Peptide probes for detection of pancreatic cancer, methods and kits

Abstract

The invention relates to new chemical compounds and combinations of chemical compounds, for medical use, more specifically for cancer diagnosis, in particular for the detection of activity specific for pancreatic cancer and in the diagnosis of pancreatic cancer. The invention relates also to an in vitro method for the detection of enzymatic activity present in an individual's body fluid sample, using such a compound or combination. The invention further relates to an in vitro method for the diagnosis of pancreatic cancer, using such a compound or combination, a kit comprising such a compound or combination, and the use of such a compound or combination for detecting enzymatic activity specific for pancreatic cancer and the use of such a compound or combination for the diagnosis of pancreatic cancer. The invention relates furthermore to such a compound or combination of compounds for use as a diagnostic marker for pancreatic cancer, and a method for the treatment of pancreatic cancer comprising the step of carrying out the method for the diagnosis of pancreatic cancer as defined above using such a compound or combination.

Description

[0001] PEPTIDE PROBES FOR DETECTION OF PANCREATIC CANCER, METHODS AND KITS

[0002] The invention relates to new chemical compounds, namely compounds comprising a tetrapeptide core and combinations of compounds having the tetrapeptide core, which can be used for medical applications, more specifically for cancer diagnosis, in particular for the diagnosis of pancreatic cancer. The invention relates also to an in vitro method for the detection of enzymatic activity present in an individual’s body fluid, in particular deriving from pancreatic cancer cells, using such compounds and combinations, an in vitro method for the diagnosis of pancreatic cancer using such compounds and combinations, kits comprising such compounds and combinations, use of such compounds and combinations for the detection of enzymatic activity specific for pancreatic cancer, the use of such compounds for the diagnosis of pancreatic cancer, such compounds and combinations for use as diagnostic markers for pancreatic cancer. The invention furthermore relates to a method for the treatment of pancreatic cancer comprising the step of carrying out the method for the diagnosis of pancreatic cancer as defined above.

[0003] Background of the invention

[0004] Pancreatic cancer is the twelfth most common cancer worldwide. At the same time, it is the sixth most common cause of cancer deaths. There are approximately half a million cases and a similar number of deaths from pancreatic cancer each year worldwide. The observed increase in incidence and mortality due to pancreatic cancer is partly related to the epidemic of obesity, diabetes and an increase in alcohol consumption, smoking being major risk factors for cancer. The incidence of new cases increases with age.

[0005] Pancreatic cancer is a highly malignant neoplasm characterised by rapid local growth and a high propensity for metastasis (e.g. to the peritoneum, lymph nodes, liver). Early pancreatic cancer is asymptomatic or uncharacteristic. The prognosis depends on the stage of the cancer at diagnosis and its differentiation. Surgical treatment is the most effective treatment for pancreatic cancer and the only one that offers a chance of cure. More than 40% of patients with operable, localised pancreatic cancer will survive five years after diagnosis, compared with 3% of patients with metastatic cancer. Diagnosis is based on imaging findings (ultrasonography, computer tomography, magnetic resonance imaging individually, endoscopic ultrasonography with biopsy). Cancer is usually diagnosed at a late stage and complete resection is possible in 15-20% of patients.

[0006] However, there are currently no commercial, non-invasive, sensitive and highly specific laboratory tests that can be used in the early diagnosis of pancreatic cancer. A number of diagnostic techniques using different diagnostic biomarkers were initially evaluated for use in the early diagnosis of pancreatic cancer. CAI 9-9, although routinely used to monitor the outcome of pancreatic cancer, is limited in the early diagnosis by its low specificity (falsepositive test results in benign pancreatic conditions) and lack of expression in 8-10% of the Caucasian population with a-b- genotype according to the Lewis group system.

[0007] GRAIL (Menlo Park, California, USA) has launched (in the Laboratory Developed Test tier) a test called Galleri™ based on technology for analysing extracellular DNA methylation levels. Its low sensitivity (72%), lack of recommendation for use and the high cost of the test are factors limiting its widespread use. A similar situation (high price, insufficient evidence of clinical efficacy - sensitivity 80%) applies to Immunovia's IMMray™ PanCan-d, a test that is an immunoproteomic microarray of dozens of protein biomarkers detected in patients' blood.

[0008] Diagnostic platforms for the detection of mucinous cysts and high grade cysts of the pancreas are known from the description of US20220381784A1. A system comprising an enzymespecific substrate for the detection or quantification of the serine protease TPP-1 is disclosed, with the resulting value corresponding to the determination of whether a mucin cyst in the pancreas is benign or malignant. A two-step diagnostic method is also disclosed, in which a sample from a pancreatic cyst is determined to be mucinous by detecting and / or quantifying aspartyl protease expression, and the cyst is then determined to be a high-grade dysplasia or malignant lesion by detecting and / or quantifying serine protease expression. Furthermore, a method for the diagnosis of a benign, malignant or pre-malignant lesion of the pancreas in an individual is described, as well as a method for the diagnosis of high-grade dysplasia or invasive carcinoma by detecting the presence, absence and / or quantity of at least one tripeptidyl peptidase I (TPP1) in a sample from an examined individual, such as a biopsy of the examined pancreatic lesion, possibly in combination with detection of cathepsin E, CEA and / or gastrin. This method therefore generally requires invasive sampling for testing and is therefore not suitable for screening tests.

[0009] Patients with chronic pancreatitis (CPP) are one of the main risk groups for pancreatic cancer. The risk of pancreatic cancer in patients with CPP is as much as 7.6-68.1 times higher than in patients without CPP. For patients with pancreatitis, the diagnosis of pancreatic cancer is difficult because clinical signs and radiological changes are difficult to distinguish. Both the misdiagnosis of pancreatic cancer and its 'overdiagnosis' (false positive results) cause serious complications for the patient. In 6-8% of cases of suspected pancreatic cancer, it turned out to be a benign lesion during surgery. This was associated with postoperative complications in more than 20% of cases. Failure to diagnose cancer in patients with pancreatitis is a reason for rapid progression of cancer leading to death.

