Linköping University Post Print How Should Thiopurine Treatment be Monitored? Methodological Aspects Svante Vikingsson, Björn Carlsson, Sven Almer and Curt Peterson N.B.: When citing this work, cite the original article. This is an electronic version of an article published in: Svante Vikingsson, Björn Carlsson, Sven Almer and Curt Peterson, How Should Thiopurine Treatment be Monitored? Methodological Aspects, 2010, Nucleosides, Nucleotides & Nucleic Acids, (29), 04-Jun, 278-283. Nucleosides, Nucleotides & Nucleic Acids is available online at informaworldTM: http://dx.doi.org/10.1080/15257771003741133 Copyright: Taylor & Francis http://www.tandf.co.uk/journals/default.asp Postprint available at: Linköping University Electronic Press http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-58361 How should thiopurine treatment be monitored? – Methodological aspects Running title: Thiopurine monitoring – methodological aspects S Vikingsson1 (Corresponding), B Carlsson1, S Almer2, C Peterson1 1Division of Drug Research, Clinical Pharmacology, Department of Medical and Health Sciences, Faculty of Health Sciences, Linköping University, Linköping, Sweden 2Division of Gastroenterology and Hepatology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden Address for correspondence: Svante Vikingsson, Clinical Pharmacology, University Hospital, SE- 58185 Linköping, Sweden Email: [email protected] Telephone: +46 733 767637, +46 10 1034487 ABSTRACT Monitoring of thiopurine metabolites is important due to a complex metabolism with large inter-individual variation, but the suitability of currently used methods has been questioned. The drawbacks include poor reproducibility, the inability to differentiate between the different analytes, as well as the use of a non-target matrix. Further research should be directed towards measuring thiopurine metabolites in mononuclear cells, measuring the different nucleotides specifically, as well as measuring the incorporation of thioguanine into DNA. The studies should not be limited to thioguanosine nucleotides but include methylthioinosine nucleotides as well. Key words: TGN, methylthioinosine monophosphate, methyl mercaptopurine, inflammatory bowel disease, childhood leukemia INTRODUCTION active metabolites during thiopurine therapy. In turn, this leads to variations The thiopurine drugs, azathioprine, in drug response resulting in lack of mercaptopurine, and thioguanine, are therapeutic effect as well as adverse widely used in the treatment of events. childhood acute lymphoblastic Low activity of the polymorphic leukemia (ALL), and inflammatory enzyme thiopurine methyltransferase bowel disease (IBD). They are pro- (TPMT) leads to abnormally high drugs with a complex metabolism with levels of thioguanosine nucleotides large inter-individual variation. This (TGN) in red blood cells (RBC), variation gives rise to substantial and especially in homozygously deficient unpredictable inter-individual patients [1], with a frequency around 1 differences in the concentrations of the in 300 in Caucasians [1, 2]. The high 1 concentrations may cause adverse WHY WE NEED NEW METHODS events that can be avoided by adjusting the dosage based on TPMT activity RBC are not the target of thiopurines determination prior to initiating and it is questionable if the metabolite treatment with thiopurines. concentration in RBC is a good However, even when the TPMT surrogate marker for drug effects. Even activity is taken into account, a though it has been shown that significant inter-individual variation in extremely high TGN concentrations in the drug response remains. To help RBC correlate with adverse events, and individualize the thiopurine treatment, that the methods are effective for monitoring of the active metabolites checking compliance, a number of has been carried out in many centers studies have failed to establish a for the last 20 years, but the clinical correlation to clinical effects[3]. In a benefit of such methods has been recent meta-analysis by Osterman et al questioned [3]. We believe that this lack [9], a correlation between remission and of success mainly might be due to TGN concentrations was found, but methodological aspects and the aim of TGN concentration only partially this article is to discuss the methods explained remission. used today, give possible explanations as to why they fail, and suggest how to WHERE DO THE PROBLEMS improve them. ARISE? THE METHODS USED TODAY There are a number of methodological disadvantages with the methodology The main strategy for the monitoring used so far that might contribute to the of thiopurine metabolites has been to lack of observed correlation. Firstly, measure TGN in RBC. A number of the acid hydrolysis methods