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DRUG METABOLISM

Extended Thiopurine Metabolite Assessment During 6-Thioguanine Therapy for Immunomodulation in Crohn's Disease

N. K. H. de Boer, L. J. J. Derijks, J. J. Keizer-Garritsen, L. H. J. Lambooy, W. Ruitenbeek, P. M. Hooymans, A. A. van Bodegraven and D. J. de Jong

From Gastroenterology and Hepatology, VU University Medical Centre, Amsterdam, the Netherlands (de Boer, van Bodegraven); Clinical Pharmacy, Maxima Medical Centre, Veldhoven, the Netherlands (Derijks); Laboratory of Pediatrics and Neurology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands (Keizer-Garritsen, Lambooy, Ruitenbeek); Clinical Pharmacy, Maasland Hospital, Sittard, the Netherlands (Hooymans); and Gastroenterology and Hepatology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands (de Jong).

Address for reprints: Address for correspondence: N. K. H. de Boer, Department of Gastroenterology and Hepatology, VU University Medical Center, PO Box 7057, 1007 MB, Amsterdam, the Netherlands.


   ABSTRACT
TOP
 ABSTRACT
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The proposed metabolic advantage of 6-thioguanine (6-TG) is the direct conversion into the pharmacologically active 6-thioguaninenucleotides (6-TGN). The authors assessed metabolic characteristics of 6-TG treatment in patients with Crohn's disease (N = 7) on therapy with 20 mg 6-TG. 6-thioguanine-monophosphate (6-TGMP), 6-thioguanine-diphosphate (6-TGDP), and 6-thioguanine-triphosphate (6-TGTP) were measured by high-performance liquid chromatography analysis in erythrocytes. Thiopurine S-methyltransferase activity and total 6-TGN levels were determined by standard methods. High interindividual variance in metabolite measurements was observed. Main metabolites were 6-TGTP (median = 531 pmol/8 x 108 red blood cells) and 6-TGDP (median = 199 pmol/8 x 108 red blood cells). Traces of 6-TGMP (median = 39 pmol/8 x 108 red blood cells) and 6-TG (2 patients) could be detected. 6-TGN levels correlated with 6-TGTP levels (r = 0.929, P = .003) and with the sum of separate nucleotides (r = 0.929, P = .003). No correlations were established between TPMT activity (median = 13 pmol/h/107) and 6-TG metabolites. The 1-step metabolism of 6-TG still leads to high interindividual variance in metabolite concentrations. Total 6-TGN level monitoring may suffice for clinical practice.

Key Words: 6-Thioguanine • Crohn's disease • thiopurine • 6-thioguaninenucleotides • metabolism • TPMT • pharmacology

Azathioprine (AZA) and 6-mercaptopurine (6-MP) are well-established immunosuppressive drugs that are regularly administered as maintenance therapy in the treatment of patients with inflammatory bowel disease (IBD). Unfortunately, up to 30% of properly indicated IBD patients do not benefit from AZA or 6-MP because of intolerance or lack of efficacy.1 The administration of another thiopurine, 6-thioguanine (6-TG), has been proposed for this nonresponsive group of patients.2,3 Potential metabolic advantages of 6-TG compared to AZA and 6-MP treatment are generation of less (toxic) metabolites and diminished interindividual—genetically determined—variance in metabolism.2 Several studies demonstrated that 6-TG therapy induced a much higher 6-thioguaninenucleotide (6-TGN) level in red blood cells (RBCs) than treatment with AZA or 6-MP.4-6 However, the interindividual variance in 6-TGN concentrations is reported to be high during 6-TG treatment.7 The metabolism of 6-TG is relatively simple compared to the multistep enzymatic metabolism of AZA and 6-MP. Via only 1 intracellular enzymatic step, 6-TG is rapidly converted into the allegedly pharmacologically active end metabolites, 6-TGN. These 6-TGN can be subdivided into 3 different phosphorylated forms: 6-thioguanine-monophosphate (6-TGMP), 6-thioguanine-diphosphate (6-TGDP), and 6-thioguanine-triphosphate (6-TGTP). 6-Thioguanine is converted to 6-TGMP via the purine salvage enzyme hypoxanthine phosphoribosyl transferase, whereas by subsequent kinase activity, 6-TGDP and 6-TGTP are produced.1 The molecular mechanism of immunosuppression by AZA and 6-MP is likely due to the specific end metabolite 6-TGTP that binds and inhibits the function of the small GTPase Rac1 in activated T lymphocytes, leading to apoptosis.8 Recently, Neurath and colleagues demonstrated that a novel assay to specifically measure 6-TGDP and 6-TGTP in erythrocytes may be more apt to monitor efficacy of AZA therapy, compared with monitoring total 6-TGN levels.9

