Simultaneous Quantification of Eleven Thiopurine Nucleotides by

Article pubs.acs.org/ac

Simultaneous Quantification of Eleven Thiopurine Nucleotides by Liquid Chromatography-Tandem Mass Spectrometry Ute Hofmann,† Georg Heinkele,† Sieglinde Angelberger,‡ Elke Schaeffeler,† Cornelia Lichtenberger,‡ Simon Jaeger,† Walter Reinisch,‡ and Matthias Schwab†,∥,* †

Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria ∥ Department of Clinical Pharmacology, Institute of Experimental and Clinical Pharmacology and Toxicology, University Hospital, Tuebingen, Germany ‡

S Supporting Information *

ABSTRACT: The prodrugs azathioprine and 6-mercaptopurine, which are well-established anticancer and immunosuppressive agents, are extensively metabolized by activating and inactivating enzymes. Whereas the 6-thioguanine nucleotides (TGN) are currently being considered as major active metabolites, methylthioinosine nucleotides seem to contribute to the cytotoxic effect as well. Thiopurine-related adverse drug reactions and thiopurine failure are frequent. Thus, therapeutic monitoring of TGN and methylthioinosine derivatives has been suggested to improve thiopurine therapy, however with limited success. To elucidate systematically underlying molecular mechanisms as potential explanation for interindividual variability of thiopurine response, we developed a novel highly specific and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantitation of eleven mono-, di-, and triphosphates of thioguanosine, methylthioinosine, methylthioguanosine, and thioinosine. Using stable isotopelabeled analogues as internal standards obtained by chemical synthesis, an intra- and interassay variability below 8% and an accuracy of 92% to 107% were achieved in spiked quality control samples with known standards. All eleven metabolites could be determined in red blood cells from patients with inflammatory bowel diseases and long-term azathioprine therapy. Thus, our novel method opens a new avenue for the understanding of the thiopurine metabolism by quantitation of all important thiopurine nucleotide metabolites in one run.

T

subsequently triggering the proteasomal degradation of the DNA methylase DNMT1.11 Thiopurine-related adverse drug reactions are frequent, ranging from 5% up to 40%.2 Patients with very low TPMT activity as a consequence of genetic variation are at increased risk for developing severe myelosuppression due to exaggerated accumulation of cytotoxic TGN.2,12,13 Dose adjustment based on TPMT activity or genetic testing prior to commencement of thiopurine therapy is recommended to avoid severe hematotoxicity.14,15 However, independent from TPMT and other candidate genes (e.g., ITPA)16 which explain in part thiopurinerelated toxicities (e.g., hematotoxicity), a substantial proportion of thiopurine-related side effects is still unpredictable. More than a decade ago, monitoring of major thiopurine metabolites (i.e., TGN and 6-methyl-mercaptopurine ribonucleotides, MMPR) in red blood cells (RBC) has been suggested to improve thiopurine therapy, but no conclusive correlation

he thiopurines azathioprine and 6-mercaptopurine are widely used as anticancer and immunosuppressive agents.1,2 They are prodrugs requiring metabolic conversion to the active 6-thioguanine nucleotides (TGN) via the purine salvage pathway by the enzymes hypoxanthine phosphoribosyltransferase (HPRT), inosine 5′-monophosphate dehydrogenase (IMPDH), and guanosine 5′-monophosphate synthetase (GMPS) (Figure 1). The predominant catabolic enzymes in the metabolism of thiopurines are the thiopurine S-methyltransferase (TPMT) and xanthine oxidase (XO). Modes of action currently known involve the incorporation of TGN into DNA or RNA3 leading to apoptosis through inhibition of replication, DNA repair mechanisms, and protein synthesis.4,5 The intermediate metabolite 6-methylthioinosine-5′-monophosphate (MeTIMP) inhibits the de novo purine synthesis.6 Furthermore, 6-thioguanosine-5′-triphosphate (TGTP) suppresses T-cell dependent pathogenic immune responses through a Rac1 dependent mechanism in patients with inflammatory bowel diseases (IBD).7,8 More recently, it has been shown that 6-thioguanine acts also as a hypomethylating agent, and this reaction is mediated by DNA methyltransferases.9,10 This epigenetic effect is explained by an attenuated expression of histone lysine-specific demethylase 1 (LSD1) © 2012 American Chemical Society

