Thiopurines: Factors influencing toxicity and response

Biochemical Pharmacology 79 (2010) 1211–1220

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Commentary

Thiopurines: Factors influencing toxicity and response Alan Kambiz Fotoohi a,b, Sally A. Coulthard c,d, Freidoun Albertioni a,* a

Department of Oncology and Pathology, Cancer Center Karolinska, Karolinska Institutet SE-171 76 Stockholm, Sweden Department of Internal Medicine, Division of Clinical Pharmacology, Karolinska University Hospital, Sweden c Division of Clinical Pharmacology, Department of Medical and Health Sciences, Faculty of Health Sciences, Linko¨ping University, Linko¨ping, Sweden d Newcastle University, Northern Institute for Cancer Research, Newcastle Upon Tyne, NE2 4HH, United Kingdom b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 5 October 2009 Accepted 12 January 2010

Thiopurines are the backbone of current anti-leukemia regimens and have also been effective immunosuppressive agents for the past half a century. Extensive research on their mechanism of action has been undertaken, yet many issues remain to be addressed to resolve unexplained cases of thiopurine toxicity or treatment failure. The aim of this review is to summarize current knowledge of the mechanism of thiopurine action in experimental models and put into context with clinical observations. Clear understanding of their metabolism will contribute to maximizing efficacy and minimizing toxicity by individually tailoring therapy according to the expression profile of relevant factors involved in thiopurine activation pathway. ß 2010 Published by Elsevier Inc.

Keywords: Acute lymphoblastic leukemia 6-Mercaptopurine 6-Thioguanine Azathioprine Hypoxanthine-guanine phosphoribosyl transferase Thiopurine methyltransferase Inosine 50 -monophosphate dehydrogenase Transport Pharmacogenetics

1. Introduction For the past half century thiopurines have earned themselves a reputation as effective anti-cancer and immunosuppressive drugs. During the same time we have witnessed dramatic improvement

Abbreviations: 6-MP, 6-mercaptopurine; 6-TG, 6-thioguanine; ALL, acute lymphocytic leukemia; AML, acute myeloid leukemia; AZA, azathioprine; TGN, thioguanine nucleotide; meMP, methylmercaptopurine; HGPRT, hypoxanthine-guanine phosphoribosyl transferase; DNPS, de novo purine synthesis; TIMP, 6-thioinosine-50 monophosphate; IMPDH, inosine monophosphate dehydrogenase; GMPS, guanosine monophosphate synthetase; meTIMP, methylthioinosine monophosphate; TGTP, 6-thioguanosine-50 -triphosphate; dTGTP, deoxy-6-thioguanosine-50 -triphosphate; MMR, mismatch repair; meMP, 6-methylmercaptopurine; meTG, 6methylthioguanine; XO, xanthine oxidase; TU, thiouric acid; AO, aldehyde oxidase; PRPP, phosphoribosyl pyrophosphate; ITPase, inosine triphosphate pyrophosphatase; MRP, multidrug resistance-associated proteins; MDR1, multidrug resistance-1; SAM, S-adenosylmethionine; medTGTP, methyl deoxythioguanosine-50 -triphosphate; TXMP, thioxanthosine monophosphate; TGMP, 6-thioguanosine-50 -monophosphate; NDPK, nucleoside diphosphate kinase; ENT, equilibrative nucleoside transporters; CNT, sodium-dependent transporters; NBTI, nitrobenzylthioinosine; meMPR, methylmercaptopurine riboside; DNMT, DNA methyltransferase. * Corresponding author. Tel.: +46 8 517 75832/70 4836536; fax: +46 8 517 750 42. E-mail address: [email protected] (F. Albertioni). 0006-2952/$ – see front matter ß 2010 Published by Elsevier Inc. doi:10.1016/j.bcp.2010.01.006

in survival rates of leukemia patients. This success is mainly due to the development of combination therapies and stratification of patients according to risk of treatment failure and relapse, rather than the discovery of new drugs. The thiopurine antimetabolites, 6-mercaptopurine (6-MP) and 6thioguanine (6-TG) are the analogues of purine nucleosides and are currently the backbone of childhood acute leukemia treatment. Both of these compounds were synthesized by Elion and Hutchins in 1951 [1] by replacing the oxygen atom at carbon 6 of hypoxanthine (6MP) or guanine (6-TG) by sulphur (Fig. 1). The first clinical trial with 6-MP involved oral administration and demonstrated a beneficial effect in the treatment of acute leukemia in children [2]. The combination of 6-MP with methotrexate (MTX) and steroids extended median survival time from 3 to 12 months. Only two years after its original synthesis, 6-MP was approved by the authorities for use in the treatment of childhood acute lymphocytic leukemia (ALL). 6-MP is now used as a routine component of all modern protocols for maintenance therapy of children with ALL and the combination of high-dose MTX and 6-MP is commonly employed for consolidation therapy of childhood ALL. In 1958, Schwartz et al. [3] demonstrated the immunosuppressive ability of 6-MP to prevent an antibody response in rabbits injected with an antigen. Shortly after that the Hitchings–Elion laboratory synthesized numerous 6-MP derivatives, including 6-

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Fig. 1. Molecular structures of endogenous bases, 6-mercaptopurine, 6-thioguanine and azathioprine. 6-Mercaptopurine is the thio analogue of hypoxanthine and 6-thioguanine is the thio analogue of guanine.

