Anti-arthritic effect of methotrexate: is it really mediated by adenosine?

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Abstract

The mechanism of action for the anti-arthritic effect of methotrexate (MTX) was investigated in rats with antigen-induced arthritis (AIA). Arthritis intensity was quantified as area under the curve (AUC) for the joint swelling. The response to MTX was in several respects similar to what is seen in the clinic. The drug reduced the AUC in a dose-dependent manner after oral weekly (2–4 mg/kg/week) or daily (0.3 mg/kg/day) dosing. This effect was not affected by supplementation with an equal dose of folate. The model thus seemed suitable for this type of study. Supplementation with folate in excess abolished the effect of MTX. A structurally similar antifolate, aminopterin, also reduced the arthritis. The effect thus seemed to be due to folate antagonism although a complete inhibition of dihydrofolate reductase (DHFR) might not be essential. Hence, it could be that the main target is a process downstream of DHFR. It has been proposed that inhibition of AICAR-transformylase induce the release of adenosine with anti-inflammatory properties. Here the adenosine antagonist R-PIA reduced the arthritis but when MTX was combined with adenosine antagonists no attenuation of the anti-arthritic effect was seen. On the contrary, three adenosine agonists (8-p-sulphophenyltheophyllamine 30 mg/kg i.p. twice daily; 3,7-dimethyl-1-propargylxanthine, p.o. 3 mg/kg/day and 8-cyclopentyl-1,3-dipropylxanthine, 1.5 mg/kg/day p.o.) potentiated MTX. The specific thymidylate synthase inhibitor 5-fluourouracil (0.3–3.0 mg/kg/day) had no anti-arthritic effect. Neither did our data support the hypotheses that syntheses of polyamines or cytokines were primary targets. It is thus possible that the mechanism of action is inhibition of a process downstream of DHFR but the release of adenosine seems not to be important.

Introduction

Methotrexate (MTX) has become the most widely used second line drug for the treatment of rheumatoid arthritis (RA). It is relatively well tolerated and has a well documented clinical efficacy, although its ability to slow down radiological progression of the disease is uncertain (Bannwarth et al., 1994, Rau et al., 1997). It thus seems likely that knowledge of the mechanism of action for the drug would provide important information on the pathophysiology of the disease. This would possibly also enable the development of new anti-rheumatic drugs. At present, however, MTX’s main targets are largely unknown.

MTX is a potent inhibitor of dihydrofolate reductase (DHFR) which subsequently inhibits de novo purine and pyrimidine synthesis. Based on these properties it was developed as a cytostatic agent. There is, however, much evidence indicating that the anti-arthritic effects of MTX are due more to an anti-inflammatory rather than anti-proliferative or immunomodulatory effect (Bannwarth et al., 1994, Cronstein, 1996). Firstly, the dosing differs from that used for treatment of oncological disease in which MTX is administered in pulses of 20–250 mg/kg; for RA treatment it is administered in weekly doses of 0.1–0.3 mg/kg (Cronstein et al., 1993). Secondly, there are reports that RA patients can be supplemented with folate in doses as high as 27.5 mg/week without compromising the drugs efficacy (Morgan et al., 1994). Although there is no consensus over this view (Stenger et al., 1992) such data suggest that complete inhibition of DHFR is not essential in rheumatological use. Interestingly it has been reported that only antifolates with a high structural similarity to MTX have anti-arthritic effect (Galivan et al., 1986, Baggott et al., 1993). It has therefore been proposed that inhibition of other folate dependent enzymes, located downstream of DHFR, such as thymidylate synthase (TS) or 5-aminoimidazole-4-carboxamide ribotide transformylase (AICAR-transformylase) are of greater importance (Baggott et al., 1993, Sandoval et al., 1995). One interesting hypothesis is that MTX exerts its anti-inflammatory effect by elevating the extracellular levels of adenosine (Cronstein et al., 1993). The line of evidence states that MTX is taken up by cells and converted to long-lived polyglutamates. AICAR-transformylase is potently inhibited by the MTX polyglutamates and subsequently the intracellular AICAR concentration will rise. High AICAR levels will inhibit adenosine degradation and hence lead to an accumulation of that mediator in the extracellular fluid (Baggott et al., 1993). Adenosine has been reported to exhibit a number of anti-inflammatory effects. Furthermore, MTX raised the adenosine and AICAR contents and inhibited the carrageenan-induced leukocyte influx in a murine air-pouch system. 3,7-Dimethyl-1-propargylxantine (DMPX), an adenosine A2 antagonist, and a adenosine degrading enzyme reversed the MTX effect but an A1 antagonist was ineffective (Cronstein et al., 1993).

