Thiopurine methyltransferase activity combined with 6-thioguanine metabolite levels predicts clinical response to thiopurines in patients with inflammatory bowel disease

BACKGROUND/AIMS: 6-Mercaptopurine and its prodrug azathioprine are effective for the treatment of inflammatory bowel disease. Thiopurine methyltransferase is important for the metabolism of thiopurines. However, there is controversy as to the clinical utility of measuring thiopurine methyltransferase enzyme activity and 6-thioguanine nucleotide levels. Our aim was to determine if thiopurine methyltransferase enzyme activity and 6-thioguanine nucleotide level monitoring would predict response to therapy with thiopurines in patients with inflammatory bowel disease. METHODS: Baseline thiopurine methyltransferase enzyme activity prior to initiation of therapy with either 6-mercaptopurine or azathioprine was determined in 39 patients with inflammatory bowel disease. The association between clinical response and thiopurine methyltransferase activity and 6-thioguanine nucleotide levels singly or in combination were analysed. RESULTS: Seventeen of 39 patients (44%) responded to 6-mercaptopurine or azathioprine therapy. Thiopurine methyltransferase enzyme activity below the mean of 30.5 U was significantly associated with clinical response. The thiopurine methyltransferase low phenotype was associated with response in 65% vs. 29% in individuals with thiopurine methyltransferase enzyme activity above 30.5 U (p = 0.05). There was no correlation between thiopurine methyltransferase activity and 6-thioguanine nucleotide levels. The maximal 6-thioguanine nucleotide levels did not predict clinical response. When combining thiopurine methyltransferase enzyme activity and 6-thioguanine nucleotide levels, the combination of thiopurine methyltransferase low/6-thioguanine nucleotide high was associated with response in 7/7 (100%) vs. only 2/8 (25%) with the combination of thiopurine methyltransferase high/6-thioguanine nucleotide low (p=0.01). CONCLUSIONS: Thiopurine methyltransferase activity inversely correlated with clinical response to thiopurine treatment in inflammatory bowel disease. Thiopurine methyltransferase enzyme activity below 30.5 U combined with a post-treatment 6-thioguanine nucleotide level > 230 pmol/8 x 10(8) erythrocytes was the best predictor of response.     

The role of thiopurine metabolite monitoring in inflammatory bowel disease

Thiopurines are the mainstay of medical management in inflammatory bowel disease (IBD), especially in the maintenance of disease remission. Given the limited IBD armamentarium it is important to optimize each therapy before switching to an alternative drug. Conventional weight based dosing of thiopurines in IBD leads to intolerance or inefficacy in many patients. More recently increased knowledge of their metabolism has allowed for dose optimization using thiopurine metabolite levels, namely 6-thioguanine nucleotides and 6-methylmercaptopurine, with the potential for improved outcomes in patients with IBD. This review will outline the current understanding of thiopurine metabolism and pharmacogenomics and will describe the clinical application of this knowledge in the optimization of thiopurines in individual patients.     

Clinical relevance of thiopurine S-methyltransferase gene polymorphisms

The therapeutic response to thiopurines may result in either severe toxic or inadequate effect based on the interindividual genetic variability. Same drug doses of various anticancer drugs cause considerable interindividual differences in the therapeutic response. Genetic factors have a major impact on effectiveness of several anticancer drugs such as mercaptopurine, 5-fluorouracil, platinum agents, and cyclophosphamide. Heredity related differences in interindividual response to thiopurine therapy represent perhaps the most compelling evidence of pharmacogenomics' usefulness in identification of patients in risk for adverse drug reactions. A number of variations in the gene for thiopurine methyltransferase (TPMT) have been associated with the low activity of this enzyme. Patients with intermediate and low activity of TPMT have a greater incidence of thiopurine toxicity. This minireview summarizes results of studies assessing the role of genetic polymorphisms in the gene encoding TPMT and their relationship to the toxicity of thiopurines.     

Azathioprine, 6-mercaptopurine in inflammatory bowel disease: pharmacology, efficacy, and safety.

6-Mercaptopurine and its prodrug azathioprine remain the mainstay of immunomodulator therapy for the maintenance of a steroid-free remission in patients with IBD. Recent evidence suggests that the cytotoxic and immunosuppressive effects of azathioprine might be mediated via the induction of lymphocyte apoptosis by its active metabolites, 6-thioguanine nucleotides. The therapeutic benefits of thiopurines have been shown to correlate with the concentration of 6-thioguanine nucleotides. Inherited differences in drug metabolism and disposition can significantly impact the safety and efficacy of these drugs. The thiopurine methyltransferase enzyme plays an important role in the metabolism of 6-mercaptopurine and azathioprine and in the determination of thiopurine cytotoxicity. By gaining an understanding of the pharmacology and metabolism of thiopurine therapy and putting it into the clinical context, clinicians will be able to optimize thiopurine therapy in IBD.     

