Azathioprine (AZA) Helen Liu Teagan Rolf von den Baumen PHM142 Fall 2017 Instructor: Dr. Jeffrey Henderson Azathioprine (AZA) Helen Liu Teagan Rolf von den Baumen Mishka Danchuk-Lauzon October 3rd 2017

General Information Brand name: Imuran Generic: Apo-Azathioprine, Mylan-Azathioprine, Teva- Azathioprine Thiopurine – class of immunosuppressive and chemotherapeutic drugs Immunosuppressant agent Discovered by George Hitchings and Gertrude Elion in 1957 Nobel prize in physiology or medicine in 1988 Azathioprine is a immunosuppressant agent that is sold as a generic drug and also as a brand name drug under the name Imuran It was discovered by

Recurrent Pericarditis Uses Crohn's disease Lupus nephritis Uveitis Multiple sclerosis Recurrent Pericarditis Labeled Indications Renal transplantation Rheumatoid arthritis Off label use

Administration and Pricing Oral : usually taken once a day after meals or in divided doses to decrease adverse GI effects I.V. : usually infused over 30 to 60 minutes but infusion time can be done in 5 minutes up to 8 hours Dosage Injection (generic) 100mg (1 injection): 300.00$ Imuran Oral 50mg (100 tablets): 803.10$ Azathioprine Oral 50mg (100 tablets): 211.09$

Dosing Dosing depends on the disease being treated Dosing also varies depending on hepatic impairment, renal impairment and other conditions Renal transplantation Following transplantation usually given as 3 mg/kg once daily Decreased to 1 to 3 mg/kg once daily for maintenance Not indicated for use in children for renal transplantation (can be taken for off-label use)

Pharmacodynamics/Kinetics Half-life of the drug is approximately 2 hours depending on the patient, it is well absorbed through the oral route The peak concentration occurs at 1 to 2 hours after oral ingestion It is primarily excreted through the urine Approximately 30% of the ingested drug becomes protein bound The initial drug response occurs in the first 30 to 90 days after beginning drug therapy with the peak response occurring 30 to 120 days after the start of drug therapy

Metabolism Hypoxanthine guanine phosphoribosyltransferase 6-thioguanine-nucleotides Xanthine oxidase 6-thiouric acid Thiopurine methyltransferase 6-methylmercaptopurine Azathioprine is first metabolized in the liver to 6-mercaptopurine via glutathione S-transferase reduction It is subsequently further metabolized in the liver and the GI tract through 3 main pathways 6-thioguanine metabolites produced during the metabolism of azathioprine mediate the drug’s immunosuppressive and toxic effects

hypoxanthine guanine phosphoribosyltransferase (HGPRT), glutathione transferase 6-thio inosine monophosphate (6-tIMP)

inosine monophosphate dehydrogenase (IMPDH) 6-thio inosine monophosphate (6-tIMP) 6-thioxanthylic acid (6-tXMP)

Successive phosphorylation by kinases (abbreviated with k) Guanosine monophosphate synthetase (GMPS) k k 6-thioguanine triphosphate 6-thioguanine monophosphate 6-thioguanine diphosphate 6-thioxanthylic acid (6-tXMP)

HGPRT Guanine 6- thioguanine (6-tG) Alternative pathway to convert 6-tG to 6-tGTP HGPRT deoxy-6-thioguanosine 5’ triphosphate (6-tGTP) Guanine 6- thioguanine (6-tG) Or 6-thioguanosine

6-tGTP Active metabolite that mimics guanine 6-tGN decreases inflammation. 6-tGN increases T cell apoptosis  

6-tGN inhibits many immune and inflammation related genes such as: tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) tumor necrosis factor receptor family 7 (TNFRSF7) α4-integrin

6-tGN incorporation in DNA 6-tGTP nucleotides incorporate itself into the T cell’s DNA T cell senses DNA damage Cell cycle is stopped and apoptosis is triggered T cell proliferation is halted and immature T cells are killed off

T cell CD28 Co-Stimulation T cell maturation relies on costimulation from the TCR and MHC, and CD28 and B7. Successful costimulation by CD28 inhibits TCR induced apoptosis. If the T cell does not receive proper costimulation, the CD28 receptor can signal for death (through apoptosis and the death receptor) One of the enzymes involved with CD28’s signal cascade is Rac1 GTPase.

