Flyer

Translational Biomedicine

  • ISSN: 2172-0479
  • Journal h-index: 12
  • Journal CiteScore: 8.06
  • Journal Impact Factor: 1.0
  • Average acceptance to publication time (5-7 days)
  • Average article processing time (30-45 days) Less than 5 volumes 30 days
    8 - 9 volumes 40 days
    10 and more volumes 45 days
20+ Million Readerbase
Indexed In
  • Open J Gate
  • Genamics JournalSeek
  • JournalTOCs
  • ResearchBible
  • The Global Impact Factor (GIF)
  • China National Knowledge Infrastructure (CNKI)
  • CiteFactor
  • Scimago
  • Electronic Journals Library
  • Directory of Research Journal Indexing (DRJI)
  • OCLC- WorldCat
  • Proquest Summons
  • Publons
  • MIAR
  • University Grants Commission
  • Geneva Foundation for Medical Education and Research
  • Google Scholar
  • Secret Search Engine Labs
  • ResearchGate
Share This Page
Recommended Webinars & Conferences

Research Article - (2022) Volume 13, Issue 12

Comprehensive Evaluation of How TPMT Genotype Influences Thiopurine Treatment

Tanya R Yakushi1*, Yong Qu1, Mike M Moradian2 and Ruan T Ramjit3
 
1Biochemical Genetics Laboratory, Regional Molecular Genetic Pathology Laboratory, SCPMG Regional Ref, Los Angeles, CA 90039, USA
2Director of Operations, Regional Molecular Genetic Pathology Laboratory, SCPMG Regional Reference Laboratories, USA
3Physician Director, Laboratory Director, Regional Molecular Genetic Pathology Laboratory, Southern California Permanente Medical Group, USA
 
*Correspondence: Tanya R Yakushi, Biochemical Genetics Laboratory, Regional Molecular Genetic Pathology Laboratory, SCPMG Regional Ref, Los Angeles, CA 90039, USA, Tel: 818- 502-5924, Email:

Received: 05-Dec-2022, Manuscript No. iptb-22-13248; Editor assigned: 07-Dec-2022, Pre QC No. iptb-22-13248; Reviewed: 19-Dec-2022, QC No. iptb-22-13248; Revised: 24-Dec-2022, Manuscript No. iptb-22-13248; Published: 30-Dec-2022, DOI: 10.36648/2172- 0479.13.12.271

Abstract

Purine analogs, 6-mercaptopurine (6-MP) and the prodrug azathioprine (Aza) are used as immunosuppressants in the treatment of many diseases including cancer, autoimmune disorders and inflammatory diseases ofthe digestive tract. Treatment with thiopurines is complicated by the high variability in response observed in a patient population. The need to titrate treatment to adequate therapeutic levels is exacerbated by the cytotoxicity that can result from overdosing patients. In this comprehensive study, we evaluated the response of 946 individuals, with known thiopurine S-methyltransferase (TPMT) genotypes, to treatment with 6-MP and Aza. We determined the allelic frequencies of the most common TPMT alleles in a diverse cohort of individuals. The TPMT*1/TPMT*1 genotype was found to occur in 92.1% of the patient population, while the TPMT*1/TPMT*3A, TPMT*1/ TPMT*3C, and TPMT*1/TPMT*2 genotypes were found to occurin 6.0%, 1.8%, and 0.1% of the patient population, respectively. We evaluated how genotype affected therapeutic response and make safe dosing recommendation based on genotype. The observations made in thisstudy,strongly suggests a need to prescribe patients with the TPMT*1/TPMT*3A genotype ~50% of the dose prescribed to wild type individuals and ~25% of the TPMT*1/TPMT*1 dosage to individuals encoding the TPMT*1/TPMT*3C genotype. The results presented are intended to serve as a guide to better understand the complex relationship between genotype and pharmaceutical response to thiopurine drugs.

Introduction

Thiopurine S-methyltransferase (TPMT) is a critical enzyme in the metabolism of thiopurine drugs, 6- mercaptopurine (6-MP) and azathioprine (Aza) [1-2]. Thiopurine are used to treat many diseases including acute lymphoblastic leukemia, autoimmune disorders such as rheumatoid arthritis and autoimmune hepatitis, and inflammatory bowel diseases such as ulcerative colitis and Crohn’s disease, as well as immunosuppressants after organ transplantation [3-6]. Thiopurines exert their cytotoxic effects through a multistep conversion into 6- thioguanine nucleotides (6-TGNs), the presence of which disrupt DNA replication and rapidly growing cells, [2,7- 8]. TPMT is a methyltransferase that covalently attaches a methyl group to thiopurine metabolites thereby shunting them from their eventual conversion into active cytotoxic 6-TGNs [1]. [Figure 1] summarizes the conversion of thiopurines into active 6-TGNs.

translational-biomedicine-metabolism

 

Individual TPMT activity varies due to the occurrence of specific single nucleotide polymorphisms that result in low or deficient TPMT activity. Previous studies have shown that polymorphisms give rise to a population in which approximately 1 in 300 individuals are deficient in TPMT activity, 10% of individuals exhibit intermediate activity, while the rest of the population displays wild type levels [9-13]. Individuals homozygous for the wild type allele (*1) exhibit normal to high TPMT activity. Heterozygous individuals carrying one functional allele and one nonfunctional allele (common nonfunctional alleles include *2, *3A, *3B, or *3C) exhibit intermediate activity, while individuals homozygous for nonfunctional alleles exhibit low to deficient activity [14-18]. Reduced TPMT activity can result in the over accumulation of cytotoxic thiopurine metabolites which lead to adverse secondary effects, such as myelosuppression, leukopenia, pancreatitis, and gastrointestinal intolerance, the effects of which are exacerbated in individuals with low or deficient activity [18-20]. Spire-Vayron de la Moureyre et al. [10] demonstrated that even within a genotype category, the phenotypic response to treatment can vary widely. Interestingly, the variability in response is greatly reduced in heterozygous individuals, indicating that these individuals may have more predictable outcomes to treatment [20-10].

