New diagnostic markers for pancreatic cancer

Abstract

The present invention provides a compound characterized by the formula (1): X1-Thr-Thr-Ala-Arg-X2, wherein cleavage of the compound into Fragment 1 containing X1 and Fragment 2 containing X2 results in a detectable signal. The present invention also provides an in vitro method for detecting protease activity in a subject's body fluid, comprising contacting the body fluid with a compound of the present invention and detecting a signal, wherein the body fluid may contain hydrolases derived from pancreatic cancer cells. The present invention also provides a kit comprising a compound of the present invention and an assay buffer. Additionally, the present invention provides the use of a compound of the present invention, an in vitro method, or a kit for detecting pancreatic cancer or for monitoring a subject suspected of having, at high risk of developing, or previously having pancreatic cancer. The present invention also provides the use of a compound of the present invention in a method for treating pancreatic cancer, comprising performing an in vitro method for detecting protease activity in a subject's body fluid and treating pancreatic cancer in the subject in whom protease activity is detected.

Description

[Technical field]

[0001] The present invention relates to compounds for use in the diagnosis of pancreatic cancer. In particular, the present invention relates to chromogenic peptides suitable for the detection of proteolytic enzymes in a sample. [Background technology]

[0002] Pancreatic cancer is usually diagnosed at a late stage and therefore often has a very poor prognosis. Due to the dynamics of this cancer, 5-year survival is rare. Approximately 250,000 cases of pancreatic cancer are diagnosed annually worldwide, 3500 cases in Poland. Unfortunately, the majority of cases are fatal (more than 80%). There is an unmet need for reliable, rapid and uncomplicated diagnostic methods for the detection of pancreatic cancer. Early diagnosis increases the chances of surgical treatment and may significantly prolong the patient's survival.

[0003] The process of initiation, growth and dissemination of cancer cells involves many factors, including several proteolytic enzymes. This group of proteins is capable of hydrolyzing proteins and peptides into smaller fragments. Proteolytic enzymes mediate the degradation of the extracellular matrix, thus allowing cancer cells to colonize new tissues, allowing the formation of new blood vessels (angiogenesis) and facilitating the efficient delivery of nutrients to the tumor. In addition, proteolytic enzymes are present as a result of the death of normal cells due to the tumor growth process. All these processes form a proteolytic enzyme profile characteristic of tumors.

[0004] Chromogenic peptide molecules have been previously described that degrade under the influence of proteolytic enzymes, causing a change or increase in color of the test solution, the coloring effect being the result of the release of a chromophore (e.g., 4-nitroanilide or 5-amino-2-nitrobenzoic acid).

[0005] Peptide derivatives of this type are known from the following publications: 1.Erlanger BF, Kokowsky N, Cohen W. The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys. November 1961;95:271-8. 2.Hojo K, Maeda M, Iguchi S, Smith T, Okamoto H, Kawasaki K. Amino acids and peptides. XXXV. Facile preparation of p-nitroanilide analogs by the solid-phase method. Chem Pharm Bull (Tokyo). November 2000;48(11):1740-4.

[0006] Methods for detecting and measuring the expression of proteases have been described previously. The disclosed methods are based on isolated antibodies that specifically bind to a particular polypeptide, and PCR of a polynucleotide encoding the particular polypeptide. Demonstration of protease activity is measured by hydrolysis of a suitable synthetic peptide substrate conjugated with a chromogenic molecule, where the protease cleaves the synthetic substrate, and the absorbance of the chromogen released during hydrolysis of the substrate is measured. (CA2425829).

[0007] Preferred compounds according to the invention have been developed such that when the compounds are contacted with urine samples from individuals with pancreatic cancer, an increase in signal, for example color in the range of 380-440 nm, can be detected, an effect that does not occur when the compounds are contacted with urine samples from healthy individuals or patients diagnosed with another type of cancer.

[0008] The inventors have discovered that such signals, e.g., chromogenic or fluorescent signals released after separation of the fluorescent donor / acceptor pair, can be used to detect the presence of proteolytic enzymes in the urine of subjects with pancreatic cancer. Enzymatic hydrolysis of the compound results in the generation of a detectable difference, e.g., the release of a free chromophore molecule (ANB-NH2-amide of 5-amino-2-nitrobenzoic acid or pNA-para-nitroaniline, respectively) that exhibits absorbance at 320-480 nm.

[0009] The compounds of the present invention provide, inter alia, (i) a rapid and non-invasive diagnostic method for the detection of pancreatic cancer, (ii) a method suitable for the early detection of pancreatic cancer, (iii) a method that can be well used for screening of pancreatic cancer, and (iv) a complete diagnostic process at an early stage of cancer development in order to introduce more effective treatments. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] CA2425 No. 829 [Non-patent literature]

[0011] [Non-Patent Document 1] Arch Biochem Biophys. November 1961;95:271-8 [Non-Patent Document 2] Chem Pharm Bull (Tokyo).2000 November;48(11):1740-4 Summary of the Invention

[0012] In a first aspect, the present invention relates to a compound characterized by formula 1: X1-Thr-Thr-Ala-Arg-X2 (formula 1), In this regard, cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 results in a detectable signal. The compound comprises the tetrapeptide Thr-Thr-Ala-Arg according to SEQ ID NO:1.

[0013] In a second aspect, the present invention relates to an in vitro method for detecting protease activity in a body fluid of a subject, the method comprising contacting the body fluid with a compound according to the first aspect of the invention and detecting a signal, the body fluid containing hydrolytic enzymes, in particular proteases, derived from pancreatic cancer cells.

[0014] In a third aspect, the present invention relates to a kit comprising a compound according to the first aspect of the invention and an assay buffer. In a fourth aspect, the present invention relates to the use of a compound according to the first aspect of the invention, a method according to the second aspect of the invention, or a kit according to the third aspect of the invention for the detection of pancreatic cancer or for monitoring a subject suspected of having, at high risk of developing, or who has had pancreatic cancer.

[0015] In a fifth aspect, the present invention relates to the use of a compound according to the first aspect of the invention in a method for treating pancreatic cancer, the method comprising the steps of: (a) carrying out a method according to the second aspect of the invention; and (b) treating pancreatic cancer in a subject in which protease activity, in particular increased protease activity, has been detected in step (a). [Brief description of the drawings]

[0016] [Figure 1]Figure 1 shows the hydrolysis rate of the substrate (1) ABZ-Thr-Thr-Ala-Arg-ANB-NH2 in urine samples from patients diagnosed with cancer (samples 1-10) and in urine from healthy individuals (11-25). Arabic numbers indicate the number of the selected urine sample. Figure 1 shows that although samples 1-10 all disintegrated, the degradation of the ABZ-Thr-Thr-Ala-Arg-ANB-NH2 substrate occurred more efficiently in samples 3 and 10 than in materials 2 or 9. This result may be due to differences in the activity and amount of enzymes involved in proteolysis. Furthermore, Figure 1 shows that incubation of a solution of the compound of formula 2 with a urine sample from a healthy individual (not diagnosed with cancer) did not result in an increase in absorbance, and therefore no hydrolysis of the test compound occurred. This result indicates the absence of proteolytic enzymes characteristic of pancreatic cancer. [Diagram 2] Figure 2 shows the hydrolysis rate of the substrate (2) ABZ-Thr-Thr-Ala-Arg-pNA in urine samples from patients diagnosed with cancer (samples 1-10) and in urine from healthy individuals (11-25). Arabic numbers indicate the numbers of the selected urine samples. Similar results as those described above for Figure 1 are obtained. [Diagram 3] FIG. 3 shows the degree of hydrolysis of the substrate (1) ABZ-Thr-Thr-Ala-Arg-ANB NH2 versus the pH environment. The tested pH values ​​are shown on the x-axis. The proteolytic activity versus the pH of the reaction environment was evaluated. As a result of this experiment, the test material was found to have at least one enzyme that exhibits maximum activity at alkaline pH. [Figure 4]Figure 4 shows reversed-phase HPLC analysis of randomly selected systems containing urine from individuals diagnosed with pancreatic cancer. Compounds (1) (ABZ-Thr-Thr-Ala-Arg-ANB-NH2, shown) and (2) (ABZ-Thr-Thr-Ala-Arg-pNA, not shown) both degrade into ABZ-Thr-Thr-Ala-Arg peptide fragments and a chromophore (ANB-NH2 or pNA, respectively). A: ABZ-Thr-Thr-Ala-Arg-ANB NH2 substrate in 200 mM Tris-HCl buffer, pH 8.0. B: Hydrolysis of ABZ-Thr-Thr-Ala-Arg-ANB NH2 substrate in urine from patients diagnosed with pancreatic cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein, as they may vary. It should also be understood that the terms used herein are only for the purpose of describing certain embodiments, and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, HGW, Nagel, B. and Kolbl, H. (eds.) (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0018] Throughout the text of this specification, several documents are cited. Each document cited herein, whether supra or infra, including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc., is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0019] To practice the present invention, unless otherwise specified, conventional chemical, biochemical, and recombinant DNA techniques are used, as described in the literature in the field (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, edited by J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0020] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0021] The elements of the present invention are described below. Although these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed to limit the invention to only the embodiments explicitly described. The description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered to be disclosed by the description of this application unless the context dictates otherwise.

[0022] Terms and abbreviations used in the patent specification and claims should be understood as follows: In the context of the present invention, a "detectable signal" refers to any signal such as that produced by any label, such as a magnetic label, a fluorescent moiety, an enzyme, a chemiluminescent probe, a metal particle, a non-metallic colloidal particle, a polymeric dye particle, a pigment molecule, a pigment particle, an electrochemically active species, a semiconductor nanocrystal, or other nanoparticles, including quantum dots or gold particles. The label can be, for example, a chromophore, a fluorophore, a quantum dot, or a radioactive label.

[0023] In the context of the present invention, the expression "chromophore" is used to refer to a compound that has "chromogenic properties". The expression "chromogenic properties" refers to the ability of a compound to form a colored product.

[0024] In the context of the present invention, the term "fluorophore" is used to refer to a chemical compound that has "fluorescence properties", which refers to the ability of a compound to form a fluorescent product.

[0025] The fluorescent dyes of the present invention include the following classes of dyes: xanthenes (e.g., fluorescein), acridines (e.g., acridine yellow), oxazines (e.g., oxazine 1), cynins (e.g., Cy7 / Cy3), styryl dyes (e.g., Alexa Fluor 350), porphines (e.g., chlorophyll B), metal-ligand complexes (e.g., PtOEPK), fluorescent proteins (e.g., APC, R-phycoerythrin), nanocrystals (e.g., QuantumDot 705), perylenes (e.g., Lumogen Red F300), and phthalocyanines (e.g., IRDYE™ 700DX), as well as conjugates and combinations of these classes of dyes.

