mercaptopurine and 6-thiouric-acid

Anemia has been frequently reported in renal transplant recipients receiving azathioprine for immunosuppression and enalapril for treatment of hypertension. During the course of a prospective trial in such patients we determined azathioprine metabolites in erythrocytes, plasma, and urine as well as erythropoietin and hemoglobin levels in order to evaluate a potential interaction between these 2 drugs, possibly leading to anemia. Two specific high performance liquid chromatography (HPLC) methods for determination of azathioprine metabolites, both employing a mercurial cellulose resin for extraction, are presented. One method using a strong anion exchange column allows detection of 6-thioguanosine di- and triphosphate (thioguanine nucleotides) in red blood cells (RBC) with a sensitivity of 30 pmol/100 microliters RBC. 6-mercaptopurine (MP) and 6-thiouric acid (TUA) in plasma and urine were analyzed simultaneously by reversed-phase HPLC with a sensitivity of 5 ng/ml. The average (median values are given) steady state concentrations of thioguanine nucleotides in erythrocytes came to 267 pmol/100 microliters RBC (range 53-613) with and to 246 pmol/100 microliters RBC (range 39-629) without concomitant enalapril medication. Mean plasma concentrations of MP and TUA 3 hours after drug intake came to 14.8 +/- 9.9 ng/ml and 398 +/- 262 ng/ml, respectively, during enalapril comedication. Withdrawal of enalapril did not influence these metabolite levels coming to 15.3 +/- 9.1 and 451 +/- 253 after stopping enalapril treatment. Thioguanine nucleotides in RBCs were neither related to the dose of azathioprine given (r = -0.113, p > 0.05) nor to hemoglobin levels (r = 0.278, p > 0.05). However, azathioprine dose/kg body weight seemed to be related to hemoglobin concentration, with and without enalapril comedication. We conclude that enalapril therapy does not influence the measured azathioprine metabolites, the reported cases of anemia may rather be due to a pharmacodynamic interaction as shown by the significant increase in erythropoietin after withdrawal of enalapril. The assays described here are suitable to study the metabolism of azathioprine in patients with various diseases.

Topics: Administration, Oral; Anemia; Antihypertensive Agents; Azathioprine; Drug Interactions; Enalapril; Erythrocytes; Hemoglobins; Humans; Immunosuppressive Agents; Kidney Transplantation; Mercaptopurine; Prospective Studies; Spectrophotometry, Ultraviolet; Uric Acid

Anticancer agent 6-mercaptopurine (6MP) has been in use since 1953 for the treatment of childhood acute lymphoblastic leukemia (ALL) and inflammatory bowel disease. Despite being available for 60 years, several aspects of 6MP drug metabolism and pharmacokinetics in humans are unknown. Molybdoflavoenzymes such as aldehyde oxidase (AO) and xanthine oxidase (XO) have previously been implicated in the metabolism of this drug. In this study, we investigated the in vitro metabolism of 6MP to 6-thiouric acid (6TUA) in pooled human liver cytosol. We discovered that 6MP is metabolized to 6TUA through sequential metabolism via the 6-thioxanthine (6TX) intermediate. The role of human AO and XO in the metabolism of 6MP was established using the specific inhibitors raloxifene and febuxostat. Both AO and XO were involved in the metabolism of the 6TX intermediate, whereas only XO was responsible for the conversion of 6TX to 6TUA. These findings were further confirmed using purified human AO and Escherichia coli lysate containing expressed recombinant human XO. Xanthine dehydrogenase (XDH), which belongs to the family of xanthine oxidoreductases and preferentially reduces nicotinamide adenine dinucleotide (NAD(+)), was shown to contribute to the overall production of the 6TX intermediate as well as the final product 6TUA in the presence of NAD(+) in human liver cytosol. In conclusion, we present evidence that three enzymes, AO, XO, and XDH, contribute to the production of 6TX intermediate, whereas only XO and XDH are involved in the conversion of 6TX to 6TUA in pooled HLC.