Due to the narrow therapeutic index, individual variation in response to treatment and potential adverse side effects resulting from toxic metabolite accumulation, it is important to monitor metabolite levels during thiopurine treatment [20- 21, 8]. Monitoring thiopurine metabolite accumulation assists in evaluating adherence to therapy, preventing cytoxicity and allows for the continued titration of therapy. Clinically, thiopurine treatment is monitored by quantifying thiopurine metabolite levels in red blood cells by liquid chromatography tandem mass spectrometry [13-22]. The complexity of the various metabolites in the pathway is reduced by an acid hydrolysis step that converts many of the metabolites into a few components. Two specific hydrolysis products are measured, 6-thioguanine (6- TG) and 6-methylmercaptopurine (6-MMP). Quantifying 6-TG levels provides a measurement of thiopurine incorporation into 6-TGNs, the active metabolites in thiopurine treatment. Dosing recommendations based on 6-TG and 6-MMP levels have been well established [23-25, 8]. Low levels of 6-TG can indicate nonadherence or the need to increase dosage. High levels of 6-TG may indicate possible toxicity and increased susceptibility to myelosuppression, and the need to decrease or change treatment. 6-MMP is measured to evaluate TPMT function and treatment adherence. Certain individuals have higher than normal TPMT activity and are considered “shunters” or hypermethylators, which results in the excessive conversion of 6-MP and Aza, directly into 6-MMP, the TPMT catalyzed methylated product of 6-MP. As a result, there is a less than expected conversion of 6-MP or Aza into active 6-TGN metabolites. High levels of 6-MMP and low levels of 6-TG indicates that the individual is a shunter and may need treatment supplemented with allopurinol or may benefit from an alternative treatment, while high levels of 6-MMP and high levels of 6-TG indicates overdosing [26-28]. Monitoring to prevent the over accumulation of 6-MMP is important in circumventing hepatotoxicity. Guidelines have been established for therapeutic levels of 6-TG and 6-MMP. The established values are as follows: 6-MMP (<5700 pmol/8x108 RBCs) and 6-TG (235- 400 pmol/8x108 RBCs) [29]. [Table 1] summarizes established thiopurine metabolite-directed dosing recommendations [25].

In this study, we have monitored 946 individuals from a diverse patient population, undergoing treatment with purine analogs, 6-MP and Aza for a wide variety of diseases. We have used genotyping and metabolite monitoring data acquired over a year, to evaluate the effect of genotype on treatment. The results obtained allowed us to make genotype specific safe dosing recommendations for patients starting treatment with thiopurine drugs.

Methods

The thiopurine metabolite quantitative assay was performed as described by Dervieux et al., [13] with modifications. The thiopurine metabolite assay is used to monitor metabolite levels in patients undergoing treatment with Imuran (azathioprine, Aza) and Purinethol (6-mercaptopurine, 6-MP) extracted from EDTA treated whole blood. Briefly, packed red blood cells were washed and hydrolyzed in the presence of the reducing agent, dithiothreitol (DTT) under boiling conditions. Hydrolyzed metabolites were fractionation by liquid chromatography on a Shimadzu liquid chromatography system and quantified using a Sciex API4000 triple quadrupole mass spectrometer by selective reaction monitoring. Quantified 6-TG and 6-MMP levels were normalized to red blood cell count and reported in units of pmol/8x108 RBCs.

6-TG Levels (235-400 pmol/ 8x108 RBCs) 6-MMP Levels (<5700 pmol/ 8x108 RBCs) Interpretation Proposed Management
Low (<235) Normal (<5700) Underdosed or Noncompliant Increase dose OR if noncompliant, then educate about compliance
Low (<235) High (≥5700) with 6-MMP/6-TG ratio
>11
6-MMP Shunter Change therapy OR add allopurinol and reduce thiopurine dose
Therapeutic (235-400) Normal (<5700) Appropriately Dosed Dose is in appropriate range
Therapeutic (235-400) High (≥5700) Treatment Refractory Change therapy OR adjust dose accordingly
High (>400) High (≥5700) or Normal Overdosed Change therapy OR reduce dose

Table 1: D osing categories based on 6-TG and 6-MMP metabolite concentrations. The Therapeutic levels adopted by the clinical community are values of 235-400 pmol/8x108 RBCs for 6-TG and <5700 pmol/8x108 RBCs for 6-MMP. Guidelines were adapted from Vande Casteele et al. (2017).