[0010] The use of the Ca 19-9 marker is of limited use in differential diagnosis. Studies show that it is not possible to make a confident distinction between benign and malignant disease on the basis of an elevated CAI 9-9 measurement. In particular, hyperbilirubinaemia is associated with a further deterioration in the specificity of the Ca 19-9 test and caution should be exercised when interpreting results in patients with jaundice, where the specificity of the test drops to 50% Currently, there is no commercially available test that specifically detects pancreatic cancer when testing patients with pancreatitis.

[0011] The differential diagnosis between pancreatic cancer and chronic pancreatitis remains a major clinical problem, often requiring invasive techniques such as endoscopic ultrasound- guided fine-needle aspiration. Because of the differences in prognosis and the therapeutic implications of correct and early diagnosis, diagnostic tools capable of differentiating between pancreatitis and pancreatic cancer are necessary.

[0012] In this field, there are known chromogenic peptide compounds that undergo enzymatic breakdown into smaller fragments resulting in a change or increase in the colour of the solution being tested. This chromogenic effect is a consequence of the release of a chromophore (e.g. 4-nitroanilide or 2-aminobenzoic acid) from a chromogenic peptide molecule.

[0013] This type of chromogenic molecules and their uses are known, for example, from the publication by 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 and 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.

[0014] From the Patent Number PAT.241174 a chromogenic chemical compound of this kind is known, finding use as a single diagnostic marker for pancreatic cancer, as well as an in vitro method for the diagnosis of pancreatic cancer using such a compound. The compound shows a sensitivity and specificity of 100% when individuals diagnosed with pancreatic cancer are compared with healthy individuals. In the case of non-specific manifestations of pancreatic diseases, there is therefore a need to differentiate between the presence of pancreatic cancer and other pancreatic diseases, including inflammatory processes of this organ, in order to eliminate false positive results of a test to detect pancreatic cancer. In this case, however, the compound disclosed in the present patent does not differentiate between such conditions effectively enough.

[0015] As the symptoms of pancreatic cancer are non-specific and manifest only when the organ is massively occupied by this type of cancer, new diagnostic markers and new diagnostic tests should enable the rapid and non-invasive detection of pancreatic cancer, also at an early stage of its development, in a highly sensitive and specific manner, allowing pancreatic cancer to be distinguished from other conditions of the organ, such as pancreatitis, thus speeding up the current diagnostic pathway and expanding therapeutic options for the patient. With early diagnosis of pancreatic cancer, many more patients will be eligible for surgery or will be able to start other treatments increasing the chances of survival.

[0016] There is therefore a continuing need in the field for new, effective diagnostic means and non- invasive diagnostic methods, free of the drawbacks known from the state of the art, suitable for highly sensitive and specific detection and diagnosis of pancreatic cancer, especially at an early stage of its development, also allowing highly sensitive and specific differentiation of pancreatic cancer from other pancreatic diseases such as pancreatitis, also suitable for use in diagnostic screening tests.

[0017] The objective of the invention is therefore to provide new, effective, sensitive and specific diagnostic means and methods, devoid of the disadvantages known from the state of the art, and suitable for reliable, non-invasive, rapid, highly sensitive and specific detection and diagnosis of pancreatic cancer, especially at an early stage of its development, allowing, at the same time, an effective differentiation between pancreatic cancer and other diseases of the pancreas, such as pancreatitis, eliminating the risk of false-positive results which can occur with state-of-the-art compounds, suitable also for use in diagnostic screening tests. These objectives have been achieved through the inventions defined in the patent claims forming part of the present application and detailed in the present description.

[0018] Summary of the invention

[0019] The subject of the present invention is a compound having a following general formula:

[0020] XI -tetrapeptide-X2 wherein

[0021] XI comprises or consists of a molecule Cl, tetrapeptide is a peptide having a sequence of four amino acids selected from: Gly-Thr-Lys- Asn (SEQ ID NO: 1), Trp-Thr-Glu-Ala (SEQ ID NO: 2), Lys-Pro-Gln-Glu (SEQ ID NO: 3), Glu-Met-Phe-Phe (SEQ ID NO: 4), Gln-Trp-Phe-Ala (SEQ ID NO: 6) and Glu-Met-Lys- Phe (SEQ ID NO: 7),

[0022] X2 comprises or consists of a molecule C2, wherein a pair of molecules Cl and C2 is a pair of a fluorescence donor and a fluorescence acceptor, and wherein the compound undergoes enzymatic cleavage into fragments 1 and 2: XI- tetrapeptide-OH (fragment 1) and X2 (fragment 2), respectively, with a generation of a measurable optical signal upon spatial separation of molecules Cl and C2.

[0023] Preferably, such a compound according to the invention undergoes hydrolytic cleavage, preferably proteolytic cleavage.

[0024] Preferably, in such a compound according to the invention, the pair of molecules Cl and C2 is selected from the group consisting of: 2-aminobenzoic acid (ABZ) / 5-amino-2- nitrobenzoic acid (ANB), (ABZ) / pNA, ABZ / ANB-NH2, ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr(3-NO2), more preferably the pair of Cl and C2 is ABZ / pNA or ABZ / ANB-NH2.

[0025] Preferably, such a compound according to the invention is selected from the group consisting of the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NH2(formula 1), ABZ-Trp-Thr- Glu-Ala-ANB-NH2(formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NH2(formula 3), ABZ-Glu- Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Gln-Trp-Phe-Ala-ANB-NH2(formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NH2(formula?).