Received: August 22, 2011 Accepted: January 5, 2012 Published: January 5, 2012 1294

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Figure 1. Metabolism of the thiopurine drugs azathioprine (AZA), 6-mercaptopurine (6-MP), and 6-thioguanine (6-TG). 6-MMP, 6-methylmercaptopurine; TGMP, 6-thioguanosine 5′-monophosphate; TGDP, 6-thioguanosine 5′-diphosphate; TGTP, 6-thioguanosine 5′-triphosphate; TIMP, 6-thioinosine 5′-monophosphate; TIDP, 6-thioinosine 5′-diphosphate; TITP, 6-thioinosine 5′-triphosphate; MeTIMP, 6-methylthioinosine 5′monophosphate; MeTIDP, 6-methylthioinosine 5′-diphosphate; MeTITP, 6-methylthioinosine 5′-triphosphate; MeTGMP, 6-methylthioguanosine 5′-monophosphate; MeTGDP, 6-methylthioguanosine 5′-diphosphate; MeTGTP, 6-methylthioguanosine 5′-triphosphate; GMPS, guanosine monophosphate synthetase; HPRT, hypoxanthine guanine phosphoribosyltransferase; IMPDH, inosine monophosphate dehydrogenase; TPMT, thiopurine S-methyltransferase; XO, xanthine oxidase; ITPA, inosine triphosphate pyrophosphatase.

MeTGMP, MeTGDP, MeTGTP, MeTIMP, MeTIDP, MeTITP, TGMP, TGDP, TGTP, TIMP, and TITP were obtained from Jena Bioscience (Jena, Germany) at 10 mmol/L concentration in water. Deuterium labeled internal standards [2H4]TGMP, [2H4]TGDP/ [2H4]TGTP), [2H3]MeTIMP, [2H3]MeTIDP/[2H3]MeTITP, [2H3]MeTGMP, and [2H3]MeTGDP/[2H3]MeTGTP were prepared by chemical synthesis as described in the Supporting Information (SI). EDTA (disodium salt, dihydrate) was prepared as a 50 mmol/L solution and adjusted to pH 10.5 with 5 N NaOH. Solutions of 1,4-dithio-D/L-threitol (DTT, 30 mg/mL in EDTA) were prepared freshly prior to any experiment. Pure water was obtained from a Milli-Q system (Millipore, Germany). Stock solutions of the labeled nucleotides were prepared in water at a final concentration of 10 mmol/L and stored at −20 °C. Working solutions of the nucleotides at final concentrations of 10 μmol/L and 1 μmol/L were prepared by dilution with EDTA. Blood Sampling. Blood samples from 18 patients with Crohn’s disease were selected from an ongoing study investigating the impact of thiopurine metabolite measurement on clinical management. Patients had been on stable azathioprine dosage for at least 6 months prior to study

between TGN levels and outcome in patients with IBD was found in numerous clinical studies.2,17 Current routine methods introduced for therapeutic monitoring of nucleotides in RBC require hydrolysis before quantitation of TGN and MMPR by HPLC analysis.18,19 Based on these methods, however, a differentiation between individual nucleotides for the respective bases (nucleoside 5′-mono-, di-, and triphosphate) is limited. Moreover lab-specific conditions for hydrolysis but also other workup procedures differ between various clinical laboratories, thereby contributing to discrepancies between test results.19 Generally, a comprehensive highly sensitive and specific method for simultaneous determination of all important thiopurine metabolites is still missing. Therefore, we aimed to establish the first LC-MS/MS method for quantitation of eleven thiopurine nucleotides in one run. Individual nucleotide levels of RBC samples from an ongoing clinical trial of IBD patients treated with azathioprine were measured and in part compared to nucleotide concentrations obtained by an independent method.18

