The thiopurine drugs, 6-mercaptopurine (6-MP) and azathioprine (AZA), are cytotoxic and immunosuppressant agents used for the treatment of leukemia and rheumatologic and autoimmune disorders; for solid organ transplant; and for second-line therapy of steroid-dependent and steroid-refractory inflammatory bowel disease (IBD). AZA is a prodrug, or derivative, of 6-MP. Clinical use of AZA and 6-MP is limited by the long onset of action (about three months) and by the toxic side effects associated with these agents, including hepatotoxicity and myelosuppression.
AZA and 6-MP act by interfering with nucleic acid metabolism and cell proliferation. Following absorption, AZA is converted to 6-MP and then to 6-methylmercaptopurine (6-MMRP) by thiopurine methyltransferase (TPMT). A complex action of several enzymes initiates an additional intracellular pathway where 6-MP is converted to the active metabolite thioinosine monophosphate (TIMP), and then to 6-thioguanine nucleotides (6-TGNs). It is thought that the incorporation of active metabolites into the ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) of cells results in both the therapeutic and immunosuppressive actions of AZA.
PHARMACOGENETICS
Thiopurine dosing was traditionally based on an individual's weight or a physician's empirical judgment. A recently discovered variation in an enzyme-producing gene has led to the understanding that inherited differences in drug metabolism may influence both the toxicity and the efficacy of the thiopurines. This variability of response and the tailoring of dosing based on genetic information is known as pharmacogenetics.
TPMT red blood cell (RBC) activity is genetically determined as a result of polymorphisms in the TPMT gene. High TPMT activity has been shown to reduce the formation of 6-TGNs. Conversely, TPMT deficiency is associated with excessively high levels of 6-TGN in hematopoietic tissues. Two alleles confer high TPMT and low TPMT activity, respectively. Homozygotes with absent or low TPMT activity (0.3 percent of the population) and heterozygotes with intermediate TPMT activity (10-15 percent of the population) generate higher 6-TGN concentrations and are more likely to experience high levels of toxicity. The remaining 85-90 percent of individuals are homozygous for a gene that results in high levels of TPMT activity. These individuals are not susceptible to toxic side effects but are more likely to be refractory to treatment.
The role of the TPMT enzyme in purine metabolism has been well documented, and polymerase chain reaction (PCR) methods reliably predict mutant alleles. TPMT genotyping, when used prior to AZA/6-MP therapy, is predictive for intermediate and low-enzyme activity in up to 95 percent of cases and may, therefore, identify individuals who are at risk for developing toxic myelosuppression with standard doses of AZA/6-MP. The measurement of TPMT activity (phenotyping) is sensitive, with a genotype correlation of 97 percent. This leads some clinicians to suggest that phenotyping provides a safer and more optimal approach to thiopurine treatment than the traditional methods of weight-based or empiric dosing.
Toxic events, some of which can be severe or even fatal, result in the discontinuation of AZA/6-MP therapy in 10-28 percent of cases. Individuals with zero TPMT activity are most likely to experience severe myelosuppression on standard AZA doses and should be considered for alternative treatment or extremely low doses of AZA. Individuals with higher-than-average TPMT activity may be refractory to the conventional dosage of AZA, and higher doses may be required, while those with intermediate TPMT activity can be safely treated with 30-50 percent of the standard dose.
Because the risk of life-threatening toxicity is high in individuals with TPMT deficiency who receive thiopurine drugs, genotyping by PCR or phenotyping by analysis of TPMT enzyme activity in erythrocytes is being advocated to identify polymorphisms in the TPMT gene. Monitoring of 6-MP metabolite level in erythrocytes has recently been investigated as a means to predict toxicity and nonresponse during treatment with AZA/6-MP.
The Mayo Clinic and St. Jude Hospital for Children genotype individuals who have acute lymphocytic leukemia (ALL) prior to administering thiopurines. Recently, a US Food and Drug Administration (FDA) Pharmacology Advisory Committee recommended that information on genetic testing be added to thiopurine labeling.
METABOLITE MONITORING
Individuals with ALL who are receiving AZA/6-MP therapy have been shown to present elevated RBC 6-TGN concentrations and leukopenia that are correlated. Therefore, routine measurement of 6-MP metabolites has been advocated as a means of assessing drug effect in the pediatric ALL population. Many recent studies concentrate on the effect of 6-MP metabolite monitoring in individuals who have IBD and autoimmune disorders. Historically, mild leukopenia has been used as the measure of appropriate therapeutic dosing for AZA/6-MP therapy, but research indicates that 6-MP metabolite levels may be a more effective guide.
Metabolite monitoring is not a universally accepted practice. For instance, the American College of Gastroenterology and the British Society of Gastroenterology advise that clinical indicators and blood tests are sufficient evidence of drug efficacy. However, they recommend that clinical implementation be delayed until the results of randomized controlled trials, which are currently in progress, are available. Others maintain that cross-sectional and retrospective studies already support the efficacy of these assays. Given the price of hospitalization and rescue therapy for individuals who experience severe effects of the therapy, as well as the costs associated with those who do not respond to therapy, metabolite monitoring may optimize treatment for certain individuals, most notably those receiving co-administration of allopurinol, nonresponders, and those in whom noncompliance is suspected.
