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BR-122026002225-A2 - METHODS FOR DIAGNOSING A DISEASE OR CONDITION AND FOR DETERMINING ACTIVE DPP3 IN A BODY FLUID SAMPLE

BR122026002225A2BR 122026002225 A2BR122026002225 A2BR 122026002225A2BR-122026002225-A2

Abstract

The present invention relates to methods for determining active DPP3 in a sample of body fluid, an assay or kit for determining active DPP3 in a sample of body fluid, a method for diagnosing a disease or condition in an individual accompanied by or related to necrotic processes, and methods for treating or preventing said disease.

Inventors

  • Andreas Bergmann

Assignees

  • 4TEEN4 Pharmaceuticals GmbH

Dates

Publication Date
20260310
Application Date
20170420
Priority Date
20160421

Claims (20)

  1. 1. Method for diagnosing a disease or condition in an individual, accompanied by or related to necrotic processes, characterized in that it comprises: (i) determining the amount of total DPP3 and/or determining the amount of active DPP3 in a sample of body fluid from said individual, (ii) comparing said determined amount of total DPP3 or active DPP3 with a predetermined limit, wherein said individual is diagnosed as having a disease or condition accompanied by or related to necrotic processes, if said determined amount is above said predetermined limit, wherein said sample is selected from the group comprising whole blood, serum and plasma.
  2. 2. Method according to claim 1, characterized in that the amount of total or active DPP3 is determined in units of concentration.
  3. 3. Method, according to claim 1 or 2, characterized in that said disease is selected from the group comprising heart failure, chronic heart failure, acute heart failure, myocardial infarction, stroke, liver failure, burns, traumatic injuries, severe infection, SIRS or sepsis, septic shock, cardiogenic shock, cancer, acute kidney injury, CNS disorders, autoimmune diseases and vascular diseases and hypotension.
  4. 4. Method, according to any one of claims 1 to 3, characterized in that the said disease is cardiogenic shock.
  5. 5. Method according to claim 3, characterized in that the serious infection is a microbial, viral or parasitic infection.
  6. 6. A method, according to any one of claims 1 to 5, characterized in that the amount of total DPP3 and/or the amount of active DPP3 is determined in a sample of bodily fluid from said individual and comprises the steps: • Contacting said sample with a capture ligand that specifically binds to complete DPP3, • Separating the DPP3 bound to said capture ligand, • Adding DPP3 substrate to said separated DPP3, • Quantifying said active DPP3 by measuring and quantifying the conversion of a DPP3 substrate.
  7. 7. Method according to claim 6, characterized in that said capture ligand is selected from the antibody group, antibody fragment or non-IgG structure.
  8. 8. Method according to claim 6 or 7, characterized in that said capture ligand is an antibody.
  9. 9. Method, according to any one of claims 6 to 8, characterized in that said trapping binder is immobilized on a surface.
  10. 10. Method, according to any one of claims 6 to 9, characterized in that said separation step is a washing step that removes ingredients from the sample that are not bound to said DPP3-capturing binder.
  11. 11. A method, according to any one of claims 6 to 10, characterized in that the conversion of the DPP3 substrate is detected by a method selected from the group comprising: fluorescence of fluorogenic substrates, color change of chromogenic substrates, luminescence of aminoluciferin-coupled substrates, mass spectrometry, HPLC/FPLC, thin-layer chromatography, capillary zone electrophoresis, gel electrophoresis followed by activity staining or western blot.
  12. 12. Method, according to any one of claims 6 to 11, characterized in that said substrate is selected from the group comprising: angiotensin II, III and IV, leu-enkephalin, met-enkephalin, endomorphin 1 and 2, valorfin, β-casomorphin, dynorphin, proctolin, ACTH and MSH, or dipeptide coupled to a fluorophore, a chromophore or aminoluciferin wherein the dipeptide is Arg-Arg.
  13. 13. Method, according to any one of claims 6 to 12, characterized in that said substrate is selected from the group comprising: a dipeptide coupled to a fluorophore, a chromophore or aminoluciferin wherein the dipeptide is Arg-Arg.
  14. 14. Method, according to any one of claims 6 to 13, characterized in that the method is conducted at least twice.
  15. 15. A method, according to any one of claims 1 to 5, characterized in that the amount of total DPP3 and/or the amount of active DPP3 is determined in a sample of bodily fluid from said individual and comprises the steps: • Contacting said sample with a first capture binder that specifically binds to full-length DPP3, • Contacting said sample with a second capture binder that specifically binds to full-length DPP3 and wherein said second capture binder is labeled, • Quantifying said DPP3 by measuring and quantifying the amount of labeled second capture binder.
  16. 16. Method according to claim 15, characterized in that said first capture ligand is linked to a solid phase.
  17. 17. Method according to claim 15 or 16, characterized in that said second capture binder is labeled with a dye, a chemiluminescent dye, a radioisotope or a reactive agent, or catalytically active moiety.
  18. 18. Method, according to any one of claims 15 to 17, characterized in that said method comprises the use of a liquid reaction mixture, wherein a first labeling component is linked to the first capture binder, wherein said first labeling component is part of a labeling system based on fluorescence quenching or amplification or chemiluminescence, and a second labeling component of said labeling system is linked to the second capture binder, and wherein after the binding of both capture binders to the full-length DPP3 a measurable signal is generated that allows the detection of the sandwich complexes formed in the solution comprising the sample.
  19. 19. Method, according to any one of claims 15 to 18, characterized in that said first and second capture ligands are selected from the antibody group, antibody fragments or non-IgG structures.
  20. 20. Method, according to any one of claims 15 to 19, characterized in that said first and second capture ligands are antibodies.

