Freidoun Albertioni

Differences between children and adults in thiopurine methyltransferase activity and metabolite formation during thiopurine therapy: Possible role of concomitant methotrexate

This study examined the role of thiopurine methyltransferase (TPMT) polymorphism in the metabolism and clinical effects of azathioprine and 6-mercaptopurine in the treatment of inflammatory bowel disease and childhood leukemia. The current hypothesis is that the cytotoxic effects of thiopurines are caused by the incorporation of thioguanine nucleotides into DNA. In this context, S-methylation catalyzed by TPMT can be regarded as a competing metabolic pathway. The authors assayed the TPMT activity in red blood cells from 122 patients treated with azathioprine or 6-mercaptopurine (83 adults with inflammatory bowel disease and 39 children with acute lymphoblastic leukemia) and in 290 untreated controls (219 adult blood donors and 71 children). The concentrations of thioguanine nucleotides and methylthioinosine monophosphate were also assayed in red blood cells from the patients. The TPMT activity and the concentrations of methylthioinosine monophosphate and thioguanine nucleotides were higher in children than in adults. All children but no adult patient received concomitant methotrexate. Interaction between methotrexate and 6-mercaptopurine has been described, and may explain the results. Low TPMT activity in adult patients with inflammatory bowel disease correlated to an increased incidence of adverse drug reactions. However, there was no correlation between TPMT activity and the red blood cell concentrations of methylthioinosine monophosphate or thioguanine nucleotides, or between the concentrations of these metabolites and the occurrence of adverse effects. The results show that the role of thiopurine metabolism for drug effects is complex.

Clinical and pharmacokinetic risk factors for high-dose methotrexate-induced toxicity in children with acute lymphoblastic leukemia--a logistic regression analysis.

The clinical and pharmacokinetic risk factors for toxicity after high-dose methotrexate (MTX) in children with acute lymphoblastic leukemia were evaluated using a multivariate statistical analysis. Plasma samples were collected after 44 24-h infusions of MTX (5 or 8 g/m2) in 13 children (age 3.3-12.9 years) and subsequently analyzed by HPLC to determine the MTX and 7-hydroxymethotrexate (7-OHMTX) concentrations. Toxicity was evaluated according to the WHO criteria. Severe toxicity was not observed. Oral mucositis (WHO grade > or = 1) was significantly related to a high plasma MTX concentration at 28 h after starting the infusion (p = 0.013), a low ratio of plasma 7-OHMTX/MTX at 66 h after starting the infusion (p = 0.049), and a slow clearance of MTX (p = 0.048). The risk of leukopenia (WHO grade > or = 2) increased significantly with the number of courses (p = 0.02). Increasing age and a long exposure to a high MTX concentration in plasma (AUC) were significant risk factors (p = 0.047 and p = 0.009, respectively) for developing elevated liver enzymes (ALAT) (WHO grade > or = 2). This study shows how a statistical model can be used to identify clinical and pharmacokinetic factors that may influence MTX-induced toxicity. The therapeutic ratio could thereby potentially be improved.

Mechanisms of anti-cancer action and pharmacology of clofarabine

Clofarabine, a next-generation deoxyadenosine analogue, was developed on the basis of experience with cladribine and fludarabine in order to achieve higher efficacy and avoid extramedullary toxicity. During the past decade this is the only drug granted approval for treatment of pediatric acute leukemia. Recent clinical studies have established the efficacy of clofarabine in treating malignancies with a poor prognosis, such as adult, elderly, and relapsed pediatric leukemia. The mechanisms of its anti-cancer activity involve a combination of direct inhibition of DNA synthesis and ribonucleotide reductase and induction of apoptosis. Due to this broad cytotoxicity, this drug is effective against various subtypes of leukemia and is currently being tested as an oral formulation and for combination therapy of both leukemias and solid tumors. In this review we summarize current knowledge pertaining to the molecular mechanisms of action and pharmacological properties of clofarabine, as well as clinical experiences with this drug with the purpose of facilitating the evaluation of its efficacy and the development of future therapies.