[0026] More preferably, the compound according to the invention undergoes hydrolytic cleavage with the generation of a fragment 1 selected from: ABZ-Gly-Thr-Lys-Asn-OH, ABZ-Trp- Thr-Glu-Ala-OH, ABZ-Lys-Pro-Gln-Glu-OH, ABZ-Glu-Met-Phe-Phe-OH, ABZ-Glu-Met- Phe-Phe-OH, ABZ-Gln-Trp-Phe-Ala-ANB-NH2and ABZ-Glu-Met-Lys-Phe-ANB-NH2and fragment 2: ANB-NH2.

[0027] The subject of the present invention is also a combination of compounds, which combination comprises three compounds of general formula:

[0028] XI -tetrapeptide-X2 wherein

[0029] XI comprises or consists of a Cl molecule, tetrapeptide means a peptide having a sequence of four amino acids selected from: Gly-Thr- Lys-Asn (SEQ ID NO: 1), Trp-Thr-Glu-Ala (SEQ ID NO: 2), Lys-Pro-Gln-Glu (SEQ ID NO: 3), Glu-Met-Phe-Phe (SEQ ID NO: 4), Thr-Thr-Ala-Arg (SEQ ID NO: 5), Gln-Trp- Phe-Ala (SEQ ID NO: 6) and Glu-Met-Lys-Phe (SEQ ID NO: 7), X2 comprises or consists of a molecule C2, wherein a pair of molecules Cl and C2 is a pair of a fluorescence donor and a fluorescence acceptor, and wherein the compound undergoes enzymatic cleavage into fragments 1 and 2: XI- tetrapeptide-OH (fragment 1) and X2 (fragment 2), respectively, with a generation of a measurable optical signal upon spatial separation of molecules Cl and C2.

[0030] Preferably, the compounds of such a combination according to the invention undergo hydrolytic, preferably proteolytic cleavage.

[0031] Preferably, in the compounds of such a combination according to the invention, the pair of molecules Cl and C2 is selected from the group consisting of: 2-aminobenzoic acid (ABZ) / 5-amino-2-nitrobenzoic acid (ANB), (ABZ) / pNA, ABZ / ANB-NEE, ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr(3-NO2), wherein more preferably the Cl and C2 pair is ABZ / pNA or ABZ / ANB-NEE.

[0032] The preferable combination according to the invention comprises 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NEE (formula 1), ABZ-Trp- Thr-Glu-Ala-ANB-NEE (formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NEE (formula 3), ABZ- Glu-Met-Phe-Phe-ANB-NEE (formula 4), ABZ-Thr-Thr-Ala-Arg-ANB-NEE (formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NH2(formula 6) and ABZ -Glu-Met-Lys-Phe- ANB -NH2(formula 7), wherein more preferably the combination according to the invention consists of 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NH2(formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NH2(formula 2), ABZ-Lys-Pro-Gln-Glu-ANB- NH2(formula 3), ABZ -Glu-Met-Phe-Phe- ANB -NH2(formula 4), ABZ-Thr-Thr-Thr-Ala- Arg-ANB-NH2(formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NH2(formula 6) and ABZ-Glu- Met-Lys-Phe-ANB-NH2(formula 7).

[0033] The subject of the present invention is also an in vitro method for the detection of enzymatic activity present in an individual’s body fluid sample, in particular activity deriving from pancreatic cancer cells, comprising: a) contacting the body fluid sample with the compound according to the invention as defined above or the combination according to the invention as defined above, and

[0034] (b) detecting of a measurable optical signal, which is generated upon spatial separation of molecules Cl and C2. Preferably, in the method for the detection according to the invention, the detected enzymatic activity is hydrolytic activity, preferably proteolytic activity.

[0035] Preferably, in the method for the detection according to the invention a combination according to the invention comprising 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NH2(formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NH2(formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB- NH2(formula 4), ABZ-Thr-Thr-Thr-Ala-Arg-ANB-NH2(formula 5), ABZ-Gln-Trp-Phe- Ala-ANB-NH2(formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NH2(formula 7), preferably the combination consisting of 3 of these compounds, is used.

[0036] Preferably, in the method for the detection according to the invention, as the said body fluid urine, preferably human urine, is used.

[0037] Preferably, in the method for the detection according to the invention, as the said measurement buffer a Tris buffer, or a buffer comprising Tris, NaCl and DMSO, is used. The subject of the present invention is also an in vitro method for the diagnosis of pancreatic cancer, wherein the presence or absence of pancreatic cancer in an individual is detected by measuring enzymatic activity specific for pancreatic cancer in a body fluid sample of an examined individual, with the use of the compound according to the invention or the combination according to the invention, the absence of the said enzymatic activity indicating the absence of pancreatic cancer and the presence of the said enzymatic activity indicating the presence of pancreatic cancer.

[0038] Preferably, in the method for the diagnosis according to the invention, the detection of enzymatic activity is carried out by means of the in vitro method for the detection of enzymatic activity present in an individual’s body fluid sample, in particular deriving from pancreatic cancer cells as defined above.

[0039] Preferably, in the method for the diagnosis according to the invention for the measurement of the said enzymatic activity the combination comprising 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NH2(formula 1), ABZ-Trp-Thr-Glu- Ala-ANB-NH2(formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NH2(formula 3), ABZ-Glu-Met- Phe-Phe-ANB-NH2(formula 4), ABZ-Thr-Thr-Ala-Arg-ANB-NH2(formula 5), ABZ-Gln- Trp-Phe-Ala-ANB-NH2(formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NH2(formula 7), more preferably the combination consisting of 3 of these compounds, is used.

[0040] Preferably, in the method for the diagnosis according to the invention, the said body fluid sample, preferably urine, more preferably human urine, is incubated with the said compound in a measurement buffer having neutral pH or alkaline pH, preferably physiological pH, within the range of sample-to-measurement buffer ratio of 1 :2 to 1 :10, preferably 1 :5.

[0041] Preferably, in the method for the diagnosis according to the invention, the concentration of the compound used is 0.1-10 mg / mL, in particular 0.25-7.5 mg / mL.