[0026] Quantum dots are semiconductors composed of atoms from groups II-VI or III-V of the periodic table (e.g., CdSe, CdTe, InP). Other alternatives include any two-dye system.

[0027] In the context of the present invention, NMP refers to N-methylpyrrolidone. In the context of the present invention, DMF refers to dimethylformamide. In the context of the present invention, DCM refers to methylene chloride.

[0028] In the context of the present invention, pNA refers to 4-nitroaniline, which is also sometimes called para-nitroaniline. In the context of the present invention, ABZ refers to 2-aminobenzoic acid.

[0029] In the context of the present invention, ANB-NH2 refers to the amide of 5-amino-2-nitrobenzoic acid. In the context of the present invention, AFC refers to 7-amido-4-trifluoromethylcoumarin.

[0030] In the context of this invention, Boc refers to a tert-butyloxycarbonyl group. In the context of the present invention, Fmoc refers to the 9-fluorenylmethoxycarbonyl group. In the context of the present invention, TFA refers to trifluoroacetic acid.

[0031] In the context of the present invention, the term "pancreatic cancer" is used in the broadest sense and refers to all cancers that originate in the pancreas. It includes exocrine cancer, endocrine cancer, pancreatoblastoma, pancreatic sarcoma, and lymphoma subtypes. Exocrine cancer includes adenocarcinoma, especially ductal adenocarcinoma, as well as cystic tumors and acinar cell carcinoma. Endocrine cancer includes gastrinoma, insulinoma, somatostatinoma, vipoma, and glucagonoma. Also included are the following stages (defined by the corresponding TNM classification (1 or more) in brackets): stage 0 (Tis, N0, M0), stage IA (T1, N0, M0), stage IB (T2, N0, M0), stage IIA (T3, N0, M0), stage IIB (T1-3, N1, M0), stage III (T4, any N, M0), and stage IV (any T, any N, M1).

[0032] The peptides according to the invention, preferably chromogenic or fluorogenic peptides, can be obtained by carrying out peptide synthesis on a solid support, as known in the art. The solid support can be in the form of a resin carrying an Fmoc group, which is removed during the course of the reaction. The resin used to carry out this process should be appropriately prepared. The preparation of this resin involves increasing its volume by repeated washing with a hydrophobic solvent.

[0033] The Fmoc protecting group must be removed from the resin by washing with a 20% solvent solution. The known process for obtaining chromogenic peptides involves the attachment of the individual components under suitable time and stoichiometric conditions. The attachment process consists of the subsequent steps of attaching the individual elements (amino acid derivatives), washing off the residues, removing the protecting groups and washing again. This cycle is repeated for each amino acid residue. The resulting peptide is separated from the resin by reaction under acidic conditions. The peptide is then precipitated from the resulting solution with a non-polar solvent after separating the solution from the resin by filtration. The peptide precipitate is then centrifuged.

[0034] Known methods for obtaining chromogenic peptides are based on processes carried out on solid supports and partly in buffers, using amide resins as solid supports and mixtures of hydrophobic solvents as solutions.

[0035] Compounds according to the invention were prepared using the methods described in Hojo et al., Chem Pharm Bull (Tokyo), 2000. Detailed descriptions of the synthesis can be found in the Examples section below.

[0036] Furthermore, the peptides according to the invention may be linked to quantum dots, which are highly luminescent unless quenched by the presence of metal nanoparticles. Viologens (e.g., methyl viologen or propyl viologen-sulfonate (PVS)) can also be used as quenchers.

[0037] When the protease cleaves the consensus sequence, the quantum dot is released and emits light. In a first aspect, the present invention provides a compound characterized by formula 1: X1-Thr-Thr-Ala-Arg-X2 (Formula 1), wherein cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 results in a detectable signal.

[0038] In a preferred embodiment, the sequence Thr-Thr-Ala-Arg is accessible to a hydrolase, in particular a hydrolase which cleaves the compound into X1-Thr-Thr-Ala-Arg-OH (fragment 1) and NH2-X2 (fragment 2).

[0039] The hydrolytic enzyme can be a protease or a combination of several proteases. The detectable signal generated by cleavage of the compound can be selected from a variety of suitable signals known to those of skill in the art, hi a preferred embodiment, the detectable signal is an optical signal.

[0040] In a preferred embodiment, X1 comprises or consists of component C1, X2 comprises or consists of component C2, and the detectable signal is provided by spatial separation of C1 and C2, i.e., by hydrolytic cleavage of the peptide Thr-Thr-Ala-Arg.

[0041] In addition to C1, X1 may, for example, include one or more amino acids on either side of C1. In addition to C2, X2 may, for example, include one or more amino acids on either side of C2. Thus, prior to cleavage, C1 and C2 may be separated by an amino acid sequence of 4-20 amino acids or 5-10 amino acids including or consisting of Thr-Thr-Ala-Arg. In a preferred embodiment, C1 and C2 are separated by 10 or fewer amino acids.

[0042] In a preferred embodiment, the detectable signal is a change in absorbance or fluorescence. The change may be an increase or a decrease. In a preferred embodiment, the detectable signal is an increase in absorbance intensity at 300 to 500 nm, particularly at 380 to 430 nm.

[0043] In a preferred embodiment, one of C1 and C2 is a chromophore having an absorption maximum 1 (AM1) at wavelength 1, and the compound has an absorption maximum 2 (AM2) at wavelength 2 that is different from wavelength 1. Thus, when the absorption is measured at wavelength 2 before and after cleavage of the compound, an increase in absorption is detected.

[0044] It is especially preferred that upon cleavage the chromophore is released in free form. Upon cleavage, fragment 1 consists of X1-Thr-Thr-Ala-Arg and fragment 2 consists only of NH2-X2. Thus, in a preferred embodiment, the chromophore is C2 and not C1. When C2 is the chromophore, it is preferred that X2 consists or essentially consists of C2. Alternatively, X1 may contain additional amino acids on either side of C1, and C1 and C2 may be separated by a longer amino acid sequence than Thr-Thr-Ala-Arg prior to cleavage.

[0045] In a preferred embodiment, the chromophore is selected from para-nitroaniline (pNA), amide of 5-amino-2-nitrobenzoic acid (ANB-NH2), 7-amido-4-trifluoromethylcoumarin (AFC) and 3-nitro L-tyrosine (Tyr3-NO2). In a preferred embodiment, the chromophore is para-nitroaniline (pNA). In a preferred embodiment, the chromophore is amide of 5-amino-2-nitrobenzoic acid (ANB-NH2).

[0046] In a preferred embodiment, C1 and C2 are a pair of fluorescent donor and fluorescent acceptor. When C1 and C2 are a pair of fluorescent donor and acceptor, and the compound is used in a method for measuring the change in fluorescence due to the cleavage of the compound, C1 and C2 are preferably separated by 10 or less amino acids to ensure efficient quenching of the fluorescent donor by the fluorescent acceptor. Those skilled in the art know that the distance between the fluorescent donor and the fluorescent acceptor is an important parameter. Therefore, a longer spacer between C1 and C2 may be possible if the amino acid sequence separating C1 and C2 is folded into a condensed or twisted secondary structure, resulting in closer proximity of C1 and C2 than in the case of a linear linker.

[0047] In a preferred embodiment, the pair C1 and C2 is selected from the group consisting of 2-aminobenzoic acid (ABZ) / pNA, ABZ / ANB-NH2, ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr(3-NO2), in particular the pair C1 and C2 is selected from ABZ / pNA and ABZ / ANB-NH2. C1 and C2 are preferably characterized by a molecular weight of less than 500 g / mol, in particular less than 400 g / mol, more particularly less than 300 g / mol, even more particularly between 100 and 200 g / mol.

[0048] The pair of C1 and C2 may also be a protein fluorescent donor and acceptor pair selected from the group consisting of BFP / GFP, BFP / CFP, BFP / YFP, BFP / DsRed, CFP / GFP, CFP / YFP, CFP / mVenus, CeFP / YFP, CeFP / mVenus, CeFP / mCitrine, CFP / dsRed, CFP / mCherry, mTurquoise / mVenus, GFP / YFP, GFP / DsRed, GFP / RFP, Clover / mRuby, Cy3 / C5, Alexa 488 / Alexa 555, and FITC / TRITC. A person skilled in the art knows how to select an appropriate protein fluorescent donor and acceptor pair based on their emission and absorbance spectra.

[0049] If C1 and C2 are a protein fluorescent donor and acceptor pair, it must be ensured that the relatively large size of the protein does not hinder the cleavage of the compound and that the sequence Thr-Thr-Ala-Arg is accessible for cleavage by hydrolases. This can be achieved, for example, by increasing the length of the amino acid sequence to include the peptide according to SEQ ID NO:1.

[0050] In a preferred embodiment, the compound is characterized by formula 2: ABZ-Thr-Thr-Ala-Arg-ANB-NH2 (Formula 2). In a preferred embodiment, the compound is characterized by formula 3: ABZ-Thr-Thr-Ala-Arg-pNA (Formula 3).

[0051] Cleavage of the compounds characterized by formula 2 or 3 results in the release of a free chromophore molecule (ANB-NH2 or pNA, respectively). Thus, an increase in absorbance intensity can be detected at 380-430 nm. In addition to this, cleavage results in the spatial separation of the fluorescent donor (ABZ) from the fluorescent acceptor (ANB-NH2 or pNA, respectively). Thus, the fluorescence emitted from ABZ is no longer quenched and an increase in fluorescence intensity can be detected at 420 nm.

[0052] In a second aspect, the present invention provides an in vitro method for detecting protease activity in a body fluid of a subject, the method comprising contacting the body fluid with a compound according to the first aspect of the invention and detecting a signal, the body fluid containing hydrolytic enzymes, particularly proteases, derived from pancreatic cancer cells.

[0053] In a preferred embodiment, the presence of protease activity in the body fluid indicates the presence of pancreatic cancer, and the absence of protease activity in the body fluid indicates the absence of pancreatic cancer. In a preferred embodiment, the present invention provides a method for diagnosing pancreatic cancer.

[0054] In a preferred embodiment, the body fluid is selected from blood or urine.In a preferred embodiment, the body fluid is urine.Unexpectedly, the inventors have found that the compound according to the first aspect of the present invention can detect hydrolase activity in urine samples.Compared to healthy subjects, subjects diagnosed with pancreatic cancer have significantly elevated hydrolase activity (Figures 1, 2).