For TS, a degree of inhibition has been demonstrated in lymphocytes from MTX-treated patients. However, 5-fluorouracil (5-FU), a specific inhibitor of TS is a much weaker immunosuppressant than MTX in animal models of transplantation (see: Baggott et al., 1993).

Another hypothesis is that MTX interferes with polyamine synthesis (Nesher and Moore, 1990, Nesher et al., 1997). Polyamines are important in immune-mediated cellular reactions. The synthesis of polyamines is dependent on availability for precursors such as methionine, which is required for the synthesis of S-adensosyl-methionine (SAM). The regeneration of methionine from homocysteine is, in turn, folate dependent and will thus be inhibited by MTX (Nesher and Moore, 1990). Finally, local cytokine release in the joints is important for the propagation of the disease and it could be that MTX inhibits this production. The relevant parts of the folate metabolism are presented in Fig. 1.

Mode of action studies for the anti-arthritic effect of MTX have mostly been performed in vitro or in non-arthritic in vivo models. There are only few reported attempts to confirm the findings in more complex disease models. This is somewhat surprising since there is an abundance of animal arthritis models and MTX seems to be effective in most of them: The drug ameliorates collagen-induced arthritis (CIA) in rats (Ekström et al., 1997) and mice (Ekström, unpublished). In the same dose interval (weekly dosing) as in the present study it is also reported to be efficient in rats with adjuvant arthritis (Suzuki et al., 1997), and in rabbits with antigen-induced arthritis (Noaves et al., 1996). In preliminary experiments we found that the compound also inhibited the development of antigen-induced arthritis (AIA). The general effect suggests that in these models the drug act on the same target. We wanted to investigate some of the hypotheses on the mechanism of action for MTX in an arthritis model. We chose AIA since this model has a high reproducibility and permits quantifications of the response with a continuous parameter (joint diameter).

Section snippets

Animals

Female rats from the Dark Agouti (DA) strain with an approximate body weight of 165 g were used in the study. They were obtained from Möllegaards Breeding Center, Denmark and kept in sawdust-covered cages with food and water ad libitum. The temperature was thermostatically maintained at +22°C, the relative humidity was 50%, and the light was on in 12 h periods from 6 a.m. to 6 p.m. The Animal Ethics Committee of Lund, Sweden approved the experiments. The rats were conditioned at least 1 week

Anti-arthritic effect of antifolates

MTX was given orally p.o., once weekly, in the doses 2, 3 or 4 mg/kg/week. Treatment was carried out Day 7 before challenge, on the day of challenge and on Day 7 after challenge. A clear dose–response relationship was seen and a statistical significant reduction of joint swelling was reached for the two highest doses (Fig. 2). The changes in body weight were similar in all groups: from the beginning to the end of the experiment the AIA+MTX 2 mg/kg/week group lost 4±2 g; AIA+3 mg/kg/week, 10±5

Verisimilitude of the model

Hypotheses regarding the mode of action for MTX have so far been mainly derived from observations made in experiments in vitro or in non-arthritic in vivo models. We wanted to investigate if any of them could be confirmed in a more complex disease model. An essential question then is whether MTX acts on the same target in humans as in the rats. Two characteristics of the response indicate that this could the case; firstly, both in the model and in RA patients, MTX is effective after weekly

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