Pharmacogenomics and Therapeutics of Hemoglobinopathies

Individual genetic constitution is an important cause of variations in the response and tolerance to drug treatment. Single nucleotide polymorphisms (SNPs) in genes located within as well as outside the human β-globin cluster have recently been shown to be significantly associated with Hb F increase in relation to hydroxyurea (HU) treatment in hemoglobinopathies patients. This article provides an update and discusses future challenges on the application of pharmacogenetic testing and pharmacogenomics for hemoglobinopathies therapeutics in relation to the current pharmacological treatment modalities for those disorders.     

Pharmacogenomics and therapeutics of hemoglobinopathies

Individual genetic constitution is an important cause of variations in the response and tolerance to drug treatment. Single nucleotide polymorphisms (SNPs) in genes located within as well as outside the human beta-globin cluster have recently been shown to be significantly associated with Hb F increase in relation to hydroxyurea (HU) treatment in hemoglobinopathies patients. This article provides an update and discusses future challenges on the application of pharmacogenetic testing and pharmacogenomics for hemoglobinopathies therapeutics in relation to the current pharmacological treatment modalities for those disorders.     

Optimizing immunomodulator therapy for inflammatory bowel disease

6-Mercaptopurine (6-MP) and its prodrug azathioprine (AZA) remain the mainstay of immunomodulator therapy for the maintenance of steroid-free remission in patients with inflammatory bowel disease (IBD). Traditional dosing strategies for initiation of thiopurines are often based on weight or empirically chosen. Dosing based on an understanding of an inherited difference in drug disposition and metabolism may provide a safer alternative. The thiopurine methyltransferase (TPMT) enzyme plays a pivotal role in the metabolism of 6-MP and AZA and is critical to the determination of thiopurine toxicity. The therapeutic benefits of thiopurines correlate best with concentration of the active 6-thioguanine (6-TGN) metabolites. Reports suggest that therapeutic response can be maximized when patients achieve therapeutic 6-TGN levels. Pharmacogenetic dosing based on TPMT and pharmacokinetic dosing based on 6-TGN levels may offer a safety and efficacy advantage over traditional dosing strategies and provide a novel mechanism for optimizing immunomodulator therapy in IBD.     

Azathioprine and 6-mercaptopurine pharmacogenetics and metabolite monitoring in inflammatory bowel disease

The thiopurine drugs azathioprine and 6-mercaptopurine (6-MP) are well-established in the treatment of inflammatory bowel disease (IBD). However, there is a wide inter- and intra-patient variation in the concentrations of active and toxic metabolites due to their complex metabolism and genetic polymorphisms in metabolizing enzymes. Serious drug toxicity leads to cessation of therapy in 9-25% of patients, and there is failure to achieve efficacy in approximately 15% of cases. Advances in the understanding of thiopurine drug metabolism have led to new genetic and metabolite tests to help clinicians optimize thiopurine use. Thiopurine methyltransferase (TPMT) enzyme activity can predict life-threatening myelotoxicity in the one in 300 patients who are TPMT-deficient. However, myelotoxicity can also occur in the presence of normal TPMT activity so blood count monitoring should remain standard practice. TPMT testing may also aid in dose individualization. 6-Thioguanine nucleotides (6-TGN) are thought to be the predominant active metabolites of the thiopurines. 6-thioguanine nucleotide concentration is correlated with bone marrow toxicity and may also correlate with efficacy in IBD. Measurement of 6-TGN and 6-methylmercaptopurine (6-MMP) concentration is most useful in determining why a patient is not responding to a standard dose of a thiopurine drug and may help in avoiding myelosuppression. The ratio of these metabolites can help distinguish non-compliance, under-dosing, thiopurine-resistant and thiopurine-refractory disease. Some of these investigations are entering routine clinical practice but more research is required to determine their optimal use in patients with IBD.     

