Genetic variants associated with methotrexate efficacy and toxicity in early rheumatoid arthritis: results from the treatment of early aggressive rheumatoid arthritis trial.

Methotrexate (MTX) has emerged as first-line therapy for early moderate-to-severe rheumatoid arthritis (RA), but individual variation in treatment response remains unexplained. We tested the associations between 863 known pharmacogenetic variants and MTX response in 471 Treatment of Early Aggressive Rheumatoid Arthritis Trial participants with early RA. Efficacy and toxicity were modeled using multiple regression, adjusted for demographic and clinical covariates. Penalized regression models were used to test joint associations of markers and/or covariates with the outcomes. The strongest genetic associations with efficacy were in CHST11 (five markers with P<0.003), encoding carbohydrate (chondroitin 4) sulfotransferase 11. Top markers associated with MTX toxicity were in the cytochrome p450 genes CYP20A1 and CYP39A1, solute carrier genes SLC22A2 and SLC7A7, and the mitochondrial aldehyde dehydrogenase gene ALDH2. The selected markers explained a consistently higher proportion of variation in toxicity than efficacy. These findings could inform future development of personalized therapeutic approaches.

Folate supplementation during methotrexate therapy for rheumatoid arthritis.

Methotrexate (MTX), an antifolate, is an anchor drug for the treatment of rheumatoid arthritis (RA). Both folic acid (FA) and folinic acid (FLN) supplements have been shown to reduce the toxicity of MTX when used in RA therapy. The effect of folate supplementation on MTX efficacy still needs to be studied. FA supplementation has been found to have a beneficial effect on homocysteine (hcy) metabolism and may prevent the formation of the less effective metabolite 7-hydroxy-MTX. The cost of FA supplements is substantially less than the cost of FLN supplements. This article reviews clinical trials related to folate supplementation during MTX therapy for RA.

Iron-dependent formation of homocysteine from methionine and other thioethers.

OBJECTIVE: We tested whether homocysteine is formed from methionine and other thioethers in vitro and in vivo, because methionine can be chemically demethylated to homocysteine. DESIGN: In in vitro studies, chemical conversions of thioethers (methionine, S-adenosylhomocysteine and cystathionine) into homocysteine were measured under various aerobic conditions. In humans, oral methionine (0.17 mmol/kg body weight) loading tests with and without an oral iron dose (ferrous sulfate, 13 mumol/kg) were performed. SETTING: A university setting in Birmingham, AL, USA. SUBJECTS: A total of five healthy adult subjects volunteered. RESULTS: The in vitro incubation of methionine, S-adenosylhomocysteine or cystathionine with chelated iron resulted in the formation of homocysteine. These conversions were iron- and pH-dependent (pH optima between 5.0 and 6.0) and it was also chelator-dependent. In humans, oral methionine loading tests resulted in a 45% increase in the area-under-the-curve for plasma total homocysteine concentrations, when iron was given together with methionine. CONCLUSION: Our data suggest that iron-dependent chemical formation of homocysteine can occur in vivo, and contribute to the plasma total homocysteine pool, since this formation can occur ceaselessly. We hypothesize that plasma total homocysteine concentrations reflect, in part, non-protein-bound iron in the body.

Effects of folate deficiency and supplementation on methylnitrosourea-induced rat mammary tumors.

