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Azathioprine and acquired aplastic anemia Morey Blinder April 29, 2005 Hematology-Oncology Grand Rounds Case report - T.F. 32 yo WM with a history of Crohn’s disease diagnosed in 1992. Complicated by abscesses/ fistulas in past. Started on Imuran 200 mg PO q.d. (azathioprine) June 2004. Traveled to Mexico early July 2004 - CBC was normal. GI clinic - 7/28/04 with 3 day history of fever, rectal pain and was thought to have perianal fistula and was admitted. CBC: Hgb 8.2; Hct 22.9; WBC count 600 (ANC 0); platelet count 42,000 Azathioprine was discontinued. Blood cultures were positive for E. coli and S. viridans Treated with antibiotics; G-CSF and blood product support. Hospital course 7/31/04 - Surgery for perianal abscess drainage and debridement of necrotic material 8/5/04 CBC: Hgb 6.4; Hct 17.9; WBC count 300 (ANC 0); platelet count 5,000 Hematology consult: Bone marrow exam: Markedly hypocellular (<10%); M:E 1:3 Consider transfer to BMT for supportive care Consider causes of severe marrow suppression Thiopurine Drugs • Thiopurine Drugs, 6-MP (right), AZA, and 6-TG are used as: - chemotherapeutic agents to treat leukemia - immunosuppressive agents to treat IBD, autoimmune diseases, and following solid organ transplantation. • The exact mechanism of the effects of AZA and 6-MP is unknown: - Interfere with DNA and RNA synthesis and chromosomal replications - Inhibit proliferation of T and B lymphocytes - Interfere with cytotoxicity of NK cells • Delayed onset of action – Mean of 17 weeks for response in CD pts • Clinical Response rates vary in steroid sparing, induction or maintenance of remission. - IBD – induces remission in ~50 – 60% pts and allows steroid withdrawal in 70% of pts • Side effects include: - Fever, chills, nausea, vomiting, anorexia, diarrhea, bone marrow suppression - Rash, hepatotoxicity • Toxicity – 10 – 20% withdraw due to adverse effects: - Severe myelotoxicity is potentially fatal and occurs early or occasionally later in treatment Efficacy of AZA in Treatment of Active CD Study # Pts Drug Dose Duration Response – Response – P Value Treatment Placebo Rhodes et al. 16 AZA 2 months 0(0%) of 9 0(0%) of 7 NS 2 mg/kg/d Willoughby 12 AZA 6 months 6(100%) of 6 1(17%) of 6 NR 2 mg/kg/d Klein et al. 26 AZA 4 months 6(46%) of 13 6(46%) of 13 NS 3 mg/kg/d Summers et al 136 AZA 17 Weeks 21(36%) of 59 20(26%) of 77 .25 2.5 mg/kg/d Present et al 72 6-MP 12 months 26(72%) of 36 5(14%) of 36 <.001 1.5 mg/kg/d Ewe et al 42 AZA 4 months 16(76%) of 21 8(38%) of 21 .03 2.5 mg/kg/d Candy et al 63 AZA 3 months 25(76%) of 33 20(67%) of 30 .6 2.5 mg/kg/d Oren et al 58 6-MP 9 months 13(41%) of 32 12(46%) of 26 NS 50 mg/d Su and Lichtenstein. Gastroenterology Clinics. 2004 Azathioprine/6-MP treatment: Early studies Study # pts. Disease % leukopenia # pancytopenia Kolle, 1969 30 JRA/Stills 17% 3 Lorenzen, 1969 40 Various 25% 4 Ginzler, 1975 73 SLE 8% 6 Singleton, 1979 59 Crohns disease 15% 0 Pollack, 1980 160 Renal transplant 25% 0 Present, 1980 68 Crohns disease 10% 2 Mertens, 1981 300 Various 5% 4 Hass, 1982 56 MS/Myasthenia NR 0 Hall, 1985 34 Renal transplant 9% 3 Kissel, 1986 64 Various 22% 1 Kvein, 1986 32 JRA NR 2 Hohfield,1988 105 Myasthenia 16% 3 Total 1021 28 (2.7%) Antsey et.al.; J. Royal Society of Med. 1992 Azathioprine Metabolism McLeod and Siva. Pharmacogenomics. 2002 Azathioprine Toxicity Baker. Reviews in Gastroenterological Disorders. 