The laboratory has monitored results for 127 batches, providing 1731 specimen results, for 946 patients with known genotypes. Patient evaluation of thiopurine metabolite values ranged from 1-11 measurements. All results obtained were categorized based on the five categories described in [Table 1]. Results in which the 6-TG values were <235 pmol/8x108 RBCs and 6-MMP values were <5700 pmol/8x108 RBCs were defined as “Underdosed”, results in which 6-TG values were <235 pmol/8x108 RBCs and 6-MMP values were ≥5700 pmol/8x108 RBCs were defined as “Shunter”, results in which 6-TG values were between 235-400 pmol/8x108 RBCs and 6-MMP values were <5700 pmol/8x108 RBCs were defined as “Appropriately Dosed”, results in which 6-TG values were between 235-400 pmol/8x108 RBCs and 6-MMP values were ≥5700 pmol/8x108 RBCs were defined as “Treatment Refractory”, and results in which 6-TG values were >400 pmol/8x108 RBCs irrespective of 6-MMP values were defined as “Overdosed”. Genotype and dosing information was obtained from electronic medical records.

Results

Thiopurine treatment is challenging due to many factors including noncompliance, concomitant use of other medication, individual response to treatment, incorrect sample collection not during a trough period, among other reasons. In addition, overdosing can lead to severe cytoxicity and secondary effects which could ultimately result in death. Using information gathered on a large and diverse patient population undergoing thiopurine treatment, we complied data on individuals with known genotypes. We used this data to evaluate the correlation between dose and genotype to suggest genotype specific safe starting dosages with the aim of improving patient outcome and compliance with thiopurine treatment.

Population Analysis

The patient population studied, 946 individuals had known genotypes. Wild type version 1 (TPMT*1) and single nucleotide polymorphisms with the highest prevalence were included in the analysis (TPMT*2, *3A, *3C). [Figure 2A] summarizes the genotype distribution observed. 92.2% of individuals were found to have the TPMT*1/TPMT*1 genotype, while 6.0% and 1.8% of individuals had the TPMT*1/TPMT*3A and TPMT*1/TPMT*3C genotypes, respectively. Only one person who accounted for 0.1% of the total patient population had the TPMT*1/TPMT*2 genotype. Prevalence observed in the mutational analysis is consistent with previously published results [30].

Response to treatment was compared to genotype and clinical results were binned into dosing categories. As expected, individuals with the TPMT*1/TPMT*1 genotype displayed varying degrees of response to treatment. 1590 results for 871 wild type individuals were categorized into the following categories: “Underdosed” (6-TG <235 pmol/8x108 RBC; 6-MMP <5,700 pmol/8x108 RBCs), “Shunter” (6-TG <235 pmol/8x108 RBC; 6-MMP ≥5,700 pmol/8x108 RBCs), “Appropriately Dosed” (6-TG 235-400 pmol/8x108 RBC; 6-MMP <5,700 pmol/8x108 RBCs), “Treatment Refractory” (6-TG 235-400 pmol/8x108 RBC; 6-MMP ≥5,700 pmol/8x108 RBCs), and “Overdosed” (6-TG >400 pmol/8x108 RBCs), summarized in [Figure 2B]. 60.1% of wild type results were found to be “Underdosed”, 6.5% were found to be in the “Shunters”, 20.6% were found to be “Appropriately Dosed”, 4.2% of results were categorized as “Treatment Refractory”, while 8.5% of results belonged to the “Overdosed” category. Of the results in the “Underdosed” category, 9.3% of results were found to have analyte levels below the Lower Limit of Quantitation (LLOQ), for both 6-TG (<30 pmol/8x108 RBCs) and 6-MMP (<300 pmol/8x108 RBCs). These results belonged to individuals who were either non-compliant, highly underdosed, tested before commencing treatment, or tested after stopping treatment. The 6.5% and 4.2% of results in the “Shunter” and “Treatment Refractory” categories, respectively, belonged to individuals expressing high levels of TPMT activity which resulted in a high conversion of 6-MP or Aza to methylated 6-MP containing byproducts. Results in which 6-TG levels were >400 pmol/8x108 RBCs, irrespective of 6-MMP levels, were classified as “Overdosed” and constituted 8.5% of the total wild type results. These individuals responded aggressively to treatment and required either lowering their dosage or changing treatment to avoid myelotoxicity.

Heterozygous individuals possessing the genotype TPMT*1/ TPMT*3A and TPMT*1/TPMT*3C produced a distribution of results very different to what was observed for wild type individuals. Since these patients have reduced TPMT activity, only one result was observed with a 6-MMP value ≥5700 pmol/8x108 RBCs, in a TPMT*1/TPMT*3A individual who was overdosed. Consequently, no results were observed for TPMT*1/ TPMT*3A and TPMT*1/TPMT*3C encoding individuals in the “Shunter” or “Treatment Refractory” categories. 99 results were obtained for individuals with the TPMT*1/TPMT*3A genotype, the second largest category whose distribution was studied. Of the results, 27.3% were categorized as “Underdosed”, 35.4% were categorized as “Appropriately Dosed” and 37.4% were categorized as “Overdosed” [Figure 2C]. 40 results were obtained for individuals with the TPMT*1/TPMT*3C genotype. Of the results, 45.0% were categorized as “Underdosed”, 35.0% were categorized as “Appropriately Dosed” and 20.0% were categorized as “Overdosed” [Figure 2D]. The distribution of results for heterozygous individuals was shifted to a higher percentage of “Appropriately Dosed” and “Overdosed” results, when compared to wild type individuals. The highest category of results for individuals with the TPMT*1/TPMT*3A genotype was found to be in the “Overdosed” category while the highest category of results for individuals with the TPMT*1/TPMT*3C genotype was found to be in the “Underdosed” category.

