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  • Writer's pictureDženita Omerkić Dautovic, MSc & Dr Edin Hamzić

🧬 💊 TPMT Deficiency, Thiopurines, Azathioprine & Mercaptopurine

This post is a follow-up of the other post that covered the TPMT gene and TPMT deficiency. You can check it here. In this one, I will cover the interaction between the set of medications, specifically thiopurines, azathioprine, and mercaptopurine, and the TPMT deficiency.


Thiopurines & TPMT Deficiency


What Are Thiopurines? What Type of Drug Is Thiopurine? 

Thiopurines are immunosuppressive drugs that deactivate key processes in T lymphocytes that lead to inflammation [citation].

There are 3 types of thiopurines that have been used in clinical practice:

  1. Azathioprine (prodrug of MP),

  2. 6-mercaptopurine (6MP), and

  3. Thioguanine (TG) [citation; citation]. 


When and Why Thiopurines Are Used? 

Thiopurines are widely used for the maintenance treatment of inflammatory bowel disease. Inter-individual variability in clinical response to thiopurines may be attributed to several factors including genetic polymorphisms, severity and chronicity of disease, comorbidities, duration of administration, compliance issues and use of concomitant medication, environmental factors, and clinician and patient preferences [citation].

MP and azathioprine are used for nonmalignant immunologic disorders. MP is used for lymphoid malignancies, and TG is used for Myeloid leukemias [citation].


How Does the TPMT Enzyme Deficiency Affect Thiopurines? 

TPMT enzyme is involved in the catabolization of therapy drugs, the S-methylation of aromatic and heterocyclic sulfhydryl compounds, including the antineoplastic agents 6-mercaptopurine (6MP), 6-thioguanine (6TG), and the immunosuppressant azathioprine (AZA) [citation].

One significant influence on thiopurine therapy is the inherited activity of TPMT. TPMT "deficiency" is associated with grossly elevated Thioguanine nucleotides (TGN) concentrations and profound toxicity after a short course of thiopurine therapy [citation].


Severe TPMT deficiency leads almost inevitably to life-threatening bone marrow depression if ‘standard’ thiopurine doses are used [citation].


How Do the TPMT Gene and Metabolism of Thiopurine Drugs Work? 

Thiopurine S-methyltransferase (TPMT) enzyme metabolizes thiopurine drugs widely used in various disciplines and leukemias [citation].

The process through which azathioprine and other thiopurines treat inflammatory diseases involves a complex set of genetic activities. Thiopurines work by deactivating key processes in T lymphocytes that lead to inflammation [citation].


What Is Thiopurine Toxicity? 

Thiopurines present toxicity at distinct levels: myelosuppression, hepatotoxicity, pancreatitis, and gastrointestinal intolerance, among others [citation].

Thiopurines are prodrugs that require external metabolism for their efficiency and cytotoxicity to be at the appropriate level.


Azathioprine & TPMT Deficiency

What Is Azathioprine? When and Why Is Azathioprine Used? 

Azathioprine (AZA) is a medication used in the management and treatment of active rheumatoid arthritis (RA) and the prevention of kidney transplant rejection [citation].


What Is the Mode of Action/Mechanism of Action of Azathioprine? 

Azathioprine is a purine analog that converts to its active metabolites, mercaptopurine (6-MP) and thioguanine (6-TGN), by the action of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and TPMT enzymes. It then inhibits purine synthesis. Its metabolites are incorporated into the replicating DNA and halt division. AZA metabolites may mediate most of its immunosuppressive and toxic effects [citation].


What Enzyme Metabolizes Azathioprine? 

Azathioprine is a prodrug that must first be activated to form thioguanine nucleotides (TGNs), the primary active metabolites. The active metabolites are metabolized and inactivated by the enzyme TPMT and Nudix hydrolase 15 (NUDT15). Thus, individuals with reduced activity of either enzyme are exposed to higher levels of thioguanine and have a higher risk of toxic side effects, including severe bone marrow suppression (myelosuppression) [citation].


What Is the Relation Between the TPMT Enzyme and Azathioprine? How does the TPMT Enzyme Deficiency Affects Azathioprine? 

Azathioprine is a prodrug that must first be activated to form thioguanine nucleotides (TGNs), the major active metabolites. The active metabolites are metabolized and inactivated by the enzyme thiopurine methyltransferase (TPMT) and the enzyme NUDT15. Thus, individuals with reduced activity of either enzyme are exposed to higher levels of thioguanine and have a higher risk of toxic side effects, including severe bone marrow suppression (myelosuppression) [citation].

