Ronney A. De Abreu

Methotrexate in rheumatoid arthritis: An updatewith focus on mechanisms involved in toxicity

OBJECTIVES To provide an update of the current knowledge of the mechanism of action of low-dose methotrexate (MTX) in the treatment of patients with rheumatoid arthritis (RA), with an emphasis on the mechanisms involved in toxicity. We also considered strategies currently used to prevent or decrease toxicity of MTX. METHODS We reviewed the literature dealing with the subjects of MTX treatment of RA, the mechanisms of action of low-dose MTX regarding efficacy and toxicity, and strategies used to prevent or decrease MTX toxicity. RESULTS MTX is a fast working and effective second-line antirheumatic agent (SLA). Its use is limited mainly because of side effects. The mechanisms of action regarding efficacy and toxicity are probably determined by different metabolic pathways. Recent data indicate that the antiinflammatory effect of MTX is mediated by adenosine. However, MTX side effects can only partly be explained by folate antagonism and may also depend on its action on other related metabolic pathways. The latter include the homocysteine-methionine-polyamine pathway and purine metabolism. Variants in these metabolic routes (ie, the C677T mutation in the methylene-tetrahydrofolate reductase [MTHFR] gene), may predispose to the development of side effects. Currently the most promising strategy to decrease or prevent toxicity of MTX is concomitant prescription of folic acid or folinic acid. Other strategies are currently under investigation. CONCLUSIONS MTX benefits a majority of RA patients. Approximately 30% of patients, however, abandon treatment because of drug-related side effects. Folic acid or folinic acid likely reduces MTX toxicity. More data, however, are needed to evaluate a potential detrimental effect on the antirheumatic efficacy of MTX.

6-Mercaptopurine: Cytotoxicity and biochemical pharmacology in human malignant T-lymphoblasts

The effects of prolonged exposure to 2 and 10 microM 6-mercaptopurine (6MP) in the human lymphoblastic T-cell line MOLT-4 were studied with respect to cell-kinetic parameters, phosphoribosyl pyrophosphate (PRPP) and purine ribonucleotide levels, formation of 6MP-nucleotides, especially methyl-thio-IMP (Me-tIMP), DNA and RNA synthesis ([32P] incorporation), and [8-14C]6MP incorporation into newly synthesized DNA and RNA. The results provided new insights into the complex mechanism of action of 6MP in human malignant lymphoblasts. Exposure to 2 microM 6MP resulted in a rapid inhibition of purine de novo synthesis (PDNS) by increased levels of Me-tIMP, resulting in increased PRPP levels and decreased purine ribonucleotides, affecting cell growth and clonal growth, and less cell death. DNA synthesis decreased, associated with an increasing delay of cells in S phase. Incorporation of thioguanine nucleotides into newly synthesized DNA resulted in an increasing arrest of cells in G2 + M phase. RNA synthesis, initially decreased, recovered partially, associated with a recovery of purine ribonucleotides. New formation of 6MP-nucleotides (tIMP) was only detected within the first 24 hr, and 6MP levels in the culture medium were already undetectable after 6 hr of exposure to 2 microM, indicating a high rate of incorporation and complete conversion of 6MP within this period. Incorporation of 6MP-nucleotides into DNA was 5 times as high as incorporation into RNA. Exposure to 10 microM 6MP resulted in early cytotoxicity at 24 hr, associated with a complete inhibition of PDNS by a large pool of Me-tIMP and lower levels of purine ribonucleotides as compared to 2 microM 6MP. A more severe delay of cells in S phase was associated with an inhibition of DNA synthesis to 14% of control within the first 24 hr, and an arrest in G2 + M phase. Further increasing levels of Me-tIMP caused an arrest of cells and late cytotoxicity in S phase at 48 hr, preventing further progression into G2 + M phase. Our data suggest that inhibition of PDNS due to Me-tIMP is a crucial event in the mechanism of 6MP cytotoxicity. It is responsible for decreased RNA synthesis and decreased availability of natural deoxyribonucleotides, causing a delay of DNA synthesis in S phase. This enhances incorporation of 6MP as thioguanine nucleotides into DNA in the S phase and subsequent late cytotoxicity in the G2 phase. However, with high concentrations of 6MP, the large pool of Me-tIMP causes severe reduction of natural deoxyribonucleotides in lymphoblasts with an active PDNS.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacogenetic and clinical aspects of dihydropyrimidine dehydrogenase deficiency.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). A deficiency of DPD is increasingly being recognized as the cause of an important pharmacogenetic syndrome. The importance of DPD deficiency in the aetiology of unexpected severe 5FU toxicity has been demonstrated by the fact that, in 39-;59% of cases, decreased DPD activity could be detected in peripheral blood mononuclear (PBM) cells. It was observed that 55% of the patients with a decreased DPD activity suffered from grade IV neutropenia compared with 13% of the patients with a normal DPD activity (P = 0·01). Furthermore, toxicity developed significantly earlier in patients with low DPD activity than in patients with normal DPD activity (10·0 ± 7·6 versus 19·1 ± 15·3 days, P < 0·05). In patients suffering from severe 5FU-associated toxicity, 11 mutations have been identified in DPYD, including one splice-site mutation (IVS14 + 1G→A), one nonsense mutation (E386X), four missense mutations (M166V, V335L, I560S, D949V) and five polymorphisms (C29R, R21Q, S534N, I543V, V732I). Considering the common use of 5FU in the treatment of cancer patients, the severe 5FU-related toxicities in patients with a low DPD activity and the high prevalence of the IVS14 + 1G→A mutation, analysis of the DPD activity in PBM cells or screening for the IVS14 + 1G→A mutation should be routinely carried out prior to the start of treatment with 5FU.