Metabolic data concerning the generation of the specific phosphorylated 6-TGN are lacking in patients with IBD during 6-TG treatment. Moreover, it is unknown whether the assessment of specific phosphorylated 6-TGN to monitor thiopurine therapy is comparable with the classical method by measuring crude 6-TGN levels.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Patient Selection
Patients were eligible for the study if they met the following criteria. Inclusion criteria were age between 18 and 75 years and confirmed Crohn's disease (CD) with an indication for immunosuppressive maintenance therapy but in whom standard AZA or 6-MP therapy failed because of adverse events. In addition, patients had to be using 6-TG for a period of at least 4 consecutive weeks to assess steady-state metabolite levels. Exclusion criteria were pregnancy, ongoing treatment with concomitant immunosuppressive drugs (eg, cyclosporine, methotrexate, or infliximab), impaired renal function (serum creatinine >2 times the upper limit of normal reference), impaired hepatic function ({gamma}-glutamyltransferase, alkaline phosphatase, aspartate aminotransferase, or alanine aminotransferase >2 times the upper limit of normal reference), and bone marrow suppression (leukocyte count less than 3 x 109/L and/or a platelet count below 100 x 109/L). Seven patients with CD were included in this study. The attending physician judged the indication for administration of 6-TG in each participating patient. The study was approved by the Medical Ethical Committee Region Arnhem-Nijmegen (the Netherlands), and informed consent was obtained from all patients.

Study Design
In all 7 patients, 6-TG (Lanvis; Glaxo Wellcome, Zeist, the Netherlands) was administered orally in a dosage of 20 mg once daily. The following data were collected: patient demographics, disease history, history of thiopurine exposure, type of thiopurine intolerance, the use of concomitant medication, blood cell counts, and liver enzymes.

Outcome Measurements
Primary outcome measures were the determination of the concentration of 6-TG metabolites 6-TGMP, 6-TGDP, and 6-TGTP and total 6-TGN in erythrocytes. In addition, the concentration of 6-TG and thiopurine S-methyltransferase (TPMT) activity were measured in erythrocytes.

Measurement of 6-TGMP, 6-TGDP, 6-TGTP, and 6-TG in Erythrocytes
Erythrocytes were isolated at the day of blood sampling, and lysates were stored at -80°C until determination. Measurement of separated nucleotides was performed as described by Keuzenkamp-Jansen and colleagues10 by high-performance liquid chromatography (HPLC). A reversed-phase column (Supelcosil LC-18-DB) was applied with a gradient of potassium biphosphate and potassium biphosphate/methanol as the mobile phase. A wavelength of 342 nm was used for detection. Pure nucleosides were included in every run to calculate the amounts of 6-TG and its phosphorylated derivatives. A detection limit of about 20 pmol/109 RBC and a day-to-day and within-day coefficient of variation of about 10% was reached by this method.

Measurement of 6-TGN in Erythrocytes
The blood samples were centrifuged to isolate erythrocytes, and after washing with phosphatebuffered saline solution, erythrocyte counts were done. Samples were stored at -20°C until required. RBC 6-TGN levels were measured in the laboratory of the Department of Clinical Pharmacy, Maasland Hospital Sittard, using a slightly modified HPLC assay (C18 column, mobile phase: 50 mM orthophosphoric acid and 0.5 mM DTT).11 An ultraviolet wavelength of 343 nm was used for detection. The detection limit of quantification of the assay was 30 pmol/8 x 108 RBC for 6-TGN levels with a run-to-run coefficient of variation of 6.6%.

