The effect of montelukast in a model of gouty arthritis induced by sodium monourate crystals. 

Loida Ponce ¹, Marjorie Arjona ¹, Gustavo Blanco ¹, Stuart Alvarez ¹, Eduardo Arcila ¹, Arnaldo Ortega ¹, Dubelis Nuñez ¹, Julie Verzura ¹, Robert Tovar ¹, Sarah Bethencourt ¹, Ricardo Riera ², Sioly Mora-Orta ¹and José Corado ¹. 

¹ Unit of Investigation in Immunology (UNIVENIN), Department of Physiological Sciences, Faculty of Health Sciences, University of Carabobo, Bárbula and

² Hospital “Dr Enrique Tejera”, Rheumatology Department. Valencia, Venezuela.

Corresponding author: Zaida Araujo. Laboratorio de Inmunología de Enfermedades Infecciosas, Instituto de Biomedicina, Universidad Central de Venezuela. Apartado 4043. Caracas 1010A, Venezuela. Teléf. +58-212- 860-4636/414-248-0215; fax: +58-212-861-1258. E-mail: zaraujogarcia@yahoo.com

Abstract.

Non-steroidal anti-inflammatory drugs (NSAIDS) are the first line of therapy in acute gouty arthritis. NSAIDs inhibit the cyclooxygenase pathway, but not the lipooxygenase activity and can have many adverse effects and thus have a limited effect on the control of inflammation in this disease. In this work we studied the effect of montelukast on the cellular inflammatory infiltrate in a model of murine arthritis induced by sodium monourate crystals (SMU), using a subcutaneous air cavity (air pouch) in BALB/c mice. Seven groups of BALB/c mice (n = 4) were distributed into five experimental groups and two inflammatory control groups, a positive and a negative one. Previous to SMU exposure, the experimental groups received montelukast (1 and 0.01 mg/Kg/w) and/or indomethacine (2.5 mg/Kg/w), followed by administration of SMU in the air pouch. The total and differential counts of inflammatory cells were analyzed after 2, 6, 12 and 24 hours. Montelukast, significantly reduced the total number of cells (p<0.05), with a predominant impact on polymorphonuclear over mononuclear cells, especially after 12 hours of the medication. The montelukast/indometacine combination showed an additive effect. Our data show that montelukast has an anti-inflammatory effect in the model of gouty arthritis. Consequently, anti-leukotrienes could represent a new and effective therapy, either isolated or combined with conventional therapy of gouty arthritis.

Efecto de montelukast en un modelo de artritis gotosa inducido por cristales de monourato de sodio.

Resumen.

En artritis gotosa aguda las drogas antiinflamatorias no esteroideas son la primera línea terapéutica. Este tratamiento no es satisfactorio porque inhibe la ciclooxigenasa sin modificar la actividad de la lipooxigenasa, y puede acompañarse de numerosos efectos adversos. Investigamos el efecto de montelukast sobre el infiltrado celular inflamatorio en un modelo de artritis múrida inducida por cristales de monourato de sodio (MUS) en el modelo experimental de la bolsa de aire (air pouch). Siete grupos de ratones BALB/c (n = 4) fueron distribuidos en cinco grupos experimentales y dos grupos controles inflamatorios: positivo y negativo. Los grupos experimentales recibieron, montelukast (1 y 0,01 mg/Kg/p) y/o indometacina (2,5 mg/Kg/p) por vía oral, previo a la administración de MUS en la bolsa del aire. El conteo absoluto y diferencial de las células inflamatorias fue analizado después de 2, 6, 12 y 24 horas de tratamiento. El tratamiento con montelukast redujo significativamente el número total de células presentes en el infiltrado inflamatorio (p < 0,05), con un efecto mayor sobre polimorfonucleares que sobre las células mononucleares, y con un máximo efecto a las 12 horas después de la administración del medicamento. La combinación montelukast/indometacina mostró un efecto aditivo. Los resultados demuestran que montelukast tiene un efecto antiinflamatorio en el modelo de la artritis gotosa. Por lo tanto, los anti-leucotrienos podrían representar una nueva y eficaz terapia, aislada o en combinación con la terapéutica convencional, para la artritis gotosa.

Received: 07-12-2009. Accepted: 07-10-2010.

INTRODUCTION

Gouty arthritis (GA) is a rheumatic condition characterized by hyperuricemia and sodium monourate crystal (SMU) deposits in one or some peripheral joints (1, 2). Once SMU crystals are deposited, joint tissues are colonized by neutrophils, monocytes/macrophages, platelets, and mast cells (3, 4). Recruitment and activation of neutrophils by SMU play a critical role in the pathogenesis and perpetuation of the acute gout (5). 

Inflammatory cells secrete proinflammatory mediators and chemotactic agents such as leukotrienes (LT), tumoral necrosis factor (TNF), complement factor C5a, and interleukin 8 (IL-8) (3, 4, 6). Leukotrienes may be very relevant in this pathology, since LTB4 is the most potent chemotactic agent for neutrophils both, in vitro and in vivo (7). Consequently transendothelial migration occurs and more neutrophils colonize synovial tissues (8, 9). Non steroidal antinflammatory drugs (NSAIDS) are the first line therapy for this disease. Colchicine and steroids, local or systemic, are also used. This therapy is partially effective, but can induce adverse reactions and/or toxicity in aproximately 80% of patients. The mechanism of action involves inhibition of the cyclooxygenase enzyme, and blockade of the synthesis of prostaglandins (PG), but they have no effect in the lipooxygenase enzyme activity, responsible for the synthesis of LTs (10, 11), which are the most important inflammatory mediators in gouty arthritis (10). In inflammatory pathologies, such as bronchial asthma (12, 13) and allergic rhinitis (13, 14), in which LTs are known to play an essential role, different selective antagonists of cysteinyl LTs are shown to have a beneficial effect.