The importance of methylthio-IMP for methylmercaptopurine ribonucleoside (Me-MPR) cytotoxicity in Molt F4 human malignant T-lymphoblasts

The importance of methyl-thioIMP (Me-tIMP) formation for methylmercaptopurine ribonucleoside (Me-MPR) cytotoxicity was studied in Molt F4 cells. Cytotoxicity of Me-MPR is caused by Me-tIMP formation with concomitant inhibition of purine de novo synthesis. Inhibition of purine de novo synthesis resulted in decreased purine nucleotide levels and enhanced 5-phosphoribosyl-1-pyrophosphate (PRPP) levels, with concurrent increased pyrimidine nucleotide levels. The Me-tIMP concentration increased proportionally with the concentration of Me-MPR. High Me-tIMP concentration also caused inhibition of PRPP synthesis. Maximal accumulation of PRPP thus occurred at low Me-MPR concentrations. As little as 0.2 microM Me-MPR resulted already after 2 h in maximal inhibition of formation of adenine and guanine nucleotides, caused by inhibition of purine de novo synthesis by Me-tIMP. Under these circumstances increased intracellular PRPP concentrations could be demonstrated, resulting in increased levels of pyrimidine nucleotides. So, in Molt F4 cells, formation of Me-tIMP from Me-MPR results in cytotoxicity by inhibition of purine de novo synthesis.

Purine de novo synthesis as the basis of synergism of methotrexate and 6-mercaptopurine in human malignant lymphoblasts of different lineages

Methotrexate (MTX) causes an inhibition of purine de novo synthesis (PDNS), resulting in increased intracellular availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) in human malignant lymphoblasts with an active PDNS. Normal bone marrow cells and peripheral blood lymphocytes lack this capacity. The increased levels of PRPP can be used for enhanced incorporation of 6-mercaptopurine (6MP), indicating a potential time-, sequence- and dose-dependent synergism of both drugs. The effects of 0.02 microM and 0.2 microM MTX on the PDNS of MOLT-4 (T-), RAJI (B-) and KM-3 (non-B-non-T-) human malignant lymphoblasts were studied with respect to PRPP levels, aminoimidazolecarboxamide ribonucleosidemonophosphate (AICAR) levels and the incorporation of labeled glycine into purine metabolites. These results were correlated with the activity of the PDNS (labeled glycine incorporation) and the purine salvage pathway (labeled hypoxanthine incorporation) in untreated cells. Inhibition of PDNS by 0.02 microM MTX was complete in KM-3 cells with a moderately active PDNS and salvage pathway. RAJI cells, with a relatively low PDNS and high salvage pathway, demonstrated an incomplete, but increasing inhibition of PDNS, whereas inhibition of PDNS in MOLT-4 cells with both pathways active was minimal and recovered in time. Treatment with 0.2 microM MTX resulted in a complete inhibition of PDNS in all cell lines. After treatment with MTX an enhanced incorporation of labeled hypoxanthine and 6MP was noticed, confirming the potential rescue from MTX cytotoxicity by hypoxanthine and a potential synergism of MTX and 6MP on cytotoxicity. The enhanced incorporation of 6MP was more obvious in RAJI and KM-3 cells in comparison with MOLT-4 cells. These data demonstrate the important role of both the activities of the PDNS and the purine salvage pathway in malignant lymphoblasts of different subclasses with respect to the synergism of MTX and 6MP.

High-performance liquid chromatographic assay for identification and quantitation of nucleotides in lymphocytes and malignant lymphoblasts

A method for the identification and quantitation of nucleotide pools in lymphocytes and leukemic blasts is described. Separation of these metabolites was performed by anion-exchange high-performance liquid chromatography using a pH and concentration gradient consisting of several linear steps. The mono-, di- and triphosphates of adenosine, cytidine, guanosine, inosine, uridine and xanthosine could conveniently be separated together with NAD+, cyclic AMP, NADP+ and uridinediphosphoglucose (UDPG). In addition, data on the accuracy and precision of the method are given and its potentials for use in the analysis of nucleotide pools in leukemic lymphoblasts are illustrated.