Azathioprine is metabolized to 6MP without enzyme function. After that, 6MP and 6TG are transformed into cytotoxic thioguanine nucleotides, which further function in acting on DNA, being incorporated into these molecules. So, by acting on an RNA or DNA molecule, these molecules create a single-strand break, exchange of sister chromatids, and DNA-protein cross-link, etc. The effect of the TPMT enzyme prevents the action of TGN. Therefore, if TPMT deficiency is detected in a patient, it is a sign of a possible unwanted consequence caused by TGN toxicity [citation].


Azathioprine-induced profound myelosuppression linked to TPMT deficiency has been documented in many patient groups, including those with Crohn's disease. At the start of azathioprine or mercaptopurine therapy, measurement of TPMT activity has a role in identifying the 1 in 300 patients at risk of severe myelosuppression when treated with standard thiopurine dosages. During the initial months of azathioprine therapy, a knowledge of TPMT status warns of early bone marrow toxicity [citation].


Can You Come Off Azathioprine? 

Given that inflammatory bowel diseases are, to date, chronic, incurable conditions, treatment should be indefinite, and only the loss of efficacy or the appearance of severe side effects may cause withdrawal. As regards efficacy and maintenance over time, evidence supports the continued use of the drug in the long term: in fact, its withdrawal very substantially increases the risk of relapse [citation].


How Toxic Is Azathioprine? Is 50mg of Azathioprine a Lot? 

These drugs are capable of causing drug-induced toxicity with the risk of death by myelosuppression. It is now known that these complications occur because of genetic polymorphisms of the TPMT enzyme responsible for its metabolism [citation]. Low-dose of AZA is calculated to be 0.6-1.2 mg/kg per day [citation].


What Are the Side Effects of Taking Azathioprine? 

Side effects of Azathioprine include:

  1. Nausea; is the most frequent side effect;

  2. Fever,

  3. Fatigue,

  4. Arthralgias/myalgia,

  5. Bone marrow suppression causing pancytopenia, thrombocytopenia, and leukopenia - there are reports of dose-dependent, life-threatening cases;

  6. Rash,

  7. Hepatotoxicity Infections,

  8. Concomitant use of AZA and steroids will increase the risk of PCP in leukopenic patients and 

  9. Hypersensitivity symptoms [citation].

Hypersensitivity symptoms include fever, chills, arthralgia/myalgia, liver abnormalities, erythema nodosum, and kidney damage [citation].


Why Is Allopurinol Given With Azathioprine?  

The xanthine oxidase inhibitor Allopurinol has been shown recently to advantageously switch thiopurine metabolism toward 6-TGN production in this subgroup of patients, and small studies have shown this switch to be safe and clinically beneficial [citation].


What Is the Blood Test for Azathioprine? 

Individualized AZA dosing based on TPMT enzyme activity is based on achieving therapeutic erythrocyte 6-TGN metabolite levels between 235–260 pmol/8×108 red blood cells [citation].


Why Is It Important to Check TPMT Levels Before Commencing Azathioprine? 

Patients with a homozygous deficiency of this enzyme have no enzyme activity and, ideally, should not be given AZA. Patients with heterozygous deficiency have 50% of enzyme activity and have been shown to respond well and tolerate half a standard dose. A patient with homozygous deficiency of TPMT who developed life-threatening neutropenic sepsis was reported, and it is strongly recommended that all patients should be tested for TPMT activity before starting AZA therapy [citation].


When Is Azathioprine Indicated to Be Used as a Medicine/Drug? 

Azathioprine is used for the treatment of: inflammatory bowel disease, Churg-Strauss syndrome, autoimmune hepatitis (for maintenance treatment along with steroids), chronic ITP (second-line agent), lupus nephritis, connective tissue disease-associated ILD, multiple sclerosis, severe myasthenia gravis, recurrent pericarditis, psoriasis, non-infectious uveitis, relapsing polychondritis, dermatomyositis/polymyositis, erythema multiforme, severe and refractory atopic dermatitis, chronic actinic dermatitis and Pyoderma gangrenosum [citation]. 


Why Is It Important to Test for the TPMT Gene if Azathioprine Is Used? 

The FDA-approved drug label states that TPMT and NUDT15 deficiency testing should be considered in individuals who experience severe bone marrow toxicities or repeated episodes of myelosuppression. The FDA recommends considering an alternative therapy for individuals who are known to have homozygous TPMT or NUDT15 deficiency, or both, and to reduce dosages for individuals who have a no-function allele, cautioning that a more substantial dose reduction may be required for individuals who are either TPMT or NUDT15 poor metabolizers [citation].