[0055] In a preferred embodiment, the subject is at high risk of developing pancreatic cancer, is suspected of having, has had, or has pancreatic cancer. In a preferred embodiment, the compound is provided in an assay buffer having a neutral or alkaline pH, preferably pH 6.8-8.5, more preferably physiological pH, at a concentration of 0.1-10 mg / mL, in particular 0.25-7.5 mg / mL, more particularly 0.5-5 mg / mL, more particularly 0.75-2 mg / mL, even more particularly about 1 mg / mL, and the body fluid sample is added to the compound in a ratio of 1:2-1:10, in particular 1:3-1:8, more particularly 1:4-1:6, even more particularly about 1:5.

[0056] In the context of this specification, the expression "neutral pH" refers to a pH of about 7.0. In the context of this specification, the expression "physiological pH" refers to a pH of about 7.4. In a preferred embodiment, detecting the signal comprises measuring absorbance or fluorescence.

[0057] In a preferred embodiment, detecting the signal involves measuring absorbance intensity at 300 to 500 nm, particularly 380 to 430 nm, at 25 to 40° C., particularly 36 to 38° C., preferably for 40 to 60 minutes.

[0058] In a preferred embodiment, an increase in absorbance indicates the presence of hydrolase activity. In a third aspect, the present invention provides a kit comprising a compound according to the first aspect of the invention and an assay buffer.

[0059] In a fourth aspect, the present invention provides the use of a compound according to the first aspect, a method according to the second aspect, or a kit according to the third aspect for the detection of pancreatic cancer or for monitoring a subject at high risk of developing, suspected of having, or having had pancreatic cancer.

[0060] Depending on the intended use of the method of the second aspect, the term "subject" may have different limitations.For example, when the method is used to detect pancreatic cancer or to screen a subject for pancreatic cancer, the subject is not known to have pancreatic cancer, i.e., may or may not have pancreatic cancer.In this example, the subject is preferably at high risk of developing pancreatic cancer, suspected to have pancreatic cancer, or has had pancreatic cancer (i.e., has been cured of detectable pancreatic cancer)."High risk" means that one or more risk factors for cancer in general or pancreatic cancer can be attributed to the subject, preferably as defined by the American Cancer Society for cancer in general or pancreatic cancer. Examples of risk factors for pancreatic cancer are: tobacco consumption (especially smoking), heavy alcohol consumption, obesity, type 2 diabetes, occupation (workplace exposure to certain chemicals used in the dry cleaning and metal industries), chronic pancreatitis, age 50 years or older (especially 65 years or older), male sex, ethnicity (especially African-American and Caribbean men of African ancestry), family history of pancreatic cancer (especially first-degree relatives), and genetic syndromes or predispositions (hereditary breast and ovarian cancer syndromes caused by mutations in the BRCA1 or BRCA2 genes; P hereditary breast cancer, caused by mutations in the ALB2 gene; familial atypical multiple nevus melanoma (FAMMM) syndrome, caused by mutations in the p16 / CDKN2A gene and associated with cutaneous and ocular melanoma; familial pancreatitis, usually caused by mutations in the PRSS1 gene; Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer (HNPCC), most often caused by defects in the MLH1 or MSH2 genes; or Peutz-Jeghers syndrome, caused by defects in the STK11 gene.

[0061] The pancreatic cancer may be of any of the subtypes and stages defined above, i.e., the presence or absence of any subtype and / or stage may be detected. In a preferred embodiment, the presence of a significant amount of protease activity, or an amount greater than the control, indicates the presence of pancreatic cancer, and the absence of a significant amount of protease activity, or an amount equal to or less than the control, indicates the absence of pancreatic cancer.

[0062] In certain embodiments, the method of the second aspect further comprises confirming the detection of pancreatic cancer by using one or more additional means for detecting pancreatic cancer. The additional means can be a cancer marker (or "biomarker") or a conventional (non-marker) detection means. The cancer marker can be, for example, a DNA methylation marker, a mutation marker (e.g., SNP), an antigen marker, a protein marker, an miRNA marker, a cancer-specific metabolite, or an expression marker. The conventional means can be, for example, a biopsy (such as a visual biopsy test with or without a staining method for a protein or expression marker), an imaging technique, or a physical, e.g., tactile test. In a preferred embodiment, the additional means for pancreatic cancer detection is selected from the group consisting of physical examination (swelling of the liver or gallbladder, jaundice, i.e., yellowing of the skin and whites of the eyes), CT scan, MRI, cholangiopancreatography (especially endoscopic retrograde cholangiopancreatography, magnetic resonance cholangiopancreatography or percutaneous transhepatic cholangiography), angiography (especially x-ray angiography, CT angiography or MR angiography), abdominal ultrasound, endoscopic ultrasound, PET scan, blood tests (especially for bilirubin, CA 19-9 or CEA) and biopsy.

[0063] The term "indicative for" or "indicate" as used herein refers to the act of identifying or designating something that is indicated. As will be understood by those skilled in the art, such assessment is preferably accurate, but may not usually be accurate for 100% of subjects. However, the term requires that an accurate indication can be made for a statistically significant portion of subjects. Those skilled in the art can easily determine whether a portion is statistically significant using several well-known statistical evaluation tools, such as determining confidence intervals, determining p-values, Student's t-test, Mann-Whitney test, etc. Details are provided in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-value is preferably 0.05, 0.01, or 0.005.

[0064] The phrase "detection method of presence or absence" used herein in relation to pancreatic cancer refers to the determination of whether a subject has cancer or not.As will be understood by those skilled in the art, such assessment is preferably accurate, but may not usually be accurate for 100% of subjects.However, this term requires that accurate indications can be made for a statistically significant portion of subjects.See above for explanation of statistical significance and appropriate confidence intervals and p-values.

[0065] The term "diagnosis" as used herein refers to the determination of whether a subject has cancer. Diagnosis by analysis of protease activity as described herein may be supplemented with additional means as described herein to confirm the cancer detected by analysis of protease activity. As will be understood by those skilled in the art, diagnosis is preferably accurate, but may not usually be accurate for 100% of subjects. However, this term requires that an accurate diagnosis can be made for a statistically significant portion of subjects. For an explanation of statistical significance and appropriate confidence intervals and p-values, see above.

[0066] The phrase "screening a population of subjects" as used herein with respect to cancer herein refers to using the method of the first aspect with samples of a population of subjects. Preferably, the subjects are at high risk of cancer, suspected of having cancer, or have had cancer. In particular, one or more risk factors listed herein may be attributed to the subjects of the population. In certain embodiments, the same one or more risk factors may be attributed to all subjects of the population. For example, the population may consist of subjects characterized by heavy alcohol and / or tobacco consumption. It should be understood that the term "screening" refers to a diagnosis as described above for the subjects of the population, preferably confirmed using further means as described herein. As will be appreciated by those skilled in the art, screening is preferably accurate, but may not usually be accurate for 100% of the subjects. However, the term requires that accurate screening results can be obtained for a statistically significant portion of the subjects. See above for an explanation of statistical significance and appropriate confidence intervals and p-values.

[0067] The term "monitoring" as used herein refers to the accompaniment of diagnosed cancer during a treatment procedure or during a certain period of time, typically at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, or any other period of time. The term "accompaniment" means that the state of cancer, in particular the change in these states of cancer, can be detected based on the amount of protease activity, in particular based on the change in the amount in any type of periodic time segment determined daily or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times per month (no more than one determination per day), over the course of treatment, which can be up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 24 months. The amount or change in amount can also be determined before and / or after a treatment-specific event, such as each treatment cycle or application of a drug / treatment. A cycle is the time from one round of treatment to the start of the next round. Cancer treatment is usually a course of treatment, not a single treatment. A course usually takes 3-6 months, but can be longer or shorter. During a course of treatment, usually 4-8 cycles of treatment are performed. Usually, a treatment cycle includes a break in treatment to allow the body to recover. As will be understood by those skilled in the art, the results of monitoring are preferably accurate, but usually may not be accurate for 100% of subjects. However, the term requires that accurate monitoring results can be obtained for a statistically significant portion of subjects. See above for an explanation of statistical significance and appropriate confidence intervals and p-values.

[0068] In a fifth aspect, the present invention provides the use of a compound according to the first aspect of the invention in a method of treating pancreatic cancer comprising carrying out a method according to the second aspect of the invention and treating pancreatic cancer in a subject in which protease activity, particularly increased protease activity, has been detected.

[0069] In another embodiment, the present invention provides a method of treating pancreatic cancer comprising carrying out a method according to the second aspect of the invention and treating pancreatic cancer in a subject in which protease activity, in particular increased protease activity, has been detected.

[0070] As used herein, the term "treatment" or "treating" in relation to cancer refers to therapeutic treatment, the goal being to reduce the progression of cancer. Beneficial or desirable clinical outcomes include, but are not limited to, release of symptoms, shortening of disease duration, stabilization of pathological condition (particularly not worsening), slowing of disease progression, improvement and / or remission (both partial and total) of pathological condition, and are preferably detectable. Successful treatment does not necessarily mean cure, but may mean prolongation of survival compared to expected survival in the absence of treatment. In a preferred embodiment, the treatment is a primary treatment, i.e., the cancer has not been previously treated. Cancer treatment includes treatment regimens.

[0071] The term "treatment regimen" as used herein refers to a method of treating a subject taking into account the disease and available procedures and medications. Non-limiting examples of cancer treatment regimens are chemotherapy, surgery and / or irradiation or a combination thereof. Early detection of cancer made possible by the present invention allows, inter alia, surgical treatment, especially curative resection. In particular, the term "treatment regimen" refers to administering one or more anti-cancer agents or treatments as defined below. The term "anti-cancer agents or treatments" as used herein refers to surgery including chemical, physical or biological agents or treatments, or combinations thereof, that have anti-proliferative, anti-tumor and / or anti-tumor properties.

[0072] The chemical anticancer agent or treatment can be selected from the group consisting of alkylating agents, antimetabolites, plant alkaloids and terpenoids, and topoisomerase inhibitors.Preferably, the alkylating agent is a platinum-based compound.In one embodiment, the platinum-based compound is selected from the group consisting of cisplatin, oxaliplatin, eptaplatin, lobaplatin, nedaplatin, carboplatin, iproplatin, tetraplatin, lobaplatin, DCP, PLD-147, JM1 18, JM216, JM335, and satraplatin.

[0073] The physical anti-cancer agent or treatment may be selected from the group consisting of radiation therapy (e.g., curative radiation therapy, adjuvant radiation therapy, palliative radiation therapy, teletherapy, brachytherapy or metabolic radiation therapy), phototherapy (e.g., using hematoporphorin or photofrin II), and hyperthermia.

[0074] The surgery can be curative, palliative, prophylactic or cytoreductive.Typically, it includes resection, such as intracapsular, marginal, wide or radical resection, as described by Baron and Valin (Rec. Med. Vet, Special Canc. 1990; 11(166):999-1007).

