mercaptopurine and 6-thioxanthine

Up to 1/5 of patients with wildtype thiopurine-S-methyltransferase (TPMT) activity prescribed azathioprine (AZA) or mercaptopurine (MP) demonstrate a skewed drug metabolism in which MP is preferentially methylated to yield methylmercaptopurine (MeMP). This is known as thiopurine hypermethylation and is associated with drug toxicity and treatment non-response. Co-prescription of allopurinol with low dose AZA/MP (25-33%) circumvents this phenotype and leads to a dramatic reduction in methylated metabolites; however, the biochemical mechanism remains unclear. Using intact and lysate red cell models we propose a novel pathway of allopurinol mediated TPMT inhibition, through the production of thioxanthine (TX, 2-hydroxymercaptopurine). In red blood cells pre-incubated with 250 μM MP for 2h prior to the addition of 250 μM TX or an equivalent volume of Earle's balanced salt solution, there was a significant reduction in the concentration of MeMP detected at 4h and 6h in cells exposed to TX (4 h, 1.68, p=0.0005, t-test). TX acts as a direct TPMT inhibitor with an apparent Ki of 0.329 mM. In addition we have confirmed that the mechanism is relevant to in vivo metabolism by demonstrating raised urinary TX levels in patients receiving combination therapy. We conclude that the formation of TX in patients receiving combination therapy with AZA/MP and allopurinol, likely explains the significant reduction of methylated metabolites due to direct TPMT inhibition.

Topics: Adult; Allopurinol; Azathioprine; Case-Control Studies; Drug Therapy, Combination; Erythrocytes; Female; Humans; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methyltransferases; Oxypurinol; Prospective Studies; Xanthines

The anticancer drug, 6-mercaptopurine (6MP) is subjected to metabolic clearance through xanthine oxidase (XOD) mediated hydroxylation, producing 6-thiouric acid (6TUA), which is excreted in urine. This reduces the effective amount of drug available for therapeutic efficacy. Co-administration of allopurinol, a suicide inhibitor of XOD, which blocks the hydroxylation of 6MP inadvertently enhances the 6MP blood level, counters this reduction. However, allopurinol also blocks the hydroxylation of hypoxanthine, xanthine (released from dead cancer cells) leading to their accumulation in the body causing biochemical complications such as xanthine nephropathy. This necessitates the use of a preferential XOD inhibitor that selectively inhibits 6MP transformation, but leaves xanthine metabolism unaffected.. Here, we have characterized two such unique inhibitors namely, 2-amino-6-hydroxy-8-mercaptopurine (AHMP) and 2-amino-6-purinethiol (APT) on the basis of IC50 values, residual activity in bi-substrate simulative reaction and the kinetic parameters like Km, Ki, kcat. The IC50 values of AHMP for xanthine and 6MP as substrate are 17.71 +/- 0.29 microM and 0.54 +/- 0.01 microM, respectively and the IC50 values of APT for xanthine and 6MP as substrates are 16.38 +/- 0.21 microM and 2.57 +/- 0.08 microM, respectively. The Ki values of XOD using AHMP as inhibitor with xanthine and 6MP as substrate are 5.78 +/- 0.48 microM and 0.96 +/- 0.01 microM, respectively. The Ki values of XOD using APT as inhibitor with xanthine and 6MP as substrate are 6.61 +/- 0.28 microM and 1.30 +/- 0.09 microM. The corresponding Km values of XOD using xanthine and 6MP as substrate are 2.65 +/- 0.02 microM and 6.01 +/- 0.03 microM, respectively. The results suggest that the efficiency of substrate binding to XOD and its subsequent catalytic hydroxylation is much superior for xanthine in comparison to 6MP. In addition, the efficiency of the inhibitor binding to XOD is much more superior when 6MP is the substrate instead of xanthine. We further undertook the toxicological evaluation of these inhibitors in a single dose acute toxicity study in mice and our preliminary experimental results suggested that the inhibitors were equally non-toxic in the tested doses.. We conclude that administration of either APT or AHMP along with the major anti-leukemic drug 6MP might serve as a good combination cancer chemotherapy regimen.

Topics: Adenine; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydroxylation; Kinetics; Mercaptopurine; Models, Chemical; Molecular Structure; Substrate Specificity; Thioguanine; Uric Acid; Xanthine; Xanthine Oxidase; Xanthines

This study describes approaches for stacking a large volume of sample solutions containing a mixture of mercaptopurine monohydrate, 6-methylmercaptopurine, thioguanine, thioguanosine, and thioxanthine in capillary electrophoresis (CE). After filling the run buffer (60 mM borate buffer, pH 8.5), a large sample volume was loaded by hydrodynamic injection (2.5 psi, 99.9 s), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (-15 kV). Monitoring the current and reversing the polarity when 95% of current recovered, the separation of anionic analytes was performed in a run buffer < 20 kV. Around 44- to 90-fold improvement of sensitivity for five analytes was achieved by large-volume stacking with polarity switching when compared with CE without stacking. This method was feasible for determination of the analytes spiked in plasma. Removing most of electrolytes from plasma is a key step for performing large-volume sample stacking. Solid-phase extraction was used for pretreatment of biological samples. To our knowledge, this study is one of few applications showing the possibilities of this stacking procedure to analyze biological samples by large-volume sample stacking with polarity switching (LVSSPS) in CE.

Topics: Electrophoresis, Capillary; Guanosine; Humans; Mercaptopurine; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Sensitivity and Specificity; Thioguanine; Thionucleosides; Xanthines

Despite widespread use of azathioprine in organ transplant recipients, the mechanism of its myelotoxicity remains unclear. The aim of this study was to assess the importance of thiopurine metabolites on bone marrow toxicity.. We investigated the relationship between intracellular concentrations of 6-thioguanine (6-TGN), 6-mercaptopurine (6-MPN) and 6-thioxanthine (6 TXN) nucleotides and the absolute count of white or red cells in forty-seven lung or heart/lung transplant patients after oral administration of azathioprine.. No significant correlation between red cell concentrations of 6-TGN or total thiopurine metabolites and white or red cell counts was found, with no difference between the sexes. Likewise, high 6-TGN levels were not related to bone marrow depletion.. These results suggest that red blood cell 6-TGN alone do not predict the haematopoietic toxicity of azathioprine.

Topics: Adolescent; Adult; Aged; Azathioprine; Bone Marrow; Female; Heart-Lung Transplantation; Humans; Immunosuppressive Agents; Lung Transplantation; Male; Mercaptopurine; Middle Aged; Thioguanine; Xanthines

A method for the analysis of thiopurine nucleotides in human transbronchial lung biopsy was developed. The sample treatment procedure is based on perchloric acid homogenisation and deproteinisation with dithiothreitol and hydrolysis of thiopurine nucleotides into their free bases by heating of the acid extract. Then, the free bases were analyzed in the gradient elution mode on a Hypersil ODS, 3-microns column using dihydrogenphosphate buffer-methanol as eluent. Mean analytical recoveries for 6-thioguanosine monophosphate and 6-thioinosinic acid from lung tissue were 97.0 +/- 2.0 and 98.0 +/- 1.8% at a concentration of 3.0 nmol/ml and the minimum detectable amounts were 3.5 and 2 pmol, respectively. The procedure described is simple and represents a suitable method for the investigation of thiopurine nucleotides in tissues.

Topics: Chromatography, High Pressure Liquid; Humans; Lung; Lung Transplantation; Mercaptopurine; Thioguanine; Xanthines