Pharmacogenomics in gastroenterologic diseases

Pharmacogenomics is the study of genetic variations that produce a modification of the response to drugs. These variations are expressed as a different capacity for the metabolism or the transport of drugs, or a variable activity of drug receptors. Drug use in gastroenterology offers different examples of the use of pharmacogenomic analysis in the identification of the appropriate drug and drug dose for each individual patient. The use of proton pump inhibitors in the treatment of gastroesophagic reflux disease and Helicobacter pylori eradication may be optimized by the analysis of polymorphisms of the CYP2C19 gene. Additionally, the study of variants of IL28 helps in the identification of patients with more chances of response to the treatment of hepatitis C with interferon and ribavirin. The analysis of polymorphisms of the gene coding for the enzyme thiopurine methyl transferase (TPMT) helps in the reduction of the risks associated with the use of azathioprine in the treatment of inflammatory bowel disease. In this way, pharmacogenomics constitute not only a therapeutic tool that already shows an impact in the individualization of drug use in gastroenterology but also a tool with a great projection in the future.     

Increasing relevance of pharmacogenetics of drug metabolism in clinical practice

Abstract
Much of the individual variation in drug response is due to genetic drug metabolic polymorphisms. Clinically relevant examples include acetylator status; cytochrome P450 2D6, 2C9 and 2C19 polymorphisms; and thiopurine methyltransferase deficiency. It is important to be aware of which drugs are subject to pharmacogenetic variability. In the future, population-based pharmacogenetic testing will allow more individualized drug treatment and will avoid the current empiricism. (Intern Med J 2001; 31: 476–478)     

Human thiopurine S-methyltransferase pharmacogenetics: variant allozyme misfolding and aggresome formation

Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs. TPMT genetic polymorphisms represent a striking example of the potential clinical value of pharmacogenetics. Subjects homozygous for TPMT*3A, the most common variant allele for low activity, an allele that encodes a protein with two changes in amino acid sequence, are at greatly increased risk for life-threatening toxicity when treated with standard doses of thiopurines. These subjects have virtually undetectable levels of TPMT protein. In this study, we tested the hypothesis that TPMT*3A might result in protein misfolding and aggregation. We observed that TPMT*3A forms aggresomes in cultured cells and that it aggregates in vitro, functional mechanisms not previously described in pharmacogenetics. Furthermore, there was a correlation among TPMT half-life values in rabbit reticulocyte lysate, aggresome formation in COS-1 cells, and protein aggregation in vitro for the three variant allozymes encoded by alleles that include the two TPMT*3A single-nucleotide polymorphisms. These observations were compatible with a common structural explanation for all of these effects, a conclusion supported by size-exclusion chromatography and CD spectroscopy. The results of these experiments provide insight into a unique pharmacogenetic mechanism by which common polymorphisms affect TPMT protein function and, as a result, therapeutic response to thiopurine drugs.     

State of Play of Pharmacogenetics and Personalized Medicine in Heart Failure

Heart failure is a common disease with high levels of morbidity and mortality. A large body of evidence guiding treatment shows prognostic benefit with beta‐blockers and angiotensin‐converting enzyme inhibitors, while diuretics are commonly prescribed for symptomatic benefit. Wide variation in drug response between clinically similar patients is a significant problem. Evidence suggests this may have a genetic component. Variation in candidate genes including the beta‐1, beta‐2, and alpha‐2 adrenergic receptors, the renin–angiotensin–aldosterone pathway and genes involved in renal electrolyte handling with diuretics may be important. Single‐nucleotide polymorphisms (SNPs) potentially influencing drug response include the Arg 389 Gly variant and the Ser 49 Gly variant in the beta‐1 adrenergic receptor, the Arg 16 Gly, Gln 27 Glu, and Thr 164 Ile polymorphisms within the beta‐2 adrenergic receptor, an insertion at the 287th base pair in the angiotensin‐converting enzyme and the Gly 264 Ala mutation in the sodium chloride co‐transporter. However, research addressing the clinical significance of these polymorphisms has yielded conflicting results that have had no influence on clinical practice. Genome‐wide association studies may provide an alternative approach to discovering genetic variations influencing drug response, a relatively unchartered area in heart failure management. If future work in this area produces a strong case that variation in drug response has a specific and clinically meaningful genetic component, this could be used to guide drug dosing based on genotype; a step forward in the journey toward personally tailored medicine.     