[0075] Biological anti-cancer agents or treatments include antibodies (e.g., antibodies that stimulate an immune response to destroy cancer cells, e.g., retuximab or alemtuzumab; antibodies that stimulate an immune response by binding to a receptor on an immune cell and inhibiting a signal that prevents the immune cell from attacking "own" cells, e.g., ipilimumab; antibodies that interfere with the action of a protein necessary for tumor growth, e.g., bevacizumab, cetuximab or panitumumab; or antibodies conjugated to a drug, preferably a cell-killing substance such as a toxin, a chemotherapeutic molecule or a radioactive molecule, e.g., Y-ibritumomab tiuxetan, I-tositumomab or ado-trastuzumab). emtansine), cytokines (e.g., interferons or interleukins, e.g., INF-alpha and IL-2), vaccines (e.g., vaccines comprising a cancer associated antigen, e.g., sipuleucel-T), oncolytic viruses (e.g., naturally occurring oncolytic viruses such as reovirus, Newcastle disease virus, or mumps virus, or genetically engineered viruses such as measles virus, adenovirus, vaccinia virus, or herpes virus that preferentially target cells bearing a cancer associated antigen), gene therapy agents (e.g., DNA or RNA that replaces an altered tumor suppressor, blocks expression of an oncogene, improves the subject's immune system, makes cancer cells more susceptible to chemotherapy, radiation therapy, or other treatments, induces cell suicide, or provides an anti-angiogenic effect), and adoptive T cells (e.g., tumor-infiltrating T cells taken from a subject selected for anti-tumor activity, or T cells taken from a subject that have been genetically modified to recognize a cancer associated antigen).

[0076] In one embodiment, the one or more anticancer agents are selected from the group consisting of abiraterone acetate, ABVD, ABVE, ABVE-PC, AC, AC-T, ADE, ado-trastuzumab emtasine, afatinib dimaleate, aldesleukin, alemtuzumab, aminolevulinic acid, anastrozole, aprepitant, arsenic trioxide, asparaginase erwinia chrysanthemum, axitinib, azacitidine, BEACOPP, belinostat, bendamustine hydrochloride, BEP, bevacizumab, bexarotene, bicalutamide, bleomycin, bortezomib, bosutinib, brentuximab Vedotin, Busulfan, Cabazitaxel, Cabozantinib-S-Malate, CAF Capecitabine, CAPOX, Carboplatin, Carboplatin-Taxol, Carfilzomib, Carmustine, Carmustine Implant, Ceritinib, Cetuximab, Chlorambucil, Chlorambucil-Prednisone, CHOP, Cisplatin, Clofarabine, CMF, COPP, COPP-ABV, Crizotinib, CVP, Cyclophosphamide, Cytarabine, Cytarabine, Liposome, Dabrafenib, Dacarbazine, Dactinomycin, Dasatinib, Daunorubicin Hydrochloride, Decitabine, Degarelix, Denileukin Diftitox, denosumab, dexrazoxane hydrochloride, docetaxel, doxorubicin hydrochloride, doxorubicin hydrochloride liposome, eltrombopag olamine, enzalutamide, epirubicin hydrochloride, EPOCH, eribulin mesylate, erlotinib hydrochloride, etoposide phosphate, everolimus, exemestane, FEC, filgrastim, fludarabine phosphate, fluorouracil, FU-LV, fulvestrant, gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, glucarpidase, goserelin acetate, HPV bivalent vaccine, recombinant HPV quadrivalent vaccine, Hyper-CVAD, ibritumomabTiuxetan, Ibrutinib, ICE, Idelalisib, Ifosfamide, Imatinib, Mesylate, Imiquimod, Iodine-131 Tositumomab and Tositumomab, Ipilimumab, Irinotecan Hydrochloride, Ixabepilone, Lapatinib Ditosylate, Lenalidomide, Letrozole, Leucovorin Calcium, Leuprolide Acetate, Liposomal Cytarabine, Lomustine, Mechlorethamine Hydrochloride, Megestrol Acetate, Mercaptopurine, Mesna, Methotrexate, Mitomycin C, Mitoxantrone Hydrochloride, MOPP, Nelarabine, Nilotinib, Obinutuzumab, Ofatumumab, Omacetaxine Mepesuxinate, OPA, OFF, OPPA, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized nanoparticle formulation, PAD, Palifermin, Palonosetron hydrochloride, Pamidronate disodium, Panitumumab, Pazopanib hydrochloride, Pegaspargase, Peginterferon alfa-2b, Pembrolizumab, Pemetrexed disodium, Pertuzumab, Plerixafor, Pomalidomide, Ponatinib hydrochloride, Pralatrexate, Prednisone, Procarbazine hydrochloride, Radium-223 dichloride, Raloxifene hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant HPV bivalent vaccine, Recombinant HPV quadrivalent vaccine, Recombinant interferon alfa-2b, Regorafenib, Rituximab, Romidepsin, Romiplostim, Ruxolitinib phosphate, Siltuximab, Sipuleucel-T, Sorafenib tosilate, STANFORD V, sunitib malate, TAC, talc, tamoxifen citrate, temozolomide, temsirolimus, thalidomide, topotecan hydrochloride, toremifene, tositumomab and I 131 iodine tositumomab, TPF, trametinib, trastuzumab, vandetanib, VAMP, VeIP, vemurafenib, vinblastine sulfate, vincristine sulfate, vincristine sulfate liposomal, vinorelbine tartrate, vismodegib, vorinostat, XELOX, Ziv-aflibercept and zoledronic acid, or salts thereof.

[0077] In another aspect, the present invention provides a method of producing a compound according to the first aspect of the invention. In a preferred embodiment, the formula ABZ 1 -Thr 2 -Thr 3 -Ala 4 -Arg 5 -ANB-NH2 6 Compounds characterized by the formula: ABZ is 2-aminobenzoic acid and ANB-NH2 is an amide of 5-amino-2-nitrobenzoic acid are produced according to a process preferably carried out on a solid support bearing an Fmoc group. Before starting the process, the solid support is prepared: its volume is increased by repeated washing with a hydrophobic solvent, preferably dimethylformamide, methylene chloride or N-methylpyrrolidone, and the Fmoc protecting group is removed by washing with a 10-30% solution of piperidine in a solvent such as dimethylformamide, methylene chloride or N-methylpyrrolidone. The process is then carried out in the following steps: a) Prior to depositing 5-amino-2-nitrobenzoic acid ANB onto the resin, the solid support is washed with a 3-6% solution of N-methylmorpholine (NMM) in DMF, followed by DMF. A solution of ANB in ​​DMF is then prepared, to which TBTU, DMAP, and finally diisopropylethylamine (DIPEA) are added in the following excess relative to the polymer deposit: ANB / TBTU / DMAP / DIPEA, 3:3:2:6. The resulting mixture is added to the resin and mixed until homogeneous, after which the resin is vacuum filtered and washed with solvents such as DMF, DCM and isopropanol. The binding of ANB to the resin is then continued using an excess of hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HATU) followed by hexafluorophosphate-O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HBTU), and once complete, the solid support is washed successively with DMF, DCM and isopropanol and gently dried. b) The attachment of amino acid residues to ANB is carried out by reaction of Fmoc-Arg(Pbf)-OH with amino acid derivatives, where at least a 5-fold molar excess of the amino acid derivative relative to the resin is dissolved in anhydrous pyridine and contacted with the resin loaded with ANB. The whole is then cooled to a temperature not lower than 20°C, and POCl3 is added in a ratio of 1:1 relative to the amount of amino acid derivative used and mixed. The mixing process is carried out at room temperature and then at elevated temperature; after the reaction is complete, the resin is filtered under reduced pressure, washed with DMF and MeOH and gently dried; the intermediate compound obtained is subjected to the acylation process.

[0078] c) Acylation of the resulting intermediate compound is carried out with an amino acid derivative, preferably Fmoc-Ala-OH, then Fmoc-Thr(tBu)-OH, followed by Fmoc-Thr(tBu)-OH and finally Boz-Abz-OH. The acylation is carried out in steps of residues 6 to 1 using diisopropylcarbodiimide as coupling agent, the coupling agent being used in excess. At the end of each step, the resin is washed with DMF and (preferably) subjected to a chloranil test to monitor the attachment of the amino acid derivative.

[0079] d) Removal of the Fmoc protecting group is carried out by washing with a 10-30% solution of piperidine in DMF, followed by washing with each of the following solvents: DMF, isopropanol and methylene chloride.

[0080] e) Cleavage of the peptide from the resin is carried out using a mixture of TFA, phenol, water and TIPS maintaining a v / v / v / v ratio of 88:5:5:2 respectively; the mixture is stirred for at least 1 hour, preferably 3 hours, the resulting precipitate is vacuum filtered, washed with diethyl ether and the resulting peptide is centrifuged.

[0081] f) The final product is prepared by dissolving the peptide in water by means of ultrasound and lyophilizing. In a preferred embodiment, ABZ 1 -Thr 2 -Thr 3 -Ala 4 -Arg5 -pNA 6 The compound characterized by the formula: ABZ is 2-aminobenzoic acid and pNA is paranitroanilide is produced according to a process preferably carried out on a solid support bearing an Fmoc group. Before starting the process, the solid support is prepared: its volume is increased by repeatedly washing with a hydrophobic solvent, preferably dimethylformamide, methylene chloride or N-methylpyrrolidone, and the Fmoc protecting group is removed by washing with a 10-30% solution of piperidine in a solvent such as dimethylformamide, methylene chloride or N-methylpyrrolidone; then the process in the subsequent steps is carried out: a) The attachment of the third amino acid residue (Fmoc-Ala) to the resin is carried out using a 9-fold molar excess of the appropriate amino acid derivative over the resin deposit dissolved in anhydrous methylene chloride. The mixture is then stirred at room temperature for at least 2 hours; after completion of the reaction, the resin is vacuum filtered, washed with DMF and MeOH, and then dried.

[0082] b) In the next step, the attachment of the amino acid residue, hereafter called acylation, is carried out using the derivative Fmoc-Thr(tBu)-OH, followed by Fmoc-Thr(tBu)-OH and Boc-ABZ-OH; before each step, the resin is washed with DMF, preferably for 5 minutes; for the subsequent attachment, a coupling agent, preferably diisopropylcarbodiimide, is used, which is used in excess. This procedure is repeated twice, after each step, the resin is washed with DMF and preferably subjected to the chloranil test, which monitors the attachment of the amino acid derivative.

[0083] c) Removal of the Fmoc protecting group is carried out by washing with a 10-30% solution of piperidine in DMF, followed by washing with each of the following solvents: DMF, isopropanol and methylene chloride.

