

39.13±3.07 21.20±0.61

++

++

++

\*

\*

\*

Table 15. Functional parameters of neutrophils measured on experimental day 7 in an experiment with coenzyme Q10 (CoQ) and methotrexate (MTX) evaluated as monotherapy and combination therapy (CoQ+MTX) of AA. For statistical analysis of data see table 1.

In summary, combined administration of CoQ10 and MTX suppressed arthritic progression in rats more effectively than did MTX alone. This finding may become a beneficial contribution to the treatment of RA. Restoration of redox homeostasis in chronic inflammatory diseases may be of significant importance in new therapeutic strategies.

In the past our research team, using the AA model, has monitored OS and inflammation in time course using different clinical and biochemical/immunological markers, and at the same time we have assessed the efficacy of the administered experimental substances with regard to their ability to reduce OS and inflammatory processes. In our experiments on AA rats we observed a beneficial effect of administration of low molecular weight antioxidants (coenzyme Q and carnosine), high molecular weight immunomodulators/antioxidants (glucomannan, Imunoglukan®) and compounds related to plants (curcumin, arbutin, pinosylvin, sesame oil, and extracts from *Boswellia serrata*, *Arctostaphylos uva-ursi* and

In light of these results, we proceeded in the search for the most suitable therapeutic substance (an antioxidant/immunomodulator) with the ability to improve the therapy of RA with MTX. The aim was to find a potential enhancement of the antirheumatic effect of MTX in particular combinations compared to monotherapy. Carnosine, coenzyme Q, pinosylvin and Imunoglukan® were selected for assessment of a combinatory therapy with MTX. The already performed experiments on arthritic rats with pinosylvin, Imunoglukan® and coenzyme Q confirmed the hypothesis about the beneficial effect of adding a suitable immunomodulator/antioxidant to the therapy with MTX. Final safety and efficacy of these approaches calls for further more detailed research not only in preclinical but also in clinical

The authors wish to thank Dr. Magda Kouřilova, PhD. for correction of the English language. Experimental work was supported by grants VEGA 2/0045/11; VEGA 2/0090/08; VEGA 2/0003/10; APVV-51-017905; APVV-3015-07, APVV-21-055205 and

**+MTX** 

46.60±7.38 ##

31.07±8.98

34.79±7.81 ##


Table 13. Protein carbonyls, HNE (4-hydroxynonenal) and MDA (malondialdehyde)-protein adducts levels in plasma measured on experimental day 28 in an experiment with coenzyme Q10 (CoQ) and methotrexate (MTX) evaluated as monotherapy and combination therapy (CoQ+MTX). Changes in all groups with arthritis were calculated compared to the control value assessed for healthy control animals on experimental day 28. For statistical analysis of data see table 1.


† - nmol 4-nitroaniline /min /g tissue

Table 14. GGT (-glutamytransferase) activity in joint, IL-1α (interleukin-1) and CoQ9 (coenzyme Q9) levels in plasma measured on experimental day 28 in an experiment with coenzyme Q10 (CoQ) and methotrexate (MTX) evaluated as monotherapy and combination therapy (CoQ+MTX). For statistical analysis of data see table 1.

The functionality of peripheral blood neutrophils in AA was evaluated by phagocytosis, oxidative burst and metabolic activity (Table 15).

Both phagocytosis and oxidative burst were increased due to arthritis. The immunosuppressive effect of MTX was demonstrated in lowering all characteristics of the functionality of peripheral blood neutrophils, not only in comparison with arthritis but also with CO. The addition of CoQ10 to MTX modulated all processes back to the level of CO. The observed immunoenhancing activity of CoQ10 may prove beneficial in MTX routine treatment. In this experiment, flow cytometric determination of the functionality of neutrophils was first applied for an experimental model on rats.


Table 15. Functional parameters of neutrophils measured on experimental day 7 in an experiment with coenzyme Q10 (CoQ) and methotrexate (MTX) evaluated as monotherapy and combination therapy (CoQ+MTX) of AA. For statistical analysis of data see table 1.

In summary, combined administration of CoQ10 and MTX suppressed arthritic progression in rats more effectively than did MTX alone. This finding may become a beneficial contribution to the treatment of RA. Restoration of redox homeostasis in chronic inflammatory diseases may be of significant importance in new therapeutic strategies.