have different methods have been described, shown poor reproducibility. In the the majority of which are based on modified Lennard and Singleton hydrolysis of the TGN back to method [4] used in our laboratory, the thioguanine using acid, possibly observed inter-day CV was more than followed by some sort of sample clean 20% at concentrations between 58 and up [4-6]. Typically, the thioguanine is 660 pmol/8x10^8 RBC [10]. detected using UV-absorbance at either The hydrolysis step is another possible around 320 nm [4], or 340 nm [6]. Most source of error. Differences in commonly used are the method hydrolysis efficiency make developed by Lennard and Singleton [4, comparisons between results obtained 7] , the method developed by by different methods difficult. This Derivieux and Boulieu[6, 8], or slightly was nicely shown by Shipkova et al[11] modified versions thereof. Briefly the in a comparison between the methods TGN are hydrolyzed using sulphuric developed by Lennard and Singleton[4, acid, in the method by Lennard and 7], and Dervieux and Boulieu[6, 8]. Singleton, or perchloric acid, in the Especially the method developed by method by Dervieux and Boulieu, and Lennard and Singleton seemed to analyzed by HPLC. In the method by suffer from incomplete hydrolysis. Lennard and Singleton, the produced Secondly, at least 9 different thioguanine is extracted by phenyl substances could possibly contribute to mercury adduct formation prior to the thioguanine reported as TGN, injection onto the HPLC. including the free base, the riboside, the three nucleotides (mono-, di-, and 2 triphosphate), as well as the deoxy Taken together, it seems likely that analogs. However, earlier studies even though the concentration of one showed that the free base [4, 10] and the or more of these substances in the riboside [10] are not present in RBC:s, target cells correlate well with the and therefore do not contribute to clinical outcome it would be very TGN. Deoxyribonucleotides are difficult to observe this when present at such low concentrations that measuring TGN in RBC. their contribution towards the total TGN probably is negligible [12]. Instead THE IDENTITY OF meTIMP AND TGN is mainly made up by ITS IMPORTANCE thioguanosine mono- (TGMP), di- (TGDP), and triphosphates (TGTP) [10, Because the thiopurines are pro-drugs, 13-15]. a number of other metabolites are It is likely that these substances differ produced. Of these, especially in their clinical effects, and therefore it methylthioinosine monophosphate is possible that individual patients (meTIMP) and methylmercaptopurine might be more or less skewed towards (meMP) as measured by various acid the more potent substances, affecting hydrolysis methods, have attracted a the clinical outcome. Some evidence research interest, especially regarding from studies comparing TGMP, adverse events. TGDP, and TGTP concentrations When acid hydrolysis methods are suggest that this is indeed the case [10, used, the analytes reported as meTIMP 13, 15]. More specifically, this indicates are the same as those reported as that the concentration of TGN might meMP. Mercaptopurine is the base in not accurately reflect the thioinosine nucleotides making concentrations of TGTP and meTIMP and meMP, indistinguishable. deoxyTGTP, which are the substances The substance is hydrolyzed back to 4- believed to be incorporated into RNA amino-5-(methylthio)carbonyl and DNA, respectively. imidazole (AMTCI) during the The third major drawback is that TGN measurement [8]. A name analogous is measured in RBC. The RBC lack with TGN is methylthioinosine inosine monophosphate nucleotides (meTIN), which will be dehydrogenase[16] and cannot form used in the reminder of this article. TGN from azathioprine or In patients treated orally, neither mercaptopurine. Instead, the TGN meMP nor meMP riboside was found present in the RBC are hypothesized to in RBC [18]. It has also been shown that be produced by phosphorylation of meTIMP does contribute to the total thioguanine absorbed from tissues meTIN, but other, not yet identified capable of converting 6- substances also contributes [10]. Earlier mercaptopurine into thioguanine[17]. research showed that very high meTIN Individual differences between patients values did predict adverse events [19], in the transport of these substances as well as that meTIN was increased might therefore give rise to differences instead of TGN in patients not in the ratio between the TGN responding to treatment [20]. Both these concentration in the targeted cells and studies indicate the importance of the TGN concentration measured in further studies of meTIN. RBC, and thus explain the weak or absent correlation between TGN WHAT ARE THE ALTERNAT- concentration and clinical