Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2001-05-16
2003-06-10
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
Reexamination Certificate
active
06576438
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to thiopurine drugs used for the treatment of inflammatory bowel disease, leukemia, and organ transplantation rejection, and more specifically to methods for determining thiopurine methyltransferase activity in order to individualize dosages of 6-mercaptopurine therapy.
2. Background Information
Mercaptopurine (6-MP or 6-thiopurine) and azathioprine [6-(1-methyl-4-nitro-5-imidazolylthio)purine] are cytotoxic drugs which are effective in the treatment of ulcerative colitis and Crohn's disease (Present et al.,
Annals of Internal Medicine
111:641-649 (1989)). Both are immunosuppressive agents that act as purine antagonists and thereby inhibit the synthesis of DNA, RNA and proteins (Lennard,
European Journal of Clinical Pharmacology
43:329-339 (1992)). 6-MP was initially used for the treatment of childhood acute lymphoblastic leukemia and for post-operative treatment of organ transplantation surgery (Burchenal et al.,
Blood
8:965-999 (1953)), and its use has since been extended to rheumatoid arthritis and inflammatory bowel disease (Kirschner,
Gastroenterology
115:813-821 (1998)).
The prodrug azathioprine (AZA) is rapidly converted to 6-mercaptopurine through non-enzymatic, nucleophilic attack by sulfhydryl-containing compounds in the circulation. 6-MP and azathioprine (AZA), which are forms of the same drug and metabolic precursors of the active components, are acted upon by at least three competing enzymatic pathways (Lennard, supra, 1992). An overview of the action of these enzymes is shown in FIG.
1
. As shown in
FIG. 2
, several major enzyme pathways are involved. Xanthine oxidase (XO) converts 6-mercaptopurine to 6-thiouric acid. Hypoxanthine phosphoribosyl transferase (HPRT) converts 6-mercaptopurine to 6-thioinosine-5′-monophosphate, which is a precursor to 6-thioguanine nucleotides. Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of 6-mercaptopurine to methylmercaptopurine (6-MMP). Thus, 6-mercaptopurine is enzymatically converted to various metabolites, including 6-thioguanine (6-TG) and 6-thioguanine nucleotides, which are the presumptive active metabolites mediating the effects of azathioprine/6-mercaptopurine drug therapy.
The interplay of the pathways described above is genetically determined and creates a highly individualized response to azathioprine/6-mercaptopurine drug therapy. The population frequency distribution of TPMT enzyme is trimodal, with the majority of individuals (89%) having high activity, 11% having intermediate activity and about 1 in 300 (0.33%) having undetectable activity (Weinshilboum and Sladek,
Amer. J. Human Genetics
32:651-662 (1980)). Such a trimodal relationship has been confirmed by direct measurements of TPMT enzyme activity by the Kröplin HPLC assay method (Kroplin et al.,
Eur. J. Clin. Pharmacol.,
54 265-271 (1998)). In contrast to variation in TPMT activity, there is very little inter-individual variation in XO activity and only limited data on HPRT activity (Lennard,
Eur. J. Clin. Pharm.,
43:329-339 (1992)).
Available evidence indicates that TPMT activity effectively modulates the concentration of 6-thioguanine by shunting 6-mercaptopurine into the production of 6-methyl-mercaptopurine. Patients who less efficiently methylate these thiopurines have more extensive conversion to 6-thioguanine nucleotides, which can lead to potentially fatal hematopoietic toxicity. Thus, patients with intermediate or low TPMT activity can be more susceptible to toxic side effects of azathioprine/6-mercaptopurine therapy (Present et al.,
Annals of Internal Medicine
111:641-649 (1989)). Such toxic side effects include allergic reactions, neoplasia, opportunistic infections, hepatitis, bone marrow suppression, and pancreatitis; in about 1 out of 300 patients, this therapy cannot be tolerated. As a consequence, many physicians are reluctant to treat patients with azathioprine/6-mercaptopurine therapy, particularly due to the risk of infection and neoplasia.