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EXPERIMENTAL SECTION Chemicals and Reagents. All chemicals and reagents used were of analytical grade. Reference compounds of the analytes 1295

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as described above. Calibration curves based on internal standard calibration were obtained by weighted (1/x, x = amount) linear regression for the peak-area ratio of the analyte to the respective internal standard against the amount of the analyte. The concentration of the analytes in unknown samples was obtained from the regression line. All standardization was performed with the MassHunter software (Agilent). The reproducibility and accuracy of the method was established by analyzing quality control samples, prepared like the calibration samples. Eight levels of calibration samples and three levels of quality controls in duplicate were analyzed with each batch of patient samples. Intra-assay precision and accuracy was assessed by measuring the concentration of the analytes in 6 aliquots of three different quality control samples extracted and analyzed on a single day. Interassay precision and accuracy was determined from the results of three different quality control samples extracted and analyzed 6-fold on three different days. The limit of quantification was determined as the lowest concentration (LLOQ) with a coefficient of variation (CV) and a bias of 3. Selectivity and specificity were determined in six randomly selected untreated RBC samples. Extracted ion chromatograms of the RBC blanks were compared to the chromatograms of the

QC high

extraction efficiency (%)

total recovery (%)

65 70 64 65 69 66 68 67 63 70 77

41 84 39 44 69 32 80 57 36 84 36

conc (μM)

matrix effect (%)

extraction efficiency (%)

total recovery (%)

8 8 8 40 40 40 8 8 8 8 8

52 118 58 58 102 49 114 85 54 116 49

65 70 65 63 69 64 69 68 65 73 64

34 83 38 36 70 31 78 58 35 85 31

blank samples spiked with the lowest calibration point (LLOQ). For nearly all analytes, no interfering peak at the expected retention time could be detected (Figure 3). Only the extracted ion chromatogram of TITP shows a large peak shortly before the expected retention time of TITP which leads to 1298

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triphosphates and an increase of the respective diphosphates was observed, which accounted for 34 ± 11% for MeTGDP, 25 ± 11% for MeTIDP, and 30 ± 11% for TGDP in eight samples investigated. Processed samples were stable for at least 24 h in the cooled (4 °C) compartment of the autosampler and for at least one week at −20 °C. Application to Patients Samples. Two independent samples from each of the 18 IBD patients on long-term azathioprine therapy were analyzed by the novel established LC-MS/MS assay. Data were compared to total TGN, MMPR, and MTGN levels determined with our previously described routine HPLC method.18 Interindividual variabilities of the eleven metabolites for all 36 samples are shown in Figure 4, and

