Nin Prapongsena, MPh.Faculty of pharmacy

Huachiew chalermprakiet University

Outline

• Introduction of thiopurines• Thiopurine metabolism and their mechanism of actions• ADR of thiopurines• How thiopurine treatment failure ???• Polymorphisms of thiopurine methyltransferase

(TPMT) gene and their clinical effects of TPMT activity• Correlation of TPMT genotype and phenotype• Limitation of TPMT genotype and phenotype

assessment• Conclusion• Pharmacist’s role

Thiopurine drugs

Azathioprine (AZA)

Pharmacological category: ImmunosuppressantIndication:

- Rheumatoid arthitis (RA)- Inflammatory bowel disease (IBD)- Psoriasis- Prevent graft rejection

ADR: - N/V- Hepatotoxicity- Leukopenia & thrombocytopenia- Cutaneous ADR

6-Mercaptopurine (6-MP): 50 mg/m2

Pharmacologic category: Antineoplastic agent & immunosuppressant

Indication: - Acute myelogenous leukemia (AML)

in induction & maintenance phase (remission ≈ 80%)

- Inflammatory bowel disease (IBD)ADR:

- Myelosuppression(onset 7 d, nadir 14-16 d, recovery 21-28 d)

- Hepatotoxicity- Cutaneous ADR

6-Thioguanine (6-TG): 40 mg/m2

Pharmacologic category: Antineoplastic agent

Indication: - Acute lymphoblastic leukemia (ALL)- Acute myelogenous leukemia (AML)

ADR: - Myelosuppression

(onset 7-10 d, nadir 14 d, recovery 21 d)- Hepatotoxicity- Cutaneous ADR

Metabolism of thiopurines

Azathioprine (AZA)

6-Mercaptopurine (6-MP)

Glutathione S- transferase (GST)

Xanthine oxidase(XO)

Thiouric acid(inactive)

*Thiopurinemethyltransferase

(TPMT)

6-Methyl mercaptopurine(antiangiogenesis)

Hypoxanthine phosphoribosyl

transferase(HPRT)

Thioinosine monophosphate(TIMP)

Metabolism of thiopurines (continue)

TIMP

*TPMT

6-Methyl thioinosinemonophosphate

(MeTIMP)

Hepatotoxicity

Inosine monophosphateDehydrogenase (IMPDH)

6-Thioguanine monophosphate

(TGMP)

Guanosine monophosphateSynthetase

Purinebiosynthesis

Metabolism of thiopurines (continue)

TGMP

6-MP 6-Thioguanine (6-TG)

HPRT

*TPMT

6-Methylthioguanine Monophosphate (MeTGMP)

Phosphokinase

Deoxythioguaninetriphosphate (TGN)

Purinebiosynthesis

Deoxythioguanine triphosphate effect

Deoxythioguaninetriphosphate (TGN)

Caused cell death& myelosuppression

ADR of thiopurines

• Found ADR 25.9% in patients who taking thiopurines

ADR of thiopurine drugs Liver abnormalities

Allergy

Pancreatitis

Bonemarrowsuppression

N/V

Other

34 %

6%9 %

***7 %

25 %

7 %

Related to dose and durationof 6-MP & 6-TG treatment in acute leukemia

How thiopurine treatment failure ? ? ?

• Drug interactions• Food interactions• Pharmacogenomics

Changed active metabolites level

Treatment failure• ADR (especially neutropenia)

• Too low dose

Thiopurine methyltransferase (TPMT) gene

• Located at chromosome 6 (6p22)

• 3.4 kb (composed of 10 exon)

• TPMT found in liver, renal, RBC, etc.

• TPMT expression α TPMT activity α 1/TGN

Weinshilboum RM., and Sladek SL

Am J Hum Genet. 1980; 32: 651-652.