[0084] d) Compound (ABZ 1 -Thr(tBu) 2 -Thr(tBu) 3 -Ala 4Steps b)-c) are repeated until -OH) is obtained; after carrying out the synthesis with residues 6-3, the obtained compound is detached from the solid support using a mixture of TFA:phenol:water:TIPS in the proportions 88:5:5:2, respectively, v / v / v / v, with stirring. After 2 hours, the contents of the flask are vacuum filtered and the precipitate is washed with diethyl ether. The precipitate is then preferably centrifuged for 20 minutes and dissolved in water by means of ultrasound, followed by freeze-drying.

[0085] e) Fmoc-Arg(Pbf)-pNA synthesis is carried out stepwise; in the first step, 2 mmol of Fmoc-Arg(Pbf) is dissolved in anhydrous tetrahydrofuran (THF) in the presence of 2 mmol of N-methylmorpholine (NMM) and the carboxyl group of the amino acid derivative is activated with 2 mmol of isobutyl chloride. After 10 minutes of activation, 3 mmol of p-nitroaniline is added and the reaction is carried out at a temperature of (preferably) -15°C for (preferably) 2 hours, followed by one day at room temperature. Once the reaction is complete, the solvent is evaporated and the dry residue is dissolved in ethyl acetate; then the resulting solution is washed successively with saturated aqueous NaCl solution, 10% citric acid, 5% sodium bicarbonate and dried over anhydrous sodium sulfate; ethyl acetate is distilled under reduced pressure and the dry residue is dried.

[0086] f) Protective peptide ABZ 1 -Thr(tBu) 2 -Thr(tBu) 3 -Ala 4 Combining -OH with paranitroanilide Arg(Pbf) is based on the following process: 1 -Thr(tBu) 2 -Thr(tBu) 3 -Ala 4 -OH is dissolved in a small amount of DCM and then activated with TFFH (tetramethylfluoroformamide) at a temperature of preferably 0° C. for preferably 30 minutes, followed by the addition of catalytic amounts of DMAP and Fmoc-Arg(Pbf) 5 -pNA 6The reaction is preferably carried out at room temperature for 24 hours, after which the solvent is evaporated and the resulting solution is poured into a mixture for removing side group protection: TFA:phenol:water:TIPS (88:5:5:2, v / v / v / v) and mixed for preferably 3 hours.

[0087] g) The final product is prepared by dissolving the peptide in water using ultrasound followed by lyophilization. EXAMPLES

[0088] The invention is illustrated by the following non-limiting examples. Example 1: Compound ABZ 1 -Thr 2 -Thr 3 -Ala 4 -Arg 5 -ANB-NH 2 6 Synthesis of 1. Preparation of Chromogenic Peptides a) The first step of the synthesis was to obtain a chromogenic peptide by solid phase synthesis on a solid support using Fmoc / tBu chemistry, i.e. using protection. The sequence ABZ 1 -Thr 2 -Thr 3 -Ala 4 -Arg 5 -ANB-NH2 6 Compounds having the formula: [wherein ABZ is 2-aminobenzoic acid, ANB-NH2 is the amide of 5-amino-2-benzoic acid, and ANB is 5-amino-2-benzoic acid] were obtained by a solid-phase chemical synthesis process using the following amino acid derivatives:

[0089] Boc-ABZ, Fmoc-Thr(tBu), Fmoc-Ala, Fmoc-Arg(Pbf), ANB The synthesis of the compound, i.e. the diagnostic marker for the detection of pancreatic cancer, where the diagnosis is linked to the hydrolysis of this compound under the influence of proteolytic enzymes, was carried out on a solid support that allows the conversion of 5-amino-2-benzoic acid to ANB-NH2 amide: for example an amide resin, for example TentaGel S RAM from RAPP Polymere (Germany), with a deposit of, for example, 0.23 mmol / g.

[0090] Other commercially available amide resins, such as Rink Amide (Germany), can also be used. The compounds were synthesized manually using a laboratory shaker. For most steps, 25 mL sintered syringes for solid-phase synthesis were used as reactors.

[0091] All the final compounds obtained contained an ABZ 2-aminobenzoic acid molecule at position 1 (i.e., N-terminus) of the sequence, and an ANB 5-amino-2-nitrobenzoic acid molecule at position 6 (C-terminus). ABZ acts as a fluorescent donor and ANB (5-amino-2-benzoic acid) acts as a fluorescent quencher and chromophore. The peptide contained at least (and preferably) one reactive site in its sequence, located at position 5 of the compound, between amino acid residues Arg-ANB-NH2. The synthesis involving the attachment of the amino acid derivative is carried out at residues 6 to 1, i.e., C-terminus to N-terminus.

[0092] b) Deposition of ANB on TentaGel S RAM resin: Peptide synthesis was carried out on TentaGel S RAM resin (Rapp Polymere) at a loading of 0.23 mmol / g. In a first step, the resin was prepared, including relaxation by washing cycles. Subsequently, the Fmoc amino group protection was removed from the solid support using a 20% solution of piperidine in NMP, followed by a solvent washing cycle. A chloranil test was performed to confirm the presence of free amino groups.

[0093] Solvent wash cycle: DMF 1×10min IsOH 1×10 min DCM 1×10 minutes Removal of Fmoc protection: DMF 1×5min 20% Piperidine in NMP 1×3 min 20% Piperidine in NMP 1×8 min Solvent wash cycle: DMF 3×2 min IsOH 3 x 2 min DCM 3×2 minutes

[0094] c) Chloranil test: The chloranil test consisted of transferring a few grains of resin from the reactor-syringe (using a spatula) into a glass ampoule and adding to it 100 μL of a saturated solution of p-chloranil in toluene and 50 μL of fresh acetaldehyde. After 10 min, a control of the color of the grains was performed.

[0095] At this step, after testing, a green particle color was obtained, indicating the presence of free amino groups. After confirming the removal of the 9-fluorenylmethoxycarbonyl protection from the resin, it was possible to proceed to the next step, the attachment of the ANB derivative (5-amino-2-nitrobenzoic acid).

[0096] d) Deposition of 5-amino-2-nitrobenzoic acid on a solid support The first step in the synthesis of the peptide library-mixture of peptides was the deposition of ANB on 1 g of resin. Prior to the attachment of the chromophore, the resin used in the reaction was washed with the following solvents: DMF, DCM and again with DMF, after which the Fmoc protection was removed from the functional groups of the solid support. One cycle of removing the Fmoc protection included the following steps: Removal of Fmoc protection: 20% Piperidine in NMP 1×3 min 20% Piperidine in NMP 1×8 min e) Cleaning DMF 3×2 min IsOH 3 x 2 min DCM 3×2 minutes f) Chloranil test for the presence of free amino groups.

[0097] The resin with free amino groups was washed with a 5% solution of N-methylmorpholine (NMM) in DMF followed by DMF. The procedure to remove the Fmoc protection and the washing cycles were carried out in a Merrifield vessel. In a separate flask, ANB was dissolved in DMF, then TBTU, DMAP and finally diisopropylethylamine (DIPEA) were added in the following excess relative to the polymer deposit: ANB / TBTU / DMA / DIPEA, 3:3:2:6. The mixture thus prepared was added to the resin and stirred for 3 hours. The resin was filtered under vacuum, washed with DMF, DCM and isopropanol and the whole acylation procedure was repeated twice. Subsequent reaction of ANB attachment to the resin was carried out using hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HATU) followed by hexafluorophosphate-O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HBTU). In the last step, the resin was washed successively with DMF, DCM and isopropanol and air-dried.

[0098] g) Attachment of the C-terminal amino acid residue to ANB The corresponding amino acid derivative (9-fold molar excess relative to the resin deposit) was dissolved in pyridine and transferred to the flask containing the ANB-deposited resin. The whole was cooled to -15°C (ice bath: 1 part by weight NH4Cl, 1 part by weight NaNO3, 1 part by weight ice). After reaching the desired temperature, POCl3 was added (in a 1:1 ratio relative to the amount of amino acid derivative used) and the whole was stirred on a magnetic stirrer at -15°C for 20 minutes, at room temperature for 30 minutes, and at 40°C for 6 hours (oil bath). After the reaction was complete, the resin was vacuum filtered, washed with DMF and MeOH, and left to dry.

[0099] In the next step, a residue (alanine) was attached at the P2 position. Before attachment of the amino acid residue, the resin was washed with DMF for 5 min each time. Diisopropylcarbodiimide was used as the coupling agent in the subsequent attachment. This procedure was repeated twice.

[0100] After each acylation, a resin washing cycle was initiated, followed by a chloranil test to monitor the attachment of amino acid derivatives to the free amino groups of the resin. Solvent wash cycle: DMF 3×2 min IsOH 3 x 2 min DCM 3×2 minutes Chloranil test: The results showed that after the first two couplings, the color of the particles was initially green and then gray, so another acylation had to be performed, which resulted in the particles of the resin tested in the chloranil test becoming colorless. This indicated the attachment of ANB to the TentaGel S RAM resin, which allowed the next step in peptide synthesis.

[0101] h) Attachment of additional protected amino acid residues: The resin, together with the attached ANB residues in the reaction vessel, was washed with DMF, after which the amino groups were deprotected with Fmoc and the protected amino acid alanine derivative was attached.

[0102] Removal of Fmoc protection: DMF 1×5min 20% Piperidine in NMP 1×3 min 20% Piperidine in NMP 1×8 min Solvent wash cycle: DMF 3×2 min IsOH 3 x 2 min DCM 3×2 minutes Chloranil test: The chloranil test gave a positive result, as evidenced by the green color of the resin particles, which allowed for moving on to the next step - attachment of the Fmoc-Thr(tBu)-OH amino acid residue.

[0103] Attachment of amino acid derivatives: Before the coupling process, the resin was washed in DMF. The composition of the coupling mixture remained unchanged when the protected glutamic acid residue was attached.

[0104] At the end of each acylation, a solvent washing cycle was performed according to a routine procedure, followed by a chloranil test for the presence of free amino groups in the solution. Solvent wash cycle: DMF 3×2 min IsOH 3 x 2 min DCM 3×2 minutes Chloranil test: After the second acylation, the resin particles under test were colorless, which made it possible to proceed to the next step of the synthesis, i.e., the introduction of other protected amino acid derivatives - threonine and 2-aminobenzoic acid molecules. The coupling process followed the procedure discussed previously.

[0105] Tests carried out after depositing the residue showed positive results: the resin particles were colorless. 2. Removal of Peptides from Solid Support After synthesis, the amide of the ABZ-Thr-Thr-Ala-Arg-ANB-NH2 peptide was removed from the solid support along with simultaneous removal of side group protection using the mixture: TFA:phenol:water:TIPS (88:5:5:2, v / v / v / v) in a round bottom flask on a magnetic stirrer.