BACKGROUND: There are metabolic and epidemiologic data consistent with the hypothesis that folate deficiency increases the likelihood of cancer. Conversely, it is also known that folate is necessary for cancer growth, but few experiments in laboratory animals have evaluated the effects of folate deficiency on the development of chemically induced cancers. PURPOSE: Our purpose was to determine the effects of nutritional folate deficiency in female Fischer 344 rats on initiation and early promotion of methylnitrosourea (MNU)-induced mammary cancer. METHODS: Rats (age, 27 days) were fed a folic acid-deficient diet (AIN-76A) supplemented with glycine and succinylsulfathiazole [FA(0)]; the FA(0) diet supplemented with 2 or 40 mg of folic acid per kilogram [FA(2) or FA(40), respectively]; or the FA(0) diet supplemented with 20 mg of folinic acid per kilogram [FL(20)]. At 57 days of age, each diet-treated group (30 rats in each group) received MNU (50 mg/kg) by intravenous injection. Immediately after MNU treatment, all animals were fed the AIN-76A complete diet containing 2 mg of folic acid per kilogram. Control groups were fed the AIN-76A complete diet throughout the entire experiment. RESULTS: After 4 weeks, folate deficiency, but not anemia or growth suppression, was documented by lower folate levels in plasma and red blood cells in the group receiving the FA(0) diet. Cancer multiplicity (i.e., number of mammary cancers per number of tumor-bearing animals) at 180 days after MNU injection was 1.32, 1.90, 2.14, and 2.73 mammary cancers per tumor-bearing animal in the FA(0), FA(2), FA(40), and FL(20) groups, respectively; the value in the FA(0) group was statistically significant compared with the values in the other groups. The time required for 50% of the rats to develop palpable mammary cancer was 170, 142, 100, and 85 days, respectively. The value of 170 days for the FA(0) group was statistically significant compared with the values of 100 and 85 days. Mammary cancer incidence was 63%, 70%, 72%, and 73%, respectively; these percentages were not significantly different. CONCLUSIONS: Folate deficiency suppresses and folate supplementation enhances initiation or early promotion of MNU-induced mammary cancer in rats, even when the folate-deficient rats do not have anemia or growth suppression. IMPLICATION: Since the rat is relatively resistant to folate deficiency anemia, other animal models should be used to test the effect of folate nutriture on carcinogenesis.

Polyglutamyl folate coenzymes and inhibitors of chicken liver glycinamide ribotide transformylase.

Chicken liver glycinamide ribotide transformylase (5,10-methenyltetrahydrofolate:5'-phosphoribosylglycinamide formyltransferase, EC 2.1.2.2), an enzyme of purine biosynthesis de novo, has greater specificity for its poly-gamma glutamyl folate coenzymes, 5,10-methenyltetrahydropteroyl(glutamate)n, where n = 3, 4, 5, 6 or 7, when compared to the monoglutamyl folate coenzyme. The relative specificity constants (V/Km) for the coenzymes 5,10-methenyltetrahydropteroyl(glutamate)n are 1.0, 1.6, 2.6, 2.4, 2.4, 4.9 and 1.5 for n = 1, 2, 3, 4, 5, 6 and 7, respectively. Pteroylpoly-gamma-(glutamate)n, where n = 3, 4, 5, 6 or 7, are much better inhibitors of this enzyme when compared to the pteroylmonoglutamate. The concentration of inhibitor required for 50% inhibition was found to be 490, 120, 58, 28, 16, 14 and 12 microM for n = 1, 2, 3, 4, 5, 6 and 7, respectively. Inhibitors with four or more glutamic acid residues gave grossly non-linear Dixon plots, in contrast to the linear plots obtained using inhibitors with three or less glutamic acid residues. The above findings make it feasible for the activity of glycinamide ribotide transformylase to be regulated by alteration in the length of the poly-gamma-glutamyl chain of its folate coenzymes and of folate inhibitors.

Bioactivity of orally administered unnatural isomers, [6R]-5-formyltetrahydrofolate and [6S]-5,10-methenyltetrahydrofolate, in humans.

