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Thiopurine Methyltransferase in the Treatment of Inflammatory

VIEWS: 3 PAGES: 30

									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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