some background peaks with an area of maximal 30% of the area of TITP at the LLOQ. Nevertheless, both accuracy and variability at the LLOQ are in the requested range with an accuracy between −17.2% and 9.8% and a reproducibility of 11.4%. Precision and accuracy was investigated at low, medium, and high concentrations of all thiopurine nucleotides. The intraday and interday coefficients of variation ranged from 0.7% to 7.5%, and the intraday and interday accuracies were between 92% and 106% for all quality control samples as shown in Table S-4. The extraction efficiency of all nucleotides was comparable ranging from 64 to 74% over the whole concentration range as shown in Table 1. The matrix effect observed for the analytes was quite variable. Only for MeTIDP and TGDP, the matrix effect was almost absent or negligible. A signal enhancement was observed for MeTGDP, TGMP, and TIMP, while the other compounds exhibited a rather high signal suppression between 33% and 51% as shown in Table 1. This leads to total recoveries between 31% and 95%. With such high and differing matrix effects, the use of stable isotope-labeled analogues as internal standards for all individual analytes is a prerequisite for a precise and accurate measurement. We have synthesized stable isotope-labeled internal standards for 9 of our analytes of interest. [2H4]TGMP was selected as internal standard for TIMP and [2H4]TGTP for TITP, since these internal standards have the highest possible chemical similarity to the analytes and show comparable retention times and matrix effects. Stability. Nucleoside 5′-phosphates are degraded very quickly and show considerable interconversion in RBC.22 The blood sampling protocol previously established for measurement of the individual 6-thioguanosine phosphates TGMP, TGDP, and TGTP20 proved to be suitable for quantification of all eleven thiopurine metabolites in the present study as shown by the systematic investigations on postphlebotomy stability using four independent patients samples (SI Figure S-3). Thus, preparation of packed RBC within 4 h after blood sampling appears to be reliable. In contrast to the workup procedure used for our previous reported HPLC method20 which includes an oxidation step with permanganate, such a reaction is not required for the LCMS/MS analysis. With LC-MS/MS analysis we observed a consistently higher percentage of TGDP compared to HPLC analysis. To prevent degradation of the triphosphates during LC-MS/MS analysis we improved the workup procedure by performing the whole sample workup on ice and including a short heating step for 5 min to 94 °C. Stability of the single nucleotides was investigated with this improved protocol (SI Table S-5). In EDTA buffer as matrix, the majority of the thiopurine nucleotides was stable with conversions less than 1%. Only MeTGMP showed a conversion of about 6% to MeTGDP when compared to the pure substance. When worked-up in RBC, TGTP showed a conversion of 5% to TGDP and 3% to TGMP, in accordance with previous investigations.20 MeTGTP also showed 4% conversion to MeTGDP. For the other nucleotides conversion to other phosphates accounted for less than 3% when compared to workup in buffer. Altogether, this demonstrates the suitability of the proposed sample workup procedure for the assessment of correct nucleotide levels. It had been shown that dephosphorylation of TGTP to TGDP in RBC samples is influenced by repeated thawing.20 This could be verified for all thiopurine triphosphates. When samples were thawed twice, dephosphorylation of the

Figure 4. Thiopurine nucleotide concentrations in two independent RBC samples of 18 patients treated with azathioprine.

values are given in SI, Table S-6. Total TGN, MMPR, and MTGN levels in RBC could be quantified in all samples. The diphosphates and triphosphates of the respective nucleosides were present at quantifiable levels by LC-MS/MS in all samples. TGMP was below the LOQ in 7 (19%) samples, MeTIMP in 3 (8%) samples, whereas MeTGMP was quantifiable only in 4 (11%) samples. The thioinosine metabolites TIMP and TITP could be determined in only 8 (22%) and 3 (8%) samples, respectively. In accordance with previous investigations which did detect neither thiopurine ribosides nor bases in RBC,23−25 we could not detect 6thioguanosine or 6-methylmercaptopurine riboside in our samples either. Comparison of total nucleotide levels to the sum of the individual nucleoside 5′-phosphates showed good correlation for TGN (r2 = 0.9543), MMPR (r2 = 0.9726), and MTGN (r2 = 0.9635). For TGN and MMPR the values obtained by HPLC were lower (Table S-5, 67% to 103% of the sum for TGN and 52% to 97% of the sum for MMPR). This might be explained by hydrolysis efficiency or by partial degradation of MMP as reported previously,19,26 indicating again the need for a more specific method for quantitation of thiopurine nucleotides. As shown in former studies, the major component of TGN is TGTP.20,26−28 There is an ongoing discussion whether MeTIMP is the major component of MMPR in RBC. The occurrence of MeTIMP, MeTIDP, and MeTITP in blood or bone marrow samples from leukemic patients treated with 6MP, or in RBC after 6-MP infusions, has only been reported in single cases.29,30 Based on our novel LC-MS/MS assay the principal metabolite of the MMPR is the triphosphate MeTITP 1299

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minimized ex vivo interconversion of the nucleoside phosphates which is mandatory for a valid assessment of single nucleoside phosphate levels. The method was applied to RBC samples from 18 patients under long-term azathioprine therapy indicating a high interindividual variability of the metabolite levels. Thus, in the future our novel sensitive and specific method enables not only profound investigations of the impact of single thiopurine nucleotides on drug response in IBD patients but also in other patient groups like children with acute lymphoblastic leukemia where thiopurines are the mainstay of therapy.