[0106] After 3 hours, the contents of the flask are filtered under vacuum in a Schott funnel and washed with diethyl ether. The precipitate obtained is centrifuged for 20 minutes in a SIGMA 2K30 Laboratory Centrifuges. The precipitate obtained after centrifugation is dissolved in water by means of ultrasound and freeze-dried.

[0107] Identity / characteristics of new compounds - HPLC analysis, MS HPLC conditions: RP Bio Wide Pore Supelco C8 250 mm 4 mm column, phase system A: 0.1% TFA in water, B: 80% acetonitrile in A), flow rate 1 mL / min, UV detection at 226 nm. It was confirmed that the compound was obtained.

[0108] Example 2: Preparation of a compound having the formula: ABZ1-Thr2-Thr3-Ala4-Arg5-pNA6 The process is carried out similarly to that described in Example 1, except that the corresponding amino acid derivatives and additional substituents are used, and the process is carried out partly in solution and partly on a solid support.

[0109] Preparation of p-nitroanilide Ala a) The first step of the synthesis was to obtain a protected peptide by solid phase synthesis using Fmoc / tBu chemistry.

[0110] ABZ 1 -Thr(tBu) 2 -Thr(tBu) 3 -Ala 4 The -OH compound, where ABZ is 2-aminobenzoic acid, was obtained by chemical synthesis on solid phase using the following amino acid derivatives: Boc-ABZ, Fmoc-Thr(tBu), Fmoc-Ala.

[0111] The compounds were synthesized on the following solid supports: -2-Chloro-chlorotriyl resin, for example from Iris BIOTECH GMBH (Germany), deposition at 1.6 mmol Cl / g basis.

[0112] The compounds were synthesized manually using a laboratory shaker. A 25 mL sintered syringe for solid-phase synthesis was used as the reactor throughout all steps. Peptide synthesis was carried out on solid support: 2-chloro-chlorotrityl resin, e.g. from Iris BIOTECH GMBH (Germany), with a loading of 1.6 mmol Cl / g groups. In a first step, the resin was relaxed with a washing cycle. Subsequently, the Fmoc amino group protection was removed from the solid support with a 20% solution of piperidine in NMP. This was followed by a solvent washing cycle. To confirm the presence of free amino groups, a chloranil test was performed.

[0113] Solvent wash cycle: DMF 1×10min IsOH 1×10 min DCM 1×10 minutes Removal of Fmoc protection: DMF 1×5min 20% Piperidine in NMP 1×3 min 20% Piperidine in NMP 1×8 min Solvent wash cycle: DMF 3×2 min IsOH 3 x 2 min DCM 3×2 minutes b) Chloranil test: The chloranil test consisted of transferring a few grains of resin from the reactor-syringe into a glass ampoule (using a spatula) and adding 100 μL of a saturated solution of p-chloranil in toluene and 50 μL of fresh acetaldehyde. After 10 min, a control of the color of the grains was performed.

[0114] This step resulted in a green particle color after testing, indicating the presence of free amino groups. After confirming the removal of the 9-fluorenylmethoxycarbonyl protection from the resin, attachment of the Fmoc-Ala derivative was initiated.

[0115] c) Embedding of Fmoc-Ala on the solid support The first step in the synthesis of the peptide library was Fmoc-Ala deposition on 1 g of resin. Prior to attachment of the amino acid derivatives, the resin used in the reaction was washed with the following solvents: DMF (dimethylformamide), DCM (methylene chloride) and again with DMF, after which the Fmoc protection was removed from the functional groups of the solid support. One cycle of removing the Fmoc protection included the following steps: Removal of Fmoc protection: 20% Piperidine in NMP 1×3 min 20% Piperidine in NMP 1×8 min Washing DMF 3×2 min IsOH 3 x 2 min DCM 3×2 minutes Chloranil test for the presence of free amino groups.

[0116] The resin with free amino groups was washed with DMF. In a separate flask, Fmoc-Ala was dissolved in DMF. Then TBTU, DMAP, and finally diisopropylethylamine (DIPEA) were added in the following excess relative to the polymer deposit: Fmoc-Pro / TBTU / DMA / DIPEA, 3:3:2:6. The resulting mixture was added to the resin and stirred for 3 hours. The resin was vacuum filtered, washed with DMF, DCM, and isopropanol, and the entire acylation procedure was repeated twice. Subsequent reaction of Fmoc-Pro attachment to the resin was carried out with hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HATU) followed by hexafluorophosphate-O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HBTU). In the final step, the resin was washed successively with DMF, DCM and isopropanol and air-dried.

[0117] c. Attachment of additional protected amino acid residues: The resin, together with the attached Fmoc-Ala residues in the reaction vessel, was washed with DMF, followed by deprotection of the Fmoc from the amino group and attachment of the protected threonine derivative.

[0118] Removal of Fmoc protection: DMF 1×5min 20% Piperidine in NMP 1×3 min 20% Piperidine in NMP 1×8 min Solvent wash cycle: DMF 3×2 min IsOH 3 x 2 min DCM 3×2 minutes Chloranil test: The chloranil test gave a positive result, as evidenced by the green color of the resin particles, which allowed for moving on to the next step - attachment of the Fmoc-Thr(tBu)-OH amino acid residue.

[0119] Attachment of amino acid derivatives: Before the coupling process, the resin was washed in DMF. The composition of the coupling mixture remained unchanged when the protected glutamic acid residue was attached.

[0120] At the end of each acylation, a solvent washing cycle was performed according to a routine procedure, followed by a chloranil test for the presence of free amino groups in the solution. Solvent wash cycle: DMF 3×2 min IsOH 3 x 2 min DCM 3×2 minutes Chloranil test: After the second acylation, the resin particles under test were colorless, which made it possible to proceed to the next step of the synthesis, i.e., the introduction of other protected amino acid derivatives - threonine and 2-aminobenzoic acid molecules. The coupling process followed the procedure discussed previously.

[0121] Tests carried out after depositing the residue showed positive results: the resin particles were colorless. d) Removal of the peptide from the solid support while maintaining side group protection After completion of the synthesis, the protected ABZ-Thr(tBu)-Th(tBu)-Ala-OH peptide was removed from the solid support and side group protection was maintained using the mixture: acetic acid:TFE (trifluoroethanol):DCM (2:2:6, v / v / v) in a round bottom flask on a magnetic stirrer.

[0122] After 2 hours, the contents of the flask were filtered under reduced pressure through a Schott funnel and washed with the astringent mixture. The solution was washed with hexane (1:10, v / v), evaporated under reduced pressure and then freeze-dried.

[0123] e) Chemical synthesis of paranitroanilide Arg derivatives Fmoc-Arg(Pbf)-pNA was synthesized using the mixed anhydride method. In the first step, 2 mmol of Fmoc-Arg(Pbf) was dissolved in anhydrous tetrahydrofuran (THF) in the presence of 2 mmol of N-methylmorpholine (NMM). The carboxyl group of the amino acid derivative was activated with 2 mmol of isobutyl chloride. After 10 min of activation, 3 mmol of p-nitroaniline was added. The reaction was carried out at -15°C for 2 h, followed by room temperature for 24 h. After completion of the reaction, the solvent was evaporated and the dry residue was dissolved in ethyl acetate. The resulting solution was washed successively with saturated aqueous NaCl, 10% citric acid and 5% sodium bicarbonate. The resulting solution was dried over anhydrous sodium sulfate, the ethyl acetate was removed under reduced pressure and the dry residue was dried over P2O5 and KOH in a vacuum desiccator.

[0124] f) Coupling of protected peptide with paranitroanilide Arg(Pbf) protection ABZ 1 -Thr(tBu) 2 -Thr(tBu) 3 -Ala 4 The -OH peptide was dissolved in a small amount of DCM and then activated with TFAH (tetramethylfluoroformamide) for 30 min at 0°C. Then catalytic amounts of DMAP and Arg(Pbf)-pNA were added. The reaction was carried out at room temperature for 24 h, after which the solvent was evaporated. The resulting solution was poured into a mixture for removing side group protection: TFA:phenol:water:TIPS (88:5:5:2, v / v / v / v) and mixed in a round-bottom flask on a magnetic stirrer for 3 h.

[0125] After this time, cold diethyl ether was added to the flask and the precipitate obtained was centrifuged in a high speed centrifuge at 5000 rpm for 20 minutes. The precipitate obtained after centrifugation was dissolved in water by means of ultrasound and then freeze-dried.

[0126] Identification points / characteristics of new compounds - HPLC analysis, MS HPLC conditions: RP Bio Wide Pore Supelco C8 250 mm 4 mm column, Phase system A: 0.1% TFA in water, B: 80% acetonitrile in A), flow rate 1 mL / min, UV detection at 226 nm. It was confirmed that the compound was obtained.

[0127] Example 3 Tests on the application of the new compound were carried out on a group of 10 patients diagnosed with pancreatic cancer. For this, the compound with formula 2: ABZ-Thr-Thr-Ala-Arg-ANB-NH2 or formula 3: ABZ-Thr-Thr-Ala-Arg-pNA was dissolved in dimethyl sulfoxide (concentration of 0.5 mg / mL); 50 μL of this solution was mixed with 120 μL of buffer (200 mM Tris-HCl, pH 8.0) and 80 μL of urine from a patient with pancreatic cancer. The measurements were carried out in a 96-well plate designed for measuring absorbance, and each sample was analyzed in triplicate at 37 °C. The measurement time was 60 min. During the measurements, the wavelength characteristic of the released chromophore (ANB-NH2 or pNA) was monitored at 405 nm (range 380-430 nm).

[0128] The results showed that all urine samples from patients diagnosed with pancreatic cancer showed an increase in the color of the solution over time. The increase in absorbance observed over time was different for each of the samples tested. The 15 samples from healthy subjects showed different results, as none of the 15 urine samples tested showed an increase in absorbance in the diagnostic range.

[0129] The analysis supports the use of the compounds according to this embodiment in the diagnosis of pancreatic cancer. The mechanism of action of the novel compounds is based on their enzymatic hydrolysis in situ, leading to the release of free chromophore molecules, ANB-NH2-amide of 5-amino-2-nitrobenzoic acid or pNA-para-nitroaniline, respectively, which show an absorbance between 320 and 480 nm, in particular between 380 and 430 nm.