It has been assumed that humans cannot utilize 5,6,7,8-tetrahydrofolates with the unnatural configuration at carbon 6, since these folates are enzymatically and microbiologically inactive. We hypothesized that orally administered unnatural [6R]-5-formyltetrahydrofolate or [6S]-5,10-methenyltetrahydrofolate is bioactive in humans. Subjects were given independent oral doses of these unnatural folates and of a natural [6S]-5-formyltetrahydrofolate. Plasma, before and after the dose for 4 h, and 2 h urine were collected. Areas under the curve for the change in plasma folate concentrations were measured microbiologically and urinary folates were measured using HPLC. Based on findings of plasma and urinary folates, the unnatural folates were estimated to be 14-50% active as compared to [6S]-5-formyltetrahydrofolate. The major plasma and urinary folate was [6S]-5-methyltetrahydrofolate in all experiments. In urine, a [6S]-5-formyltetrahydrofolate peak was observed only after a [6S]-5-HCO-H4folate dose and peaks of unnatural [6S]-10-formyltetrahydrofolate and 5-formyltetrahydrofolate were identified after a [6R]-5-formyltetrahydrofolate dose. A possible pathway that explains our findings is discussed. This pathway includes the oxidation of the unnatural [6S]-10-formyltetrahydrofolate to 10-formyl-7,8-dihydrofolate which can be further metabolized by 5-amino-4-imidazolecarboxamide-ribotide transformylase producing dihydrofolate. Dihydrofolate can then be metabolized to [6S]-5-methyltetrahydrofolate by well-established metabolism.

Inhibition of human serine proteases by SPAAT, the C-terminal 44-residue peptide from alpha1-antitrypsin.

SPAAT has previously been shown to be a competitive inhibitor of the model serine protease, chymotrypsin. We now present evidence that SPAAT is likewise a competitive inhibitor of human neutrophil elastase and cathepsin G with Ki's of 15-20 and 40 microM, respectively. The mechanism of this inhibition was investigated by comparing the relative effectiveness of the 23-residue N-terminal fragment of SPAAT (N-SPAAT) to inhibit chymotrypsin and human neutrophil elastase. N-SPAAT, which does not contain the primary chymotrypsin cleavage site, was approximately 10-fold less effective as an inhibitor of chymotrypsin than SPAAT (Ki of 65 microM versus 7.5 microM). In contrast, this fragment, which contains the primary human neutrophil elastase cleavage site, was found to competitively inhibit human neutrophil elastase with a Ki of 24 microM which was comparable to that of SPAAT (Ki = 15-20 microM). Thus it appears that SPAAT is a reversible inhibitor of these enzymes rather than an irreversible, stoichiometric one like its parent protein, AAT. Such fragmentation of AAT, however, might provide a mechanism whereby a cascade of decreasingly potent, but increasingly specific SPAAT-related inhibitory peptides could be generated. These results further substantiate the view that SPAAT may play a role in vivo in the protection of extracellular proteins from inappropriate attack by proteases which are elevated during various pathophysiological conditions.

Homocysteine levels in patients with rheumatoid arthritis treated with low-dose methotrexate.

Plasma homocysteine levels were determined in patients who participated in a randomized, double-blind placebo-controlled trial of folate supplementation (1 mg/day) during methotrexate therapy for rheumatoid arthritis. Plasma and red blood cell folate levels before methotrexate therapy were significantly negatively correlated with homocysteine levels. Homocysteine levels were not significantly correlated with the initial C1 index (an assay that measures the folate status of blood mononuclear cells) or the C1 index during methotrexate therapy. There was no significant difference in homocysteine levels between pretreatment and levels drawn at 3 or 6 months. Initial homocysteine levels were predictive of toxicities, such as gastrointestinal intolerance and elevations of liver enzymes in the placebo group. There was no significant correlation between occurrence of toxicity and initial homocysteine levels in the folic acid-supplemented group. Homocysteine levels were not predictive of the efficacy of methotrexate therapy. We conclude that plasma homocysteine levels are correlated with plasma and red blood cell folate levels before methotrexate therapy but is not correlated with folate status in blood mononuclear cells.

Folate conjugase activity in the plasma and tumors of breast-cancer patients.