which accounts for 54 to 88% of the sum of all methylthioinosine phosphates. MeTIMP and MeTIDP represent 3% to 38%, or 8% to 19%, of the sum, respectively. For MeTIMP similar low concentrations have been reported (about 20% of total MMPR).26 Methylated thioguanosine metabolites (MTGN) are the main metabolites formed by TPMT after treatment of patients with 6-thioguanine18,31−33 but can be also detected at lower levels after azathioprine therapy.34 Recent studies have demonstrated mutagenic properties of methylthioguanine, which could lead to a potential carcinogenic mechanism associated with chronic thiopurine administration.35 Of note, methylated thioguanosine nucleotides may not be incorporated into nucleic acids since the methylation in nucleic acids occurs via spontaneous reaction of DNA 6-thioguanine with Sadenosylmethionine,4 and this reaction appears to be of minor significance in leukemia cells treated with 6-thioguanine.36 However, quantitation of methylated thioguanosine nucleotides may be important since this reaction is TPMTdependent and subjects with very high TPMT activity may be at risk for poor response. In our study MeTGTP was the main component with 67% to 87% of total MTGN levels, followed by MeTGDP. We did not find a correlation between levels of isolated methylated thioguanosine or thioinosine nucleotides and individual TPMT activities (Table S-2) which may be explained by the small samples size of the study population and TPMT activity levels missing very low or very high values. Moreover, we have included the 6-thioinosine phosphates TIMP and TITP in our assay. TIMP is the precursor for the formation of both MeTIMP and TGMP, and its formation by IMPDH is regarded as the rate-limiting step in thiopurine bioactivation.37 Accumulation of TITP levels has been suggested as a cause for adverse events under azathioprine therapy due to polymorphic deficiency of the enzyme inosine triphosphate pyrophosphohydrolase (ITPase).38−40 In the majority of RBC samples both TIMP and TITP were below the LLOQ which may be explained by a very low IMPDH activity in RBC41 in contrast to other cells like PBMC where higher levels of TIMP and TITP may be possible. Taken together, our novel LC-MS/MS assay allows for the first time a reliable and systematic quantitation of eleven thiopurine metabolites to elucidate comprehensively the variability of thiopurine metabolites in vivo with consequences on drug response including adverse events. Thiopurines are still an important drug class in treatment of patients with inflammatory bowel diseases sometimes in combination with other drugs (e.g., TNF α blocker).1 Since quantitation of individual thioguanosine phosphates may provide better prediction of thiopurine response as we have previously shown,20,28 one can assume that a systematic profile of 11 individual thiopurine metabolites will majorly improve our understanding of thiopurine metabolism. Thus, the ultimate goal is the establishment of definitive cutoff levels of thiopurine metabolites by a prospective clinical trial.

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ASSOCIATED CONTENT

* Supporting Information S

Chemical synthesis of deuterium labeled internal standards, with data on composition and purity and the reaction schemes, the patients’ characteristics, mass spectrometer parameters for the determination of the thiopurine metabolites and their internal standards with LC-MS/MS, product ion spectra of the thionucleotides, validation data, data on stability of thiopurine nucleotides in RBC and post phlebotomy in blood samples, and the concentration of thiopurine metabolites in RBC of patients treated with azathioprine. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone ++49 711 8101 3700. Fax ++49 711 859295. E-mail: [email protected].

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ACKNOWLEDGMENTS The skillful and excellent technical assistance of Monika Seiler is acknowledged. This work was in part supported by the Robert-Bosch Stiftung, Stuttgart, Germany and by the Federal Ministry for Education and Research (BMBF, Berlin Germany; FKZ 03 IS 2061C). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Part of the work was presented at the Joint Annual Meeting of the German, Swiss, and Austrian Societies for Clinical Pharmacology and Toxicology, Zurich, Switzerland, October 2011.

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CONCLUSIONS Our novel LC-MS/MS assay is the first comprehensive method for simultaneous quantitation of 11 relevant nucleotide metabolites of thiopurine drugs in RBC. Using stable isotopelabeled analogues of the metabolites, obtained by chemical synthesis, enabled a reproducible and accurate determination of all analytes. Investigations on the stability of the analytes demonstrated that the optimized sample workup procedure 1300

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