[0130] [Table 1]

[0131] [Table 2]

[0132] [Table 3]

[0133] The present invention further relates to 1. Compound - a diagnostic marker of the following general formula: ABZ 1 -Thr 2 -Thr 3 -Ala 4 -Arg 5 -X 6 (Formula 1) [In formula: ABZ is 2-aminobenzoic acid, X is ANB-NH2 or pNA; In this regard: ANB-NH2 is the amide of 5-amino-2-benzoic acid, ANB is 5-amino-2-nitrobenzoic acid; and pNA is para-nitroaniline].

[0134] 2. Compounds - Diagnostic markers: ABZ 1 -Thr 2 -Thr 3 -Ala 4 -Arg 5 -ANB-NH2 6 , (Equation 2) [In formula: ABZ is 2-aminobenzoic acid, ANB-NH2 is the amide of 5-amino-2-nitrobenzoic acid. The method for obtaining is based on the fact that the process is carried out on a solid support, preferably carrying an Fmoc group. Before starting the process, the solid support is prepared: its volume is increased by repeatedly washing with a hydrophobic solvent, preferably dimethylformamide, methylene chloride or N-methylpyrrolidone, and the Fmoc protecting group is removed by washing with a 10-30% solution of piperidine, preferably in a solvent such as dimethylformamide, methylene chloride or N-methylpyrrolidone. The process is then carried out in the following steps: a) Prior to depositing 5-amino-2-nitrobenzoic acid ANB onto the resin, the solid support is washed with a 3-6% solution of N-methylmorpholine (NMM) in DMF, followed by DMF. A solution of ANB in ​​DMF is then prepared, to which TBTU, DMAP, and finally diisopropylethylamine (DIPEA) are added in the following excess relative to the polymer deposit: ANB / TBTU / DMAP / DIPEA, 3:3:2:6. The resulting mixture is added to the resin and mixed until homogeneous, after which the resin is vacuum filtered and washed with solvents such as DMF, DCM and isopropanol. The binding of ANB to the resin is then continued using an excess of hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HATU) followed by hexafluorophosphate-O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HBTU), and once complete, the solid support is washed successively with DMF, DCM and isopropanol and gently dried. b) The attachment of amino acid residues to ANB is carried out by reaction of Fmoc-Arg(Pbf)-OH with amino acid derivatives, where at least a 5-fold molar excess of the amino acid derivative relative to the resin is dissolved in anhydrous pyridine and contacted with the resin loaded with ANB. The whole is then cooled to a temperature not lower than 20°C, and POCl3 is added in a ratio of 1:1 relative to the amount of amino acid derivative used and mixed. The mixing process is carried out at room temperature and then at elevated temperature; after the reaction is complete, the resin is filtered under reduced pressure, washed with DMF and MeOH and gently dried; the intermediate compound obtained is subjected to the acylation process.

[0135] c) Acylation of the resulting intermediate compound is carried out with an amino acid derivative, preferably Fmoc-Ala-OH, then Fmoc-Thr(tBu)-OH, followed by Fmoc-Thr(tBu)-OH and finally Boz-Abz-OH. The acylation is carried out in steps of residues 6 to 1 using diisopropylcarbodiimide as coupling agent, the coupling agent being used in excess. At the end of each step, the resin is washed with DMF and (preferably) subjected to a chloranil test to monitor the attachment of the amino acid derivative.

[0136] d) Removal of the Fmoc protecting group is carried out by washing with a 10-30% solution of piperidine in DMF, followed by washing with each of the following solvents: DMF, isopropanol and methylene chloride.

[0137] e) Cleavage of the peptide from the resin is carried out using a mixture of TFA, phenol, water and TIPS maintaining a v / v / v / v ratio of 88:5:5:2 respectively; the mixture is stirred for at least 1 hour, preferably 3 hours, the resulting precipitate is vacuum filtered, washed with diethyl ether and the resulting peptide is centrifuged.

[0138] f) The final product is prepared by dissolving the peptide in water by means of ultrasound and lyophilizing.

[0139] 3. Compounds - Diagnostic markers: ABZ 1 -Thr 2 -Thr 3 -Ala 4 -Arg 5 -pNA 6 (Formula 3) [In formula: ABZ is 2-aminobenzoic acid, pNA is paranitroanilide] The method for obtaining is based on the fact that the process is carried out on a solid support, preferably carrying an Fmoc group. Before starting the process, the solid support is prepared: its volume is increased by repeated washing with a hydrophobic solvent, preferably dimethylformamide, methylene chloride or N-methylpyrrolidone, and the Fmoc protecting group is removed by washing with a 10-30% solution of piperidine, preferably in a solvent such as dimethylformamide, methylene chloride or N-methylpyrrolidone; then the process in the subsequent steps is carried out: a) The attachment of the third amino acid residue (Fmoc-Ala) to the resin is carried out using a 9-fold molar excess of the appropriate amino acid derivative over the resin deposit dissolved in anhydrous methylene chloride. The mixture is then stirred at room temperature for at least 2 hours; after completion of the reaction, the resin is vacuum filtered, washed with DMF and MeOH, and then dried.

[0140] b) In the next step, the attachment of the amino acid residue, hereafter called acylation, is carried out using the derivative Fmoc-Thr(tBu)-OH, followed by Fmoc-Thr(tBu)-OH and Boc-ABZ-OH; before each step, the resin is washed with DMF, preferably for 5 minutes; for the subsequent attachment, a coupling agent, preferably diisopropylcarbodiimide, is used, which is used in excess. This procedure is repeated twice, after each step, the resin is washed with DMF and preferably subjected to the chloranil test, which monitors the attachment of the amino acid derivative.

[0141] c) Removal of the Fmoc protecting group is carried out by washing with a 10-30% solution of piperidine in DMF, followed by washing with each of the following solvents: DMF, isopropanol and methylene chloride.

[0142] d) Compound (ABZ 1 -Thr(tBu) 2 -Thr(tBu) 3 -Ala 4Steps b)-c) are repeated until -OH) is obtained; after carrying out the synthesis with residues 6-3, the obtained compound is detached from the solid support using a mixture of TFA:phenol:water:TIPS in the proportions 88:5:5:2, respectively, v / v / v / v, with stirring. After 2 hours, the contents of the flask are vacuum filtered and the precipitate is washed with diethyl ether. The precipitate is then preferably centrifuged for 20 minutes and dissolved in water by means of ultrasound, followed by freeze-drying.

[0143] e) Fmoc-Arg(Pbf)-pNA synthesis is carried out stepwise; in the first step, 2 mmol of Fmoc-Arg(Pbf) is dissolved in anhydrous tetrahydrofuran (THF) in the presence of 2 mmol of N-methylmorpholine (NMM) and the carboxyl group of the amino acid derivative is activated with 2 mmol of isobutyl chloride. After 10 minutes of activation, 3 mmol of p-nitroaniline is added and the reaction is carried out at a temperature of (preferably) -15°C for (preferably) 2 hours, followed by one day at room temperature. Once the reaction is complete, the solvent is evaporated and the dry residue is dissolved in ethyl acetate; then the resulting solution is washed successively with saturated aqueous NaCl solution, 10% citric acid, 5% sodium bicarbonate and dried over anhydrous sodium sulfate; ethyl acetate is distilled under reduced pressure and the dry residue is dried.

[0144] f) Protective peptide ABZ 1 -Thr(tBu) 2 -Thr(tBu) 3 -Ala 4 Combining -OH with paranitroanilide Arg(Pbf) is based on the following process: 1 -Thr(tBu) 2 -Thr(tBu) 3 -Ala 4 -OH is dissolved in a small amount of DCM and then activated with TFFH (tetramethylfluoroformamide) at a temperature of preferably 0° C. for preferably 30 minutes, followed by the addition of catalytic amounts of DMAP and Fmoc-Arg(Pbf) 5 -pNA 6The reaction is preferably carried out at room temperature for 24 hours, after which the solvent is evaporated and the resulting solution is poured into a mixture for removing side group protection: TFA:phenol:water:TIPS (88:5:5:2, v / v / v / v) and mixed for preferably 3 hours.

[0145] g) The final product is prepared by dissolving the peptide in water using ultrasound followed by lyophilization.

[0146] 4. The method for diagnosing pancreatic cancer is based on the following process: a compound of general formula 1 in a concentration range of 0.1-10 mg / mL (preferably 1 mg / mL) is incubated with a small amount of human urine in a ratio range of 1:2-1:10 (preferably 1:5 urine sample to measurement buffer) in a measurement buffer of neutral or alkaline pH, preferably physiological pH, and the absorbance intensity in the range of 300-500 nm (preferably 380-430 nm) is measured over a period of 40-60 minutes at a temperature in the range of 25-40°C (preferably 36-38°C).

[0147] In addition, the present invention relates inter alia to the following: 1. A compound characterized by formula 1: X1-Thr-Thr-Ala-Arg-X2 (formula 1), In this regard, cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 results in a detectable signal.

[0148] 2. The compound according to item 1, wherein the sequence Thr-Thr-Ala-Arg is accessible to a hydrolase, in particular a hydrolase which cleaves the compound into X1-Thr-Thr-Ala-Arg-OH (fragment 1) and NH2-X2 (fragment 2).

[0149] 3. A compound according to item 1 or 2, wherein X1 comprises or consists of component C1 and X2 comprises or consists of component C2, and the detectable signal is provided by spatial separation of C1 and C2.

[0150] 4. A compound according to any of items 1 to 3, wherein one of C1 and C2, in particular C2, is a chromophore having an absorption maximum 1 (AM1) at wavelength 1, and the compound has an absorption maximum 2 (AM2) at wavelength 2 different from wavelength 1.

[0151] 5. The compound according to any of items 1 to 4, wherein C1 and C2 are a fluorescent donor and fluorescent acceptor pair. 6. The compound according to any of claims 1 to 5, wherein the pair C1 and C2 is selected from the group consisting of 2-aminobenzoic acid (ABZ) / pNA, ABZ / ANB-NH2, ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr(3-NO2), in particular the pair C1 and C2 is selected from ABZ / pNA and ABZ / ANB-NH2.

[0152] 7. An in vitro method for detecting protease activity in a body fluid of a subject, comprising contacting the body fluid with a compound according to any one of items 1 to 6 and detecting a signal, the body fluid may contain hydrolases, in particular proteases, derived from pancreatic cancer cells.

[0153] 8. An in vitro method according to item 7 for detecting the presence or absence of pancreatic cancer in a subject, wherein the presence of protease activity in the body fluid indicates the presence of pancreatic cancer, and the absence of protease activity in the body fluid indicates the absence of pancreatic cancer.