What Is Allopurinol? When and Why Allopurinol Is Used? 

Allopurinol is an FDA-approved urate-lowering medication for managing gout, preventing tumor lysis syndrome, and preventing recurrent calcium nephrolithiasis in patients with hyperuricosuria [citation].

Medicines that affect thiopurine metabolism are 5-aminosalicylic acid (5-ASA) and allopurinol. Allopurinol, a potent xanthine oxidase (XO) and TPMT inhibitor, reduces 6-MMP formation [citation].

Allopurinol is used in patients who do not respond adequately to thiopurine. For example, 15% of patients are in the group that corresponds to the allopurinol prescription. Before prescribing the drug, metabolites are measured to determine possible drug tolerance and prevent hepatotoxicity. In such cases, 100 mg/day is the recommended allopurinol dose [citation].


What Is the Interaction Between the TPMT Enzyme and Allopurinol? 

Allopurinol, prescribed to the patient to help control gout flares, acts through the blockade of xanthine oxidase and TPMT. When co-administered with azathioprine, this shunts azathioprine away from the production of inactive metabolites and towards the production of active metabolites, which leads to myelosuppression and apoptosis of white blood cells [citation]. 


How Does the TPMT Enzyme Deficiency Affect Allopurinol? 

Azathioprine is a prodrug that must first be activated to form thioguanine nucleotides (TGNs), the major active metabolites. The active metabolites are metabolized and inactivated by the TPMT and the enzyme NUDT15. Thus, individuals with reduced activity of either enzyme are exposed to higher levels of thioguanine and have a higher risk of toxic side effects, including severe bone marrow suppression (myelosuppression) [citation]. Therefore, allopurinol increases the bioavailability of mercaptopurine (MP), which results from an accelerated breakdown of thiouric acid. A combination of allopurinol and thiopurine is used in patients with a TPMT enzyme deficiency, resulting in hepatotoxicity and accompanying side effects. However, this medicine is safe, and dangerous side effects are reported in very few numbers [citation].


Does Allopurinol Inhibit TPMT? 

Up to 1/5 of patients with wild-type TPMT activity prescribed azathioprine [citation]or mercaptopurine demonstrate a skewed drug metabolism in which mercaptopurine is preferentially methylated to yield methylmercaptopurine (MeMP). This is known as  thiopurine hypermethylation [citation], and is associated with drug toxicity [citation], and treatment non-response. 

Co-prescription of allopurinol [citation], with low dose AZA/MP (25–33%) circumvents this phenotype and leads to a dramatic reduction in methylated metabolites; however, the biochemical mechanism remains unclear. Therefore, the mechanism by which allopurinol affects the reduction of methylated thiopurine metabolites needs to be investigated more [citation].

A decrease in 6-MMP levels after allopurinol therapy was reported. However, in vitro and in vivo scientific research found that the lack of inhibition of erythrocyte TPMT activity was also evaluated, which calls the previous claim into question. Some logical explanations of the confirmed research are that allopurinol and purines share the same transport systems of the erythrocyte membrane and are mutually competitive transport inhibitors [citation].


Using intact and lysate red cell models Blaker and colleagues propose a novel pathway of allopurinol-mediated TPMT inhibition through the production of thioxanthine (TX, 2-hydroxymercaptopurine) [citation].


What Medicines Should Not Be Taken With Allopurinol? 

Using this medicine with any of the following medicines is not recommended but may sometimes be required, i.e., first-line immunosuppressive azathioprine in patients with inflammatory bowel disease (IBD) is marked by low beneficence due to high rates of adverse events, but in combination with allopurinol it increases tolerance [citation].

 The doctor may decide not to treat the patient with this medication or change some other medication prescriptions. Following medicines should not be combined with allopurinol:

  • Acenocoumarol

  • Azathioprine

  • Capecitabine

  • Captopril

  • Didanosine

  • Enalapril [citation].

Which Drugs Show Significant Interaction With Allopurinol? 

Several drug interactions exist with allopurinol. The most important drug interaction is with azathioprine and 6-mercaptopurine, both metabolized by the enzyme xanthine oxidase. Using any xanthine oxidase inhibitor, such as allopurinol, in a patient on azathioprine or 6-mercaptopurine can cause severe agranulocytosis and pancytopenia [citation]. 

Optimization strategy for patients with IBD is to combine low-dose thiopurine with allopurinol [citation].


Mercaptopurine & TPMT Deficiency


What Is Mercaptopurine? 