Measurement of TPMT Activity in Erythrocytes
A validated and published HPLC technique was used for the measurement of TPMT activity in erythrocytes. The enzymatic activity was measured by the amount of 6-methylmercaptopurine formed, using 6-MP as substrate and S-adenosylmethionine as cosubstrate.12

Statistical Analysis
Data are given descriptively and, when appropriate, expressed as median and range. Correlations between parameters were determined using the Spearman test. P values of less than .05 were considered significant. SPSS for Windows version 11.0 was used for statistical analysis.


   RESULTS
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 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Patients
Demographic data of the 7 CD patients are depicted in Table I. Reasons for withdrawal of previous treatment with AZA or 6-MP were gastrointestinal complaints (n = 3), allergic reactions (n = 3), myelotoxicity (n = 2), arthralgia (n = 2), and general malaise (n = 1). Some patients developed more than 1 adverse event on AZA or 6-MP therapy. Laboratory parameters were within reference limits in all patients (median levels: hemoglobin 7.8 g/L, range 7.7-8.8; leucocytes 8.4 x 109/L, range 3.8-13.7; platelets 372 x 109/L, range 136-438; creatinine 75 µmol/L, range 63-115; {gamma}-glutamyltransferase 19 U/L, range 14-114; alkaline phosphatase 66 U/L, range 48-87; aspartate aminotransferase 19 U/L, range 11-22; alanine aminotransferase 18 U/L, range 6-32; and amylase 151 U/L, range 120-203).


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  		Table I Patient Demographics

 

Metabolic Characteristics of 6-TG
High interindividual variances in all metabolite concentrations were observed after 6-TG administration (20 mg/d; Table II). The main metabolites were 6-TGTP (median, 531 pmol/8 x 108 RBC; range, 118-1316 pmol/8 x 108 RBC; mean, 630 pmol/8 x 108 RBC; SD, 464 pmol/8 x 108 RBC) and 6-TGDP (median, 199 pmol/8 x 108 RBC; range, 0-286 pmol/8 x 108 RBC; mean, 189 pmol/8 x 108 RBC; SD, 118 pmol/8 x 108 RBC). In 5 of the 7 patients (71%), 6-TGTP was found to be the major metabolite with by far the highest concentration. Crude 6-thioguaninenucleotide levels correlated with 6-TGTP levels (r = 0.929, P = .003) and with 6-TGDP levels (r = 0.786, P = .036). In addition, 6-TGN correlated with {Sigma} 6-TGXP, being the sum of 6-TGMP, 6-TGDP, and 6-TGTP (r = 0.929, P = .003). Furthermore, {Sigma} 6-TGXP correlated with the 6-TGTP level (r = 0.893, P = .007). The {Sigma} 6-TGXP (median, 783 pmol/8 x 108 RBC; mean, 857 pmol/8 x 108 RBC; SD, 521 pmol/8 x 108 RBC) was higher than the 6-TGN concentrations in all 7 patients, with the 6-TGN values varying between 48% and 95% of the {Sigma} 6-TGXP values. In 5 of 7 patients, 6-TGDP concentrations of more than 15% of {Sigma} 6-TGXP were found. Two patients had active disease, of which 1 had 52% of 6-TGDP. Of the remaining 5 patients, who were all in remission, percentages of 6-TGDP varied between 0% and 60%. Median TPMT activity was 13 pmol/h/107 RBC (range, 10-22 pmol/h/107 RBC), concomitant use of 5-ASA did not influence the in vitro TPMT activity. The levels of 6-TGN, 6-TGMP, 6-TGDP, and 6-TGTP were not correlated with TPMT activity, 6-TG dosages per kilogram bodyweight, disease location, or concomitant use of corticosteroids or 5-aminosalicylates.