Topics: Adult; Aged; Aldehyde Oxidase; Cytosol; Escherichia coli; Female; Humans; Liver; Male; Mercaptopurine; Metabolic Detoxication, Phase I; Middle Aged; Recombinant Proteins; Uric Acid; Xanthine Dehydrogenase; Xanthine Oxidase; Young Adult

To develop a method that prospectively assesses adherence rates in paediatric patients with acute lymphoblastic leukaemia (ALL) who are receiving the oral thiopurine treatment 6-mercaptopurine (6-MP).. A total of 19 paediatric patients with ALL who were receiving 6-MP therapy were enrolled in this study. A new objective tool (hierarchical cluster analysis of drug metabolite concentrations) was explored as a novel approach to assess non-adherence to oral thiopurines, in combination with other objective measures (the pattern of variability in 6-thioguanine nucleotide erythrocyte concentrations and 6-thiouric acid plasma levels) and the subjective measure of self-reported adherence questionnaire.. Parents of five ALL patients (26.3%) reported at least one aspect of non-adherence, with the majority (80%) citing "carelessness at times about taking medication" as the primary reason for non-adherence followed by "forgetting to take the medication" (60%). Of these patients, three (15.8%) were considered non-adherent to medication according to the self-reported adherence questionnaire (scored > or = 2). Four ALL patients (21.1%) had metabolite profiles indicative of non-adherence (persistently low levels of metabolites and/or metabolite levels clustered variably with time). Out of these four patients, two (50%) admitted non-adherence to therapy. Overall, when both methods were combined, five patients (26.3%) were considered non-adherent to medication, with higher age representing a risk factor for non-adherence (P < 0.05).. The present study explored various ways to assess adherence rates to thiopurine medication in ALL patients and highlighted the importance of combining both objective and subjective measures as a better way to assess adherence to oral thiopurines.

Topics: Adolescent; Antimetabolites, Antineoplastic; Child; Child, Preschool; Female; Humans; Male; Medication Adherence; Mercaptopurine; Parents; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Thioguanine; Uric Acid

The thiopurines, azathioprine (AZA) and mercaptopurine are extensively used in Crohn's discase (CD). Thiopurine bioactivation can be diverted by either thiopurine methyltransferase (TPMT), or by xanthine oxidase/dehydrogenase (XOD) which forms 6-thiouric acid (6TU).. To investigate whether chronic inflammation could influence small intestinal XOD activity using urinary excretion of 6TU as a surrogate marker of XOD activity.. 6-Thiouric acid excretion was compared between 32 CD patients and nine dermatology patients (control group), on AZA. Six CD patients were interesting: five with low TPMT activity (one deficient, four intermediate), and one receiving AZA/allopurinol co-therapy.. There was no statistical difference in 6TU excretion between the CD and control group. CD location, severity or surgery did not affect excretion. The TPMT-deficient patient excreted 89% of daily AZA dose as 6TU, but excretion by TPMT carriers was essentially normal. Concurrent 5-aminosalicylic acid therapy increased 6TU excretion significantly (median 32.9%), consistent with inhibiting TPMT. 6TU was undetectable in the patient on AZA/allopurinol co-therapy.. The results refuted our hypothesis, but fitted a model where most of an oral thiopurine dose effectively escapes first-pass metabolism by gut XOD, but is heavily catabolized by TPMT. Bioavailability of thiopurines may be competitively inhibited by dietary purines.

Topics: Adult; Biological Availability; Case-Control Studies; Crohn Disease; Female; Humans; Immunosuppressive Agents; Intestine, Small; Mercaptopurine; Methyltransferases; Uric Acid; Xanthine Oxidase

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

There have been very few published studies that have evaluated exposure to myelotoxic drugs among production workers in pharmaceutical plants. Previous studies have focussed mainly on nurses and evaluated exposure to cytotoxic drugs using urine mutagenicity as a marker of exposure. The aim of this study was to evaluate the exposure of workers involved in the production of chloramphenicol and azathioprine. Exposure was evaluated utilising biological monitoring, biological effect monitoring and environmental monitoring. Biological monitoring included plasma chloramphenicol levels, plasma 6-mercaptopurine and urine 6-thiouric acid levels. These were analysed using high performance liquid chromatography. Myelotoxic effect was assessed by measuring the haematological indices of bone marrow function. The exposed 17 workers were compared to matched controls of equal numbers. Neither substance could be detected in serum nor urine by the analytical methods employed. However, haematological indices demonstrated a significantly decreased mean reticulocyte and neutrophil count in the azathioprine exposed group. Industrial hygiene measurements demonstrated contamination of the air inside the airhood of exposed workers. In conclusion, it is evident that workers involved in the production of both these drugs are at risk of developing adverse health effects. Furthermore, more sensitive analytical methods need to be developed to evaluate absorption of myelotoxic chemicals among occupationally exposed workers.