translational-biomedicine-patient

 

Genotype Specific Dosing Recommendations

Correlations between optimal thiopurine dosing and genotypes were made, for 6-MP and Aza. Aza is an imidazole derivative of 6-MP, first metabolized in the liver, to produce active 6-MP. Aza and 6-MP are indicated to treat the same diseases, however conversion of Aza to therapeutically active 6-TGN metabolites is slowed due to the additional metabolic step needed to convert Aza to 6-MP [21, 31-32]. Individuals in the proper therapeutic range of 235-400 pmol/8x108 RBCs for 6-TG and <5700 pmol/8x108 RBCs for 6-MMP, with a wild type genotype (TPMT*1/TPMT*1) were evaluated. 1437 results were obtained for 787 individuals with known dosing information. Of the 1437 results, 328 results produced values that were consistent with an “Appropriately Dosed” category, 803 results were consistent with the “Underdosed” category, and 135 results were consistent with the “Overdosed” category. Of the remaining values, 95 results were below the LLOQ for both the 6-TG and 6-MMP analytes, while 171 results produced values consistent with the “Shunter” or “Treatment Refractory” categories. Please note results obtained below the LLOQ for both analytes, were excluded from the dosage distribution analysis. Individuals with this result typically had been tested prior to starting treatment, after stopping treatment or had documented non-compliance; therefore including these results would have inaccurately reflected the effect of an active treatment on response. Categorized results were compared to Aza and 6-MP dosages in the absence of the TPMT mediated inhibitor, allopurinol. The largest percentage of “Appropriately Dosed” results was achieved when Aza was administered at a dose of 100 mg/day [Figure 3A], while the largest percentage of “Appropriately Dosed” results with 6-MP was achieved at a concentration of 50 mg/day [Figure 3B]. These results are consistent with previously published findings that suggest using Aza at approximately 2- fold the amount of 6-MP [33]. A straightforward shift to the “Overdosed” category upon continued increase in dosing was not observed. In some individuals increasing the dose resulted in a sharp increase in 6-MMP levels without a proportional increase in the 6-TG value, causing a shift to the “Shunter” or “Treatment Refractory” categories instead of the “Appropriately Dosed” category.

Allopurinol (Allo) is often used in conjunction with 6-MP and Aza when treating individuals who exhibit high TPMT activity [34-36, 8]. Allo mediates the inhibition of TPMT, promoting the conversion of 6-MP and Aza into therapeutically active 6-TGNs and results in a decreased accumulation of 6-MMP [37, 28]. A simplified mechanism of action is depicted in Figure 3D. The use of Allo during treatment requires a concomitant decrease in the prescribed dosage of 6-MP and Aza [34, 8]. [Figure 3C and 3E] summarize the distribution of results observed in wild type individuals. Using Allo required decreasing the dose of 6-MP and Aza by 25-50%, consistent with previously published findings [34-36].

image

translational-biomedicine-distribution

 

As expected, individuals with a heterozygous genotype did not have results categorized in either the shunter or refractory categories, due to their intermediate thiopurine methyltransferase activity. A total of 90 results were obtained for 50 individuals encoding the TPMT*1/TPMT*3A genotype with known dosing information. The largest percentage of “Appropriately Dosed” TPMT*1/TPMT*3A individuals (20.0%) was achieved when a dose of 50 mg/day of Aza was administered [Figure 4A]. Results obtained when 6-MP was administered were mixed [Figure 4B]. 8.0% of TPMT*1/TPMT*3A individuals treated with 6-MP were “Appropriately Dosed” when 25 mg/day was prescribed and 10.0% were “Appropriately Dosed” when 50 mg/day was prescribed. It is important to note that although the largest percentage of “Appropriately Dosed” was observed when 50 mg/ day was prescribed, the ratio of “Overdosed” to “Appropriately Dosed” was 1.8:1, indicating that at this dosage the patient is almost twice as likely to be overdosed at this concentration, than “Appropriately Dosed”. It stands to reason that the safer starting dose for individuals with a TPMT*1/TPMT*3A genotype is 25 mg/ day when 6-MP is selected.

Results for 14 TPMT*1/TPMT*3C individuals were evaluated in 35 analyses. When Aza was prescribed, an equal percentage (10.0%) of TPMT*1/TPMT*3C heterozygous individuals achieved “Appropriately Dosed” levels at 25-28.6 mg/day and 50 mg/ day [Figure 4C]. However, at a dosage of 50 mg/day, TPMT*1/ TPMT*3C individuals were 3x as likely to be “Overdosed”, when compared to 25 mg/day, indicating that the safer starting dosage for TPMT*1/TPMT*3C individuals prescribed Aza is 25 mg/day. Prescribing 6-MP to TPMT*1/TPMT*3C individuals resulted in the largest percentage of “Appropriately Dosed” results being achieved at dosages below 10 mg/day [Figure 4D]. Since fewer measurements were made for TPMT*1/TPMT*3C individuals, additional measurements are still needed to confirm these results. It is important to note that in all dosing situations analyzed, the need to use twice the concentration of Aza to 6-MP is consistent with previously published dosing recommendations [33].

