Patent Application
20160160264
The invention relates to an in vitro method for determining the enzyme activity of thiopurine S-methyltransferase in body fluids. In particular, the invention relates to such a method in which the urothione content and/or the jukathione content is determined in vitro in plasma, serum and/or urine in order to determine the enzyme activity of thiopurine S-methyltransferase. Further, the invention relates to a diagnostic in vitro method using urothione and/or jukathione as a biomarker.
Thiopurine S-methyltransferase (hereinafter mostly referred to as TPMT) is a key enzyme in the drug metabolism in the treatment of various forms of leukemia and chronic inflammatory diseases. TPMT transforms cytostatic drugs, such as 6-mercaptopurine, into the clinically inactive methylated forms (e.g. 6-methylmercaptopurine), which are then excreted renally. In spite of the pharmacological significance of TPMT, which is presented below, its physiological substrate and the function of TPMT in the human metabolism is unknown.
Thiopurines, such as 6-thioguanine (6-TG), 6-mercaptopurine (6-MP) or azathioprine (AZA), are purine antimetabolites that are widely used in the treatment of leukemia, such as acute lymphoblastic leukemia, autoimmune diseases (e.g. Crohn's disease, rheumatoid arthritis), and in recipients of organ transplants. They are analogs of the naturally occurring nucleic acid base guanine. Other indications for thiopurines are, for instance, non-Hodgkin lymphoma, ulcerative colitis, polycythemia vera, psoriatic arthritis, various autoimmune diseases, including systemic lupus erythematosus, Behçet's disease, other forms of vasculitis, autoimmune hepatitis, atopic dermatitis, myasthenia gravis, neuromyelitis optica (Devic's disease), restrictive lung disease.
TPMT is an enzyme which methylates thiopurine compounds. More specifically, TPMT catalyzes the S-methylation of the thiopurine agents. The methyl donor is S-adenosyl-L-methionine, which in turn is transformed into S-adenosyl-L-homocysteine by the reaction. That is, TPMT metabolizes thiopurine agents using S-adenosyl-L-methionine as an S-methyl donor while forming S-adenosyl-L-homocysteine as a byproduct.
Genetic polymorphisms that affect the enzymatic activity of TPMT correlate with the response and the side effects of thiopurine drugs in the treatment of patients.
Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) is a cytosolic enzyme that catalyzes the S-methylation of thiopurine drugs, such as azathioprine (AZA) and 6-mercaptopurine (6-MP). The enzyme activity shows itself to be extremely variable, with about 1 in 200 Caucasians having a complete deficiency, while 11% exhibit an intermediary and 89% a normal TPMT activity in red blood cells (RBCs) (Schaeffeler et al. 2004). In addition, 1-2% of Caucasians deviate from this trimodal distribution and exhibit a very high level of TPMT activity (Schaeffeler et al. 2004). Since thiopurine-based immunosuppressives afford only a relatively limited therapeutic range, the TPMT polymorphism influences the ratio between the two methylated metabolites 6-methylmercaptopurine (methylated ribonucleotide) and thioguanine nucleotide (so-called 6-TGN) significantly, and thus determines the effectiveness and toxicity of the thiopurine therapy in patients with inflammatory bowel diseases, acute lymphoblastic pediatric leukemia and other diseases (summarized in Teml et al. 2007). For example, individuals with a reduced or lacking TPMT activity haven an increased risk for myelosuppression during therapy with the standard dose of azathiopurine, or 6-MP, because excessively high 6-TGN levels are present in these cases. Therefore, these patients require an individually adapted dosage in their thiopurine treatment (Kaskas et al Gut 2002).
In contrast, individuals with very high levels of TPMT activity are in danger of developing resistances to therapy (due to 6-TGN levels that are too low), because an intensive methylation of thiopurine metabolites occurs in these cases and the active 6-TGN metabolites are formed in smaller amounts for this reason. In addition, liver damage may be caused by high 6-methylmercaptopurine ribonucleotide levels (Dubinsky et al. 2000, Nygaard et al. CPT 2008). Pediatric patients with acute lymphoblastic leukemia and high levels of TPMT activity are subject to an increased risk of relapse under standard treatment conditions (Stanulla et al. 2005).
The molecular fundamentals of TPMT deficiency are well understood. TPMT constitutes one of the few examples in which a pharmacogenetic phenomenon was translated into generally recognized diagnostic routine tests for the adjustment of optimum thiopurine doses. These recommendations were recently updated (Relling et al. 2013), even though the endogenous substrate of TPMT is still not known today.
TPMT activity is generally determined in red blood cells (RBCs). Cut-off values for very low, intermediary and normal activities that are strongly dependent upon the measuring method used were defined (e.g. radiochemical measurement (Weinshilboum et al. 1978) or HPLC-based methods (Kroplin et al. 1998)).
