Anti-inflammatory potential of orally administrated bioflavonoid-robinin, active sub-stance of original drug Flaroninum™ (FL), was investigated in the combination with methotrexate (MTX) and in monotherapy in rats suffering from adjuvant-induced arthritis (AA). Robinin (kaempferol-3-O-robinoside-7-O-rhamnoside) was isolated from the aerial parts of Astragalus falcatus Lam. The monotherapy with robinin was not efficient in alleviating symptoms of AA. The combination of MTX with robinin was similarly active as MTX alone in reducing the hind paw volume and change of body weight during the whole experiment. The combination, however, reduced plasma levels of Interleukin-17Aand activity of gamma-glutamyl transferase in joint more efficiently then MTX alone. Our results demonstrate that the novel combination of robinin and MTX mildly improved the reduction of inflammation in experimental arthritis.

Micro- and nanoemulsions are potential drug solubilizers and penetration enhancers through the high surfactant/co-surfactant content. This study aimed to evaluate the influence of minoxidil (MXD) solubilized in the microemulsions (MEs) on drug release by in vitro/ex vivo diffusion through the semi-permeable membrane Spectra/Por^® (Spectrum Laboratory, Gardena, CA, USA) and porcine ear skin. Moreover, a residual amount of drug in the skin after ex vivo diffusion was evaluated. The reference ME_R, lecithin-containing ME_L, and gelatin-containing ME_G were characterized in terms of their size, polydispersity index, density, viscosity, electrical conductivity and surface tension. Based on the in vitro diffusion, it can be argued that ME_L slowed down the drug release, while ME_R and ME_G have no significant effect compared to the sample, in which propylene glycol (PG) was used as a solubilizer. Determination of the residual drug amount in the skin after 6 h of the ex vivo permeation was demonstrated as the most valuable method to evaluate the effectiveness of the ME’s application. The results indicate that the most optimal MXD permeation enhancers in alginate gel were the natural surfactants containing MEs. MXD solubilization in ME_G and ME_L had caused more than 5% of the drug remaining in the skin, which is almost a 1.5-fold higher amount compared to the reference gel.

Many of disease‐modifying anti‐rheumatic drugs often have side effects at high doses and/or during long‐term administration. Increased efficacy without increased toxicity is expected for combination therapy of rheumatoid arthritis (RA). The aim of the study was to examine the effect of N‐feruloylserotonin (N‐f‐5HT) and methotrexate (MTX) in monotherapy and in combination therapy on disease progression and inflammation in arthritic rats. Adjuvant arthritis was induced by intradermal injection ofMycobacterium butyricumin incomplete Freund′s adjuvant in Lewis rats. The experiment included healthy animals, arthritic animals without any drug administration, arthritic animals with administration of N‐f‐5HTin the oral daily dose of 15 mg/kg b.w., arthritic animals with administration ofMTXin the oral dose of 0.3 mg/kg b.w. twice a week and arthritic animals treated with the combination of N‐f‐5HTandMTX. N‐f‐5HTin monotherapy reduced only activation ofNF‐κB and did not have any significant effect on other parameters monitored. Low‐dose treatment ofMTXdecreased the level ofIL‐1β andMCP‐1 on day 14 and activation ofNF‐κB in liver without significant effect on other parameters. N‐f‐5HTandMTXcombination showed both the anti‐arthritic (hind paw volume and arthritic score) and anti‐inflammatory effect (plasmatic levels ofIL‐1β,IL‐17,MCP‐1,CRP, and activation ofNF‐κB in liver). In combination withMTX, N‐f‐5HTmarkedly potentiated the therapeutic effect ofMTXlow dose, which resulted in significant improvement of all parameters measured. The findings showed that the combination therapy simultaneously decreased multiple markers of inflammation, a result crucial for future therapy ofRA.