Topics: Adult; Air Pollutants, Occupational; Azathioprine; Blood Cell Count; Case-Control Studies; Chloramphenicol; Drug Industry; Environmental Monitoring; Humans; Male; Mercaptopurine; Occupational Exposure; South Africa; Uric Acid

6-Chloropurine (CP) has antitumor activity against animal and human neoplasms, but the mechanism is unclear. Recently, we have shown that S-(6-purinyl)glutathione (PG), a putative metabolite of CP, is metabolized in vivo to yield the antitumor drug, 6-mercaptopurine (6-MP). In this study, CP metabolism to PG and 6-MP was investigated in an effort to provide further insights into the mechanism of CP antitumor activity. Rat hepatic and renal glutathione S-transferases metabolized CP to PG; Vmax values for liver and kidney cytosol were 166 and 24 nmol/mg of protein/min, respectively. PG was isolated and characterized by fast atom bombardment mass spectrometry from the bile of rats given CP. When rats were given CP (14 mumol/kg), PG excretion was linear with time for up to 1 hr; nearly 80% of the PG excreted at 2 hr was excreted at 1 hr. Rats given CP (10-1200 mumol/kg) excreted at 1 hr into bile nearly 18% of the dose as PG; rats given CP (400-1200 mumol/kg) excreted at 24 hr into urine nearly 4% of the dose as 6-MP and its further metabolites, 6-methylthiopurine and 6-thiouric acid. CP, PG, 6-MP, 6-methylthiopurine and 6-thiouric acid were also detected in plasma, liver and kidney of rats given CP (1200 mumol/kg); in these tissues, maximum CP concentrations were observed at 30 min, as compared to 60 to 180 min, and plasma CP concentrations were higher than those detected in liver or kidney. Liver or kidney CP metabolite concentrations at 30 to 120 min were, however, higher than those detected in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bile; Biliary Tract; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Glutathione; Glutathione Transferase; Kidney; Liver; Male; Mercaptopurine; Microsomes; Proteins; Purines; Rats; Rats, Sprague-Dawley; Time Factors; Uric Acid

The plasma concentrations and tissue distribution of thiopurines were studied in mice after oral administration of 50 mg/kg azathioprine (AZA) using HPLC analysis. Peak concentrations of AZA and three other thiopurine metabolites in plasma [thiouric acid (TUA) greater than 6-mercaptopurine (6-MP) greater than AZA greater than 8-hydroxy-AZA] were observed as early as 10 min after drug application, thus indicating fast absorption and extensive metabolism of AZA, and were followed by a rapid decline. The extraction of thiopurines from organs (intestinal mucosa, liver, kidney, testes, spleen, and bone marrow) and from red blood cells (RBCs) was preceded by an acid hydrolysis procedure resulting in the release of thiopurine bases from their corresponding ribonucleotides. 6-MP, 6-thioxanthene (6-TX), 6-thioguanine (6-TG), TUA, and 8-hydroxy-6-MP (8-OH-6-MP) were extracted from the organs, whereas only 6-MP and 8-OH-6-MP were found in the processed RBCs. Initially, high concentrations of TUA, the endpoint of metabolic AZA degradation, were detected in the intestinal mucosa and in the liver. This provides evidence for a first-pass metabolism of AZA in these two organs. The initial concentrations of 6-MP extracted from the organs were about 10-fold those found in plasma. This indicates rapid cellular uptake of 6-MP and an accumulation of 6-MP derivatives that can be explained by formation of the 6-MP ribonucleotide thioinosine monophosphate (TIMP). With the exception of plasma and RBCs, 6-TG, which may originate from intracellular 6-thioguanosine nucleotides (TGNs), was extracted from all organs examined in the study. From the sequence of appearance of 6-MP, 6-TX, and 6-TG extracted from spleen and bone marrow homogenates, it can be assumed that formation of TGN occurs via the nucleotide interconversion pathway TIMP----6-thioxanthosine monophosphate----6-thioguanosine monophosphate. The highest concentrations of 6-TG derivatives were found in the spleen and bone marrow. This correlates with the clinical and experimental observation that AZA cytotoxicity mainly affects bone-marrow stem cells and lymphocytes and supports the hypothesis (derived from in vitro experiments) that the incorporation of TGN into DNA is the cytotoxic mechanism of AZA and 6-MP.

