"Azathioprine - More than a drug information"

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Life stories of Nobel Laureate:

The student life of Elion was struggling, she completed her master's in Chemistry in 1941 and joined Johnson & Johnson in 1944 as a research chemist later positioned working with nucleic acids alongside Hitchings at Burroughs Wellcome Company (now GlaxoSmithKline). She never did Ph.D. but received an honorary doctorate degree.
 
Elion was a great scientist and an inspiration

George H. Hitchings who was born in Hoquiam in 1905 also impacted by the death of his father at a young age from a prolonged illness. He was highly influenced by Louis Pasteur's research and practicals. He earned his Ph.D. in 1933; but he believes his actual career began after 1942 after joining Wellcome Research Laboratories in Tuckahoe, New York, as head and sole member of the Biochemistry Department (now GlaxoSmithKline). The interim period was difficult both in terms of financial and intellectually.

Elion and Hitchings set out on an unorthodox course of creating medicines by studying the chemical composition of diseased cells. Rather than relying on trial-and-error methods, they preferred rational drug design i.e to create drugs that specifically interfere in pathways contributing to cell homeostasis. This technique is preferred nowadays for creating drugs.
They aimed to interfere with the purine pathway to reduce the production of adenine and guanine nucleotides which consequently lead to decreased DNA synthesis.
Hitchings and his team were known for innovative methods of drug designing and their contributions were - new drug therapies for malaria (pyrimethamine), leukemia (6-mercaptopurine and thioguanine), gout (allopurinol), organ transplantation (azathioprine), and bacterial infections (cotrimoxazole) (trimethoprim), major antiviral drugs for herpes infections (acyclovir) and AIDS (zidovudine).

Here we will discuss Azathioprine & related compound in detail.

Azathioprine & 6- mercaptopurine  :

As we all are aware, most of the conventional agents used in rheumatology are not primarily designed for rheumatological conditions; this is rather a serendipity.
Azathioprine is a prodrug of 6 Mercaptopurine containing an additional imidazole group.
Azathioprine seemed to be better tolerated than 6 - mercaptopurine and favorable therapeutic index. Azathioprine is a purine analog and is included in the antimetabolite family of medication. It is discovered in 1957. It was initially investigated as a chemotherapeutic drug for Leukemia.
Later in 1958, Robert Schwartz described its anti-inflammatory effects on rabbits.
Sir Roy Calne, the British pioneer in transplantation was the first to use this drug as transplantation medicine. Later it has been investigated and found to be effective in various immune-mediated rheumatic diseases.

Pharmacokinetics & Mechanism of Action: 

The main therapeutic effect of azathioprine relies on its metabolism to cytotoxic thioguanine nucleotides, which inhibit de novo purine synthesis, thereby decreasing leukocyte proliferation.
Azathioprine is absorbed from the whole intestinal tract.
Dosing for AZA (2- 2.5 mg/kg) and 6- MP (1-1.5 mg/kg).
AZA is converted rapidly to 6 MP (peaks 1-3 hours after AZA administration)(Half-life of 6 MP is 1 -2 hours). 6 - MP converted to an active metabolite (6-TGN) has a half-life of 1-2 weeks.
Thiopurines have a relatively slow onset of action, with a therapeutic response typically realized after a minimum of 8–12 weeks, although some patients may take longer.
 
In the intestinal wall, liver, and red blood cells, it gets converted to 6-mercaptopurine via a glutathione-mediated non-enzymatic process that involves the removal of an imidazole group.
Subsequent metabolism of 6-MP to its active thioguanine nucleotides (TGNs) metabolite requires multiple enzymes:
1. Thiopurine-S methyltransferase (TPMT) which catalyzes the methylation of 6-MP to an inactive metabolite 6-methylmercaptopurine (6-MMP)
2. Xanthine oxidase (XO) which catalyzes 6-MP to inactive 6-thiouric acid (6-TU),
3. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) which converts 6-MP into the active metabolite 6-TGN.
Inactive metabolites and 1 % of unchanged 6 MP excreted via urine.



This active thiopurine metabolite acts to inhibit purine and protein synthesis in lymphocytes mainly the de novo pathway; hence inhibiting proliferation.