Thus, there is a need for a method of optimizing the dose of 6-mercaptopurine by determining the level of thiopurine methyltransferase activity in a patient. Such a method would be valuable for optimizing therapeutic efficacy of azathioprine/6-mercaptopurine therapy while minimizing undesirable side effects. The present method satisfies this need and provides related advantages as well.
SUMMARY OF THE INVENTION
The present invention provides a method of determining thiopurine methyltransferase (TPMT) activity in a subject. The method includes the steps of reacting sample obtained from the subject with a thiopurine derivative that is not 6-mercaptopurine to produce a methylated purine product; contacting the reacted sample with acid, thereby precipitating proteinaceous material from the reacted sample; separating supernatant from the precipitated proteinaceous material; and detecting in the supernatant the methylated purine product, where the amount of the methylated purine product indicates a level of thiopurine methyltransferase activity in the subject. In a method of the invention, the subject can be, for example, an inflammatory bowel disease patient. In one embodiment, the acid used to precipitate proteinaceous material is perchloric acid, for example, 70% perchloric acid. In another embodiment, the thiopurine derivative used as a substrate is 6-thioguanine. In a further embodiment, the methylated purine product is detected by fluorescence, which can be combined, if desired, with high performance liquid chromatography (HPLC).
REFERENCES:
Breithaupt et al., “Quantitative high pressure liquid chromatography of 6-thioguanine in biological fluids,”J. Chromatogr. Sci. 19:496-499 (1981).
Burchenal et al., “Clinical Evaluation of a New Antimetabolite, 6-Mercaptopurine in the treatment of leukemia and allied diseases,”Blood8:965-999 (1953).
Boulieu et al., “Methylated and Non Methylated Thiopurine Nucleotide Ratio (ME6-MPN/6-TGN) : Usefulness in the Monitoring of Azathiopurine Therapy?”Purine and Pyrimidine Metabolism in Man X, 361-367 (Kluwer Academic/Plenum Publishers 2000).
Capdeville et al., “Interactions between 6-mercaptopurine therapy and thiopurine-methyl-transferase (TPMT) activity,”Eur. J. Clin. Pharmacol. 46:385-6 (1994).
Dervieux and Boulieu, “Simultaneous determination of 6-thioguanine and methyl 6-mercaptopurine nucleotides of azathioprine in red blood cells by HLPC,”Clinical Chemistry44:551-555 (1998).
Dubinsky et al., “Pharmacogenomics and metabolite measurement for 6-mercaptopurine therapy in inflammatory bowel disease,”Gastoenterology118:705-13 (2000).
Ganiere-Monteil et al., “Thiopurine methyl transferase activity: new extraction conditions for high-performance liquid chromatographic assay”J Chromatograph B.727:235-9 (1999).
Jacqz-Aigrain et al., “Thiopurine methyltransferase activity in a French population: h.p.l.c. assay conditions and effects of drugs and inhibitors,”Br. J Clin. Pharmacol.38:1-8 (1994).
Keuzenkamp-Jansen et al., “Thiopurine methyltransferase: a review and a clinical pilot study,”J Chromatogr B Biomed Appl.678:15-22 (1996).
Kirschner, “Safety of azathioprine and 6-mercaptopurine in pediatric patients with inflammatory bowl disease,”Gastroenterology115:813-821 (1998).
Kröplin et al., “Inhibition of thiopurine S-methyltransferase activity by impurities in commercially available substrates: a factor for differing results of TPMT measurements,”Eur. J. Clin. Pharmacol.55:285-91 (1999).
Kroöplin et al., “Thiopurine S-methyltranferase activity in human erythrocytes: A new HPLC method using 6-thioguanine as substrate,”Eur. J. Clin. Pharmacol.54:265-71 (1998).
Kröplin et al., “Determination of thiopurine methyltransferase activity in erythrocytes using 6-thioguanine as the substrate,”Adv. Exp. Med. Biol.431:741-5 (1998).
Krynetski et al., “Methylation of Mercaptopurine, Thioguanine, and Their Nucleotide Metabolites by Heterologously Expressed Human Thiopurine S-Methyltransferase,”Mol Pharmacol.47:1141-7 (1995).
Lennard and
Prometheus Laboratories Inc.
Saucier Sandra E.
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