Mercaptopurine pharmacogenetics: monogenic inheritance of erythrocyte thiopurine methyltransferase activity

Undetected(0.3%)

Intermediate or LowActivity (11%)

Normal activity (89%)

X-axis: RBC TPMT activity (U/mL)6-mp 4mM

Y-a

xis:

% o

f su

bje

cts

RBC TPMT activity

Polymorphisms of thiopurinemethyltransferase (TPMT) gene

Proc. Natl. Acad. Sci. USAVol. 94, pp. 6444–6449, June 1997Medical Sciences

Enhanced proteolysis of thiopurine S-transferase(TPMT) encoded by mutant alleles in human TPMT*3A, TPMT*2): mechanisms for the genetic polymorphism of TPMT activity

Tai HL., Krynetski EY., Yanishevski Y., and Evans WE

mRNA of TPMT

RBC TPMT activity

Stability of TPMT

Polymorphisms of thiopurinemethyltransferase (TPMT) gene

Normal actvityT1/2: 18 h

Low TPMT activityT1/2: 15 min

Low TPMT activity

Polymorphism of TPMT gene in Thailand

200 unrelated samples

Clinical effects of TPMT activity

• High TPMT activity (If TPMT level > 65 U/mL):

- High meTIMP level

- Low TGN level

Inhibited purine synthesis

Hepatotoxicity

Risk to failure in ALL treatment

• How to solve this problems ?

Recommend increasing dose of 6-MP or 6-TG thanusual dose and closely hepatic enzymes monitoring

Clinical effects of TPMT activity

• Intermediate TPMT activity (25 U/mL > TPMT level < 45 U/mL):

- High TGN level Myelosuppression

Increased risk of 2nd malignancy

• How to solve this problems ?

Recommend reducing dose to 50-80 % of standard dose

Clinical effects of TPMT activity

• Low TPMT activity or TPMT deficiency (If TPMT level < 25 U/mL):

- Very high TGN level Severe myelosuppression

High risk of 2nd malignancy

• How to solve this problems ?

Recommend reducing dose to 10-20 % of standard dose

Correlation of TPMT genotype and phenotype

Assessment of Thiopurine S-MethyltransferaseActivity in Patients Prescribed Thiopurines: A Systematic Review

Ann Intern Med. 2011;154:814-823.

Ronald A. Booth, PhD; Mohammed T. Ansari, MBBS, MMedSc, MPhil; Evelin Loit, PhD; Andrea C. Tricco, PhD; Laura Weeks, PhD; Steve Doucette, MSc; Becky Skidmore, MLS; Margaret Sears, PhD; Richmond Sy, MD; and Jacob Karsh, MDCM

Have False positive(Specificity is not 100%): cause low dose of 6-MP/6-TG usage

Limitation of TPMT phenotype and genotype testing

Genotype testing

Missed target:pseudogene onchromosome 18

Phenotype testing

• Factors affected to TPMT activity:

- Age- Blood transfusion

• Loss sample between testing

Investigated both genotype and phenotypeof TPMT are more utility

Cost effective for screening TPMT mutation

J Am Acad Dermatol 2000;42: 628-632

Cost effective for screening TPMT mutation

Conclusion• TPMT gene mutation affected to TPMT level, activity

and stability• TPMT expression α TPMT activity α 1/TGN level,

TGN level α myelosuppression & 2nd malignancy • Only TPMT*3C found in Thais (≈ 9%)• Dose adjustment:

- High TPMT (> 65 U/mL): dose- Intermediate TPMT (25-45 U/mL): dose 20-50%- Low or absence TPMT (< 25 U/mL): dose 80-90%

•TPMT genotype correlated with phenotype but not100% (Many factors affected to TPMT activity)

• There are limitation of genotype and phenotype testing, then investigated both are more utility.

Case report

Cutaneous ADR occured

Pharmacist’s role

Prevented

Screening TPMT genotype Testing TPMT activity

Considered appropriate initial dose

Monitor CBC and hepatic enzymes

Toxicity or ADR occurred:Adjusted dose or delayed dose± antibiotic (NCCN guideline)

± myelosuppressive treatment (NCCN guideline)and closely liver enzymes monitoring

Corrected

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