2003 Pharmacogenetics – TPMT • Polymorphisms in a single gene – Thiopurine S-Methyltransferase (TPMT) - influence metabolism of thiopurine-based drugs • Various TPMT alleles with point mutation(s) in the ten exons and nine introns as well as the 5’ promoter region have been associated with deficient, intermediate, or normal/high activity. • Trimodal pattern of inheritance – Autosomal Codominant: - 0.3% of the population = Low/No TPMT activity - 11% of the population = Intermediate TPMT activity - 89% of the population = Normal/High TPMT activity • Nomenclature: Wild type allele TPMT*1 Silent Variant alleles TPMT*1s Mutant Alleles TPMT*2 – TPMT*18 TPMT • TPMT is a cytosolic enzyme which catalyzes the S-methylation of the immunosuppressive/cytotoxic thiopurine drugs: 6-Thioguanine (6-TG), 6-Mercaptopurine (6-MP) and Azathioprine (AZA). • No natural substrate is known for TPMT, and it has no known involvement in endogenous metabolic pathways. • Multiple transcripts (3.2Kb, 1.7Kb, 1.0Kb) are expressed in lung, liver, skeletal muscle, kidney, RBCs, and WBCs • In the absence of exposure to thiopurines, TPMT deficiency has no known effect on human health. TPMT Mutant Alleles Schaeffeler et al. Pharmacogenetics. 2004 Ethnic Variations of TPMT Alleles McLeod and Siva. Pharmacogenomics. 2002 Influence of TPMT genotype on duration of Azathioprine Therapy • 61 pts wild type • 5 pts heterozygous (TPMT*3A) Median = 39 wks Median = 2 wks P = 0.018 Black, A. J. et. al. Ann Intern Med 1998;129:716-718 Correlation Between Genotype and Toxicity • Evaluated 23 pts referred to clinic for excessive toxicity while receiving thiopurine drugs • 6 TPMT homozygous mutants and 11 heterozygotes – 65% (10% normal population) • Several other studies have also demonstrated that 60 – 70% of patients suffering from thiopurine induced toxicity have one or two mutant TPMT alleles Evans et al J. Clin. Onc. 2001 Thiopurine Dosing and Polymorphisms Evans et al. J. Clin Onc. 2001 TPMT Polymorphisms and Toxicity Evans. Pharmacogenetics.2002 Testing for TPMT Polymorphisms • TPMT enzyme activity assay • Analysis of RBC Thiopurine metabolites • Molecular genetic analysis RBC TPMT Activity TPMT Phenotype was traditionally determined by a nonchelated radiochemical assay. • Gold Standard for phenotyping TPMT Activity RBC lysates +/- 6-MP • Activity in RBCs correlates with activity in other tissues [14C-methyl]- S-adenosylmethionine • In patients who have had a recent (30 – 60 days) RBC 6-[14C-methyl]-mercaptopurine (MMP) transfusion, TPMT activity in Extract with toluene/ RBCs can be spurious. isoamyl alcohol 14C-MMP Counted TPMT Activity by HPLC TPMT Activity (nmol 6-MTG/gHb/h) RBC lysates None +/- 6-TG (0.8) S-adenosylmethionine Low 6-methylthioguanine (6-MTG) (17) Normal (36) Quantification of fluorescent 6-MTG by HPLC High (76) TPMT Polymorphisms – RBC Metabolites 6-TGN 6-MMPN * 6-TGN and 6-MMPN Metabolite concentration are measured by HPLC When to Measure Metabolites Recommendations by Prometheus Laboratories 1. Following initiation of thiopurine drug therapy – (after 3 wks to establish steady metabolic state) 2. Following any dose adjustment – (Intended “therapeutic range” 235 – 450 pmol/8x108 RBC) 3. When utilizing a steroid-sparing strategy 4. Any time of disease flare 5. After six months of treatment – (TPMT activity may increase due to an “induction effect”) 6. At the time of an adverse event - bone marrow or hepatic toxicity 7. Whenever expected response is not occurring (To measure effects of concomitant drug use) 8. Twice yearly to measure against baseline (Therapeutic drug monitoring) Correlation between 6-TG and Genotype Dubinsky et al. Gastroenterology. 