(1-methyl-4-nitro-5-imidazolyl) thiopurine, azathioprine (AZA) (Fig. 1), a pro-drug that is reduced non-enzymatically to 6-MP in vivo (Fig. 2). The first comparison of AZA and 6-MP as immunosuppressors was performed on 10 dogs that had received renal transplants; AZA had the better effect in this system and was soon shown to be an effective immunosuppressive agent in humans as well. These drugs, like many cytotoxic agents, have a relatively narrow therapeutic index, with potential life-threatening druginduced toxicity primarily in the form of myelosuppression. The

other major toxic effect of thiopurines is hepatotoxicity which is related to the amounts of thioguanine nucleotides (TGNs) or methylmercaptopurine (meMP) in erythrocytes, as well as accumulation of 6-MP and its metabolites in the liver. After numerous observations of AZA association with an increased risk of various malignancies, i.e. acute myeloid leukemia (AML) it was recognized as a human carcinogen by the International Agency for Research on Cancer. Nevertheless, the beneficial effects of thiopurines outweigh the toxic side effects and they continue to be widely used to treat childhood ALL, inflammatory bowel disease

Fig. 2. Metabolism of thiopurines. Thiopurines are catabolized by xanthine oxidase (XO), guanase and aldehyde oxidase (AO) in the extracellular space. When inside the cell 6TG is converted directly by hypoxanthine–guanine phosphoribosyl transferase (HGPRT) through addition of ribose-5-phosphate to 6-thioguanosine-50 -monophosphate (TGMP), 6-MP is converted first to 6-thioinosine-50 -monophosphate (TIMP) by HGPRT then to 6-thioxanthine-50 -monophosphate (TXMP) by inosine monophosphate dehydrogenase (IMPDH) and finally to TGMP by guanosine monophosphate synthetase (GMPS). Both 6-MP and 6-TG and their respective monophosphates (TIMP and TGMP) are extensively inactivated inside the cell by thiopurine-S-methyltransferase (TPMT). Methylthioinosine monophosphate (meTIMP) is a strong inhibitor of DNPS. The remaining TGMP is converted to 6-thioguanosine-50 -diphosphate (TGDP), reduced to deoxy-6-thioguanosine-50 -diphosphate (dTGDP) by ribonucleotide reductase (RR) and phosphorylated by nucleoside diphosphate kinase (NDPK) to dTGTP, a DNA polymerase substrate.

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and autoimmune diseases, as well as to prevent rejection of organ transplants. Treatment success for leukemia is impressive with 80% cure rates for ALL, yet the remaining 20% at risk of relapse and chemotherapy toxicities is a substantial problem. Understanding the mechanism of action and resistance to thiopurines in different patients and consolidating this knowledge with pharmacogenetic characteristics of individual patients has the potential for improving survival rates. In this review we summarize the current knowledge on thiopurines 6-MP and 6-TG mechanism of action and resistance, emphasizing issues which need to be addressed to improve the therapeutic potential of those drugs in leukemia patients. 2. Thiopurine metabolism pathways Thiopurines are inactive pro-drugs that exert their cytotoxicity after they have been metabolized within the cell. Intracellular metabolites contribute to the effect of thiopurines either by inhibiting de novo purine synthesis (DNPS) or by being incorporated into DNA [4]. Both 6-MP and 6-TG require activation by hypoxanthine-guanine phosphoribosyl transferase (HGPRT, E.C. 2.4.2.8) followed by a multi-step metabolism to TGNs that can be incorporated into DNA or RNA or to methylated products which inhibit DNPS (Fig. 2). The first step of 6-MP metabolism is conversion to 6thioinosine-50 -monophosphate (TIMP) by HGPRT. TIMP is then metabolized to 6-thioguanosine-50 -monophosphate (TGMP) in a two-step process involving inosine monophosphate dehydrogenase (IMPDH, E.C. 1.1.1.205) and guanosine monophosphate synthetase (GMPS, E.C. 6.3.4.1) [4]. TIMP can then be methylated by thiopurine methyltransferase (TPMT, E.C. 2.1.1.67) to form methylthioinosine monophosphate (meTIMP) a strong inhibitor of DNPS [5]. 6-TG, on the other hand, is directly converted by HGPRT into TGMP, which is then phosphorylated further by two kinases to yield 6-thioguanosine-50 -triphosphate (TGTP), which can either be incorporated into RNA or, after enzymatic reduction to deoxy-6thioguanosine-50 -triphosphate (dTGTP), into DNA (Fig. 2). Incorporation of dTGTP into DNA triggers cell cycle arrest and apoptosis by a process involving the mismatch repair (MMR) pathway [6]. The thiopurines 6-MP and 6-TG can be metabolized by four enzymes: HGPRT as described above to form TIMP; TPMT to form inactive metabolites 6-methylmercaptopurine (meMP) and 6-methylthioguanine (meTG); xanthine oxidase (XO, E.C. 1.17.3.2) to form inactive thiouric acid (TU) (though this does not occur to any great extent in haematopoietic tissue) and finally aldehyde oxidase (AO, E.C. 1.2.3.1) which converts meMP [7] and 6-TG [8] into their hydroxlated metabolites (Fig. 2). In clinical practice, however, it is recommended to avoid milk products when administering 6-MP since they contain XO which could compromise the treatment [9]. Together, the effects of XO and AO activity leaves only about 16% of the total dose of 6-MP available for systemic distribution. Because of the lower cytotoxic potency of meMP, methylated metabolites were considered at first to be less cytotoxic than the parent drug. Subsequently, several groups established that certain methylated metabolites are actually more cytotoxic and it is now clear that methylation can contribute to the cytotoxic effects of thiopurines, both in vivo and in vitro [10,11]. One of the methylated metabolites of 6-MP, meMPR, is a well-characterized and potent inhibitor of DNPS and cytotoxic agent that contributes to the cytocidal effects of 6-MP [5]. In the case of oral administration of AZA, humans can metabolize as much as 12% of this compound to hypoxanthine and methyl-4-nitro-5-thioimidazole, which may contribute to their immunosuppressive effects [12].