Description

[001] The present invention is directed to methods for determining active DPP3 in a sample of body fluid, an assay or kit for determining active DPP3 in a sample of body fluid, a method for diagnosing a disease or condition in an individual accompanied by or related to necrotic processes, and methods for treating said disease. State of the Art [002] Dipeptidyl peptidase 3 - also known as dipeptidyl aminopeptidase III, dipeptidyl arylamidase III, dipeptidyl peptidase III, enkephalinase B or red blood cell angiotensinase; short name: DPP3, DPPIII - is a metallopeptidase that removes dipeptides from physiologically active peptides, such as enkephalins and angiotensins. [003] DPP3 was first identified and its activity measured in bovine anterior pituitary extracts purified by Ellis & Nuenke 1967. The enzyme, which is classified as EC 3.4.14.4, has a molecular mass of approximately 83 kDa and is highly conserved in prokaryotes and eukaryotes (Prajapati & Chauhan 2011). The amino acid sequence of the human variant is presented in SEQ ID NO 1. Dipeptidyl peptidase III inhibitors are primarily cytosolic peptidase that is ubiquitously expressed. Despite the lack of a signal sequence, some studies have reported membrane activity (Lee & Snyder, 1982). [004] DPP3 is a zinc-dependent exo-peptidase belonging to the M49 peptidase family. It has a broad substrate specificity for oligopeptides of three/four to ten amino acids of various compositions and is also capable of cleaving after proline. DPP3 is known to hydrolyze N-terminal dipeptides of its substrates, including angiotensin II, III and IV; Leu- and Mct-enkephalin; endomorphin 1 and 2. The DPP3 metallopeptidase has its optimal activity at a pH of 8.09.0 and can be activated by the addition of divalent metal ions such as Co2+ and Mg2+. Structural analysis of DPP3 revealed the catalytic motifs HELLGH (hDPP3 450-455) and EECRAE (hDPP3 507-512), as well as the following amino acids, which are important for substrate binding and hydrolysis: Glu316, Tyr318, Asp366, Asn391, Asn394, His568, Arg572, Arg577, Lys666, and Arg669 (Prajapati & Chauhan 2011; Kumar et al. 2016; numbering refers to the human DPP3 sequence, see SEQ ID No. 1). Considering all known amino acids or sequence regions involved in substrate binding and hydrolysis, the active site of human DPP3 can be defined as the area between amino acids 316 and 669. [005] DPP3 activity can be inhibited by nonspecific inhibitors of proteases in general (e.g., PMSF, TPCK), sulfhydryl reagents (e.g., PHMB, DTNB) and metal chelators (EDTA, o-phenanthroline; Abramic et al., 2000). [006] DPP3 activity can be specifically inhibited by different types of compounds: an endogenous DPP3 inhibitor is the peptide spinorphine. Several synthetic spinorphine derivatives, for example, tynorphine, have been produced and shown to inhibit DPP3 activity to varying degrees (Yamamoto et al., 2000). Other published peptide inhibitors of DPP3 are propioxatin A and B (US4804676) and propioxatin A analogs (Inaoka et al., 1988). [007] DPP3 can also be inhibited by small molecules such as fluostatins and benzimidazole derivatives. Fluostatins A and B are antibiotics produced in Streptomyces sp. TA-3391 that are non-toxic and strongly inhibit DPP3 activity. To date, 20 different benzimidazole derivatives have been synthesized and published (Agic et al. 2007; Rastija et al. 2015), of which the two compounds 1’ and 4’ show the strongest inhibitory effect (Agic et al. 2007). For a complete list of DPP3 inhibitors see Table 2. [008] The exact biological function of DPP3 in cellular physiology is not understood, but recent findings indicate its role not only in protein metabolism, but also in blood pressure regulation, pain modulation, and inflammatory processes (Prajapati & Chauhan 2011). [009] DPP3 has been shown to be a promising biomarker in several publications, all of which refer to intracellular DPP3. DPP3 activity has been shown to be elevated in homogenates of ovarian and endometrial tumors. DPP3 activity further increases with the severity/malignancy of these tumors (Simaga et al. 1998 and 2003). Immune histology and Western blot analysis of glioblastoma cell lines have also revealed elevated DPP3 levels (Singh et al. 2014). [0010] Intracellular or membranous DPP3 has also been proposed to be a potential arteriorisk marker (US2011008805) and a marker for rheumatoid arthritis (US2006177886). Patent application WO2005106486 claims: expression and activity of DPP3 as a diagnostic marker and DPP3 as a therapeutic target in all types of diseases, due to the ubiquitous expression of DPP3 in or on the cell surface. EP 1498480 mentions the possibility of diagnostic and therapeutic use of hydrolytic enzymes, including DPP3. [0011] Not only has DPP3 been proposed as a potential biomarker, but also as a potential therapeutic target due to its ability to cleave several bioactive peptides. Overexpression of DPP3 protects neuroblastoma cells from oxidative s