Other Mechanism: Azathioprine can directly promote apoptosis and inhibit proliferation pathways. Possibly because of directly inhibiting RAC1 (RAS-related C3 botulinum toxin substrate 1 - GTP binding protein) activity which in turn reduces BCL XL (f B cell lymphoma-extra large - a transmembrane molecule) having anti-apoptotic property.

Pharmacogenomics: It is the branch of genetics concerned with the way in which an individual's genetic attributes affect the likely response to therapeutic drugs.
 
Altered thiopurine metabolism has been associated with variations in clinical effectiveness.
Factors attributing :

1. TPMT Genotype/Phenotype :
Genetic polymorphisms in TPMT enzyme activity are inherited and can be both qualitatively and quantitatively measured.
TPMT enzyme deficiency is an autosomal recessive condition. 
The overall prevalence of TPMT deficiency is similar between different ethnic groups, the frequency of variant alleles differs between different populations. Three variant alleles, TPMT*2, TPMT*3A, and TPMT*3C, account for 80–95% of intermediate or low activity cases.

Individuals having :

% Population

High/normal activity

90 %

Intermediate activity

10 %

Low/ absent activity

0.3%

Low and intermediate TPMT enzyme activity leads to decrease metabolism and  more preferential production of active TGN which lead to myelotoxicity, hence it should be avoided in low (homozygous) genotype,
High TPMT enzyme activity tends to shunt 6-MP towards preferential 6-MMP production (inactive metabolite), hence decreased effectiveness.

Low or Intermediate TPMT Activity à More active metabolite (TGN) à More Myelotoxicity

High TPMT Activity à More Inactive metabolites (6- MMP) à Less therapeutic efficacy

 - Should we need to do Pre Medication TPMT testing?

 In 2004, the FDA approved a TPMT enzyme diagnostic test and made recommendations regarding the inclusion of advice about TPMT testing in thiopurine drug packaging, but deferred from making testing compulsory.
Another concern is it does not predict all adverse events related to the drug. But still, it gives a clue to one of the hazardous and life-threatening complications i.e Myelotoxicity.
Alternative to TPMT testing :
Most of the doctor believes pre-TPMT testing might not be very necessary and we can overcome adverse event by gradually up titrating the dosage of azathioprine and vigilantly monitoring the blood counts.
TARGET Study (TPMT: Azathioprine Response to Genotyping and Enzyme Testing) is an ongoing study. Its aim to establish the clinical utility and cost-effectiveness of TPMT genotyping in reducing the number of ADRs associated with prescribing azathioprine.
2.  Other genetic polymorphisms associated with inosine triphosphate pyrophosphatase (ITPA) and NUDT15 has also been implicated in thiopurine metabolism. NUDT15 encodes an enzyme that hydrolyses 6-thioguanine nucleotides into inactive metabolites.

Thiopurine metabolites Reference range

Therapeutic Range:

6-TGN: 230–450 pmol/8 × 108 RBC

6-MMP/6-TGN ratio: 5–25

Toxic Range:

6-TGN >450 pmol/8 × 108 RBC

6-MMP >5700 pmol/8 × 108 RBC


American biochemist and pharmacologist Gertrude B. Elion born in New York in 1918. She won a Nobel Prize for Medicine in 1988 together with George Hitchings and Sir James Black. 

Although TPMT enzyme testing is commercially available; but cost remains a concern. So evaluating for phenotype associated with high-risk groups is a better cost-effective measure. 

 

Clinical Indication : 

These drugs are mainly used by Rheumatologists, Gastroenterologist, Hematooncologist, Dermatologists, and Ophthalmologists.

Rheumatological Indication: It is mainly used for CTD's rather than RA. It is preferred for the treatment of maintenance therapy and as glucocorticoid sparing therapy for Lupus Nephritis, Vasculitis after Cyclophosphamide induction. It is also used in the treatment of Inflammatory Myositis, Uveitis, Bechet's disease, and CTD-associated ILD.

It is used by gastroenterologists for Autoimmune Hepatitis, IBD's.

Adverse Events : 

1. Reversible myelosuppression

2. Liver Enzyme Abnormality

3. Acute hypersensitivity syndrome - rare, within 2 weeks of starting therapy.

4. Infections - less common

Drug Interaction :

The most Concerning and fatal interactions are with Xanthine Oxidase Inhibitors i.e Allopurinol and Febuxostat. It increases active metabolite concentration.




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