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3. Main actors in metabolism of thiopurines 3.1. Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) HGPRT is a purine salvage enzyme that catalyzes the conversion of hypoxanthine and guanine to their respective mononucleotides. A partial deficiency in this enzyme, often caused by a single base mutation, can result in overproduction of uric acid leading to hyperuricemia, hereditary gouty arthritis and nephrolithiasis. However, the absence of HGPRT activity as seen in patients suffering from the X-linked recessive Lesch–Nyhan syndrome is characterized by hyperuricemia, mental retardation, choreoathetosis and compulsive self-mutilation. AZA and 6-MP are not cytotoxic in these patients. HGPRT is expressed widely throughout the human body, with highest levels in the central nervous system, 4–8-fold lower levels in erythrocytes and lymphocytes, and 16–20fold lower levels in the liver, kidney and spleen [13]. A low level or absence of HGPRT activity is the most extensively characterized mechanism underlying the resistance of leukemic cells to 6-MP and 6-TG, but this mechanism is rarely observed in leukemic cells obtained from patients with ALL. In a study involving 83 children with untreated ALL, low HGPRT activity was correlated with a poorer prognosis in those patients with precursor B-ALL [14]. Notable differences in HGPRT activity were found between boys and girls with ALL which is believed to be due to the X-linked property of the enzyme and its co-substrate phosphoribosyl pyrophosphate (PRPP). When children with leukemia were compared to healthy donors the leukemic children demonstrated a markedly higher HGPRT activity though this did not correlate to the production of 6-TGNs [15]. Another report demonstrated a 100-fold variation in HGPRT activity in the lymphoblasts of children with ALL at the time of relapse [16]. However, no currently available evidence supports a significant contribution of this mechanism to clinical activity to thiopurines, probably because a very low level of HGPRT activity can generate sufficient levels of the cytotoxic nucleotides of thiopurines. 3.2. Thiopurine methyltransferase (TPMT) TPMT is a cytosolic methylating enzyme whose physiological role, despite extensive investigations remains unclear. However, this enzyme is known to catalyze S-methylation of aromatic and heterocyclic compounds, preferentially thio compounds such as 6MP and 6-TG. It has a molecular mass of 26 kDa and is expressed in the liver, kidneys, intestine, erythrocytes, leukocytes and a number of other tissues (reviewed by Coulthard and Hogarth [17] and references therein). The discovery that levels of TPMT activity in human tissues are influenced by a common genetic polymorphism represents the most important example of the influence of pharmacogenetics on anti-cancer therapy as well as one of the best examples of the potential importance of pharmacogenetics for clinical medicine in general [18]. Specifically, it is now known that a reduction in TPMT activity, caused by genetic polymorphism, results in severe and sometimes fatal haematological toxicity in patients treated with standard doses of thiopurines such that the dose must be decreased for patients with heterozygous or homozygous polymorphisms in the TPMT gene. Conversely, patients with very high TPMT activity may be undertreated. Pronounced inherited variations in TPMT activity, ranging from high to virtually undetectable levels were first observed in human tissues more than two decades ago. The frequency of distribution of erythrocyte TPMT activity in 298 control subjects was found to be trimodal. Approximately 1 in 300 individuals (0.3%) had low or undetectable levels of TPMT activity, with intermediate levels in approximately 10% of individuals [18].

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The level of TPMT activity in erythrocytes reflects the corresponding levels in lymphocytes. Cloning and characterization of the human TPMT cDNA and gene revealed that these phenotypic variations were primarily from variation in the sequence of the gene itself [19]. Numerous genetic polymorphisms have been identified which are or may be associated with decreased levels of TPMT enzyme activity and/or enhanced toxicity of thiopurines [20]. Variant human alleles of TPMT proven to be associated with decreased catalytic activity can involve point mutations in the open-reading frame or at intron/exon splice sites. The wild-type allele, TPMT*1, encodes the fully active enzyme; while TPMT*2 (238 G > C), TPMT*3A (460 G > A, 719 A > G) and TPMT*3C (719 A > G) are the most prevalent in Caucasians, accounting together for 80–95%, of the polymorphic alleles that lead to a significant reduction in enzyme activity due to enhanced rates of proteolysis of the mutant proteins [21]. TPMT*3B (460G > A) alone is very rare in Caucasians, whereas in other ethnic populations the frequency of these alleles varies. Other more-rare TPMT variants can be considered to be family-specific (‘private’) mutations (reviewed by Sahasranaman et al. [22]). Patients that inherit one wild-type allele and one of the mutant alleles have intermediate TPMT activity while those that are homozygous for mutant alleles are TPMT deficient [21,23–26]. Other alterations detected in the TPMT gene include deletion of exons six and nine [27] and polymorphisms in the variable number of tandem repeats [28–31]. A complete list of all detected TPMT genotype alternatives and a short description of each variant phenotype is summarized in Table 1 with relevant references [20,21,23,25,26,28,30–44]. Pharmacokinetic and pharmacodynamic studies of 6-MP and 6TG in TPMT knockout mice with high, medium and no TPMT activity indicated that 6-MP was significantly more affected by TPMT polymorphisms than 6-TG [45]. Those studies corroborated earlier research by Dervieux et al. [46] who found that elevation of TPMT activity in human CCRF-CEM cell lines by retroviral gene transfer rendered the cells less sensitive to 6-TG, but more sensitive to 6-MP. This effect was also seen in another study by Coulthard et al. [10] in which human embryonic kidney cells were transfected with TPMT cDNA under the control of an inducible system in which they showed a 4.4-fold increase in sensitivity to 6MP and a 1.6-fold decrease in sensitivity to 6-TG. However, these observations have to be confirmed in patients to determine if they have a clinical importance. TPMT activity is inversely related to TGN concentrations in the erythrocytes of children treated for leukemia [47]. High erythrocyte concentrations of TGNs are correlated with the degree of leucopenia and a good prognosis [48] whereas low concentrations are associated with an increased risk for relapse [49]. The role of TPMT activity on the risk of second malignancies and survival rates has been investigated by several groups. At first the results appear to be conflicting, however, these anomalies are most likely to be protocol dependent. Essentially, for patients treated with the St. Jude Children’s Hospital protocols [50] or the NOPHO protocols [51], low TPMT activity is associated with higher risk of secondary cancers, in contrast to patients treated with the BFM protocols [52] where no such association was found. 3.3. Inosine 50 -monophosphate dehydrogenase (IMPDH) IMPDH catalyzes the first rate-limiting step in guanine nucleotide biosynthesis, i.e. the conversion of inosine monophosphate to xanthosine monophosphate. Human IMPDH activity is regulated by two separate 56-kDa enzymes; types I and II, which have virtually identical catalytic activities, substrate affinities and ki values and are 84% identical at the amino acid level. Since TIMP, the major intracellular metabolite of 6-MP, is a substrate for IMPDH, the activity of this enzyme may play an important role in treatment of

