Folic acid, but not folinic acid, inhibited rabbit liver AO. Di and trinuclear heterocyclic aromatic compounds and folic acid, celecoxib, and prednisone have been reported to inhibit human liver AO in vitro
). Our data suggests that folic acid binds to rabbit liver AO tighter than MTX (i.e. lower Ki for folic acid than Km for MTX).
The capacity to catabolize MTX to 7-OH-MTX varied considerably between patients. We had reported that the percent of dose excreted as 7-OH-MTX varied from 0.94 to 13.2% in patients with RA in a 72-hour urine (2
). The catabolic capacity was not a function of body/organ mass since the urinary excretion of 7-OH-MTX corrected for creatinine varied by a factor of 10 and was bimodal (). Others have reported variable capacity to catabolize MTX to 7-OH-MTX in patients with RA (references in 2) including a report that 2 of 16 patients had levels of 7-OH-MTX below detection limits (i.e. 1×10−9
M) in serum following oral MTX dosing (8
). Variability in the in vitro
activity of human liver AO has been reported (references in 2) including a 48-fold range of activities in human liver cytosols (9
). Inhibitors of AO and xanthine oxidase (i.e. allopurinol) both reduce the formation of 7-OH-MTX in human liver cytosol (10
). It is possible that both enzymes catalyze the formation of 7-OH-MTX.
Patients with a lower capacity to catabolize MTX to 7-OH-MTX also respond better to MTX. Our clinical data was collected after 6 and 7 weeks of MTX therapy and response may have improved with therapy duration. We could not calculate the DAS-28 score which is a better measure of efficacy, which limits the interpretation of these results.
Folic acid supplements lower the in vivo
formation of 7-OH-MTX when compared to folinic acid supplements. This may be due to inhibition of AO, since with dihydrofolate reductase inhibited by MTX; folic acid should be relatively trapped as such in vivo
. On the other hand, folinic acid can be metabolized to other folate coenzymes independent of the reductase and folinic acid and its tetrahydrofolate metabolites have little effect on AO. Polyglutamylation of folic acid may lower its inhibition of AO (4
), but daily dosing may maintain a pool of folic acid as the monoglutamate.
The more 7-OH-MTX excreted, the more MTX is excreted in the same individual (). We previously observed that when MTX catabolism to 7-OH-MTX is reduced by cyclosporine, the amount of MTX excreted was also reduced, although it was not statistically significant (11
). This suggests that 7-OH-MTX competes with MTX for protein binding sites. One critical site would be the polyglutamyl synthetase. If relatively large amounts of 7-OH-MTX are formed, they may be polyglutamylated and sequestered inside cells at the expense of MTX which is excreted in greater quantities. The data in confirms that the more 7-OH-MTX excreted in 24-hours, the less of the MTX dose is retained during the 24 hours after the dose.
We hypothesize that there is a phenotype which produces relatively large amounts of 7-OH-MTX which excludes MTX from the polyglutamyl synthetase, forms more 7-OH-MTX polyglutamates (7-OH-MTX Glun), and excretes relatively more MTX and 7-OH-MTX in urine. A second phenotype produces relatively little 7-OH-MTX and 7-OH-MTX Glun and relatively little MTX and 7-OH-MTX is excreted in urine and retains more MTX Glun.
This, the consequences of excessive 7-OH-MTX formation would be not only to reduce the in vivo
MTX pool but also to promote MTX excretion, lower in vivo
MTX retention and possibly its efficacy. Others have reported that concentrations of MTX polyglutamates in red blood cells of patients after 6 months of therapy was variable and that lower concentrations were found in patients with a poorer clinical response (12
). It has been reported in ten patients that one had no detectable pentaglutamates of MTX in red blood cells after 40 weeks of therapy and in nine others the time to detect this polyglutamate ranged from 1 to 28 weeks of therapy (13
). The variability of red blood cell MTX polyglutamate concentrations could be a function of MTX catabolism to 7-OH-MTX. 7-OH-MTX could also displace MTX from the active site of other folate metabolizing enzymes (14
), reducing the amount of MTX retained. The changes plotted in use the patients as their own control, thus, 7-OH-MTX formation and in vivo
MTX retention can change in the same patient in one week. With stable MTX doses, it is likely that the patients' in vivo
AO activity could be altered by changes in normobiotic activators or inhibitors, nutrients, amounts of other medications and changes in the amount of AO. .
In conclusion, the urinary excretion of 7-OH-MTX and the extent of catabolism of MTX to 7-OH-MTX were not normally distributed in patients and this catabolism is likely to alter MTX efficacy and the amount of the MTX dose retained in vivo. Folic acid supplements may inhibit in vivo AO and therefore the catabolism of MTX to 7-OH-MTX and potentiate the efficacy of MTX. There may be three reasons to use folic acid supplements in MTX treated patients with RA: 1) lower MTX toxicity, 2) lower blood homocysteine, and 3) reduce 7-OH-MTX formation.