translational-biomedicine-heterozygous

 

The results suggesting that TPMT*1/TPMT*3C individuals benefit from a starting dose that is approximately half the dose needed by TPMT*1/TPMT*3A individuals to achieve appropriate dosing levels, prompted us to compare the distribution of 6-MMP analyte levels in our pool of results. Our goal was to determine if a difference in accumulated 6-MMP existed between TPMT*1/ TPMT*3A and TPMT*1/TPMT*3C individuals, which might indicate a difference in overall TPMT activity. When results were evaluated, we observed that TPMT*1/TPMT*3A [Figure 5A] individuals had a wider distribution of 6-MMP values compared to individuals with the TPMT*1/TPMT*3C [Figure 5B] genotype. 74% of all 6-MMP quantities measured for TPMT*1/TPMT*3C individuals had 6-MMP concentrations of ≤100 pmol/8x108 RBCs, whereas 36% of all quantities measured for TPMT*1/ TPMT*3A individuals had 6-MMP concentrations of ≤100 pmol/8x108 RBCs. In the case of TPMT*1/TPMT*3C individuals, no results were obtained with 6-MMP values between 200- 1300 pmol/8x108 RBCs and only 4 values were observed >1300 pmol/8x108 RBCs, 2 of which were observed in overdosed individuals and 2 were observed in patients with 6-TG values in the 300-400 pmol/8x108 RBCs range. The distribution observed in TPMT*1/TPMT*3A individuals was more evenly distributed supporting the observation that TPMT activity is likely higher in TPMT*1/TPMT*3A individuals, which is why they require twice the starting dose needed to reach “Appropriately Dosed” levels compared to TPMT*1/TPMT*3C individuals.

Through the course of this study only one individual with the TPMT*1/TPMT*2 genotype was identified and only two measurements were performed for this individual. Therefore, not enough information was acquired on this genotype group to draw conclusions or make dosing recommendations at this time.

translational-biomedicine-observed

 

Discussion

Taking a personalized approach to medicine is the future of health care. Harnessing genetic information to inform therapeutic approaches is paving the way to tailored medicine. In this study we employed a classical pharmacogenetic example, treatment of wild type and heterozygous individuals with purine analogs 6-MP and Aza, added an additional layer of understanding to how heterozygous individuals carrying one of the two most prevalent single nucleotide polymorphisms TPMT*3A and TPMT*3C respond to treatment. This work was carried out using a diverse and expansive patient population of 946 individuals with known genotypes, of which 787 TPMT*1/TPMT*1, 50 TPMT*1/TPMT*3A, and 14 TPMT*1/TPMT*3C individuals had a known treatment and displayed analyte levels above the LLOQ. Patients with values below detectable levels were included in the population studies but were excluded from the dosing distribution analysis. Since many of the patients with values below detectable levels were tested prior to commencing treatment, after concluding treatment, or had documented instances of treatment noncompliance, inclusion of these individuals would have unfairly skewed the results. Evaluating the results in a wholistic manner, it is clear that achieving therapeutic levels is difficult due to many factors. Of the patient population studied, only 20.6% of TPMT*1/TPMT*1 individuals are “Appropriately Dosed”, while 35.4% and 35.0% of TPMT*1/TPMT*3A and TPMT*1/TPMT*3C individuals, respectively, are “Appropriately Dosed”. Factors that influence those low numbers include individual TPMT enzymatic activity, noncompliance, concomitant use of other medication, concerns due to increased susceptibility to infections as a result of using immunosuppressants, severe side effects resulting from overdosing and many other reasons [8, 33, 38-39]. Additionally, studies have shown that a lower therapeutic cut off (125 pmol/8x108 RBCs) is effective in treating patients with gastrointestinal disorders prescribed a combination therapy of thiopurines and anti-TNF agents, which may have contributed to a larger than expected group of patients with results in the “underdosed” category [40-41].

Genotype Azathioprine (Aza) Mercaptopurine (6-MP)
TPMT*1/TPMT*1 100 mg/day 50 mg/day
TPMT*1/TPMT*3A 50 mg/day 25 mg/day
TPMT*1/TPMT*3C 25 mg/day 12.5 mg/day

Table 2: Genotype specific safe starting dose recommendations. Dosing recommendations were Determined based on the results of the analysis presented in this study. These recommendations are suggested to improve compliance and minimize the potential secondary effects that are observed when overdosing occurs.

To improve compliance and prevent life threatening secondary effects associated with the use of purine analogs, it is essential to start at a safe lower dosage and titrate up. With this in mind, we evaluated the response of individuals with the three most prevalent genotypes, TPMT*1/TPMT*1, TPMT*1/TPMT*3A, and TPMT*1/TPMT*3C, to specific doses of Aza and 6-MP. Using the large amount of data acquired, we suggest genotype specific starting dosages, summarized in [Table 2]. The results presented clearly point to a lower overall TPMT activity in individuals carrying the TPMT*1/TPMT*3C genotype when compared to individuals with the TPMT*1/TPMT*3A genotype, which translates to requiring a lower dose of Aza and 6-MP during treatment. These results are consistent with earlier findings that suggest a lower overall activity in TPMT*1/TPMT*3C individuals [10]. Acquisition of future data will help to further refine the concentrations listed in this study. Furthermore, the information included here is presented as a starting point for treatment, weight-based calculation were not performed since studies have shown that weight-based dosing does not necessarily correlate with achieving therapeutic levels in thiopurine treatment [42-43].