patients with purine analogues. Variation in the expression of either of the enzyme forms can be expected to exert a significant influence on thiopurine metabolism, with increased activity promoting toxicity and reduced activity predicting a poor clinical response. IMPDH activity increases with cell proliferation and transformation [53] and is higher in acute leukemic blasts than in a mixture of normal bone-marrow cells [54]. Inhibition of this enzyme in HL60 myeloid cells results in a low intracellular concentration of GTP and terminal differentiation of the cells [55] and micromolar concentrations of IMPDH inhibitors inhibits cell growth [56]. Approximately 9% of patients with inflammatory bowel disease who are resistant to AZA may carry mutations in enzymes involved in drug metabolism, including IMPDH. Indeed a 9-bp insertion into the IMPDH1 P3 promoter was detected in one patient who exhibited severe resistance to AZA [57]. However, to date, no significant correlation between altered IMPDH activity and resistance to thiopurines has been demonstrated, either in vitro or in vivo. 3.4. Guanine monophosphate synthetase (GMPS) GMPS catalyzes the amination of xanthosine 50 -monophosphate to guanosine monophosphate, and like IMPDH, is a crucial enzyme in the de novo biosynthesis of guanine nucleotides. Within the context of thiopurines, GMPS converts TXMP to TGMP. The level of GMPS mRNA expression is substantially higher in rapidly proliferating, such as neoplastic and regenerating tissues. Inhibition of GMPS could be an enzyme drug target for immunosuppression and cancer chemotherapy as decreased levels of GMPS result in a depletion of guanine nucleotides and cell proliferation in lymphocytes [58]. However, the effect of reducing activity of this enzyme with efficacy of 6-MP has not been investigated. 3.5. Other factors Systematic consideration of each of the enzymes involved in the degradation of thiopurines reveals a number of plausible candidate enzymes whose levels of expression might vary as a result of allelic polymorphism. Among these, a deficiency in 50 -nucleotidase enzymes is associated with enhanced thiopurine toxicity [59], but the genetic basis for this phenomenon has not been determined. Inhibition of XO and AO by allopurinol also potentiates thiopurine toxicity [60], but genetic deficiencies in this enzyme, manifested as xanthinuria, are very rare. However, the level of expression of XO in the liver does vary considerably between individuals and an as yet undiscovered polymorphism may be involved. Inosine triphosphate pyrophosphatase (ITPase) converts inosine triphosphate (ITP) back to IMP, thereby preventing accumulation of ITP. Marinaki et al. [61] predicted that in ITPase-deficient patients treated with thiopurine drugs, the metabolite 6-thio-ITP would accumulate and give rise to toxicity. Indeed, these investigators found that an ITPA 94C > A heterozygous genotype was significantly associated with adverse effects of AZA, especially flu-like illness, pancreatitis and rashes. More recently, the effect of the ITPase variant P32T was assessed by Stocco et al. [62] in children with ALL receiving 6-MP in the context of TPMT genotype. When 6-MP dose was not adjusted for TPMT genotype, TPMT genotype had a greater role in toxicity than ITPase P32T; however, where 6-MP dose was adjusted, the ITPase P32T variant was significantly associated with the incidence of febrile neutropenia, suggesting that the effect of ITPase activity has to be considered in the context of TPMT activity and dose of 6-MP prescribed. When the 6-MP dose was not adjusted for TPMT genotype, TPMT genotype affected toxicity more than ITPase P32T, however where 6-MP dose was adjusted, the ITPase P32T variant was significantly associated with the incidence of febrile neutropenia,

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Table 1 TPMT polymorphisms and their impact on TPMT enzymatic activity. Allele

Mutation

Description

Ref.

TPMT*1 TPMT*1A

Wild type 178C > T

TPMT*1S TPMT*2

474T > C (Ile158Ile) 238G > C (Ala80Pro)

Wild type, normal TPMT activity. Polymorphism in un-translated exon 1, unaltered TPMT activity. Silent mutation, unaltered TPMT activity. TPMT mRNA levels comparable to wild type (TPMT*1), but approximately a 100-fold reduction in S-methylation activity. Substitution of rigid proline for a flexible alanine changes the tertiary structure of protein leading to protein instability and decreased catalytic activity.