Topics: Administration, Oral; Animals; Azathioprine; Chromatography, High Pressure Liquid; Erythrocytes; Male; Mercaptopurine; Mice; Mice, Inbred Strains; Purines; Thioguanine; Time Factors; Tissue Distribution; Uric Acid

A sample preparation technique and a high-performance liquid chromatographic method for 6-mercaptopurine (6-MP) that is simple, sensitive and without interference from its metabolites is described. 6-Thioguanine (6-TG) is added as an internal standard to the plasma sample, which is then treated with an aqueous solution of aluminum perchlorate to denature the plasma proteins and form complexes with 6-TG, 6-MP and its major metabolite, 6-thiouric acid (6-TUA). These complexes coprecipitate with proteins on centrifugation. 6-MP and its analogues are then extracted from the precipitate with perchloric acid containing sodium hydrosulfite and the extract is chromatographed on an Ultrasphere ODS column eluted with 0.1 M phosphoric acid and 0.001 M dithiothreitol in deionized water. The eluate is monitored at 340 nm. No interfering peak was encountered in over 300 clinical plasma samples. 6-TUA was separated from 6-MP and was found to be present in much higher concentration than 6-MP itself throughout the sampling time (6 h) following oral administration of the drug.

Topics: Aluminum; Cations; Chelating Agents; Chromatography, High Pressure Liquid; Humans; Mercaptopurine; Spectrophotometry, Ultraviolet; Thioguanine; Uric Acid

Despite extensive clinical experience with azathioprine (AZA), the disposition of various AZA metabolites remains obscure. We therefore evaluated the pharmacokinetics of three AZA metabolites: 6-mercaptopurine (6-MP), the immediate metabolite; 6-thiouric acid (6-TU), the final end product; and 6-thioguanine nucleotides (TGN), the active moiety; in eight renal transplant patients after oral administration of AZA. The low peak plasma 6-MP level of 73.7 +/- 23.7 ng/mL (mean +/- SD) and the short half-life (t1/2) of 1.9 +/- 0.6 hours suggest rapid conversion of 6-MP to other metabolites. A peak plasma 6-TU concentration of 1210 +/- 785 ng/mL was observed at 3.5 +/- 0.6 hours after the AZA dose. The strong correlation between 6-TU t1/2 and serum creatinine (r = 0.98, P = .0008) supported our previous work showing that 6-TU is primarily excreted by the kidneys. The total TGN levels in red blood cells (RBCs) in each patient remained largely unchanged over 24 hours with the intraindividual coefficient of variation ranging from 4.4% to 29.8%. In comparison, the mean TGN level varied considerably between patients, and ranged from undetectable to 413 pmol per 8 X 10(8) RBCs. However, there was no apparent correlation between white cell counts on day 0 (P greater than .5), day 7 (P greater than .5), or day 14 (P greater than .5) and RBC TGN level. The persistence of TGN in body tissues thus provides a pharmacokinetic rationale for the conventional once or twice daily AZA regimen.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Administration, Oral; Adult; Azathioprine; Chromatography, High Pressure Liquid; Data Collection; Female; Humans; Kidney Transplantation; Male; Mercaptopurine; Middle Aged; Thioguanine; Time Factors; Uric Acid