[0042] Preferably, in the method for the diagnosis according to the invention, the measurement of the said enzyme activity involves the measurement of the absorbance intensity in the range 300-500 nm, preferably 380-430 nm, in particular 410 nm, for a period of 40-60 minutes, at a temperature in the range 25-40° C, preferably 36-38° C.

[0043] The subject of the present invention is also a kit comprising at least one compound according to the invention as defined above or the combination according to the invention as defined above and a measurement buffer.

[0044] Preferably, the kit according to the invention comprises the combination according to the invention comprising 3 compounds selected from the following compounds: ABZ-Gly-Thr- Lys-Asn-ANB-NH2 (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NH2 (formula 2), ABZ-Lys- Pro-Gln-Glu-ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ- Thr-Thr-Ala-Arg-ANB-NH2 (formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NH2 (formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NH2 (formula 7), more preferably the combination according to the invention consisting of 3 of the said compounds.

[0045] The further subject of the present invention is use the compound according to the invention as defined above or the combination as defined above for detecting enzymatic activity specific for pancreatic cancer.

[0046] The subject of the present invention is also a compound according to the invention as defined above for use in a method for the diagnosis of pancreatic cancer.

[0047] Preferably, in such use or for such use, the compound according to the invention is the compound selected from: ABZ-Gly-Thr-Lys-Asn-ANB-NH2 (formula 1), ABZ-Trp-Thr- Glu-Ala-ANB-NH2 (formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NH2 (formula 3), ABZ-Glu- Met-Phe-Phe-ANB-NH2 (formula 4), ABZ-Gln-Trp-Phe-Ala-ANB-NH2 (formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NH2(formula 7).

[0048] The subject of the present invention is also the combination according to the invention as defined above for use in a method for the diagnosis of pancreatic cancer.

[0049] Preferably, such a combination for use according to the invention is the combination comprising 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn- ANB-NH2 (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NH2 (formula 2), ABZ-Lys-Pro-Gln- Glu-ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Thr-Thr- Ala-Arg-ANB-NH2 (formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NFB (formula 6) and ABZ- Glu-Met-Lys-Phe-ANB-NH2 (formula 7), more preferably the combination consisting of 3 of these compounds.

[0050] The subject of the present invention is furthermore use in an in vitro method for the diagnosis of pancreatic cancer of the compound according to the invention as defined above, preferably selected from the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NFB (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NH2 (formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NFB (formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Gln-Trp-Phe-Ala-ANB-NH2(formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NFG (formula 7).

[0051] The subject of the present invention is furthermore use in an in vitro method for the diagnosis of pancreatic cancer the combination according to the invention as defined above, preferably the combination comprising 3 compounds selected from the following compounds: ABZ- Gly-Thr-Lys-Asn-ANB-NH2 (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NFB (formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Thr-Thr-Ala-Arg-ANB-NFB (formula 5), ABZ-Gln-Trp-Phe-Ala-ANB- NH2 (formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NFT (formula 7), more preferably the combination consisting of 3 of these compounds.

[0052] Preferably in such uses and for use according to the invention, diagnosis of pancreatic cancer includes detection of primary pancreatic cancer, detection of residual disease after surgical resection of pancreatic cancer and / or detection of recurrence of pancreatic cancer.

[0053] More preferably in such uses and for use according to the invention, the combination comprises 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn- ANB-NH2 (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NFB (formula 2), ABZ-Lys-Pro-Gln- Glu-ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Thr-Thr- Ala-Arg-ANB-NH2 (formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NFB (formula 6) and ABZ- Glu-Met-Lys-Phe-ANB-NH2 (formula 7), more preferably the combination consists of 3 of these compounds.

[0054] The subject of the present invention is furthermore the compound according to the invention as defined above or the combination according to the invention as defined above for use as a diagnostic marker for the detection of pancreatic cancer.

[0055] Preferably, such a combination for use according to the invention as a diagnostic marker is the combination comprising 3 compounds selected from the following compounds: ABZ- Gly-Thr-Lys-Asn-ANB-NH2 (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NFB (formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2 (formula 4), ABZ-Thr-Thr-Ala-Arg-ANB-NFB (formula 5), ABZ-Gln-Trp-Phe-Ala-ANB- NH2 (formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NFB (formula 7), more preferably the combination consisting of 3 of these compounds.

[0056] The subject of the present invention is also a method for the treatment of pancreatic cancer, wherein

[0057] (a) the presence of enzymatic activity specific for pancreatic cancer is detected by means of the method for the detection according to the invention as defined above in a body fluid sample from an examined individual, preferably urine, preferably human urine, and

[0058] (b) when the said enzymatic activity is found in the said sample, treatment of the individual's pancreatic cancer is undertaken.

[0059] Preferably in such a method according to the invention, after completion of the treatment in accordance with point b), monitoring of the said enzymatic activity specific for pancreatic cancer is carried out at defined time intervals.

[0060] Preferably, in such a method according to the invention in step a), the compound selected from the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NFB (formula 1), ABZ-Trp- Thr-Glu-Ala-ANB-NH2 (formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NFB (formula 3), ABZ- Glu-Met-Phe-Phe-ANB-NH2 (formula 4), ABZ-Gln-Trp-Phe-Ala-ANB-NFB (formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NH2 (formula 7), or the combination comprising 3 of the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NFB (formula 1), ABZ-Trp-Thr-Glu- Ala-ANB-NH2 (formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NFB (formula 3), ABZ-Glu-Met- Phe-Phe-ANB-NH2 (formula 4), ABZ-Thr-Thr-Ala-Arg-ANB-NFB (formula 5), ABZ-Gln- Trp-Phe-Ala-ANB-NH2 (formula 6) and ABZ-Ghi-Met-Lys-Phe-ANB-NJL (formula 7), more preferably the combination consisting of 3 of these compounds, is used.