[26] [42] [36,43,44]

Vmax/Km values are 10% of the wild-type TPMT. Enhanced degradation of protein. TPMT*3A

460G > A (Ala154Thr), 719A > G (Tyr240Cys)

TPMT*3B

460G > A (Ala154Thr)

TPMT*3C TPMT*3D

719A > G (Tyr240Cys) 292G > T (Glu98Stop), 719A > G, 460G > A

TPMT*4

G!A

TPMT*5

146T > C (Leu49Ser)

TPMT*6 TPMT*7

539A > T (Tyr180Phe) 681T > G (His227Glu)

TPMT*8

644G > A (Arg215His)

TPMT*9 TPMT*10 TPMT*11 TPMT*12

356A > C (Lys119Thr) 430G > C (Gly144Arg) 395G > A (Cys132Tyr) 374C > T (Ser125Leu)

TPMT*13

83A > T (Glu28Val)

TPMT*14

1A > G (Met1Val)

TPMT*15 TPMT*16

_1G ! A 488G > A (Arg163His)

TPMT*17 TPMT*18 TPMT*19 TPMT*20a TPMT*21

124C > G (Gln42Glu) 211G > A (Gly71Arg) 365A > C (Lys122Thr) 106G > A (Gly36Ser) 712A > G (Lys238Glu) 205C > G (Leu69Val)

TPMT*22 TPMT*23 TPMT*24a

488G > C (Arg163Pro) 500C > G (Ala167Gly) 537G > T (Gln179His) 106G > A (Gly36Ser)

TPMT*25

634T > C (Cys212Arg)

TPMT*26 Tandem repeats

777T > C (Phe208Leu) VNTR3–VNTR9

a

TPMT mRNA levels comparable to wild type, but TPMT protein levels were about 400-fold lower, with no detectable catalytic activity. Enhanced degradation of protein. 9-fold reduction in catalytic activity. Rare ‘‘private’’ mutation. Enhanced degradation of protein. 1.4-fold reduction in catalytic activity. Enhanced degradation of protein. Low enzyme activity. First reported allele for low TPMT activity as a result of a mutation within an intron 9 leading to a splicing error. Undetectable enzyme activity as well as considerably lower expression level of TPMT. Suspected deficient enzyme activity. Mis-sense mutation in exon 10 resulting in non-functional allele. Vmax/Km values are 10% of the wild-type TPMT. Identified in one heterozygous African-American with intermediate enzyme activity. No significant change in catalytic activity. Mis-sense mutation resulting in deficient enzyme activity. Deficient enzyme activity. Mis-sense mutation resulting in significant decrease in intrinsic clearance values, retaining about 30% of the wild-type enzyme [33]. Considerably lower expression level of TPMT is observed [33,44]. Mis-sense mutation resulting in decreased in intrinsic clearance values, retaining about 57% of the wild-type enzyme. Considerably lower expression level of TPMT as well as lower enzymatic activity. Splicing defect in intron 7 resulting in low enzymatic activity. Significantly decreased intrinsic clearance value (by 3-fold). Located in a highly conserved region of the human TPMT protein. Vmax/Km values are 10% of the wild-type TPMT. Slightly reduced expression of TPMT. No significantly decrease in activity compared to wild-type TPMT. Reduced enzyme activity [40]. Decreased TPMT activity [41]. Enzyme with significantly decreased intrinsic clearance characterizing non-functional allele. Decreased enzyme activity. Considerably lower expression level of TPMT. Decreased enzyme activity. Almost undetectable enzyme activity. Mis-sense mutations. No significant change in catalytic activity [32] Vmax/Km values are 10% of the wild-type TPMT [44]. Mis-sense mutation. Enzyme with significantly decreased intrinsic clearance characterizing non-functional allele. Decreased enzyme activity. Polymorphisms in the variable number of tandem repeats in the promoter region with mostly minor decrease in the level of enzyme activity. VNTR6 leads to moderately reduced enzymatic activity.

[36,42,43]

[36,42,43] [21,25,43] [25,26]

[25,26] [20,26,44] [25,26] [20,26,44] [23] [32] [20,39] [39] [33,44]

[33] [37,44] [37] [34] [44] [44] [34] [40,41] [41] [41] [38] [32,40,44] [32] [35] [28,30,31]

More than one SNP reported with the same nomenclature.

suggesting that the effect of ITPase activity has to be considered in the context of TPMT activity and dose of 6-MP prescribed. 4. Uptake of thiopurines Initial studies indicated that cellular uptake of 6-MP occurs primarily by passive diffusion, but it was discovered that facilitated diffusion of this compound can be competitively inhibited by hypoxanthine. 6-MP is rapidly transported into cells and

phosphoribosylated, after which the main rate-determining step in its incorporation into nucleic acids is further conversion to TIMP. Since the chemical structures of 6-MP and 6-TG are very similar to those of hypoxanthine and guanine, these drugs can be expected to be taken up into and exported from cells by the same transporters that translocate these nucleotides. The most extensively characterized families of transporters that mediate uptake of nucleosides and nucleobases are the nucleoside transporters, which can be subdivided into two major classes: equilibrative (facilitated)