Mechanisms of antifolate resistance and methotrexate efficacy in leukemia cells

Antifolates are the first class of antimetabolites introduced to clinic about 6 decades ago. Now, after several years of administration of antifolates against malignancies and particularly leukemia, we are still trying to achieve a full understanding of the mechanisms of action and resistance to these agents. The present article covers different factors able to influence efficacy of antifolates on leukemic cells, the known mechanisms of resistance to methotrexate (MTX) and strategies to overcome these mechanisms. The dominant factors that are contributed to tolerance to cytocidal effects of MTX including pharmacokinetic factors, impaired transmembrane uptake as the most frequent rote of provoking resistance to MTX, augmented drug efflux, impaired intracellular polyglutamation as a determining process of drug efficacy, alterations in expression or activity of target enzymes and increased intracellular folate pools; and finally role of 7-hydroxymethotrexate on response or resistance to MTX will be discussed in more detail. Finally, strategies to overcome resistance to antifolates are discussed.

Thiopurines: factors influencing toxicity and response.

Thiopurines are the backbone of current anti-leukemia regimens and have also been effective immunosuppressive agents for the past half a century. Extensive research on their mechanism of action has been undertaken, yet many issues remain to be addressed to resolve unexplained cases of thiopurine toxicity or treatment failure. The aim of this review is to summarize current knowledge of the mechanism of thiopurine action in experimental models and put into context with clinical observations. Clear understanding of their metabolism will contribute to maximizing efficacy and minimizing toxicity by individually tailoring therapy according to the expression profile of relevant factors involved in thiopurine activation pathway.

Regulation of p53R2 and its role as potential target for cancer therapy

p53R2, a recently discovered small subunit of human ribonucleotide reductase, is believed to play essential roles in DNA repair, mtDNA synthesis, and protection against oxidative stress. Because of the positive correlation between the level of this protein and drug sensitivity and tumor invasiveness, it constitutes a potential target for anticancer drugs as well as a diagnostic marker in cancer.

Pharmacological basis for cladribine resistance in a human acute T lymphoblastic leukaemia cell line selected for resistance to etoposide

Cross‐resistance between different classes of anti‐neoplastic agents can jeopardize successful combination cancer chemotherapy. In this study, we observed an unexpected cross‐resistance between the podophyllotoxine derivative etoposide (VP) and the nucleoside analogue cladribine (CdA) in CCRF‐CEM cells developed for resistance to VP. The resistant cells also displayed 14‐ and twofold resistance to cytarabine (ara‐C) and gemcitabine respectively. Closer analysis of these cells showed that they contained lower amounts of topoisomerase (topo) IIα (P < 0·001) and β protein (P < 0·026), formed substantially lower amounts of the topo II–DNA complex, and had a markedly decreased level of Fas (CD95/APO‐1)‐ligand mRNA expression. Interestingly, Fas expression in the resistant cells did not differ from that in the parental cell line. No differences were observed in the accumulation/efflux of daunorubicin or in the gene expressions of P‐glycoprotein, multidrug resistance‐associated protein and the lung resistance‐related protein. The activity of deoxycytidine kinase (dCK), responsible for activation of CdA and ara‐C, was the same for resistant and wild‐type cells. However, there was an increase in the activity of the cytosolic 5′‐nucleotidases (5′‐NT), responsible for deactivation of nucleotides, amounting to 206% (P < 0·001) for the high Km and 134% (P < 0·331) for the low Km 5′‐NT in resistant cells. The high Km 5′‐NT is probably responsible for the decreased amount of the active metabolite CdA 5′‐triphosphate [40% decreased (P < 0·045)], as well as for other purine ribonucleosides and deoxyribonucleosides triphosphates in the resistant cells. In contrast, a significantly higher deoxycytidine triphosphate (dCTP) level (167%, P < 0·001) was observed in the resistant cells. Thus, this study suggests that the major cause of resistance to the nucleoside analogues CdA and ara‐C in cells selected for resistance to VP is a result of metabolic alterations producing increased activity of 5′‐NT and higher dCTP levels. Furthermore, these results indicate that there is a common factor in the regulation of nucleotide‐degrading enzymes and DNA topoisomerases, which may be altered in cross‐resistant cells.