[0061] Detailed description of the invention

[0062] It is to be understood that the present invention is defined in the appended claims. The present description illustrates various, non-limiting embodiments and examples of the invention. The present invention is not limited to any particular methodology, protocol or reagents used to carry it out, unless indicated otherwise. The terms as well as scientific and technical expressions as used herein have meanings commonly known and used by persons skilled in the art of the present invention. For the sake of clarity however, the following expressions / terms and acronyms used in the patent shall be understood as follows:

[0063] A chromogenic compound or a chromogenic molecule means a compound having chromogenic properties. Chromogenic properties mean the ability of a compound to form a coloured product. A fluorescent compound or a fluorescent molecule means a compound having fluorogenic properties. Fluorogenic properties mean the ability of a compound to form a product emitting fluorescence.

[0064] NMP stands for A-methylpirrolidone; DMF stands for dimethylformamide; DCM stands for methylene chloride or dichloromethane; pNA stands for 4-nitroaniline or para-nitroaniline (in a free form) or para-nitroanilide (in a form incorporated into a peptide molecule) ; ABZ stands for 2-aminobenzoic acid, ANB-NH2 stands for amide of 5-amino-2-nitrobenzoic acid; Boc stands for tert-butyloxycarbonyl group; Fmoc stands for 9-fluorenylometoxycarbonyl group; and TFA stands for trifluoroacetic acid.

[0065] In the context of the present invention, enzymatic activity specific for pancreatic cancer means that such activity is only present in samples from individuals with pancreatic cancer, and is not present in samples from healthy individuals or individuals diagnosed with another pancreatic disease such as pancreatitis. Such activity is preferably hydrolytic activity, especially proteolytic activity. Methods of determining such activity in samples, such as body fluid samples such as urine, are well known in the field.

[0066] In the context of the present invention, the term pancreatic cancer is to be understood as a primary carcinoma (malignant tumour) of the pancreas developing from tissues within the pancreas. 95% of pancreatic cancers originate from the exocrine part, the remaining 4% of cancers are those originating from the endocrine part (neuroendocrine tumours). Adenocarcinoma, originating from the endocrine part, is the most common pancreatic neoplasm. The term pancreatic cancer, as used herein, therefore includes any malignant tumour of the pancreas that develops from tissues within the pancreas.

[0067] In the context of the present invention, the term diagnosis of pancreatic cancer is to be understood as the diagnosis of this disease, in particular at an early stage in which other diagnostic methods are too insensitive and / or specific enough, while at the same time in a sufficiently specific manner to allow differentiation from other pancreatic diseases, e.g. pancreatitis. As used herein, the diagnosis of pancreatic cancer also includes the detection of residual disease after surgical resection of pancreatic cancer and the detection of pancreatic cancer recurrence after previously completed pancreatic cancer treatment.

[0068] In the context of the present invention, the term treatment of pancreatic cancer is to be understood as treatment at an early stage of the disease, allowing a significant increase in survival time and an improvement in the quality of life of patients.

[0069] In the context of the present invention, the term monitoring is to be understood as the diagnosis of Minimal Residual Disease (MRD) - the presence of a small number of surviving tumour cells in the body (during treatment or in remission), in quantities not detected by standard diagnostic methods.

[0070] In the context of the present invention, the term individual is to be understood as a human or mammal suspected of having pancreatic cancer, alternatively as a human or mammal belonging to a group at increased risk of pancreatic cancer, or a human or mammal after surgical excision of pancreatic cancer or after completed treatment for pancreatic cancer. The individual is preferably a human being.

[0071] The compounds according to the invention, comprising the tetrapeptide core, have chromogenic properties due to the presence of a chromophore and fluorogenic properties, i.e. they contain fluorescence donor and acceptor molecules. Due to their structure, which is designed in such a way that a colour increase in the wavelength range 380-440 nm is observed specifically as a result of contact between an examined sample of body fluid from an examined individual and pancreatic cancer, and, at the same time, no such effect is observed when reacting with a sample of body fluid from a healthy individual or with a diagnosis of another type of cancer or other pancreatic disease, these compounds allow the detection of enzymatic activity specific for pancreatic cancer and, in particular, the diagnosis of pancreatic cancer in a sensitive and specific manner also at an early stage of its development. The examined individual is preferably a human being. The sample from the patient is a body fluid sample. The term of body fluid is well known in the field. An example of a body fluid is urine, such as human urine.

[0072] In the diagnostic process, the key parameters are not only sensitivity, but also specificity. Specificity of a test or assay means the ability to correctly identify the absence of disease where there is none. In the case of pancreatic cancer, a specific test makes it possible to distinguish samples with cancer not only from samples from healthy individuals, but also from samples from individuals with other diseases, including diseases of the pancreas, and in particular pancreatitis. The compounds according to the invention and their combinations are characterised as diagnostic markers by an extremely high sensitivity and specificity, allowing the reliable detection of pancreatic cancer, making it possible to distinguish pancreatic cancer from other diseases of the pancreas, including pancreatitis, while eliminating the risk of false-positive results in the case of samples from individuals with other types of pancreatic diseases, such as pancreatitis.

[0073] In a first aspect of the present invention, there are provided new chemical compounds having general formula:

[0074] XI -tetrapeptide-X2 wherein

[0075] XI comprises or consists of a molecule Cl, tetrapeptide means a peptide having a sequence of four amino acids selected from: Gly-Thr- Lys-Asn- (SEQ ID NO: 1), Trp-Thr-Glu-Ala (SEQ ID NO: 2), Lys-Pro-Gln-Glu (SEQ ID NO: 3), Glu-Met-Phe-Phe (SEQ ID NO: 4), Gln-Trp-Phe-Ala (SEQ ID NO: 6) and Glu- Met-Lys-Phe (SEQ ID NO: 7),

[0076] X2 comprises or consists of a molecule C2, wherein a pair of molecules Cl and C2 is a pair of a fluorescence donor and a fluorescence acceptor, and wherein the compound undergoes enzymatic cleavage into fragments 1 and 2: XI- tertapeptide-OH (fragment 1) and X2 (fragment 2), respectively, with a generation of a measurable optical signal upon spatial separation of molecules Cl and C2.