Since LTs are important inflammatory mediators in gouty arthritis (15 Rae) and drugs such as montelukast (Mk) act as a competitive inhibitor of LTs by joining selectively to LT receptors (16), especially to the LTD4 receptor, we sought to investigate the effect of montelukast in a model of experimental arthritis induced by SMU crystals deposits in the air pouch model in BALB/c mice (17). 

METHODS 

Animals 

One hundred and five male BALB/c mice, 10 weeks old, on a regular diet, ad libitum, under strict conditions of hygiene and temperature (25-26°C) were chosen for the experimental protocol. Surgical procedures and animal treatments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (FONACIT).

Experimental protocol 

BALB/c mice were treated to produce an air pouch on their backs, by applying a subcutaneous injection of 5 mL of sterile air on day one, and 3 mL 3 days later (17). Thereafter, mice were distributed in five experimental groups (15 each) and two control groups (15 each). The experimental groups were designated as A, B, C, D, E, a positive control group for inflammation (F) and a negative control group (G). All the experimental groups received Mk and/or indomethacine (Indo), orally as follows: Mk 0.01 mg/Kg/w and 1 mg/Kg/w group A and group B respectively; Indo 2.5 mg/Kg/w group C; groups D and E received both drugs at same doses. Groups F and G received, buffer phosphate saline (PBS, Dako) orally, as control. One hour later, groups A-F were injected in the air pouch with 2 mL of SMU (5 mg/mL) prepared in PBS according to the Denko method (18). Group G received only PBS pH 7.2. Each animal group received an overdose of ether and was then sacrificed at 2, 6, 12 and 24 hours, to obtain samples for its analysis.

Sample collection and processing 

The air pouch was washed with 2 mL PBS pH 7.2, plus 20 units of sodium heparine (Lilly^Ò). Samples from all groups were obtained at 2 (n = 3), 6 (n = 4), 12 (n = 4) and 24 (n = 4) hours, and centrifuged at 1.200 rpm, at 25°C, for 5 minutes. The pellet was resuspended in 1 mL of PBS. Total and differential cellular counts were done using hematoxiline/eosine in a Neubauer chamber, with an optical microscope (400X).

Statistical analysis 

Data are expressed as mean ± 2 SD (n = 4) and the Student’s test was used to analyze the results. A P value of less than 0.05 was considered statistically significant. 

RESULTS 

Effect of Mk on the inflammatory exudate induced by SMU in the air pouch of experimental mice

The total cell number in the inflammatory exudates of SMU-treated mice progressively increased, reaching a value of 1,264,583 ± 434,771 cell/mL after 2 hours and a peak of 6,071,250 ± 1,043,857 cell/mL at 12 hours, followed by a decline to 3,795,000 ± 1,724,824 cell/mL at 24 hours. In the negative inflammatory control (–) the total cell number reached a peak of 200 ± 50 cel/mL. At doses of 0.01 and 1 mg/Kg/w, Mk significantly decreased the total cell number in inflammatory exudates (p < 0.05). When comparing both doses of Mk, it was found that Mk 1mg/Kg/w had a significantly greater effect than 0.01 mg/Kg/w, but only at 24 hours (Fig. 1). This reduction reached 46.5% and 47.8% at 2 hours; 78% and 69% at 6 hours; 82.1% and 86.9% at 12 hours and 73.1% and 87.5% at 24 hours, respectively. At the dose of 2.5 mg/Kg/w, Indo significantly decreased the total cell count at all times tested (p < 0.05) (Fig. 2). The inhibitory effect was 61.6% at 2 hours, 86.4 % at 6 hours, 87.4% at 12 hours and 86.7% at 24 hours (data not shown).

Fig. 1. Effect of montelukast on total cell count (mean ± 2D) of the inflammatory exudates induced by SMU crystals in the air pouch model in BALB/c mice. Dashed and solid lines indicate treatment with Mk 1 mg/Kg/w and 0.01 mg/Kg, respectively. Dotted line indicates without treatment. *p < 0.05.

Fig. 2. Effect of indometacine on total cell count (mean ± 2D) of the inflammatory exudates induced by SMU crystals in the air pouch model in BALB/c mice. The pointed line indicates treatment with SMU, the dashed line indicates treatment with indometacine (2.5 mg/Kg). *p < 0.05.

The combination of Mk (0.01 mg/Kg/w) plus Indo (2.5 mg/Kg), and Mk (1 mg/Kg/w) plus Indo (2.5 mg/Kg) showed a stronger inhibitory effect at 12 hours when comparing with Mk at either dose alone (Fig. 3). When Mk 0.01mg/Kg/w and Indo (2.5 mg/Kg) were used in combination the reduction reached 92.7 % and 94.6% (Fig. 3).

Fig. 4. Montelukast effect in the differential cell count (mean + 2D) in inflammatory exudate of gouty arthritis model at 12 hours. Values represent the percentage of polimorphonuclear cells (gray histogram) and mononuclear cells (white histogram).