Identification and expression analysis of novel LAGE-1 alleles with single nucleotide polymorphisms in cancer patients

Objective The purpose of this study is to identify single nucleotide polymorphisms (SNPs) in the coding region alleles of the X chromosomal LAGE-1 gene, and investigate the frequency of such SNPs in both cancer patients and healthy controls, and thus determine the potential significance of these SNPs with respect to cancer vaccine therapy. Methods In this study, different mRNAs transcribed from the LAGE-1 gene were identified by RT-PCR from healthy donors and cancer patients samples. Results A new LAGE-1 allele containing three coding region SNPs (69A/G, 317C/G, and 397T/G) were identified. The allele is highly expressed as the LAGE-1a mRNA variant AY679089 in some of the cancer patients. The three SNPs altered the LAGE-1 gene sequence to that of NYESO-1 at both the nucleotide and amino acid level. Conclusion There is a high frequency of the LAGE-1 gene allele with SNPs in coding regions in cancer patients. There was a clear relationship between the variant AY679089 and gastric cancer. The SNPs may lead to accelerated progress of poorly differentiated gastric cancer. The SNPs found in these alleles may also alter the immunological characteristics of LAGE-1a and should be taken into account if this antigen is adopted as a cancer vaccine component. Yi Shao and Zhen-yuan Sun contributed equally to this article.     

Thiopurines in inflammatory bowel disease: pharmacogenetics, therapeutic drug monitoring and clinical recommendations

There is a growing interest in the use of thiopurines (azathioprine, 6-mercaptopurine and 6-thioguanine) for the management of inflammatory bowel disease. The genetically controlled thiopurine (S)-methyltransferase enzyme is involved in the metabolism of these agents and is hypothesised to determine the clinical response to thiopurines. Diminished activity of this enzyme decreases the methylation of thiopurines, theoretically resulting in potential overdosing, while high thiopurine (S)-methyltransferase status leads to overproduction of toxic metabolites and ineffectiveness of azathioprine and 6-mercaptopurine. In practice, controversies exist regarding the utility of standard thiopurine (S)-methyltransferase pheno- and genotyping. Current pharmacogenetic insights suggest that another enzyme system may participate in the efficacy and toxicity of thiopurines; inosine triphosphate pyrophosphatase. Other topics discussed in this review are the utilisation of therapeutic drug monitoring and the experimental use of 6-thioguanine in the treatment of inflammatory bowel disease. 6-Thioguanine has a less genetically controlled metabolism and skips genetically determined metabolic steps. On theoretical basis, 6-thioguanine might therefore have a more predictable profile than azathioprine and 6-mercaptopurine. However, the use of 6-thioguanine has been associated with an increased risk of nodular regenerative hyperplasia of the liver and veno-occlusive disease. Further research is warranted before 6-thioguanine can be considered as a treatment option for inflammatory bowel disease.     

Some cases demonstrating the clinical usefulness of therapeutic drug monitoring in thiopurine-treated inflammatory bowel disease patients

The thiopurines azathioprine and 6-mercaptopurine (6-MP) are effective drugs in steroid-dependent and refractory inflammatory bowel disease patients. Therapeutic drug monitoring (TDM) is a new concept to improve drug efficacy and prevent toxic adverse events. As thiopurine metabolism is influenced by genetic polymorphisms of methylating enzymes, metabolite levels may vary considerably, enabling significant adverse effects. In the present paper five patients are described to demonstrate the clinical usefulness of TDM when applying thiopurines for inflammatory bowel disease. Emphasized are patients with liver function test abnormalities and myelosuppression due to inappropriate 6-MP metabolite levels, and subsequently the treatment of these events. In addition, sophisticated 6-MP metabolite level-guided therapy, including non-compliance, is demonstrated. These cases demonstrate that TDM may improve effectivity and safety of thiopurine treatment.     

Safety of thiopurines in the treatment of inflammatory bowel disease

BACKGROUND: Thiopurines have proven efficacy in inflammatory bowel disease. However, concerns regarding toxicity have limited the use of these agents as first line of medical therapy. METHODS: Review of the literature regarding metabolism, efficacy and side effects. RESULTS: In clinical trials, up to 15% of patients discontinued 6-mercaptopurine or its pro-drug azathioprine prematurely due to adverse events. These events may be divided into dose-independent idiosyncratic reactions and dose-related, pharmacologically explainable toxicity. Dose-independent reactions include skin rash, fever, diarrhoea and pancreatitis. Most frequently observed dose-dependent adverse events are nausea, malaise and myelotoxicity. Furthermore, dose-dependent and dose-independent hepatotoxicity may occur. Recent insights obtained by therapeutic drug monitoring in patients on azathioprine or 6-mercaptopurine have led to strategies to reduce toxicity. One strategy is to detect poor metabolisers of thiopurines by establishing the activity of the key enzyme thiopurine methyltransferase. However, the clinical relevance of this strategy is still a point of debate. Another strategy is to administer 6-thioguanine, which is an agent close to the effective 6-thioguanine nucleotides. CONCLUSION: Therapeutic drug monitoring of thiopurines resulted in strategies to reduce toxicity. The value of these strategies has yet to be proven in prospective randomized trials.     