[0077] Such enzymatic cleavage is preferably hydrolytic, more preferably proteolytic.

[0078] Preferably, in the compound according to the invention, the pair of molecules Cl and C2 is selected from the group consisting of: 2-aminobenzoic acid (ABZ) / 5-amino-2-nitrobenzoic acid (ANB), (ABZ) / pNA, ABZ / ANB-NH2, ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr(3-NO2). More preferably, the pair of Cl and C2 is ABZ / pNA or ABZ / ANB-NEE, even more preferably ABZ / ANB-NEE.

[0079] More preferably, the compound according to the invention is the compound selected from the group consisting of: ABZ1-Gly2-Thr3-Lys4-Asn5-ANB6-NH2 (formula 1), ABZkTrp2- Thr3-Glu4-Ala5-ANB6-NH2 (formula 2), ABZ1-Lys2-Pro3-Gln4-Glu5-ANB6-NH2 (formula 3), ABZ1-Glu2-Met3-Phe4-Phe5-ANB6-NH2(formula 4), ABZ1-Gln2-Trp3-Phe4-Ala5-ANB6- NH2(formula 6) and ABZ1-Glu2-Met3-Lys4-Phe5-ANB6-NH2 (formula 7).

[0080] In accordance with the present invention, the formula notations of the compounds according to the invention may be used without the numbering of the residues or interchangeably with the Arabic numerals in superscript. The numerals in superscript indicate the consecutive positions of the residues in the compound according to the invention. The core of all compounds according to the invention is a tetrapeptide with the indicated sequence of 4 amino acids selected from the following sequences: Gly-Thr-Lys-Asn (G-T-K-N) (SEQ ID NO: 1), Trp-Thr-Glu-Ala (W-T-E-A) (SEQ ID NO: 2), Lys-Pro-Gln-Glu (K-P-Q-E) (SEQ ID NO: 3), Glu-Met-Phe-Phe (E-M-F-F) (SEQ ID NO: 4), Gln-Trp-Phe-Ala (Q-M-FA) (SEQ ID NO: 6) and Glu-Met-Lys-Phe (E-M-K-F) (SEQ ID NO: 7), the notation in triletter amino acid abbreviation format being equivalent to the notation in single-letter amino acid abbreviation format given in brackets. The above sequences are shown in the sequence listing as sequences Nos. 1-4, 6-7, respectively. Such compounds can be used, for example, to detect enzymatic activity specific to pancreatic cancer, and therefore also as diagnostic markers, more specifically cancer (bio)markers, especially pancreatic cancer biomarkers.

[0081] In accordance with the invention a compound having formula 5 is also described herein, which can be used in combinations according to the invention. This compound has the same general formula, i.e. Xl-tetrapeptide-X2, as the compounds according to the invention, preferably compounds having formulas 1-4, 6-7, wherein the tetrapeptide core of this compound having formula 5 is the sequence Thr-Thr-Ala-Arg (T-T-A-R) (Sequence No. 5). This sequence is shown in the sequence listing as SEQ ID NO: 5.

[0082] The compounds described herein undergo enzymatic cleavage into 2 fragments: XI- tetrapeptide-OH (fragment 1) and X2 (fragment 2) with the generation of a measurable optical signal upon spatial separation of the molecules Cl and C2. The sequences of the amino acid residues of the tetrapeptides in the compounds described herein, preferably compounds having formulas 1-7, are as follows: Gly-Thr-Lys-Asn (SEQ ID NO: 1), Trp- Thr-Glu-Ala (SEQ ID NO: 2), Lys-Pro-Gln-Glu (SEQ ID NO: 3), Glu-Met-Phe-Phe (SEQ ID NO: 4), Thr-Thr-Ala-Arg (SEQ ID NO: 5), Gln-Trp-Phe-Ala (Q-M-FA) ((SEQ ID NO: 6) and Glu-Met-Lys-Phe (E-M-K-F) ((SEQ ID NO: 7). Such a measurable optical signal is measured by a means of measuring the change in absorption / fluorescence after enzymatic cleavage of the compound. Preferably, molecules Cl and C2 are separated from each other by not more than 10 amino acid residues, which ensure efficient quenching of the fluorescence donor by the fluorescence acceptor. It is obvious for the skilled person that the key factor is the distance between the fluorescence donor and acceptor. Therefore, where the amino acid sequence separating molecules Cl and C2 is folded into a twisted or condensed secondary structure, resulting in a proximity of molecules Cl and C2 relative to the primary structure, the distance between molecules Cl and C2 can be greater than 10 amino acid residues.

[0083] Such compounds as defined above, due to their chromogenic properties and the presence of a reactive site at position 5 enabling enzymatic (preferably proteolytic) cleavage into smaller fragments, are particularly suitable for use, for example, for detecting enzymatic activity specific to pancreatic cancer (in other words, deriving from pancreatic cancer), and therefore also for use as a diagnostic biomarker and, in particular in combination, as a highly specific diagnostic biomarker for pancreatic cancer, particularly for use in the early detection of pancreatic cancer, including the effective differentiation of pancreatic cancer from other pancreatic diseases such as pancreatitis. They allow false positives to be eliminated and appropriate treatment to be implemented quickly, which increases the patient's chances of a successful outcome.

[0084] The compounds according to the invention can be obtained by known methods. For example, they can be obtained using a method which consists in carrying out the process on a support in the form of a resin having an Fmoc group, which is removed in the course of the reaction. For example, it can be an amide resin, e.g. Teenage S RAM or RinkAmide, but any other commercially available resin can also be used. The resin used to carry out the process should be properly prepared. The preparation of the resin consists in increasing its volume by repeated washing with hydrophobic solvents. Preferably, a resin with a substitution of 0.23 mmol / g is used. The Fmoc protecting group must be removed from the resin by washing it with a 20% solvent solution.