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transporters (the SLC29 family) which mediate the net flux of nucleoside molecules across the plasma membrane only down a concentration gradient; and concentrative or Na+-dependent transporters (the SLC28 family) which use an electrochemical ion gradient to drive active uptake even against a concentration gradient [63]. The human family of equilibrative transporters (ENT) contains four members for which the well-characterized ENT1 and ENT2 exhibited similar broad specificities for purine and pyrimidine nucleosides, with ENT2 possessing the ability to transport nucleobases as well [64]. The family of concentrative nucleoside transporters consists of three subtypes of sodium-dependent transporters: CNT1 transports pyrimidine nucleosides preferentially, CNT2 prefers purine nucleosides, and CNT3 transports both these types of nucleosides across the cell membrane [65]. Zaza et al. [66] identified 60 species of mRNA whose level of expression was significantly correlated with accumulation of TGNs in ALL patients treated with 6-MP. Down-regulation of ENT1 was associated with low intracellular concentrations of TGNs and inhibition of ENT1 by nitrobenzylthioinosine caused a significant reduction of the level of TGNs in ALL cells in vitro, suggesting that this transporter protein plays a role in clinical response to 6-MP therapy. In a recent study [67], two thiopurine-resistant sub-lines of MOLT4 cells showed neither of the well-characterized mechanisms of resistance to thiopurines, i.e. absence of HGPRT activity or altered TPMT activity. Instead, defective cellular uptake was found to be the primary mechanism underlying this resistance. Quantification of the levels of mRNA encoding nucleoside transporters revealed significant reductions with respect to CNT3 and ENT2 in the thiopurineresistant cells compared to the wild-type parental cells. In order to verify the involvement of these nucleoside transporters in cellular uptake of 6-MP, targeting of the genes encoding these transporters in wild-type MOLT4 cells with siRNAs resulted in a significant reduction in the initial rate of 6-MP transport as well as an enhanced resistance to this agent. High concentrations of NBTI and dipyridamole, well-known inhibitors of ENT1, did not influence the uptake of 6-MP by wild-type MOLT4 cells, ruling out involvement of this transporter in such uptake. In contrast, uptake was significantly attenuated in the absence of Na+ ions, further verifying the involvement of a concentrative nucleoside transporter in this process. This study provided the first evidence that impairment of transport as a consequence of decreased expression of CNT3 and ENT2 can in itself confer resistance to thiopurines. More recently, an in vitro study on CCRF-CEM cells with acquired resistance to 6-MP showed decrement of expression of ENT1, CNT2 and CNT3 in comparison to wild-type CCRF-CEM cells [68]. The family of multidrug resistance-associated proteins (MRPs) now consists of nine multi-specific drug transporters, designated, MRP1–9. Although the ability of several of these trans-membrane proteins transport a wide variety of anti-cancer drugs out of cells and their expression by many different types of tumor cells make them likely to be the cause of unexplained cases of drug resistance, proof that they contribute to clinical drug resistance is still lacking. Overexpression of the related MRP4 [69] and MRP5 [70] does confer a certain degree of resistance against 6-MP and 6-TG. Peng et al. recently showed that up-regulation of MRP4 together with downregulation of nucleoside transporter proteins play a major role in reduction of intracellular accumulation of 6-MP and its metabolites, and resistance to this agent [68]. Furthermore, the resistance of human embryonic kidney cells to 6-MP and 6-TG resulting from transfection with MRP4 and MRP5 cDNAs appears to reflect extrusion of metabolites of 6-MP by these transporters [71]. The organic anion transporter-3 expressed by cells of the blood–brain barrier is likely to be involved in the transport of anionic drugs such as 6-MP and acyclovir [72]. This may be one mechanism underlying the limited accumulation of these drugs in the brain, and could contribute to the proliferation of leukemic

cells in this organ and relapse during chemotherapy [73]. Contribution of the MDR1 gene pharmacogenetics to the efficacy of AZA [74] suggests a possible role for this ATP-dependent effluxing protein in transport of the active metabolites and response to thiopurines. 5. Mechanisms of thiopurine cytotoxicity Despite extensive research and clinical use of thiopurines for over 50 years, there is still uncertainty about their exact mechanisms of cytotoxicity in individual patients. This is evident from the number of crucial enzymes known to be involved in their metabolism in vitro, yet there is lack of understanding on their role in clinical setting. At present incorporation of dTGTPs into DNA and RNA is considered to be the primary mechanism of thiopurine cytotoxicity. In addition a number of studies indicate that DNPS inhibition, which is yet to be shown conclusively in patients, and MMR status may also contribute to thiopurine cytotoxicity. dTGTP is the ultimate cytotoxic product of 6-TG and 6-MP intracellular metabolism, and is incorporated by DNA polymerases into DNA as a fraudulent base, causing various forms of DNA damage. The highest level of cytotoxicity is exhibited after at least one Sphase during which there is incorporation of dTGTP which is then methylation non-enzymatically in the DNA by S-adenosylmethionine (SAM) and futile attempts of MMR to remove the fraudulent base. When Chinese hamster ovary fibroblasts are exposed to 6-TG, specific and drastic morphological changes in the chromosomes in the G2-phase of the cell cycle become evident 28 h later [75]. This effect is dose dependent, which indicates that unilateral chromatid damage may play a central role in the delayed cytotoxicity of 6-TG [76]. However, no such gross chromosomal deformation occurred when this same cell line was exposed to 6-MP. Furthermore the delay in 6-MP-induced cytotoxicity is associated with arrest in the G1 or G1/S-phase, whereas in the case of 6-TG this delay is associated with arrest in the late S/G2-phase [77]. During DNA replication, methyl deoxythioguanosine-50 -triphosphate (medTGTP) directs incorporation of either thymine or cytosine into the growing complementary DNA strand and the resulting medTGTP-T pairs are recognized and attempted to be repaired by the post-replicative mismatch system [6]. MMR introduces potentially lethal lesions into the DNA with anomalous bases and consequently triggers cell cycle arrest and apoptosis [78]. In cell lines such as MOLT4, CCRF-CEM and Jurkat it is well established that an inactive MMR system leads to strong thiopurine resistance. In 2003, a novel protein complex (HMGB1, HMG2, HSP70, ERp60, and GAPDH) was shown to bind preferentially to DNA with medTGTP-T mismatchpair, and deficiency in HMGB1 was associated with 6-MP resistance in model organisms [79]. In a recent study collateral enhancement in sensitivity to the toxicity of meMPR, a metabolite of 6-MP in thiopurine-resistant cells [67], has been explained. Characterization of MOLT4 cells with acquired resistance to 6-MP and 6-TG revealed that the size of ribonucleoside triphosphate pools and rate of DNPS in these resistant cells were reduced by greater than 50%. In addition, uptake of methylmercaptopurine riboside (meMPR) by these cells was normal, which rendered these cells more sensitive to inhibition of DNPS by meMPR. This finding may indicate that administration of meMPR to patients with ALL experiencing relapse or resistance could be beneficial. DNA with incorporated dTGTP becomes prone to oxidation due to high reactivity of thiobase. Specifically when exposed to UVA, the predominant component of solar radiation destabilizes the double helix and sensitizes cells to the mutagenic effects of sunlight the consequence of which is believed to be one of the causes for thiopurine-therapy-related cancer. Another explanation