In the 1980s, the genetic foundations of TPMT deficiency were laid (Weinshilboum et al. 1980). Today, 27 single nucleotide polymorphisms (SNP) associated with altered TPMT activities are described (Appell et al. 2013). The co-inventors Dr. Schaeffeler and Dr. Schwab investigated the TPMT genotype-phenotype correlation in a large cohort (1222 persons) of healthy, voluntary test subjects. They were able to confirm the trimodal distribution of the TPMT phenotypes and found a TPMT deficiency in 0.6% (1:180) of the individuals, an intermediary level of TPMT activity in 10.2%, and normal or high levels in 89.2% (Schaeffeler et al Pharmcogenetics 2004).
The concordance between the TPMT genotypes and phenotypes is 98.4% with a sensitivity and specificity for positive and negative predictions of greater than 90% when using the genetic analysis for predicting the correct TPMT phenotype. Nevertheless, there are significant limitations in the activity determination in RBCs and the genetic examination of TPMT in the clinical routine: (1) The determination of the correct TPMT phenotype in patients that received blood transfusions within the last 6-8 weeks due to disease-related anemia is subject to a high risk of misclassification (Cheung et al. 2003; Ford et al. 2004, Schwab et al. 2001). Therefore, further genetic analysis is of the essence in all these cases. (2) In daily routine, the genetic analysis of the TPMT genotype is not carried out by means of sequencing techniques and therefore detects only the frequent mutations that are already known. Therefore, a misclassification due to rarely occurring alleles that are not analyzed may occur.
For the above reasons, the identification of an endogenous substrate that is methylated exclusively by TPMT is of the greatest significance and of great value for improved clinical diagnostics. Such a direct biomarker would remove all of the above-mentioned limitations and would be detectable not only in the blood, but also in urine, which would significantly simplify clinical diagnostics.
Since the TPMT analysis is necessary in the routine treatment of children with acute lymphoblastic leukemia treated with 6-MP (Relling et al. 2013), a simpler urine analysis in this group of patients would be very advantageous indeed. Furthermore, approximately 1-2% of the individuals are characterized by an increased TPMT activity; the underlying genetic cause is still not known today. The determination of the endogenous reaction product of TPMT would also efficiently identify patients with very high levels of TPMT activity (Schaeffeler et al. 2004) and thus reduce or remove the risk of a poorer response of the azathioprine or 6-MP therapy due to a dosage that is too low.
Furthermore, TPMT deficiency is associated with the toxicity of cisplatin, and therefore, a genetic analysis of TPMT is also recommended prior to a cisplatin treatment (Ross et al. 2009). These patients would also benefit from a simple and fast determination of the endogenous TPMT product.
Because of the risk of side effects, pediatric leukemia patients, prior to therapy, are examined with respect to their TPMT activity by means of genetic and enzymatic analysis. These tests involve a lot of effort and, at present, are therefore routinely carried out mostly only in particularly endangered patient groups, e.g. in pediatric patients, whereas other patient groups, such as patients with chronic inflammatory diseases, are most frequently treated with suboptimum doses at first, in order to avoid toxicities. By increasing the dose in, amongst others, patients with a reduced and non-lacking TPMT activity, one gradually approaches empirically the maximum thiopurine dose tolerated by the patient. In the process, there is, on the one hand, always the danger of severe or even lethal side effects due to overdosing, and on the other hand, there is the danger of underdosing, which jeopardizes the success of the treatment.
Thus, there is a demand for a simple, cost-effective and fast method for determining the TPMT activity in the respective patient in order to be able to determine an effective thiopurine dose without the occurrence of side effects during the therapy.
This problem is solved by the method for determining the enzyme activity of TPMT defined in claim 1. Preferred embodiments are presented in the dependent claims. Furthermore, protection is sought for a diagnostic in vitro method using urothione and/or jukathione as a biomarker for an increased susceptibility with regard to one or more particular diseases.
This invention is based upon the insight of the inventors that TPMT catalyzes an essential step in the catabolism of the molybdenum cofactor (Moco).
The catabolic final product and excretion product of Moco is urothione of the following structure (Goto and Sakurai, 1969):
Urothione is also known under the IUPAC name 2-amino-7-(1,2-dihydroxyethyl)-6-methylsulfanyl-1H-thieno[3,2-g]pteridin-4-one or the term 2-amino-7-(1,2-dihydroxyethyl)-6-methylthio-1H-thieno[3,2-g]pteridin-4-one. It is a sulfur-containing pteridine derivative isolated from human urine.
It is still unknown today which enzymes take part in the transformation of Moco into urothione. The inventors have demonstrated for the first time that TPMT catalyzes the methylation of Moco. The present invention is based on this finding.