[0085] Subsequently, the known processes for the preparation of chromogenic peptides involve the attachment of the individual components under appropriate time and stoichiometric conditions. This attachment process consists of successive steps in which the individual components (amino acid derivatives) are attached, the residues are washed off and protecting groups are successively removed and washed again. This cycle is repeated for each amino acid residue. The resulting peptide is separated from the resin by reaction under acidic conditions. The solution is then separated from the resin in a filtration process, and the peptide is then precipitated from the resulting solution with a non-polar solvent. The resulting peptide precipitate is centrifuged.

[0086] An exemplary, detailed but non-limiting, method for the synthesis of the compounds described according to the invention is described below and in Example 1, using the exemplary, representative compound according to the invention, i.e. a compound having formula 3 ABZ-Lys-Pro-Gln-Glu-ANB-NFB (used preferably as substrate 3) (Example 1 A) and an analogous compound that can be used as substrate 3, comprising instead of the ANB- NH2 chromophore molecule a pNA chromophore molecule (ABZ-Lys-Pro-Gln-Glu-pNA) (Example I B).

[0087] The method for the synthesis of compounds according to the invention, using the compound ABZ-Lys-Pro-Gln-Glu-ANB-NFE as an example, consists in that the process is carried out on a support in the form of a resin preferably having an Fmoc group, wherein the support being prepared before the process starts by increasing its volume by repeated washing with hydrophobic solvents, preferably dimethylformamide, methylene chloride or N- methylpyrrolidone, and removing the Fmoc group, preferably by washing with a 10 - 30 % piperidine solution, in solvents such as dimethylformamide, methylene chloride or N- methylpyrrolidone.

[0088] The method of synthesis is then carried out in the following steps: a) attachment of 5-amino-2-nitrobenzoic acid ANB (or any other chromophore suitable for use according to the invention as defined in the claims) to the resin is preceded by washing the support with a 3-6% solution of N-m ethylmorpholine (NMM) in DMF, followed by DMF, after which a solution of ANB in DMF is prepared, to which TBTU, DMAP, and finally diisopropylethylamine (DIPEA) are added successively in the following excesses relative to the polymer substitution: ANB / TBTU / DMAP / DIPEA, 3:3:2:6, the mixture thus prepared is added to the resin and is mixed until homogenous, after which the resin is filtered off under reduced pressure and washed with solvents such as DMF, DCM and isopropanol, and the attachment of ANB to the resin is continued by using hexafluorophosphate-O-(7- azabenzotriazol-l-yl)-A,A,A’,7V’-tetramethyluronium (HATU), and then hexafl uorophosphate-(9-(benzotri azol - 1 -yl)-A, N, N ’N ’-tetramethyluronium (HBTU) in excess amounts, and upon completion the support is washed successively with DMF, DCM and isopropanol and gently dried; b) attachment of the amino acid residue to ANB is carried out by reaction with the amino acid derivative Fmoc-Glu(OtBu)-OH, wherein at least five times the molar excess of the amino acid derivative relative to the resin is dissolved in anhydrous pyridine and contacted with the resin with ANB deposited, after which the whole is cooled to not less than -20°C, and then POCh is added in the ratio of 1 : 1 to the amount of amino acid derivative used and the whole is stirred, after which the stirring process is carried out at room temperature and then at elevated temperature, and at the end of the reaction the resin is drained under reduced pressure, washed with DMF and MeOH and gently dried, after which the intermediate compound obtained is acylated by successively attaching the Lys-Pro-Gln fragment; c) acylation of the intermediate compound obtained is carried out using an amino acid derivative, preferably Fmoc-Gln(Trt) OH, and successively Fmoc-Pro-OH, then Fmoc- Lys(Boc)-OH and in the final synthesis step Boc-Abz-OH, wherein the acylation being carried out in steps from residue 6 to 1 using diisopropylcarbodiimide as a coupling agent, which is used in excess, and at the end of each step the resin is washed with DMF, and preferably subjected to a chloroanil test (free amino group test) in which the attachment of the amino acid derivative is monitored; d) removal of the Fmoc protecting group is carried out by washing with a solution of 10 - 30% piperidine in DMF and subsequent washes with each solvent: DMF, isopropanol and methylene chloride, e) cleavage of the peptide from the resin is carried out using a TFA:phenol:water:TIPS mixture at ratios of 88:5:5:2, N / N / N / N, respectively, wherein the mixture is stirred for at least one hour, preferably three hours, and the precipitate obtained is filtered off under reduced pressure, followed by washing with diethyl ether and the peptide obtained is centrifuged; f) preparation of the finished product is carried out by dissolving the peptide in water by means of ultrasound, and then it is subjected to lyophilisation.

[0089] In a second aspect of the present invention, there is provided a combination of compounds having general formula:

[0090] XI -tetrapeptide-X2 wherein

[0091] XI comprises or consists of a molecule Cl, tetrapeptide means a peptide having a sequence of four amino acids selected from: Gly-Thr- Lys-Asn (SEQ ID NO: 1), Trp-Thr-Glu-Ala (SEQ ID NO: 2), Lys-Pro-Gln-Glu (SEQ ID NO: 3), Glu-Met-Phe-Phe (SEQ ID NO: 4), Thr-Thr-Ala-Arg (SEQ ID NO: 5), Gln-Trp- Phe-Ala (Q-M-FA) (SEQ ID NO: 6) and Glu-Met-Lys-Phe (E-M-K-F) (SEQ ID NO: 7), X2 comprises or consists of a molecule C2, wherein a pair of molecules Cl and C2 is a pair of a fluorescence donor and a fluorescence acceptor, and wherein the compound undergoes enzymatic cleavage into fragments 1 and 2: XI- tetrapeptide-OH (fragment 1) and X2 (fragment 2), respectively, with a generation of a measurable optical signal upon spatial separation of molecules Cl and C2.