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for thiopurine-therapy-related cancer again involves MMR system, where somatic inactivation of MMR in myeloid precursor cells of patients treated with thiopurines leads to outgrowth of drugresistant cells [29]. Feedback inhibition of DNPS by TIMP and TGMP and their methylated metabolites reduces endogenous levels of purines. meTIMP, the predominant intracellular methylated metabolite of 6-MP, inhibits PRPP amidotransferase, the first enzyme in DNPS, much more potently than TIMP. Since inhibition of this enzyme gives rise to accumulation of PRPP, and PRPP is a co-substrate for the conversion of 6-MP to TIMP, such inhibition might enhance production of the cytotoxic TIMP. The other mechanism in which inhibition of DNPS by meTIMP might contribute to cytotoxicity involves depletion of intracellular pools of nucleotides, which are vital for the survival of rapidly proliferating cells. Diminished pools of purine nucleotides can also lead to elevated pyrimidine synthesis, leading to imbalanced cell growth and, finally, cell death. However clinical data do not support in vitro studies since elevated TPMT levels, which should lead to higher levels of methylated thiopurine metabolites and thus increased inhibition of DNPS do not sensitize cells to thiopurine cytotoxicity. Metabolites of 6-MP can also inhibit DNA-dependent RNApolymerase [80]. Moreover, in 1980 Lee et al. [81] reported that TIMP potentiates the inhibition of DNA synthesis induced by 1beta-D-arabinofuranosyl-ATP (ara-ATP). This phenomenon may be the result of selective inhibition of the proofreading 30 ! 50 exonuclease activity of DNA polymerase by the metabolite of 6MP, thus preventing removal of newly incorporated araAMP from the 30 -termini of elongating DNA chains. Direct induction of apoptosis by TGN via a mitochondrial pathway has recently been described. This induction requires costimulation with CD28 and is mediated by specific inhibition of Rac1 activation by the binding of TGTP instead of GTP to Rac1. In this manner activation of Rac1 target genes, such as mitogenactivated protein kinase, NF-kB, and bcl-x(L), is suppressed by AZA, giving rise to apoptosis via a mitochondrial pathway [82]. Several researchers have reported a paradoxical decrease in the cytotoxicity of thiopurines in several different cell lines, with increasing drug concentration [83–85]. There are three possible primary explanations for this fascinating phenomenon: first, thiopurine may induce a concentration-dependent blockage of progression through cell cycle which results in decreased incorporation of this agent into DNA at high concentrations. Secondly, depletion of intracellular ATP may limit the ATPdependent conversion of 6-thioxanthosine-50 -monophosphate (TXMP) to 6-thioguanosine-50 -monophosphate (TGMP) at high concentrations. And thirdly, desulphuration of this drug at high concentrations may not only result in detoxification, but also generate an antidote in the form of the naturally occurring purine [86]. Since in human cells, the reduction in 6-MP cytotoxicity occurs at very high concentrations of 6-MP and 6TG (i.e. >100 mM) that were never achieved following administration to patients, this phenomenon is of no clinical significance. 6. The clinical pharmacology of thiopurines as anti-cancer agents 6-MP is the common component of regimen for intensification/ consolidation as well as long-term continuation therapy of childhood ALL. 6-TG on the other hand is commonly applied for blocks of intensive treatment, and was initially developed as a component of induction and remission maintenance therapy of myeloid leukemias. The routine dose of 6-MP employed for chemotherapeutic maintenance of ALL is 75 mg/m2 of body surface area. Following