Evolving Role of Thiopurines in Inflammatory Bowel Disease in the Era of Biologics and New Small Molecules

In recent years, with the increasing availability of biologic therapies and due to safety concerns, the role of thiopurines in the management of inflammatory bowel disease has been questioned. While acknowledging that the benefit/risk ratio of biologic therapies is very high, they are expensive and are not required by a majority of patients. Therefore, thiopurines do retain an important role as steroid-sparing and maintenance agents when used as monotherapy, and in combination therapy with biologics due to their clinical and pharmacokinetic optimization of anti-tumor necrosis factor agents in particular. Safety concerns with thiopurines are real but also relatively rare, and with careful pre-treatment screening and ongoing monitoring thiopurine benefits outweigh risks in the majority of appropriately selected patients. Measurement of newer pharmacogenomic markers such as nudix hydrolase 15 (NUDT15), when combined with knowledge of existing known mutations (e.g., thiopurine S-methyltransferase—TPMT), will hopefully minimize the risk of potentially life-threatening leukopenia by allowing for pre-treatment dosing stratification. Further optimization of thiopurine dosing via measurement of thiopurine metabolites should be performed routinely and is superior to weight-based dosing. The association of thiopurines with malignancies including lymphoproliferative disorders needs to be recognized in all patients and individualized in each patient. The decrease in lymphoma risk after thiopurine cessation provides an incentive for thiopurine de-escalation in appropriate patients after a period of prolonged deep remission. This review will summarize the current role of thiopurines in inflammatory bowel disease management and provide recommendations for commencing and monitoring therapy, and when to consider de-escalation.

     

Is thiopurine methyltransferase genetic polymorphism a major factor for withdrawal of azathioprine in rheumatoid arthritis patients?

OBJECTIVE: To determine whether the presence of thiopurine methyltransferase (TPMT) alleles associated with reduced or absent activity of thiopurine methyltransferase is a major factor for withdrawal of azathioprine (AZA) in rheumatoid arthritis (RA) patients. METHODS: The TPMT genotype, including the variable number of tandem repeats (VNTR) pattern in the 5' untranslated region, was analysed in 111 patients with long-standing RA. Azathioprine (AZA) therapy was used in 40 patients (36%) as a disease-modifying anti-rheumatic drug. RESULTS: Seven out of 111 RA patients (6.3%) were carriers of a mutant allele, TPMT3A (G(460)-->A, A(719)-->G) being the mutant allele observed most frequently. In the group of 40 AZA-treated patients, therapy was discontinued in six patients because of side-effects and in 26 patients because of lack of efficacy. Three patients presented moderate side-effects and were homozygous for the wild-type TPMT allele, whereas the remaining three patients, who developed gastrointestinal effects with severe nausea and vomiting, were TPMT3A carriers. CONCLUSION: In this observational study, the absence of response, probably due to the low-dose scheme used, was the major cause of AZA withdrawal in our series of RA patients. TPMT genotyping may allow the use of high doses of AZA in patients with normal TPMT alleles to improve the efficacy of this immunosuppressive drug. Our data support the relationship between gastrointestinal intolerance and thiopurine metabolic imbalance.     

Myelotoxicity and hepatotoxicity during azathioprine therapy

Azathioprine is a frequently used immunosuppressant for managing inflammatory bowel disease (IBD). In recent years the hepatotoxic profile of thiopurines has been recognised. We report the case of a 40-year-old man with Crohn's disease treated with azathioprine. After taking azathioprine (2.2 mg/kg daily) for two years, his liver function tests were found to be elevated. Moreover, a myelodepression was established as platelet and leucocytes counts were lowered. The 6-thioguaninenucleotide level was 738 picomoles/8 x 10(8) per red blood cell, which is well above the proposed upper limit of efficacy and associated with an increased risk of developing a myelodepression. Genotyping of the enzyme thiopurine methyltransferase revealed no mutant alleles. The ultrasonography and CT scan showed signs of portal hypertension (spleen 17 cm and widened splenic vein). A liver biopsy was performed and an incomplete septal liver cirrhosis was found. An upper endoscopy revealed oesophageal varices (grade 2 to 3). Autoimmune and viral liver diseases were ruled out by laboratory parameters. After cessation of therapy, all laboratory parameters normalised. Therefore, azathioprine is believed to be the causative factor for inducing the liver cirrhosis. Continuous monitoring of patients taking thiopurines is mandatory. The role of 6-thioguaninenucleotide levels in inducing myelotoxicity and hepatotoxicity is discussed.