2000 • Individuals were genotyped using Allele specific PCR • 8 pts were heterozygous for the low TPMT activity allele (TMPTH/TPMTL) • 84 were homozygous wild type (TMPTH/TMPTH) • Median 6-TG concentrations in RBCs are significantly higher in TPMT heterozygotes. While there is no correlation between 6- * P < 0.0001 MMPN concentrations and genotype. Measuring RBC Metabolites for Therapeutic drug monitoring of IBD Patients on AZA • 170 IBD patients treated with AZA or 6-MP. Wanted to correlate 6-TGN with diseae activity as measured by IBDQ and Leukopenia. • Inflammatory Bowel Disease questionnaire (IBDQ) previously validated with higher scores = better quality of life. Patients in Clinical remission have a minimal score of 170 • Median 6-TGN concentrations were similar in (139 vs. 131 pmol/8 X 108) 56 pts with active disease and 114 pts in remission. • Questionable usefulness of therapeutic drug monitoring with 6-TGN Lowry et al. Gut. 2001 TPMT Genotyping – Genetic Mutational Analyses • Genotyping has been traditionally performed by allele specific PCR of most common mutations: 1. TPMT*3A (Caucasian) 2. TPMT*3C (African and Asian) • Other methods may be used to evaluate many more mutations Genotype/Phenotype Correlation Schaeffeler et al. Pharmacogenetics. 2004. Investigated the genotype-phenotype correlation of 1214 healthy blood donors to determine accuracy of genotyping in predicting TPMT phenotype. Phenotyping – HPLC Genotyping – HPLC + Sequencing Recommendations for Genotyping • DNA based genotyping offers a clinically important strategy to prospectively diagnose TPMT deficiency and minimize the risk of hematologic toxicity in patients treated with Thiopurine medications. • Despite encouraging data – the routine use of TPMT genotyping to make treatment decisions is still limited. Genotype-specific 6-MP starting doses for childhood ALL McLeod and Siva. Pharmacogenomics. 2002 Available laboratory testing Laboratory tests sent to Mayo clinic laboratory: Volume Name Measurement Cost (1/04-12/04) PRO-predict 6-MMP,6-TGN $273 36 TPMT enzyme assay TPMT $127 9 TPMT genotyping TPMT*2 $385 6 TPMT*3A/3C Laboratory testing Samples sent to Prometheus Laboratories (San Diego, CA) 1. PRO-Predict EnzAct (Enzyme activity) Result: 3.5 EU < 6.7 low activity 6.7 – 23.6 Intermediate activity > 23.6 High activity 2. PRO-Predict Metabolites (6-TGN and 6-MMPN) Result: 6 -TGN: 1673 (230 – 400) 6 -MMPN: undetectable (< 5700) 3. PRO-Predict TPMT (Genotyping) Result: Homozygous TPMT*3A Hospital course 8/6/04 Severe exfoliating rash thought to be due to Imipenem 8/9/04 Throat culture (+) HSV 8/10/04 Perirectal abscess - debridement with necrotic ulcer; acute and chronic inflammation; (+) C. difficile 8/11/04 Oral (+) HSV 8/19/04 Transferred to BMT floor 9/3/04 Discharged (day 38): 16 U pRBCs; 19 U platelets CBC: Hgb 10.7; Hct 30.0; WBC count 2,700 (ANC 1,100); platelet count 15,000 9/10/04 CBC: Hgb 11.6; Hct 33.2; WBC count 3,800 (ANC 1,900); platelet count 40,000 2/8/05 CBC: Hgb 13.1; Hct 37.8; WBC count 3,000 (ANC 2,100); platelet count 129,000 Conclusions • Thiopurine S-methyltransferase (TPMT) plays a major role in metabolizing thiopurine medications like AZA, 6-MP, and 6-TG • TPMT activity is polymorphic, 10% heterozygous and 1/300 have low/deficient activity • Patients with low or deficient TPMT activity are at a significantly higher risk of rapidly developing hematologic toxicity when given a standard does of thiopurine drug. • TPMT genotype correlates well with in vivo enzyme activity in erythrocytes. • TPMT genotyping provides clinicians with a reliable method (especially when RBC phenotyping is not available) for identifying TPMT-deficient patients who can benefit from low doses of thiopurine drugs in order to reduce risk of toxicity. • The availability of CLIA-certified TPMT genotyping has made routine clinical genotype testing a feasible approach for optimizing thiopurine therapy. • Routine monitoring of TMPT genotype and/or phenotype prior to induction of thiopurine therapy is a cost-effective measure and provides one of the best established applications for pharmacogenetics in clinical practice to date.
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