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oral administration, peak plasma concentrations of 0.3–1.8 mM achieved after a mean of 2.2 h. This drug has a poor and variable bioavailability (5–37%) and a short half-life. When 6-TG is administered orally at the usual dose of 20 mg/ m2 to patients with AML, peak plasma levels of 0.03–5 mM are observed 2–4 h later [87]. The bioavailability of this drug ranges between 14 and 46% and its half-life in plasma is 90 min. Because of the pronounced inter-individual variation in response to 75 mg/m2 6-MP administered either orally or intravenously, as well as variations in concentration with time observed in one and the same patient who receives the same dose on repeated occasions [88], it has been difficult to develop a reliable strategy for determining the dosage of 6-MP required to achieve the desirable effect while avoiding both relapse and severe myelotoxicity. Prolonged intravenous administration of a high-dose of 6-MP (50 mg/m2 for 48 h) to children with refractory cancers, which can be tolerated, yields a mean steady-state plasma concentration of 6.9 mM, a concentration within the cytotoxic range in vitro with little inter-patient variation. Unfortunately, this approach did not produce a therapeutic response in a phase II paediatric trial involving 40 children with ALL [89]. As 6-MP requires extensive metabolism to TGNs to cause its cytotoxic effect it is more relevant to optimize therapy on the basis of TGN concentration rather than the plasma concentration of the parent drug, 6-MP. This observed lack of therapeutic response when 6-MP was administered i.v. was further reinforced by a recent clinical study with de novo ALL and lymphoblastic nonHodgkin’s lymphoma patients in which monthly addition of intravenous 6-MP to conventional continuation therapy of weekly oral MTX and daily 6-MP had a detrimental effect [90]. Exact explanation of this phenomenon is still under investigation. In general, oral daily administration of 6-MP is preferred over weekly intravenous administration to maintain high intracellular concentrations. In a randomized trial comparing the toxicity and efficacy of 6MP vs. 6-TG in childhood ALL, 6-TG caused excess toxicity without an overall benefit. Yet the intracellular TGNs formation was more reliable after 6-TG administration indicating its potentially greater efficacy. Better understanding of the cause of 6-TG excess toxicity could allow identification of patients who can be given the drug safely. Until then 6-MP remains the choice for continuing therapy. The most important factor influencing intracellular accumulation of TGN after administration of 6-MP is the variation in activity of TPMT resulting from well-characterized genetic polymorphisms (see above). There is a clear negative correlation between TPMT activity and intracellular concentration of TGNs, with high activity and the associated lower concentration being connected with a higher risk of failure in treatment of children with ALL [47,91]. Since determination of this concentration in lymphoblasts in the bone-marrow is difficult, erythrocyte concentrations of TGNs and TPMT activities are measured as a reflection of the corresponding parameters in the target leukemic cells. Today, these erythrocyte values serve as prognostic markers of 6-MP metabolism and TPMT activity in children with ALL. Patients with a low concentration of TGNs might be at higher risk for relapse [92], while patients with high levels may experience myelosuppression and hepatotoxicity. For patients that are homozygous for a non-functional variant, 6–10% of the standard thiopurine dose is recommended while for heterozygous patients full dose can be given initially but would most likely require dose reduction to avoid toxicity [93]. The cutoff for high and intermediate TPMT activity is 10.70 nmol/h/ml per red blood cell (pRBC) [94]. Since better outcomes were observed in children with intermediate or low TPMT activity than in homozygous for wild-type allele, increasing 6-MP doses in children with wild-type phenotypes could potentially improve

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treatment outcome [93]. The TPMT activity at the end of 6 months treatment does not correlate with the ratio of meMP/6-TGN ratio indicating that determination of TPMT activity after the initiation of thiopurine therapy could be unproductive [95]. 7. Conclusions Studies of pharmacogenetics designed to improve drug safety and efficacy have been already proven to be advantageous in the case of the influence of genetic polymorphisms in the TPMT gene on response to thiopurines. However, the 15–28% occurrence of adverse reactions in patients receiving AZA or 6-MP [96–98] is more frequent than the prevalence of such polymorphisms, indicating that additional pharmacogenetic factors may be involved in this context. Gene array technology should aid researchers in finding new targets for thiopurines and may help predict the response to these agents through comparison of the expression profiles of responsive and non-responsive patients. Application of HapMap tools or specially designed biochips to genotype TPMT already show great promise and could be modified to determine other important players affecting thiopurine efficacy. Since several phosphorylating enzymes are involved in the activation of thiopurines and their consequent incorporation into DNA and RNA, it is highly likely that alterations in the activities of certain kinases and phosphatases may influence the efficacy of thiopurine therapy profoundly. 50 -Nucleotidases are well known to deactivate nucleoside analogues, which require phosphorylation in order to exert their cytotoxic effects and a deficiency in these enzymes is associated with enhanced thiopurine cytotoxicity [59]. Interestingly, when doses of 6-MP were adjusted for TPMT activity the efficacy of ALL treatment in children appeared to be influenced by glutathione-S-transferase M1 and thymidylate synthetase genotypes [99]. There is also emerging evidence that these drugs are responsible for changes in methylation levels within the cells. The thiopurine drugs 6-MP, and meMRP caused a reduction in DNA methylation in the T-cell acute lymphoblastic leukemic cell line MOLT-4 [100]. Hogarth et al. [11] have shown that there is a decrease in demethyltransferase (DNMT) activity and protein after thiopurine treatment which was influenced by TPMT for 6-TG, but not 6-MP in human embryonic kidney cells. As demethylating agents are known to be active in leukemia it is possible that inhibition of DNA methylation by the thiopurine drugs may also contribute to their cytotoxic affects. Ultimately, stratification of patients according to their general pharmacogenetic characteristics through application of expression profiling techniques could further improve treatments outcomes by providing individually tailored combination therapy. Acknowledgments This work was supported by the Children Cancer Foundation, The Cancer and Allergy Foundation, The Cancer Society in Stockholm, The King Gustaf V Jubilee Fund, Swedish Medical Society, Swedish Cancer foundation and the Karolinska Institute Foundations. SAC was supported by the Swedish Research Council ¨ stergo¨tland. and the County Council of O References [1] Elion GB, Hitchings GH, Vanderwerff H. Antagonists of nucleic acid derivatives. VI. Purines. The Journal of Biological Chemistry 1951;192:505–18. [2] Burchenal JH, et al. Clinical evaluation of a new antimetabolite, 6-mercaptopurine, in the treatment of leukemia and allied diseases. Blood 1953;8:965–99. [3] Schwartz R, Stack J, Dameshek W. Effect of 6-mercaptopurine on antibody production. Proceedings of the Society for Experimental Biology and Medicine Society for Experimental Biology and Medicine (New York NY) 1958;99:164–7.

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