6-tGN and CD28 Signaling 6-tGTP will bind to Rac1 in the T cell. (Rac1 normally binds to GTP) Rac1’s pathway is now blocked CD28’s costimulatory signal to the T cell is converted into an apoptotic signal Activated T cells are killed off

XO Pathway Xanthine oxidase 6-Mercaptopurine Prodrug 6-thiouric acid Inactive metabolite Another inactivating metabolic pathway of AZA is the Xanthine oxidase (XO) pathway, which forms oxidized metabolites from 6-Mercaptopurine 6-MP is catabolized by XO to the inactive metabolite 6-thiouric acid (6TU) 6-thiouric acid is then eliminated in the urine XO is expressed in many tissues, but not in hematopoietic cells (i.e. WBCs), making them more vulnerable to this toxicity

Allopurinol X Allopurinol, a common gout medication is contraindicated in patients taking AZA Co-prescribing requires significant dose reduction Allopurinol blocks the XO inactivation pathway of 6MP, leading to accumulation and toxicity

Allopurinol + AZA Cases where pharmacists and health teams who have failed to check drug risks has lead to deaths

Genetic Considerations: TPMT polymorphisms and 6-MP toxicity  TPMT (Thiopurine S-methyltransferase) full or parital deficiency is associated with increased IC level of 6-TGNs An increase in the active metabolite, 6-TGN can cause life-threatening bone marrow suppression when patients are treated with standard doses of 6MP or AZA The lack of the XO inactivation pathway in hematopoietic cells makes them most vulnerable to toxicity A deficiency in the TPMT pathway would make both inactivation pathways null, causing an accumulation of active metabolite and suppression of DNA synthesis here TPMT deficiency associated with increased intracellular levels of 6-thioguanine nucleotides (active metabolite)

TPMT Polymorphisms Extensive metabolizers: high enzyme activity 2 WT alleles Intermediate metabolizers: intermediate enzyme activity Heterozygotes: 1 variant, 1 WT allele 11% of population Poor metabolizers: poor or complete lack of enzyme activity 2 variant alleles Normal dose can be life-threatening or fatal 1/300 individuals 3 phenotypes for TPMT activity: Extensive metabolizers, intermediate metabolizers, and poor metabolizers. ~1/300 individuals in the population are intermediate or poor metabolizers, and for them a ‘normal’ dose can be life threatening Poor metabolizers require a 10-fold lower dose or alternative drug if possible The occurrence of severe hematopoietic toxicity is minimized with genetic-based dosing Results of TPMT phenotyping may not be accurate following recent blood transfusions Genetic-based dose adjustments aim to equalize levels of IC drug concnetration, therefore preventing toxicity while allowing the drug to still be efficacious

Summary Slide Azathioprine is first metabolized in the liver to 6-mercaptopurine via glutathione S-transferase reduction Azathioprine is further metabolized in the liver and the GI tract by the following enzymes: hypoxanthine guanine phosphoribosyltransferase, xanthine oxidase and thiopurine methyltransferase This immunosuppressant is important in preventing rejection of the transplanted organ since it lowers the efficacy of the recipient’s immune system by targeting white blood cells 6-tGN plays many important roles: 1) It inhibits many inflammation related genes 2) it incorporates itself into T cell DNA, halts the cell cycle and triggers apoptosis of immature T cells- stopping t cell proliferation. 3) It further increases apoptosis of T cells by modifying the CD28 costimulatory response into an apoptotic signal. Therefore also deleting activated T cells. It is important to take into consideration genetic factors when administering this drug since toxicity can occur in patients who have TPMT polymorphisms Taking Azathioprine and Allopurinol is contraindicated because Allopurinol blocks the XO inactivation pathway of 6MP leading to toxicity