The activity of folate conjugase (pteroylpolyglutamate hydrolase, EC 3.4.22.12) was measured in plasma from normal subjects and patients with breast cancer using pteroylglutamyl-gamma-glutamyl-gamma-(U14C) glutamate as the substrate. Conjugase assays also were performed on samples of breast-tumor tissue, normal breast, and fibroadenoma. When assayed at pH 4.5 and 7.4, mean plasma conjugase activity was significantly (p less than 0.05) elevated in a group of patients with anatomically proven metastatic disease (n = 21) when compared with control subjects (n = 12) and a group of patients (n = 13) with no clinical evidence of disease after mastectomies. Mean plasma conjugase activity assayed at pH 4.5 also was significantly higher in the metastatic disease group when compared with breast cancer patients before mastectomy (n = 9) and fibroadenoma patients before biopsy (n = 3). The specific activity of tissue conjugase assayed at pH 4.5 was significantly higher (p less than or equal to 0.05) in infiltrating ductal carcinoma than in normal adjacent breast tissue according to the Wilcoxon test for paired samples (n = 10).

Effects of tenidap on superoxide-generating enzymes. Non-competitive inhibition of xanthine oxidase.

The anti-rheumatic drug tenidap has been shown previously to attenuate superoxide production by activated neutrophils. Given the importance of leukocyte as well as endothelial cell derived superoxide in mediating inflammatory responses, the effects of tenidap on mammalian enzymes capable of generating superoxide were determined. Tenidap had no effect on the generation of superoxide by NADPH oxidase reconstituted from fractionated neutrophil lysates. However, significant inhibition of superoxide production by mixtures of hypoxanthine and xanthine oxidase was observed in the presence of 3-30 micrograms/mL tenidap. The kientics of xanthine oxidase inhibition by tenidap were non-competitive; the Ki of tenidap for xanthine oxidase was 11 micrograms/mL (34 microM). No inhibition of xanthine oxidase was observed in the presence of other known inhibitors of cyclooxygenase. Inhibition of xanthine oxidase may be a heretofore unrecognized mechanism of the antirheumatic effects of tenidap.

Methotrexate in rheumatoid arthritis: folate supplementation should always be given.

Methotrexate is now the disease-modifying antirheumatic drug prescribed most frequently for the treatment of rheumatoid arthritis. Methotrexate is an antifolate that inhibits methylation reactions and reactions of amino acid, purine and pyrimidine metabolism. Toxic manifestations of methotrexate administration for rheumatoid arthritis (at relatively low doses compared with those used in cancer chemotherapy) include cytopenias, gastrointestinal intolerance, liver disease, pulmonary injury, central nervous system dysfunction, skin rashes and nodulosis. Delayed wound healing and increased risk for infections with opportunistic organisms also occur. Some of these toxic manifestations respond to supplementation with folates [folic acid or folinic acid (calcium folinate)]. The folate status of patients has been shown to be impaired after prolonged treatment with methotrexate, and poor baseline folate status is an independent risk factor for subsequent toxicity. Numerous studies have now documented that folic acid, even in high doses, and moderate doses of folinic acid are beneficial in preventing methotrexate toxicity without affecting efficacy. In this article we present guidelines and rationale for monitoring methotrexate therapy, and guidelines for folate supplementation during methotrexate therapy for rheumatoid arthritis. It is our recommendation that folic acid should be empirically supplemented in all patients at the initiation of methotrexate therapy. This regimen is associated with a high benefit : risk ratio and is likely to be cost effective.

Urinary adenosine and aminoimidazolecarboxamide excretion in methotrexate-treated patients with psoriasis.