It is important to note that only 3.8% of wild type individuals treated with Aza and 8.8% of wild type individuals treated with 6-MP were undergoing treatment supplemented with allopurinol to achieve “Appropriately Dosed” levels. The larger scale use of allopurinol by clinicians in the future may prove beneficial in assisting more individuals shift from the “Shunter” and “Treatment Refractory” categories to the “Appropriately Dosed” category.

Lastly, this study lends credence to the need to measure TPMT activity prior to patients starting treatment. The data presented clearly indicates that within the larger TPMT*1/TPMT*1 genotype category, individuals required a wide range of doses to reach “Appropriately Dosed” levels. In the case of TPMT*1/ TPMT*1 individuals treated with Aza, concentrations between 50-200 mg/day were needed. A four-fold difference between individual dosing needs is very large and demonstrates that a more direct measurement of TPMT activity would prove more useful in guiding treatment, than genotype alone. Spire-Vayron et al. (10) demonstrated that TPMT*1/TPMT*1 individuals can vary in TPMT activity between 10-50 U/ml RBC, which is equivalent to a 5-fold difference between the lowest to highest TPMT activity measured. This observation is consistent with the wide range of doses observed in this study, required to achieve therapeutic levels. In the three TPMT genotype categories studied >8.5% of results in each grouping were overdosed, increasing the risk of these patients to myelotoxicity and hepatoxicity. Measuring TPMT enzymatic activity prior to initiating treatment would provide physicians an extra layer of granularity in the decisionmaking process and would allow them to determine starting doses more accurately for patients with low to moderate TPMT activity. Knowing if a patient expresses high TPMT activity would allow physicians to treat patients with combination treatments, such as 6-MP and allopurinol, at the onset of treatment, greatly reducing the risk of hepatoxicity. Genotype alone does not allow physicians to identify patients with high TPMT activity but measuring enzymatic activity does. Understanding biochemically how a patient will respond prior to treatment will reduce treatment risk and lead to improved patient compliance.

Acknowledgments

We would like to thank Dr. Jefferey Rauch, M.D. from the Department of Gastroenterology, for his careful review of this article and for the valuable feedback he provided. We would also like thank the Kaiser Biochemical Genetics Department for their contributions.

Conflict of Interest

The authors declare no conflict of interest in the preparation of this article.

References

  1. Ford LT, Berg JD (2010) Thiopurine S-methyltransferase (TPMT) assessment prior to starting thiopurine drug treatment; a pharmacogenomics test whose time has come. J Clin Pathol. 63: 288-295.
  2. Indexed at, Google Scholar, Crossref

  3. Zaza G, Cheok M, Krynetskaia N, Thorn C, Stocco G, et al. (2010) Thiopurine pathway. Pharmacogenet Genomics. 20: 573–574.
  4. Indexed at, Google Scholar, Crossref

  5. Bayoumy AB, Simsek M, Seinen ML, Mulder CJJ, Ansari A, et al. (2020) The continuous rediscovery and the benefit-risk ratio of thioguanine, a comprehensive review. Expert Opin Drug Metab Toxicol. 16: 111-123.
  6. Indexed at, Google Scholar, Crossref

  7. Nielsen OH, Vainer B, Rask-Madsen J (2001) Review article: the treatment of inflammatory bowel disease with 6-mercaptopurine or azathioprine. Aliment Pharmacol Ther. 15: 1699-1708.
  8. Indexed at, Google Scholar, Crossref

  9. Bischoff S, Yesmembetov K, Antoni C, Sollors J, Evert M, et al. (2020) Autoimmune hepatitis: a review of established and evolving treatments. J Gastrointestin Liver Dis. 29: 429-443.
  10. Indexed at, Google Scholar, Crossref

  11. Harmand PO, Solassol J (2020) Thiopurine drugs in the treatment of ulcerative colitis: identification of a novel deleterious mutation in TPMT. Genes (Basel). 11: 1212.
  12. Indexed at, Google Scholar, Crossref

  13. Tiede I, Fritz G, Strand S, Poppe D, Dvorsky R, et al. (2003) CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes. J Clin Invest. 111: 1133-1145.
  14. Indexed at, Google Scholar, Crossref

  15. González-Lama Y, Gisbert JP (2016) Monitoring thiopurine metabolites in inflammatory bowel disease. Frontline Gastroenterol. 7: 301-307.
  16. Indexed at, Google Scholar, Crossref

  17. Weinshilboum RM, Sladek SL (1980) Mercaptopurine pharmacogenetics: monogenic inheritance of erythrocyte thiopurine methyltransferase activity. Am J Hum Genet. 32: 651-662.
  18. Indexed at, Google Scholar

  19. Spire-Vayron de la Moureyre C, Debuysere H, Mastain B, Vinner E, Marez D, et al. (1998) Genotypic and phenotypic analysis of the polymorphic thiopurine S-methyltransferase gene (TPMT) in a European population. Br J Pharmacol. 125: 879-887.
  20. Indexed at, Google Scholar, Crossref