References Wong, D., Derijks, L., Den Dulk, M., Gemmeke, E., & Hooymans, P. (2007). The role of xanthine oxidase in thiopurine metabolism: a case report. Ther Drug Monit, 845-848. doi:10.1097/FTD.0b013e31815bf4dc Sanderson, J. D. (2015). TPMT Testing Before Starting Azathioprine or Mercaptopurine: Surely Just Do It? Gastroenterology, 149(4), 850-852. doi:10.1053/j.gastro.2015.08.040 Wang, L., & Weinshilboum, R. (2006). Thiopurine S-methyltransferase pharmacogenetics: insights, challenges and future directions. Oncogene, 25, 1629-1638. doi:10.1038/sj.onc.1209372 Liew, D., & Booth, J. (2017). Fatal azathioprine toxicity. Aust Prescr, 40(3), 109. doi:10.18773/austprescr.2017.035 Connell, W. R., Kamm, M. A., Ritchie, J. K., & Lennard-Jones, J. E. (1993). Bone marrow toxicity caused by azathioprine in inflammatory bowel disease: 27 years of experience. Gut, 34, 1081-1085. doi:10.1136/gut.34.8.1081 VanLoon, J., Weinshilboum, RM. (1982). Thiopurinemethyltransferase biochemical genetics human lymphocyte activity. BiochemGenet, 20, 637-58.

References 2 Tiede, I., Fritz, G., Strand, S., Poppe, D., Dvorsky, R., Strand, D., … Neurath, M. F. (2003). CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes. Journal of Clinical Investigation, 111(8), 1133–1145. http://doi.org/10.1172/JCI200316432 Dumont, C., Corsoni-Tadrzak, A., Ruf, S., de Boer, J., Williams, A., Turner, M., … Tybulewicz, V. L. J. (2009). Rac GTPases play critical roles in early T-cell development. Blood, 113(17), 3990–3998. http://doi.org/10.1182/blood-2008-09-181180 Anna L. Taylor, Christopher J.E. Watson, J. Andrew Bradley, Immunosuppressive agents in solid organ transplantation: Mechanisms of action and therapeutic efficacy, In Critical Reviews in Oncology/Hematology, Volume 56, Issue 1, 2005, Pages 23-46, ISSN 1040-8428, https://doi.org/10.1016/j.critrevonc.2005.03.012. (http://www.sciencedirect.com/science/article/pii/S1040842805000843) Keywords: Immunosuppression; Solid organ transplant STARZL, T. E., MARCHIORO, T. L., & WADDELL, W. R. (1963). THE REVERSAL OF REJECTION IN HUMAN RENAL HOMOGRAFTS WITH SUBSEQUENT DEVELOPMENT OF HOMOGRAFT TOLERANCE. Surgery, Gynecology & Obstetrics, 117, 385–395. Elion G.B., Hitchings G.H. (1975) Azathioprine. In: Sartorelli A.C., Johns D.G. (eds) Antineoplastic and Immunosuppressive Agents. Handbuch der experimentellen Pharmakologie / Handbook of Experimental Pharmacology (Heffter-Heubner / New Series), vol 38 / 2. Springer, Berlin, Heidelberg University of Toronto IMM250 Notes

References 3 Azathioprine [PDF]. (n.d.). Ipharmacist. Booth, R. A. (n.d.). Introduction. Retrieved September, 2017, from https://www.ncbi.nlm.nih.gov/books/NBK55938/ George Hitchings and Gertrude Elion. (2016, September 13). Retrieved September, 2017, from https://www.chemheritage.org/historical-profile/george-hitchings-and-gertrude-elion Physiology or Medicine 1988 - Press Release. (n.d.). Retrieved September, 2017, from https://www.nobelprize.org/nobel_prizes/medicine/laureates/1988/press.html Images: http://www.achievement.org/achiever/gertrude-elion/ http://www.medicinenet.com/kidney_failure/article.htm https://www.webmd.com/drugs/2/drug-13771/azathioprine-oral/details