BACKGROUND: We hypothesized that low-dose methotrexate treatment for patients with psoriasis would block purine biosynthesis at the step catalyzed by aminoimidazolecarboxamide (AICA) ribotide transformylase and would inhibit adenosine metabolism as evidenced by increased urinary levels of AICA and adenosine, respectively. Eight patients collected a 24-hour urine specimen on the day before their methotrexate dose and the next day during their methotrexate dose. Eight age- and sex-matched controls also collected a 24-hour urine sample. Urinary AICA and adenosine were assayed by spectrophotometric and radioimmune assays, respectively; means are reported as micromole per millimole of creatinine and were compared by the paired t test (1-tailed). OBSERVATIONS: Mean AICA excretion increased from 1.30 micromol/mmol on the day before to 1.85 micromol/mmol on the day during methotrexate dosing (P<.01). Mean adenosine values increased from 0.68 to 1.07 micromol/mmol, (P<.03). Controls had mean AICA and adenosine levels of 1.29 and 0.50 micromol/mmol, respectively. During the day of methotrexate dosing, patients had higher mean AICA and adenosine levels when compared with controls (P<.01). Mean AICA levels increased from 1.36 to 2.06 micromol/mmol (P<.025), and mean adenosine levels increased from 0.72 to 1.25 micromol/mmol (P<.025) in 5 patients showing improvement in clinical disease activity. In contrast, 3 patients with no change or worsening in clinical disease activity had smaller increases. CONCLUSIONS: Methotrexate treatment of patients with psoriasis inhibits AICA ribotide transformylase and adenosine metabolism. Since adenosine is a T-lymphocyte toxin, it may be partially responsible for the immunosuppressive effect.

Antifolates in rheumatoid arthritis: a hypothetical mechanism of action.

The antifolates, methotrexate, aminopterin, 10-deazaaminopterin and sulfasalazine are clinically useful in the treatment of rheumatoid arthritis. Toxicity, rather than efficacy, appears to the the major factor limiting the usefulness of the classical antifolates (i.e., methotrexate and 10-deazaaminopterin). The fact that folate supplementation of methotrexate-treated rheumatoid arthritis patients reduces toxicity without altering efficacy also suggests that inhibition of the drug's target enzyme, dihydrofolate reductase, is not complete and not essential for efficacy. Since polyglutamates of methotrexate are direct inhibitors of thymidylate synthase and folate dependent enzymes of purine biosynthesis, the efficacy of this agent may involve blockade of these pathways. We hypothesize that blockage of aminoimidazole carboxamide ribotide transformylase, the folate dependent enzyme responsible for the insertion of carbon 2 into the purine ring, produces an immunosuppression mediated by secondary inhibition of adenosine deaminase, and S-adenosyl homocystein hydrolase by aminoimidazolecarboxamide metabolites. This mechanism of immunosuppression may explain the clinical effect of methotrexate, 10-deazaaminopterin, and possibly sulfasalazine. Since purine biosynthesis is a fundamental process, blockading this pathway may also decrease leukotriene production and interleukin-1 expression, which also could contribute to the efficacy of methotrexate.

Iron compounds catalyze the oxidation of 10-formyl-5,6,7,8 tetrahydrofolic acid to 10-formyl-7,8 dihydrofolic acid.

We have previously demonstrated that 10-formyl-7,8-dihydrofolic acid (10-HCO-H2folate) is a better substrate for mammalian aminoimidazolecarboxamide ribotide transformylase (EC 2.1.2.3) than is 10-formyl-5,6,7,8-tetrahydrofolic acid (10-HCO-H4folate) (J.E. Baggott, G.L. Johanning, K.E. Branham, C.W. Prince, S.L. Morgan, I. Eto, W.H. Vaughn, Biochem. J. 308, 1995, 1031-1036). Therefore, the possible metabolism of 10-HCO-H4folate to 10-HCO-H2folate was investigated. A spectrophotometric assay for the oxidation of 10-HCO-H4folate to 10-HCO-H2folate which measures the disappearance of reactant (decrease in absorbance at 356 nm after acidification of aliquots of the reaction solution), is used to demonstrate that iron compounds catalyze the oxidation of 10-HCO-H4folate to 10-HCO-H2folate in the presence and absence of ascorbate. Chromatographic separation of the 10-HCO-H2folate product from the reaction mixture, its UV spectra, a microbiological assay and an enzymatic assay established that the iron-catalyzed oxidation product of 10-HCO-H4folate was 10-HCO-H2folate; without substantial side reactions. The inhibition of this iron-catalyzed oxidation by deferoxamine, apotransferrin and mannitol and the stimulation by citrate and EDTA indicated of a mechanism involving a reaction of 10-HCO-H4folate with hydroxyl radicals (*OH) generated by Fenton chemistry. The presence of "free iron" (e.g., Fe3+ citrate) in bile, cerebrospinal fluid and intracellularly suggest that this oxidation could occur in vivo and that 10-HCO-H4folate may be a *OH scavenger.