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  		Table II Metabolic Characteristics

 


   DISCUSSION
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 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 REFERENCES
 
Pharmacokinetic and metabolic data concerning 6-TG use in patients with IBD are lacking, despite the fact that IBD patients were treated with 6-TG as early as in 1966.13 The proposed advantage of administering 6-TG compared to AZA or 6-MP is its relatively simple metabolism leading to high 6-TGN levels and less potentially toxic metabolites. 6-Thioguaninenucleotides derived from 6-TG are formed in a single step compared to multiple steps when originating from AZA or 6-MP. Moreover, 6-TG is less affected by TPMT and is a poor substrate for xanthine oxidase when compared to 6-MP. Genetic polymorphisms of these metabolizing enzymes seem to be of less influence.14 Despite this straightforward monostep metabolism, our data show for the first time that a wide interindividual variance exists not only in total 6-TGN concentrations but also in 6-TGMP, 6-TGDP, and 6-TGTP concentrations during 6-TG therapy, not explained by different 6-TG dosages per kilogram bodyweight or TPMT activity. However, other factors, such as absorption capacity, individual metabolism, or disease activity, may have contributed to the observed variance in metabolite levels. Derijks and colleagues have already demonstrated that absolute (mg) or relative (mg/kg) 6-TG dosages do not correlate with crude 6-TGN levels.15 As only small concentrations of 6-TGMP were detected, active 6-TGTP and its presumed inactive precursor 6-TGDP are probably the main metabolites within the total nucleotide pool of 6-TGN. The remarkable discrepancy between the 6-TGN level and the sum of 6-TGMP, 6-TGDP, and 6-TGTP is probably explained by the hydrolysis of nucleotides as for the total 6-TGN analysis, erythrocytes have not always been isolated on the same day blood sampling occurred. However, differences in extraction procedure or method of detection may also have influenced the outcomes.16,17 The significant correlations among the 6-TGN level, 6-TGTP level, and the sum of nucleotides may favor the determination of the total 6-TGN concentration in clinical daily practice as this assay is the current reference method, easier to perform, and less time consuming. However, the determination of separate phosphorylated thiopurine nucleotides by HPLC seems to be the method of choice for further detailed studies on the mechanism of action of thiopurines, as this technique allows evaluation of the role of 6-TGTP as a pharmacological active end metabolite.9 Moreover, it may be interesting to determine 6-TGDP and 6-TGTP levels in patients with adequate 6-TGN levels but without proper response to thiopurine therapy as low 6-TGTP levels may provide essential information for treatment failure. The influence of TPMT on the different 6-TG-derived metabolites seems limited as no correlations could be established. This finding provides support to the idea of administering 6-TG to IBD patients who developed intolerable side effects during AZA or 6-MP therapy caused by TPMT polymorphisms.2 Nevertheless, 6-TG-induced myelotoxicity due to TPMT polymorphisms has been reported.18

The absorption of orally administered 6-TG is known to be incomplete and highly variable. Studies demonstrated that after 6 hours of administration, 6-TG becomes undetectable in plasma as it is rapidly transported into cells in which 6-TG is immediately metabolized to 6-TGN.19 However, our data demonstrate that this may not be the case in all patients, as traces of 6-TG were found in 2 of our patients after 3 and 11 hours of administration. The explanation for this delayed absorption or conversion is unclear. The clinical importance, however, seems limited as no more than traces of 6-TG were found and 6-TG itself has no pharmacological activity.

In conclusion, the 1-step metabolism of 6-TG is still characterized by a high interindividual variance in the concentration of different 6-TG metabolites that could be explained by other factors such as absorption capacity, disease activity, or individual metabolism. The standard determination of 6-TGN levels seems sufficient for routine clinical practice as the 6-TGTP level as well as the sum of nucleotides are significantly correlated with 6-TGN level.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
REFERENCES
 
Financial disclosure: None declared.

DOI: 10.1177/0091270006294403


   REFERENCES
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 

1. Al Hadithy AF, de Boer NK, Derijks LJ, Escher JC, Mulder CJ, Brouwers JR. Thiopurines in inflammatory bowel disease: pharmacogenetics, therapeutic drug monitoring and clinical recommendations. Dig Liver Dis. 2005;37: 282-297.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

2. de Jong D, Mulder CJ, van Sorge AA. Why measure thiopurine methyltransferase activity? Direct administration of 6-thioguanine might be the alternative for 6-mercaptopurine or azathioprine. Gut. 2001;49: 874.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