  21. Ameyaw MM, Collie-Duguid ES, Powrie RH, Ofori-Adjei D, McLeod HL (1999) Thiopurine methyltransferase alleles in British and Ghanaian populations. Hum Mol Genet. 8: 367-370.
  22. Indexed at, Google Scholar, Crossref

  23. McLeod HL, Pritchard SC, Githang'a J, Indalo A, Ameyaw MM, et al. (1999) Ethnic differences in thiopurine methyltransferase pharmacogenetics: evidence for allele specificity in Caucasian and Kenyan individuals. Pharmacogenetics. 9: 773-776.
  24. Indexed at, Google Scholar, Crossref

  25. Dervieux T, Meyer G, Barham R, Matsutani M, Barry M, et al. (2005) Liquid chromatography-tandem mass spectrometry analysis of erythrocyte thiopurine nucleotides and effect of thiopurine methyltransferase gene variants on these metabolites in patients receiving azathioprine/6-mercaptopurine therapy. Clin Chem. 51: 2074-2084.
  26. Indexed at, Google Scholar, Crossref

  27. Krynetski EY, Schuetz JD, Galpin AJ, Pui CH, Relling MV, et al. (1995) A single point mutation leading to loss of catalytic activity in human thiopurine S-methyltransferase. Proc Natl Acad Sci U S A. 92: 949-953.
  28. Indexed at, Google Scholar, Crossref

  29. Szumlanski C, Otterness D, Her C, Lee D, Brandriff B, et al. (1996) Thiopurine methyltransferase pharmacogenetics: human gene cloning and characterization of a common polymorphism. DNA Cell Biol. 15: 17-30.
  30. Indexed at, Google Scholar, Crossref

  31. Tai HL, Krynetski EY, Yates CR, Loennechen T, Fessing MY, et al. (1996) Thiopurine S-methyltransferase deficiency: two nucleotide transitions define the most prevalent mutant allele associated with loss of catalytic activity in Caucasians. Am J Hum Genet. 58: 694-702.
  32. Indexed at, Google Scholar

  33. Tai HL, Krynetski EY, Schuetz EG, Yanishevski Y, Evans WE (1997) Enhanced proteolysis of thiopurine S- methyltransferase (TPMT) encoded by mutant alleles in humans (TPMT*3A, TPMT*2): mechanisms for the genetic polymorphism of TPMT activity. Proc Natl Acad Sci U S A. 94: 6444-6449.
  34. Indexed at, Google Scholar, Crossref

  35. Wang L, Weinshilboum R (2006) Thiopurine S-methyltransferase pharmacogenetics: insights, challenges and future directions. Oncogene. 25: 1629-1638.
  36. Indexed at, Google Scholar, Crossref

  37. Lennard L, Van Loon JA, Weinshilboum RM (1989) Pharmacogenetics of acute azathioprine toxicity: relationship to thiopurine methyltransferase genetic polymorphism. Clin Pharmacol Ther. 46:149-154.
  38. Indexed at, Google Scholar, Crossref

  39. Benkov K, Lu Y, Patel A, Rahhal R, Russell G, et al. (2013) NASPGHAN Committee on Inflammatory Bowel Disease. Role of thiopurine metabolite testing and thiopurine methyltransferase determination in pediatric IBD. J Pediatr Gastroenterol Nutr. 56(3): 333-340.
  40. Indexed at, Google Scholar, Crossref

  41. Gearry RB, Barclay ML (2005) Azathioprine and 6-mercaptopurine pharmacogenetics and metabolite monitoring in inflammatory bowel disease. J Gastroenterol Hepatol. 20:1149-1157.
  42. Indexed at, Google Scholar, Crossref

  43. Asadov C, Aliyeva G, Mustafayeva K (2017) Thiopurine S-Methyltransferase as a pharmacogenetic biomarker: significance of testing and review of major methods. Cardiovasc Hematol Agents Med Chem. 15: 23-30.
  44. Indexed at, Google Scholar, Crossref

  45. Relling MV, Gardner EE, Sandborn WJ, Schmiegelow K, Pui CH, et al. (2011) Clinical Pharmacogenetics Implementation Consortium. Clinical Pharmacogenetics Implementation Consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing. Clin Pharmacol Ther. 89(3): 387-91.
  46. Indexed at, Google Scholar, Crossref

  47. Abaji R, Krajinovic M. (2017) Thiopurine S-methyltransferase polymorphisms in acute lymphoblastic leukemia, inflammatory bowel disease and autoimmune disorders: influence on treatment response. Pharmgenomics Pers Med. 10: 143-156.
  48. Indexed at, Google Scholar, Crossref

  49. Vande Casteele N, Herfarth H, Katz J, Falck-Ytter Y, Singh S (2017) American Gastroenterological Association Institute technical review on the role of therapeutic drug monitoring in the management of inflammatory bowel fiseases. Gastroenterology. 153: 835-857.
  50. Indexed at, Google Scholar, Crossref

  51. Dubinsky MC, Yang H, Hassard PV, Seidman EG, Kam LY, et al. (2002) 6-MP metabolite profiles provide a biochemical explanation for 6-MP resistance in patients with inflammatory bowel disease. Gastroenterology. 122: 904-915.
  52. Indexed at, Google Scholar, Crossref