The immunosuppressive activity of azathioprine (AZA) is unpredictable and depends on the formation of intracellular thiopurine ribonucleotides. However, the quantification of these active thiopurines presents difficult analytical problems. It has recently been postulated that plasma concentrations of 6-thiouric acid (6-TU) and 6-mercapto-purine (6-MP), metabolites of AZA, may provide more readily measurable indices of the pharmacologic activity of AZA. In order to evaluate the utility of 6-TU and 6-MP plasma concentrations in monitoring AZA therapy, we studied their pharmacokinetics in 6 renal transplant patients, and their in vitro immunosuppressive potency in a mixed lymphocyte proliferation assay. A peak plasma 6-TU concentration of 710.7 ng/ml was observed at 3.8 h after oral dosing. Good correlation was observed between the elimination t1/2 of 6-TU and serum creatinine, and between AUC over 24 h and serum creatinine. However, we did not observe a second peak in plasma 6-TU concentration that could be attributed to the degradation of active AZA metabolites. 6-MP plasma concentrations in the patients were low (mean peak concentration 36.0 ng/ml) and rapidly disappeared within 8 h. In vitro immunosuppressive activity could not be demonstrated for 6-TU over a concentration range of 1.25 ng/ml to 0.25 mg/ml. We conclude that 6-TU is pharmacologically inert and is primarily eliminated by the kidneys. Our findings currently do not support the use of plasma concentrations of 6-TU or 6-MP to monitor AZA therapy. In order to optimize AZA therapy, analytical techniques that are technically feasible and that can directly quantify the active intracellular thiopurines are being explored.

Topics: Adult; Azathioprine; Female; Humans; Kidney Transplantation; Lymphocyte Culture Test, Mixed; Male; Mercaptopurine; Middle Aged; Time Factors; Transplantation, Homologous; Uric Acid

We present a rapid, sensitive and specific high-performance liquid chromatographic method for the analysis of 6-mercaptopurine (6MP), 6-thioguanine nucleotides (6TGN) and 6-thiouric acid (6TU), with excellent chromatographic separation of the thiopurines. Thiopurines in plasma and erythrocytes were extracted by mercuricellulose and re-eluted with beta-mercaptoethanol. For quantitative detection a reverse phase column (Lichrosorb RP-18 4 mm X 30 cm) and a UV detector were used. Detection limits were 20 nmol/l for 6MP in plasma, 250 nmol/l for 6TGN in erythrocytes, and 10 nmol/l and 15 nmol/l, respectively, for 6MP and 6TU in urine diluted 1:100 with beta-mercaptoethanol. Within-run and between-run variations were less than 10%. Recovery of 6MP added to plasma, and 6TGN monophosphate added to haemolysed erythrocytes were 91% and 81%, respectively.

Topics: Chromatography, High Pressure Liquid; Humans; Mercaptopurine; Thioguanine; Uric Acid

Topics: Animals; Azathioprine; Chromatography, High Pressure Liquid; Indicators and Reagents; Kinetics; Male; Mercaptopurine; Mice; Mice, Inbred C57BL; Thioguanine; Thiouracil; Uric Acid

A specific and highly sensitive fluorimetric method was developed for the determination of 6-mercaptopurine (6MP) in serum. The method is based on the enzymatic oxidation of 6MP with xanthine oxidase to the oxypurine, followed by oxidation with acidic chromate to the corresponding 6-sulfonate. The fluorescent product has excitation and emission maxima at 330 and 400 nm, respectively. The limit of sensitivity was approximately 22 pg/ml for 6MP in water. The sensitivity limit for 6MP in serum containing azathioprine was approximately 2.2 ng/ml. The rate constants for conversion of 6MP into the final product (6-thiouric acid) and the apparent Michaelis constant were also determined by a nonlinear regression analysis based on the integrated Michaelis-Menten equation using the ultraviolet absorbance data and the simplified complementary tristimulus colorimetry.

Topics: Chromatography, High Pressure Liquid; Humans; Kinetics; Mercaptopurine; Oxidation-Reduction; Spectrometry, Fluorescence; Uric Acid; Xanthine Oxidase

Topics: Animals; Azathioprine; Chemical Precipitation; Chlorides; Chromatography, Ion Exchange; Dogs; Female; Glutathione; Humans; Male; Mercaptopurine; Mercury; Spectrophotometry, Ultraviolet; Sulfhydryl Compounds; Uric Acid