Metabolism of 10-formyldihydrofolate in humans.

The metabolism of 10-formyldihydrofolate is reviewed in this article. It had been the dogma that only tetrahydrofolates participate in enzyme-catalyzed one-carbon transfer reactions, until we showed in 1986 that 10-formyldihydrofolate serves as a substrate for aminoimidazolecarboxamide ribotide (AICAR) transformylase. Our data from studies in humans, cultured cells and bacteria as well as in vitro experiments indicate that the oxidation of 10-formyltetrahydrofolate to 10-formyldihydrofolate takes place, and 1 0-formyldihydrofolate is subsequently converted to dihydrofolate by AICAR transformylase. Dihydrofolate is then reduced to tetrahydrofolate and further metabolized by the well-established enzyme reactions. We believe that a new folate metabolic map is needed which incorporates the oxidation of 10-formyltetrahydrofolate and the utilization of 10-formyldihydrofolate by AICAR transformylase.

Racial or ethnic differences in allele frequencies of single-nucleotide polymorphisms in the methylenetetrahydrofolate reductase gene and their influence on response to methotrexate in rheumatoid arthritis.

BACKGROUND: The anti-folate drug methotrexate (MTX) is commonly used to treat rheumatoid arthritis. OBJECTIVE: To determine the allele frequencies of five common coding single-nucleotide polymorphisms (SNPs) in the methylenetetrahydrofolate reductase (MTHFR) gene in African-Americans and Caucasians with rheumatoid arthritis and controls to assess whether there are differences in allele frequencies among these ethnic or racial groups and whether these SNPs differentially affect the efficacy or toxicity of MTX. METHODS: Allele frequencies in the 677, 1298 and 3 additional SNPs in the MTHFR coding region in 223 (193 Caucasians and 30 African-Americans) patients with rheumatoid arthritis who previously participated in one of two prospective clinical trials were characterised, and genotypes were correlated with the efficacy and toxicity of MTX. Another 308 subjects with rheumatoid arthritis who participated in observational studies, one group predominantly Caucasian and the other African-American, as well as 103 normal controls (53 African-Americans and 50 Caucasians) were used to characterise allele frequencies of these SNPs and their associated haplotypes. RESULTS: Significantly different allele frequencies were seen in three of the five SNPs and haplotype frequencies between Caucasians and African-Americans. Allele frequencies were similar between patients with rheumatoid arthritis and controls of the same racial or ethnic group. Frequencies of the rs4846051C, 677T and 1298C alleles were 0.33, 0.11 and 0.13, respectively, among African-Americans with rheumatoid arthritis. Among Caucasians with rheumatoid arthritis, these allele frequencies were 0.08 (p<0.001 compared with African-Americans with rheumatoid arthritis), 0.30 (p = 0.002) and 0.34 (p<0.001), respectively. There was no association between SNP alleles or haplotypes and response to MTX as measured by the mean change in the 28-joint Disease Activity Score from baseline values. In Caucasians, the 1298 A (major) allele was associated with a significant increase in MTX-related adverse events characteristic of a recessive genetic effect (odds ratio 15.86, 95% confidence interval 1.51 to 167.01; p = 0.021), confirming previous reports. There was an association between scores of MTX toxicity and the rs4846051 C allele, and haplotypes containing this allele, in African-Americans, but not in Caucasians. CONCLUSIONS: : These results, although preliminary, highlight racial or ethnic differences in frequencies of common MTHFR SNPs. The MTHFR 1298 A and the rs4846051 C alleles were associated with MTX-related adverse events in Caucasians and African-Americans, respectively, but these findings should be replicated in larger studies. The rs4846051 SNP, which is far more common in African-Americans than in Caucasians, can also be proved to be a useful ancestry informative marker in future studies on genetic admixture.