Mercaptopurine is a medication used in the management and treatment of acute lymphoblastic leukemia [citation].


What Type of Drug Is Mercaptopurine? Is Mercaptopurine a Thiopurine? 

Mercaptopurine or 6-MP (brand names Purinethol, Purixan) is an immunosuppressant and anti-neoplastic agent that belongs to the drug class of thiopurines [citation]. It is in the class of medications known as purine antagonists [citation].


What Is the Mode/Mechanism of Action of Mercaptopurine?

Mercaptopurine and azathioprine are prodrugs of a purine analog hypoxanthine that works as an antagonist to endogenous purines required for DNA replication during the S-phase of the cell cycle and inhibition of RNA and protein synthesis. Azathioprine breaks down into 88% mercaptopurine and 12% other thiopurine metabolites. 6-MP requires conversion into 6-thioguanine nucleotides (6TGN) to become active and exert antileukemic effects [citation].


When Is Mercaptopurine Indicated to Be Used as a Medicine/Drug? 

Mercaptopurine (6-MP) was approved by the FDA for use in acute lymphoblastic leukemia in children and adults as part of combination therapy [citation]. Azathioprine and 6-mercaptopurine are the two most commonly used thiopurine compounds in IBD management [citation].


What Is the Relation Between Mercaptopurine and the TPMT Gene/Enzyme? 

TPMT can affect the inactivation of thiopurine, so in order to prevent unwanted consequences, enzyme testing is recommended [citation]. The FDA-approved drug label states that the initial dose of mercaptopurine should be reduced in individuals who are known to lack TPMT [citation].


Why Is It Important to Test for the TPMT Gene if Mercaptopurine Is Used? 

The American Gastroenterological Association recommends testing for TPMT enzyme activity before prescribing thiopurine drugs. The dose is determined based on the enzyme status [citation]. In individuals who have reduced TPMT enzyme activity (heterozygous deficiency), the label states that the dose of mercaptopurine should be reduced based on tolerability [citation].

The reason for this is that TPMT can affect the inactivation of thiopurine so as to prevent unwanted consequences, for example, toxicity. That is why such drugs are not recommended for people with TPMT deficiency, or if they are necessary, they are prescribed in a smaller dose (<10%). Heterozygotes or individuals with intermediate enzyme activity should be given half of the usual dose [citation].


What Is Thioguanine? What Type of Drug Is Thioguanine? 

Thioguanine (TG) is a thiopurine that has been used for patients with inflammatory bowel disease (IBD), who have failed azathioprine (AZA) or mercaptopurine (MP) due to adverse events or suboptimal response [citation]. TG was introduced in 1950 as a treatment for leukemia and about five decades later, clinicians started using it for treating IBD. Modern cases suggest that TG offers better outcomes in terms of efficacy (51–60%) as rescue therapy, low incidence of adverse events and possibly a more rapid onset of action compared to conventional thiopurines [citation].


What Is the Mode/Mechanism of Action of Thioguanine? 

Thioguanine is a thiopurine, a purine analog and antimetabolite. It is a derivative of mercaptopurine (2-amino-6-mercaptopurine) and, like its parent molecule, inhibits purine metabolism, thus blocking DNA, RNA and subsequent protein synthesis [citation].


When Is Thioguanine Indicated to Be Used as a Medicine/Drug? 

Thioguanine (also referred to as 6-thioguanine) is a purine analogue used in the therapy of acute and chronic myelogenous leukemias. Thioguanine therapy is associated with minor, usually transient and asymptomatic elevations in serum aminotransferase levels and has also been linked to rare instances of cholestatic acute liver injury and to chronic liver injury, resulting in portal hypertension due to nodular regenerative hyperplasia [citation].


What Is the Relation Between Thioguanine and the TPMT Gene/Enzyme? 

The active metabolites are metabolized and inactivated by the enzyme thiopurine methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15). Individuals with reduced activity of either enzyme are exposed to higher levels of thioguanine and have a higher risk of toxic side effects, including severe bone marrow suppression (myelosuppression) [citation].


Why Is It Important to Test for the TPMT Gene if Thioguanine Is Used?

The FDA-approved drug label states that TPMT and NUDT15 deficiency testing should be considered in individuals who experience severe bone marrow toxicities or repeated episodes of myelosuppression. The label also includes dosing recommendations for when TPMT or NUDT15, or both, genotypes are known. For individuals with a pharmacogenetic deficiency in either enzyme, the initial dose of thioguanine should be reduced, and individuals who have a deficiency in both enzymes may require more substantial dose reductions [citation].

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