  53. Yarur AJ, Abreu MT, Deshpande AR, Kerman DH, Sussman DA (2014) Therapeutic drug monitoring in patients with inflammatory bowel disease. World J Gastroenterol. 20: 3475-3484.
  54. Indexed at, Google Scholar, Crossref

  55. Deswal S, Srivastava A (2017) Role of allopurinol in optimizing thiopurine therapy in patients with autoimmune hepatitis: a review. J Clin Exp Hepatol. 7: 55-62.
  56. Indexed at, Google Scholar, Crossref

  57. Dubinsky MC, Lamothe S, Yang HY, Targan SR, Sinnett D, et al. (2000) Pharmacogenomics and metabolite measurement for 6-mercaptopurine therapy in inflammatory bowel disease. Gastroenterology. 118: 705-713.
  58. Indexed at, Google Scholar, Crossref

  59. Schaeffeler E, Fischer C, Brockmeier D, Wernet D, Moerike K, et al. (2004) Comprehensive analysis of thiopurine S-methyltransferase phenotype-genotype correlation in a large population of German-Caucasians and identification of novel TPMT variants. Pharmacogenetics.14: 407-417.
  60. Indexed at, Google Scholar, Crossref

  61. Kaplowitz N (1976) enzymatic thiolysis of azathioprine in vitro. Biochem Pharmacol. 25: 2421-2426.
  62. Indexed at, Google Scholar, Crossref

  63. Morris PJ, Knechtle SJ (2014) Chapter 15 – Azathioprine. In: Morris PJ, Knechtle SJ editors. Kidney Transplantation-Principles and Practice: Seventh Edition. Saunders Elsevier: Philadelphia. pp 216-220.
  64. Google Scholar

  65. Dubinsky MC. Azathioprine, 6-mercaptopurine in inflammatory bowel disease: pharmacology, efficacy, and safety. Clin Gastroenterol Hepatol. 2004;2:731-43.
  66. Indexed at, Google Scholar, Crossref

  67. Sparrow MP, Hande SA, Friedman S, Lim WC, Reddy SI, et al. (2005) Allopurinol safely and effectively optimizes tioguanine metabolites in inflammatory bowel disease patients not responding to azathioprine and mercaptopurine. Aliment Pharmacol Ther. 22: 441-446.
  68. Indexed at, Google Scholar, Crossref

  69. Ansari A, Patel N, Sanderson J, O'Donohue J, Duley JA, et al. (2010) Low-dose azathioprine or mercaptopurine in combination with allopurinol can bypass many adverse drug reactions in patients with inflammatory bowel disease. Aliment Pharmacol Ther. 31:640-647.
  70. Indexed at, Google Scholar, Crossref

  71. Appell ML, Wagner A, Hindorf U (2013) A skewed thiopurine metabolism is a common clinical phenomenon that can be successfully managed with a combination of low-dose azathioprine and allopurinol. J Crohns Colitis. 7: 510-513.
  72. Indexed at, Google Scholar, Crossref

  73. Blaker PA, Arenas-Hernandez M, Smith MA, Shobowale-Bakre EA, Fairbanks L, et al. (2013) Mechanism of allopurinol induced TPMT inhibition. Biochem Pharmacol. 86: 539-547.
  74. Indexed at, Google Scholar, Crossref

  75. Warner B, Johnston E, Arenas-Hernandez M, Marinaki A, Irving P, et al. (2018) A practical guide to thiopurine prescribing and monitoring in IBD. Frontline Gastroenterol. 9: 10-15.
  76. Indexed at, Google Scholar, Crossref

  77. Hanauer SB, Sandborn WJ, Lichtenstein GR (2019) Evolving Considerations for Thiopurine Therapy for Inflammatory Bowel Diseases-A Clinical Practice Update: Commentary. Gastroenterology. 156: 36-42.
  78. Indexed at, Google Scholar, Crossref

  79. Yarur AJ, Kubiliun MJ, Czul F, Sussman DA, Quintero MA, et al. (2015) Concentrations of 6-thioguanine nucleotide correlate with trough levels of infliximab in patients with inflammatory bowel disease on combination therapy. Clin Gastroenterol Hepatol. 13: 1118-1124.
  80. Indexed at, Google Scholar, Crossref

  81. Nguyen DL, Flores S, Sassi K, Bechtold ML, Nguyen ET, et al. (2015) Optimizing the use of anti-tumor necrosis factor in the management of patients with Crohn's disease. Ther Adv Chronic Dis. 6: 147-154.
  82. Indexed at, Google Scholar, Crossref

  83. Haines ML, Ajlouni Y, Irving PM, Sparrow MP, Rose R, et al. (2011) Clinical usefulness of therapeutic drug monitoring of thiopurines in patients with inadequately controlled inflammatory bowel disease. Inflamm Bowel Dis. 17: 1301-1307.
  84. Indexed at, Google Scholar, Crossref

  85. Holt DQ, Strauss BJ, Moore GT (2016) Weight and body composition compartments do not predict therapeutic thiopurine metabolite levels in inflammatory bowel disease. Clin Transl Gastroenterol. 7(10): e199.
  86. Indexed at, Google Scholar, Crossref

Citation: Yakushi TR, Qu Y, Moradian MM, Ramjit RT (2022) Comprehensive Evaluation of How TPMT Genotype Influences Thiopurine Treatment. Transl Biomed, Vol.13 No. 12: 271.