Simultaneous quantitation of methotrexate and its two main metabolites in biological fluids by a novel solid-phase extraction procedure using high-performance liquid chromatography

We have developed an assay for the simultaneous determination of methotrexate (MTX) and its main metabolites, 7-hydroxymethotrexate (7-OHMTX) and 2,4-diamino-N10-methylpteroic acid (DAMPA) in plasma, urine and saliva meeting the requirement of rapidity for routine use in high-dose MTX therapy and the requirement of sensitivity for its potential use in therapeutic drug monitoring in low-dose MTX therapy. Sample preparation is based on solid-phase extraction using C8 Isolute cartridges. Chromatographic separation was achieved with a reversed-phase column (C18), and quantitation by subsequent exposure to UV light of 254 nm, which converted MTX and its two metabolites by photolytic oxidation to fluorescent products. The recoveries of MTX, 7-OHMTX and DAMPA from plasma at 100 nmol/l were 85.8, 91.1 and 102.3%, respectively. The limits of detection for MTX, 7-OHMTX and DAMPA in plasma and saliva were 0.1 nmol/l. In urine the limit of detection was 10 nmol/l for all compounds. The limits of quantitation in plasma and saliva were 0.5 nmol/l for all compounds.

Pharmacological basis for cladribine resistance.

The inherent or acquired resistance of leukemic cells to cytostatic agents is a major clinical challenge. The purpose of this review was to elucidate and analyse the available data concerning mechanisms of resistance of cladribine with emphasis on recent advances in the characterization of activating and inactivating enzymes in the induction of resistance to cladribine. All available in vitro and clinical data on cladribine was undertaken. Cladribine, unlike many other drugs, is toxic to both dividing and indolent lymphoid malignancies. Cladribine is a prodrug and must be phosphorylated intracellularly to cladribine-monophosphate (MP) by the nuclear/cystosol enzyme deoxycytidine kinase (dCK) and the mitochondrial enzyme deoxyguanosine kinase. The cytotoxicity mainly depends on the accumulation of cladribine-triphosphates (TP) after phosphorylation of cladribine-MP by nucleoside monophosphate kinase and nucleoside diphosphate kinase. 5′-Nucleotidase (5′-NT) dephosphorylates cladribine-MP and the accumulation of cladribine-TP depends on the ratio of dCK and 5′-NT in the cells. The mechanisms underlying cladribine resistance are multifactorial, e.g. decreased nucleoside transport, decreased activity or deficiency of dCK, altered intracellular pools of competing nucleotides, altered regulation of ribonucleotide reductase and increased drug inactivation by 5′-NT. Finally, cladribine resistance may be a consequence of a defective induction of apoptosis. In spite of the fact that more than one mechanism can contribute to a cladribine resistance phenotype, a reduction in dCK activity is probably the major determinant of cladribine resistance. Insight into the mechanism of action and resistance to cladribine is crucial for its optimal use as well as for the development of newer analogues.

Gene expression profiling of leukemia T-cells resistant to methotrexate and 7-hydroxymethotrexate reveals alterations that preserve intracellular levels of folate and nucleotide biosynthesis ☆

In vitro treatment of human T-cell leukemia cells with 7-hydroxymethotrexate, the major metabolite of methotrexate resulted in acquired resistance as a result of the complete loss of folypolyglutamate synthetase (FPGS) activity. This was in contradistinction to the major modality of antifolate resistance of impaired drug transport in leukemia cells exposed to methotrexate. To identify the genes associated with methotrexate and 7-hydroxymethotrexate resistance, we herein explored the patterns of genome-wide expression profiles in these antifolte-resistant leukemia sublines. mRNA levels of the reduced folate carrier, the primary influx transporter of folates and antifolates, were down-regulated more than two-fold in methotrexate-resistant cells. The dramatic loss of FPGS activity in 7-hydroxymethotrexate-resistant cells was associated with alterations in the expression of various genes aimed at preserving reduced folates and/or enhancing purine nucleotide biosynthesis, e.g. methylene tetrahydrofolate reductase, glycinamide ribonucleotide formyltransferase, adenosine deaminase, cystathionine beta synthase, as well as the ATP-dependent folate exporters BCRP/ABCG2 and MRP1/ABCC1. The observed changes in gene expression were generally not paralleled by acquired DNA copy numbers alterations, suggesting transcriptional regulatory mechanisms. Interestingly, gene expression of DNA/RNA metabolism and transport genes were more profoundly altered in methotrexate-resistant subline, whereas in 7-hydroxymethotrexate-resistant cells, the most profoundly affected groups of genes were those encoding for proteins involved in metabolism and cellular proliferation. Thus, the present investigation provides evidence that 7-hydroxymethotrexate induces gene expression alterations and an antifolate resistance modality that are distinct from its parent drug methotrexate.