3. Dubinsky MC, Hassard PV, Seidman EG, et al. An open-label pilot study using thioguanine as a therapeutic alternative in Crohn's disease patients resistant to 6-mercaptopurine therapy. Inflamm Bowel Dis. 2001;7: 181-189.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

4. Derijks LJ, de Jong DJ, Gilissen LP, et al. 6-Thioguanine seems promising in azathioprine- or 6-mercaptopurine-intolerant inflammatory bowel disease patients: a short-term safety assessment. Eur J Gastroenterol Hepatol. 2003;15: 63-67.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

5. Dubinsky MC, Feldman EJ, Abreu MT, Targan SR, Vasiliauskas EA. Thioguanine: a potential alternate thiopurine for IBD patients allergic to 6-mercaptopurine or azathioprine. Am J Gastroenterol. 2003;98: 1058-1063.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

6. Herrlinger KR, Deibert P, Schwab M, et al. Remission maintenance by tioguanine in chronic active Crohn's disease. Aliment Pharmacol Ther. 2003;17: 1459-1464.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

7. Deibert P, Dilger K, Fischer C, et al. High variation of tioguanine absorption in patients with chronic active Crohn's disease. Aliment Pharmacol Ther. 2003;18: 183-189.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

8. Tiede I, Fritz G, Strand S, et al. CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes. J Clin Invest. 2003;111: 1133-1145.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

9. Neurath MF, Kiesslich R, Teichgraber U, et al. 6-thioguanosine diphosphate and triphosphate levels in red blood cells and response to azathioprine therapy in Crohn's disease. Clin Gastroenterol Hepatol. 2005;3: 1007-1014.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

10. Keuzenkamp-Jansen CW, De Abreu RA, Bokkerink JP, Trijbels JM. Determination of extracellular and intracellular thiopurines and methylthiopurines by high-performance liquid chromatography. J Chromatogr B Biomed Appl. 1995;672: 53-61.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

11. Derijks LJ, Gilissen LP, Engels LG, et al. Pharmacokinetics of 6-mercaptopurine in patients with inflammatory bowel disease: implications for therapy. Ther Drug Monit. 2004;26: 311-318.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

12. Keizer-Garritsen JJ, Brouwer C, Lambooy LH, et al. Measurement of thiopurine S-methyltransferase activity in human blood samples based on high-performance liquid chromatography: reference values in erythrocytes from children. Ann Clin Biochem. 2003;40: 86-93.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

13. Bean RHD. Treatment of ulcerative colitis with antimetabolites. Br Med J. 1966;1: 1081-1084.[Free Full Text]

14. Lennard L, Davies HA, Lilleyman JS. Is 6-thioguanine more appropriate than 6-mercaptopurine for children with acute lymphoblastic leukaemia? Br J Cancer. 1993;68: 186-190.[Web of Science][Medline] [Order article via Infotrieve]

15. Derijks LJJ, Gilissen LPL, Engels LGJB, et al. Pharmacokinetics of 6-thioguanine in patients with inflammatory bowel disease. Ther Drug Monit. 2006;28: 45-50.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

16. Shipkova M, Armstrong VW, Wieland E, Oellerich M. Differences in nucleotide hydrolysis contribute to the differences between erythrocyte 6-thioguanine nucleotide concentrations determined by two widely used methods. Clin Chem. 2003;49: 260-268.[Abstract/Free Full Text]

17. Armstrong VW, Shipkova M, Von Ahsen N, Oellerich M. Analytic aspects of monitoring therapy with thiopurine medications. Ther Drug Monit. 2004;26: 220-226.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

18. McBride KL, Gilchrist GS, Smithson WA, Weinshilboum RM, Szumlanski CL. Severe 6-thioguanine-induced marrow aplasia in a child with acute lymphoblastic leukemia and inherited thiopurine methyltransferase deficiency. J Pediatr Hematol Oncol. 2000;22: 441-445.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]

19. Lancaster DL, Patel N, Lennard L, Lilleyman JS. 6-Thioguanine in children with acute lymphoblastic leukaemia: influence of food on parent drug pharmacokinetics and 6-thioguanine nucleotide concentrations. Br J Clin Pharmacol. 2001;51: 531-539.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
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