[0154] 9. An in vitro method according to item 7 or 8 for the diagnosis of pancreatic cancer. 10. An in vitro method according to any of paragraphs 7 to 9, wherein the body fluid is urine. 11. The method according to any of items 7 to 10, wherein the compound is provided in a measurement buffer having a neutral or alkaline pH, preferably a physiological pH, at a concentration of 0.1 to 10 mg / mL, in particular 0.25 to 7.5 mg / mL, more particularly 0.5 to 5 mg / mL, more particularly 0.75 to 2 mg / mL, even more particularly about 1 mg / mL, and the body fluid sample is added to the compound in a ratio of 1:2 to 1:10, in particular 1:3 to 1:8, more particularly 1:4 to 1:6, even more particularly about 1:5.

[0155] 12. The method according to any of items 7 to 11, wherein detecting the signal comprises measuring absorbance or fluorescence, in particular measuring absorbance intensity at 300 to 500 nm, more particularly at 380 to 430 nm, at 25 to 40°C, in particular at 36 to 38°C, preferably over a period of 40 to 60 minutes.

[0156] 13. A kit comprising a compound according to any one of items 1 to 6 and a measurement buffer. 14. Use of a compound according to any one of items 1 to 6, a method according to any one of items 7 to 12, or a kit according to item 13 for detecting pancreatic cancer or for monitoring a subject suspected of having, at high risk of developing, or having had pancreatic cancer.

[0157] 15. A compound according to any one of items 1 to 6 for use in a method for treating pancreatic cancer, the method comprising: a. carrying out a method according to any of items 7 to 12; and b. Treating pancreatic cancer in a subject in which protease activity, particularly increased protease activity, is detected in step a. Includes. This specification includes the disclosure of the following inventions: [1] A compound characterized by formula 1: X1-Thr-Thr-Ala-Arg-X2 (formula 1), In this regard: Cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 results in a detectable signal, X1 comprises or consists of component C1, X2 comprises or consists of component C2, and the detectable signal results from the spatial separation of C1 and C2. [2] The compound according to [1], wherein the sequence Thr-Thr-Ala-Arg is accessible to hydrolases, in particular hydrolases which cleave the compound into X1-Thr-Thr-Ala-Arg-OH (fragment 1) and NH2-X2 (fragment 2). [3] A compound according to either [1] or [2], wherein one of C1 and C2, particularly C2, is a chromophore having an absorption maximum 1 (AM1) at wavelength 1, and the compound has an absorption maximum 2 (AM2) at wavelength 2 different from wavelength 1. [4] The compound according to any one of [1] to [3], wherein C1 and C2 are a pair of a fluorescent donor and a fluorescent acceptor. [5] The compound according to any one of [1] to [4], wherein the pair of C1 and C2 is selected from the group consisting of 2-aminobenzoic acid (ABZ) / pNA, ABZ / ANB-NH2, ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, and ABZ / Tyr(3-NO2), in particular, the pair of C1 and C2 is selected from ABZ / pNA and ABZ / ANB-NH2. [6] An in vitro method for detecting protease activity in a body fluid of a subject, comprising contacting the body fluid with a compound and detecting a signal, the body fluid may contain hydrolases, particularly proteases, derived from pancreatic cancer cells; The compound is characterized by formula 1: X1-Thr-Thr-Ala-Arg-X2 (formula 1), In this regard, cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 results in a detectable signal. [7] An in vitro method for detecting the presence or absence of pancreatic cancer in a subject according to [6], wherein the presence of protease activity in the body fluid indicates the presence of pancreatic cancer, and the absence of protease activity in the body fluid indicates the absence of pancreatic cancer. [8] The in vitro method described in [6] or [7] for the diagnosis of pancreatic cancer. [9] The in vitro method described in any one of [6] to [8], wherein the body fluid is urine.

[10] The method according to any of [6] to [9], wherein the compound is provided in a measurement buffer having a neutral or alkaline pH, preferably a physiological pH, at a concentration of 0.1 to 10 mg / mL, in particular 0.25 to 7.5 mg / mL, more particularly 0.5 to 5 mg / mL, more particularly 0.75 to 2 mg / mL, even more particularly about 1 mg / mL, and the body fluid sample is added to the compound in a ratio of 1:2 to 1:10, in particular 1:3 to 1:8, more particularly 1:4 to 1:6, even more particularly about 1:5.

[11] The method according to any of [6] to

[10] , wherein detecting the signal comprises measuring absorbance or fluorescence, in particular measuring absorbance intensity at 300 to 500 nm, more specifically 380 to 430 nm, at 25 to 40°C, in particular 36 to 38°C, preferably for 40 to 60 minutes.

[12] A kit comprising the compound according to any one of [1] to [5] and a measurement buffer solution.

[13] For the detection of pancreatic cancer or for monitoring subjects suspected of having, at high risk of developing, or who have had pancreatic cancer; A compound characterized by formula 1: X1-Thr-Thr-Ala-Arg-X2 (formula 1), In this regard, cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 results in a detectable signal; [6] -

[11] ; or A kit comprising the compound and an assay buffer Use of.

[14] A method for treating pancreatic cancer, comprising: a. carrying out the method according to any one of [7] to

[12] ; and b. Treating pancreatic cancer in a subject in which protease activity, particularly increased protease activity, is detected in step a. The method comprising:

Claims

1. A compound characterized by formula 1: X1-Thr-Thr-Ala-Arg-X2 (Formula 1), [In the formula, Cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 results in a detectable signal; X1 consists of a moiety C1 and X2 consists of a moiety C2, the detectable signal being provided by hydrolytic cleavage of said compounds due to the spatial separation of C1 and C2, and C1 and C2 are fluorescent donor and fluorescent acceptor pairs selected from the group consisting of 2-aminobenzoic acid (ABZ) / pNA, ABZ / ANB-NH 2 , ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr(3-NO 2 )].

2. The compound of claim 1, wherein the sequence Thr-Thr-Ala-Arg is accessible to hydrolases.

3. The compound according to claim 2, wherein the hydrolase is a hydrolase that cleaves the compound into X1-Thr-Thr-Ala-Arg-OH (fragment 1) and NH 2 -X2 (fragment 2).

4. The compound according to any one of claims 1 to 3, wherein one of C1 and C2 is a chromophore having an absorption maximum 1 (AM1) at wavelength 1, and the compound has an absorption maximum 2 (AM2) at wavelength 2 different from wavelength 1.

5. The compound described in claim 4, wherein C2 is a chromophore having an absorption maximum 1 (AM1) at wavelength 1, and the compound has an absorption maximum 2 (AM2) at wavelength 2 different from wavelength 1.

6. The compound according to claim 1, wherein the pair of C1 and C2 is selected from ABZ / pNA and ABZ / ANB-NH 2 .

7. 1. An in vitro method for detecting protease activity in a bodily fluid of a subject, the method comprising contacting the bodily fluid with a compound and detecting a signal, the bodily fluid may contain hydrolytic enzymes derived from pancreatic cancer cells; The compound is characterized by formula 1: X1-Thr-Thr-Ala-Arg-X2 (Formula 1), [In the formula, Cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 results in a detectable signal; X1 consists of a moiety C1 and X2 consists of a moiety C2, the detectable signal being provided by hydrolytic cleavage of said compounds due to the spatial separation of C1 and C2, and C1 and C2 are fluorescent donor and fluorescent acceptor pairs selected from the group consisting of 2-aminobenzoic acid (ABZ) / pNA, ABZ / ANB-NH 2 , ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr(3-NO 2 )].

8. The in vitro method of claim 7, wherein the hydrolase is a protease.

9. 9. An in vitro method as described in claim 7 or 8 for detecting the presence or absence of pancreatic cancer in a subject, wherein the presence of protease activity in the body fluid indicates the presence of pancreatic cancer and the absence of protease activity in the body fluid indicates the absence of pancreatic cancer.

10. An in vitro method according to any one of claims 7 to 9 for diagnosing pancreatic cancer.

11. The in vitro method according to any one of claims 7 to 10, wherein the body fluid is urine.

12. 12. The method according to any one of claims 7 to 11, wherein the compound is provided in a concentration of 0.1 to 10 mg / mL in an assay buffer having a neutral or alkaline pH, and the body fluid sample is added to the compound in a ratio of compound:body fluid sample = 1:2 to 1:10 v / v.

13. The method of claim 12, wherein the compound is provided at a concentration of 0.25 to 7.5 mg / mL.

14. The method of claim 13, wherein the compound is provided at a concentration of 0.5 to 5 mg / mL.

15. The method of claim 14, wherein the compound is provided at a concentration of 0.75 to 2 mg / mL.

16. The method of claim 15, wherein the compound is provided at a concentration of 1 mg / mL.

17. The method of claim 12, wherein the measurement buffer has a physiological pH.

18. The method of any of claims 12 to 17, wherein the body fluid sample is added to the compound in a ratio of 1:3 to 1:8 v / v.

19. The method of claim 18, wherein the ratio is 1:4 to 1:6 v / v.

20. The method of claim 19, wherein the ratio is 1:5 v / v.

21. The method of any one of claims 7 to 20, wherein detecting the signal comprises measuring absorbance or fluorescence.

22. The method according to claim 21, wherein the absorbance measurement comprises measuring the absorbance intensity at 300 to 500 nm.

23. The method according to claim 22, wherein the measurement of absorbance is performed by measuring absorbance intensity at 380 to 430 nm.

24. The method of any of claims 21 to 23, wherein detecting the signal comprises measuring absorbance or fluorescence at 25 to 40°C for 40 to 60 minutes.

25. The method of claim 24, wherein detecting the signal comprises measuring absorbance or fluorescence at 36-38°C.

26. The method of claim 25, wherein detecting the signal comprises measuring absorbance at 36-38°C.

27. A method according to any of claims 12 to 26 for detecting pancreatic cancer or for monitoring a subject suspected of having, at high risk of developing, or having had pancreatic cancer.

28. A kit comprising the compound according to any one of claims 1 to 6 and an assay buffer.

29. For the detection of pancreatic cancer or for monitoring a subject suspected of having, at high risk of developing, or who has had pancreatic cancer; A compound characterized by formula 1: X1-Thr-Thr-Ala-Arg-X2 (Formula 1), [In the formula, Cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 results in a detectable signal; X1 consists of a moiety C1 and X2 consists of a moiety C2, the detectable signal being provided by hydrolytic cleavage of said compounds due to the spatial separation of C1 and C2, and C1 and C2 are a fluorescent donor and fluorescent acceptor pair selected from the group consisting of 2-aminobenzoic acid (ABZ) / pNA, ABZ / ANB-NH 2 , ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr(3-NO 2 ); or A kit comprising the compound and an assay buffer Use of.

30. 1. A method for identifying a subject in need of treatment for pancreatic cancer, comprising: a. carrying out a method according to any one of claims 12 to 27; and b. Selecting subjects in which protease activity is detected in step a. The method comprising:

31. A method for identifying a subject described in claim 30, wherein the protease activity in step b is increased protease activity.

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