Animal models of rheumatoid arthritis (RA) are widely used for testing potential new therapies for RA. The most commonly used models of human RA are adjuvant-induced arthritis (AIA) and collagen-induced arthritis in rats and mice. In this chapter, we will focus on inflammatory and oxidative stress (OS) processes during the development of AIA. OS is a result of increased production of reactive oxygen species (ROS) or a reduction in the body’s endogenous antioxidant defense system. ROS and reactive nitrogen species (RNS) can contribute to the pathogenesis of RA by the induction of membrane oxidation, irreversible damage to proteins and DNA, cartilage damage, and induction of bone resorption. ROS/RNS can also modulate a variety of signaling events that control gene expression and affect cellular processes that participate in chronic inflammation. Our research team has been studying the course of OS during the development of rat AIA for more than a decade. We have analyzed the course of OS using markers of lipid peroxidation (malondialdehyde, 4-hydroxy-2-nonenal, and F-2 isoprostanes), protein carbonyls, antioxidant enzymes (heme oxygenase and gamma-glutamyl transferase), and levels of endogenous antioxidants (coenzyme Q10 and Q9, gamma-tocopherol) in plasma and different tissues (joint, liver, lung, skeletal muscle, and spleen).

Oxygen metabolism has an important role in the pathogenesis of rheumatoid arthritis. A certain correlation was observed between oxidative stress, arthritis and the immune system. Reactive oxygen species produced in the course of cellular oxidative phosphorylation and by activated phagocytic cells during oxidative burst, exceed the physiological buffering capacity and result in oxidative stress. The excessive production of ROS can damage protein, lipids, nucleic acids, and matrix components. Patients with rheumatoid arthritis have an altered antioxidant defense capacity barrier. In the present study the effect of substances with antioxidative properties, i.e. pinosylvin and carnosine, was determined in monotherapy for the treatment of adjuvant arthritis (AA). Moreover carnosine was evaluated in combination therapy with methotrexate. Rats with AA were administered first pinosylvin (30 mg/kg body mass daily per os), second carnosine (150 mg/kg body mass daily per os) in monotherapy for a period of 28 days. Further, rats with AA were administered methotrexate (0.3 mg/kg body mass 2-times weekly per os), and a combination of methotrexate+carnosine, with the carnosine dose being the same as in the previous experiment. Parameters, i.e. changes in hind paw volume and arthritic score were determined in rats as indicators of destructive arthritis-associated clinical changes. Plasmatic levels of TBARS and lag time of Fe2+- induced lipid peroxidation (tau-FeLP) in plasma and brain were specified as markers of oxidation. Plasmatic level of CRP and activity of γ-glutamyltransferase (GGT) in spleen and joint were used as inflammation markers. In comparison to pinosylvin, administration of carnosine monotherapy led to a significant decrease in the majority of the parameters studied. In the combination treatment with methotrexate+carnosine most parameters monitored were improved more remarkably than by methotrexate alone. Carnosine can increase the disease-modifying effect of methotrexate treatment in rat AA.

Background: Combination therapy with methotrexate (MTX) is the most common therapeutic strategy used for the treatment of patients with rheumatoid arthritis (RA). In this study, we combined the natural compound carnosic acid (CA) with MTX to reduce inflammation and oxidative stress in adjuvant arthritis (AA). Methods: AA was induced in 6–8 rats per group. MTX was administrated twice a week at a dose of 0.3 mg/kg b.w., while CA was administered daily at a dose of 100 mg/kg both in monotherapy and in combination with MTX. Plasma samples were collected on the 14th, 21st, and 28th day. Body weight and hind paw volume were measured once a week. Results: We found that, mainly, the CA + MTX combination significantly reduced the hind paw swelling, the levels of IL-17A, MMP-9, and MCP-1 in plasma, and GGT activity in joint homogenates. The mRNA expression of HO-1, catalase, and IL-1β in the liver were significantly improved by CA + MTX only. Our results indicate that adding CA to MTX treatment could be a good therapeutic option for patients suffering from RA. Conclusions: The addition of CA to methotrexate treatment significantly improved its efficacy in decreasing the development of AA by inhibiting the markers of inflammation and oxidative stress.