TPMT is capable of transforming Moco into urothione in an S-adenosylmethionine-dependent reaction in the presence of a phosphatase. The direct reaction product of TPMT is the phosphorylated form of urothione (jukathione), which is then transformed into urothione by a phosphatase not yet known, as shown in the following diagram. The steps that result in the formation of the substrate of 2-thiopurine methyltransferase (TPMT) are unknown. In an S-adenosylmethionine-dependent reaction TPMT forms jukathione.
The content of urothione in body fluids, e.g. urine, correlates with the activity of TPMT and can therefore be used as a reliable biomarker for determining the TPMT activity. Therefore, the determination of the urothione content can replace other less accurate methods, such as the determination of the activity by other substrates or genetic tests.
The use of urothione in the manner described makes possible:
Thus, urothione and jukathione are very reliable biomarkers for the therapeutic monitoring under thiopurine therapy as an alternative for the determination of thiopurine metabolites in blood.
The following biochemical analyses prove that TPMT participates in the catabolism of urothione:
1. The in vitro synthesis of urothione in kidney protein extracts is dependent on S-adenosylmethionine and Moco. This is shown in
2. The S-adenosylmethionine-dependent synthesis of urothione is dependent on the quantity of Moco and the presence of a phosphatase. In vitro, alkaline phosphatase is able to catalyze this reaction, as becomes clear from
3. In vitro transformation of Moco by means of purified TPMT in urothione: The comparative analysis with recombinantly expressed and purified TPMT shows that Moco from a denatured Moco enzyme (human sulfite oxidase) as a substrate can be transformed into urothione in the presence of a phosphatase, as is shown in
4. The in vitro transformation of Moco by means of liver extracts into urothione in the presence and absence of the TPMT inhibitor 3,4,5-triiodo benzoic acid is shown in
5. The in vitro transformation of Moco by purified TPMT into jukathione in the presence and absence of the TPMT inhibitor 3,4,5-triiodo benzoic acid is shown in
6.
7. The in vitro transformation of Moco by protein extracts from RBCs of individuals with a normal TPMT activity, compared with persons with heterozygous mutations in the TPMT gene locus, shows an activity in the urothione synthesis that is reduced to about 70%. As is apparent from
8. The determination of the urothione content in the urine of individuals with a normal TPMT activity, in comparison with persons with heterozygous mutations in the TPMT gene locus, shows no significant differences in the urothione content, while no urothione could be detected in two persons with homozygous mutations in the TPMT gene locus (
Urothione detection: 10 μM Moco (isolated from heat-denaturated human sulfite oxidase) are incubated in 50 mM Tris/HCl, pH 7.2, 1 mM SAM, 250 μM dithiothreitol and 1.7 μg/μL liver protein extract or 3.4 μg/μL RBCs for 4 h at 37° C. After adding 33 units of alkaline phosphatase, 20 mM MgCl2 and 0.1 M Tris/HCl, pH 8.3, incubation is carried out for at least 4 h (37° C.) and the reaction is stopped by 15 minutes of incubation at 80° C. The HPLC analysis of urothione took place on a YMC C18 Hydrosphere®, 250×4 mm column with a 5 μm particle size in 20 mM formic acid and elution in a methanol gradient (0-25% 20 min, 1 mL/min).
A final concentration of 3 ng/μL enzyme was used for the reaction kinetics with purified TPMT. The reaction was stopped with 3,4,5-triiodo benzoic acid. The analysis took place as described above.
In order to detect urothione in urine, a two-stage solid phase extraction with Florisil® (500 mg matrix/mL urine, elution with 50% acetone) and an aminopropyl matrix was carried out (500 mg matrix/mL urine, elution with 20 mM acetic acid). A C18 Reprosil® 100, 250×3 mm column with 5 μm particles (Dr. Maisch GmbH) was used for the subsequent HPLC analysis. 20 mM formic acid served as eluent, and elution took place in a methanol gradient (0-25% 20 min, 1 mL/min). Creatinine was determined according to Vasiliades.
According to the invention, the urothione level is determined in vitro in body fluids or cellular extracts and correlated with the TPMT activity. Using the data obtained in this manner, cut-off values for urothione levels can be determined for the stratification of TMPT-deficient individuals, individuals with a reduced TPMT activity, individuals with a normal TPMT activity and individuals with an extremely high level of TPMT activity. Then, these cut-off values serve for the dose-adjusted therapy of patients with thiopurines, in analogy to the current procedure using the measurement of the TPMT activity in RBCs or the genetic diagnostics for the TPMT variants.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2013 011 995.2 | Jul 2013 | DE | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/064999 | 7/14/2014 | WO | 00 |