Sequence-, time- and dose-dependent synergism of methotrexate and 6-mercaptopurine in malignant human T-lymphoblasts☆

Methotrexate (MTX) and 6-mercaptopurine (6MP) are common drugs in the oral maintenance therapy of acute lymphoblastic leukemia (ALL). On the basis of their biochemical effects on cell metabolism, a sequence-dependent synergism might be anticipated. In order to investigate this hypothesis, MOLT-4 human malignant T-lymphoblasts were incubated with various concentrations of MTX. The time at which maximal increase of intracellular 5-phosphoribosyl-1-pyrophosphate (PRPP) levels was found correlated with the concentrations of MTX used. Determination of aminoimidazolecarboxamide ribonucleoside monophosphate (AICAR) levels and labeled glycine incorporation into purine metabolites revealed an incomplete inhibition of purine de novo synthesis after incubation with 0.02 microM MTX, and a complete inhibition with 0.2 microM MTX. After prolonged periods of incubation, glutamine exhaustion of the medium caused inhibition of purine de novo synthesis in MTX-untreated cells, with a concomitant increase of PRPP levels. Addition of glutamine to the medium prevented this phenomenon. The increased availability of PRPP after pretreatment with MTX can be used for enhanced intracellular incorporation of hypoxanthine and 6MP in their respective nucleotides. The time- and dose-dependent effects of MTX on PRPP levels correlated with the enhanced incorporation of hypoxanthine and 6MP. The data presented in this study demonstrate that a synergistic action of the combination of MTX and 6MP can be anticipated in malignant lymphoblasts with an active purine de novo synthesis depending on the concentration of MTX and on the time and sequence of administration of both drugs.

Reference values for amino acids in cerebrospinal fluid of children determined using ion-exchange chromatography with fluorimetric detection

One thousand specimens of CSF were collected from subjects ranging in age from newborn to 18 yr, who were undergoing a diagnostic lumbar puncture. Sixty-two samples were judged retrospectively as being suitable for calculating reference age-related values. The analyses were performed by an amino acid analyser using ion-exchange chromatography with fluorimetric detection giving a tenfold increase in sensitivity, thereby enhancing the diagnostic capabilities. As many as 36 known compounds could be detected, additionally 10 we could not identify. In children older than 3 yr nine of the identified compounds showed age-dependency. We found 22 amino acids to be significantly higher in infants younger than 1 yr, with only gamma-aminobutyric acid being significantly lower in infants. Alpha-aminoadipic acid showed a sex difference, being slightly higher in girls.

High-performance liquid chromatographic determination of purine and pyrimidine bases, ribonucleosides, deoxyribonucleosides and cyclic ribonucleotides in biological fluids

A method is presented for the separation and quantitative determination of compounds normally related to purine and pyrimidine metabolism in biological material. The retention behaviour of nucleobases, ribonucleosides, deoxyribonucleosides and cyclic ribonucleotides has been systematically investigated by reversed-phase high-performance liquid chromatography using a non-linear gradient. Ultimately a separation of the purine and pyrimidine compounds was achieved in a 35-min run with an average detection limit of 5-10 pmol per injection. Recoveries of standards added to urine, plasma or serum were 96 +/- 5%.

A biochemical basis for synergism of 6-mercaptopurine and mycophenolic acid in Molt F4, a human malignant T-lymphoblastic cell line

6-Mercaptopurine (6MP) cytotoxicity was studied in Molt F4 cells, a T-cell acute lymphoblastic leukemia (ALL) cell line. The effects on cytotoxicity were concentration-dependent. Measurements of intracellular thionucleotide intermediates of 6MP demonstrated a rapid rise of thio-IMP (tIMP) levels, and subsequently a rapid decrease. Thio-GMP (tGMP) and methyl-thio-IMP (Me-tIMP) appeared later in time, and persisted longer. Mycophenolic acid (MPA), a specific inhibitor of IMP dehydrogenase (IMPDH), was used to inhibit the conversion of tIMP into tGMP, thereby decreasing the incorporation of 6MP into DNA. A synergistic effect on cell viability and cell growth was observed when cells were treated with a combination of 2 microM 6MP and 0.5 microM MPA. Also, intracellular Me-tIMP increased 5 times with the combination. Based on the increase of Me-tIMP concentration and the observed synergism between 6MP and MPA, we conclude that methylation of tIMP into Me-tIMP is an important alternative route for 6MP cytotoxicity.