[0092] The combination of compounds according to the invention can be used for diagnostic purposes, in particular for the diagnosis of cancer, in particular diagnosis of pancreatic cancer, and in particular for the highly sensitive and specific diagnosis of pancreatic cancer, allowing the differential diagnosis of pancreatic cancer, i.e. the effective differentiation of pancreatic cancer from other pancreatic diseases such as inflammation. This means that the combination of compounds according to the invention can be used for detecting activity specific to pancreatic cancer, as well as as a biomarker combination (diagnostic biomarker combination), since the compounds therein can be used as diagnostic biomarkers, in particular diagnostic biomarkers for pancreatic cancer. The combination according to the invention preferably comprises any 3 compounds selected from the compounds described herein having general formula as defined above, preferably 3 of the compounds having formulas 1-7, more preferably consisting of 3 such compounds, which allows an extremely sensitive and specific detection of activity specific for pancreatic cancer, or otherwise derived from pancreatic cancer cells, and thus an extremely sensitive and specific diagnosis of pancreatic cancer, also at an early stage, allowing effective differentiation of pancreatic cancer from other pancreatic diseases, including pancreatitis (inflammation of pancreas), thus eliminating diagnostic problems and therapeutic complications associated with falsepositive results, and, as a result, allows for the implementation of appropriate treatment and a successful outcome.

[0093] In a third aspect of the present invention, there is provided an in vitro method for the detection of enzymatic activity present in an individual’s body fluid sample, in particular deriving from pancreatic cancer cells, in other words specific for pancreatic cancer, comprising:

[0094] (a) contacting a sample of body fluid, preferably urine, more preferably human urine, with the compound according to the invention as defined above, or the combination according to the invention as defined above, and

[0095] (b) detecting a measurable optical signal, which is generated upon spatial separation of molecules Cl and C2.

[0096] Preferably in the method for the detection according to the invention, the detected enzymatic activity is hydrolytic activity, more preferably proteolytic activity.

[0097] Preferably, in the method for the detection according to the invention the combination according to the invention comprising 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NH2(formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NH2(formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB- NH2(formula 4), ABZ-Thr-Thr-Ala-Arg-ANB-NH2(formula 5), ABZ-Gln-Trp-Phe-Ala- ANB-NH2(formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NH2(formula 7), more preferably the combination consisting of 3 of these compounds, is used.

[0098] Preferably, in the method for the detection according to the invention, as the said measurement buffer a Tris buffer, or a buffer comprising Tris, NaCl and DMSO, is used. However, any known measurement buffer suitable for measuring the change in absorbance may be used.

[0099] The method for the detection of enzymatic activity according to the invention can, for example, be used in an in vitro method for diagnosing pancreatic cancer according to the invention. In a fourth aspect of the present invention, there is provided an in vitro method for the diagnosis of pancreatic cancer, wherein the presence or absence of pancreatic cancer in an individual is detected by measuring enzymatic activity specific for pancreatic cancer in a body fluid sample of an examined individual, using the compound according to the invention or a combination of compounds according to the invention, wherein the absence of the said enzymatic activity indicates the absence of pancreatic cancer, while the presence of the said enzymatic activity indicates the presence of pancreatic cancer.

[0100] Preferably, in the method for the diagnosis of pancreatic cancer according to the invention, the detection of enzymatic activity specific for pancreatic cancer is carried out by the method for the detection of enzymatic activity according to the invention as defined above.

[0101] Preferably, in such a method for the diagnosis of pancreatic cancer according to the invention, for the measure of the said enzymatic activity the combination comprising 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NBL (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NHj (formula 2), ABZ-Lys-Pro-Gln-Glu-ANB- NH2 (formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NIB (formula 4), ABZ-Thr-Thr-Ala-Arg- ANB-NH2 (formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NIB (formula 6) and ABZ-Glu-Met- Lys-Phe-ANB-NH2 (formula 7), more preferably the combination consisting of 3 of these compounds, is used, as will be discussed in more detail below and in the accompanying examples, what allows an extremely sensitive and specific detection of enzymatic activity specific for pancreatic cancer and reliable diagnosis of this type of cancer.

[0102] Preferably in such a method for the diagnosis of pancreatic cancer according to the invention, the said body fluid sample is incubated with the said compound in a measurement buffer having neutral pH or alkaline pH, preferably physiological pH, within the range of sample- to-measurement buffer ratio of 1 :2 to 1 : 10, preferably 1 :5.

[0103] Preferably, in the method for the diagnosis of pancreatic cancer according to the invention, the said compound is used at a concentration of 0.1-10 mg / mL, in particular 0.25-7.5 mg / mL. Preferably, in the method for the diagnosis of pancreatic cancer according to the invention, the measurement of the said enzymatic activity involves the measurement of the absorbance intensity in the range 300-500 nm, preferably 380-430 nm, particularly 410 nm, for a time of 40-60 minutes, at a temperature in the range 25-40° C, preferably 36-38° C. The above parameters of the method allow the best measurement readings to be obtained.

[0104] For example, the method for the diagnosis of pancreatic cancer according to the invention can be carried out, preferably but not limited to, by means of the method for the detection of enzymatic activity specific for pancreatic cancer according to the invention, in other words activity deriving from pancreatic cancer cells, as follows, using the following, preferable, reagents as substrates (i.e. combinations according to the invention, preferably combinations of compounds having formulas 1-7) or analogously using individual (single) compounds according to the invention, preferably compounds having formulas 1-4 and 6-7.

[0105] 1. Reagents

[0106] Detection of the said enzymatic activity specific for pancreatic cancer using the compound or combination according to the invention (i.e. substrates of the enzymatic reaction to be tested) is carried out by the method according to the invention with a body fluid sample to be tested, preferably human urine, in a measurement buffer. Such a measurement buffer has a pH neutral or alkaline, preferably physiological, more preferably as given in Table 1 below. Such measurement buffers are known in the field, wherein preferable, but non-limiting, examples of buffers being, for example, those given in Table 1 below. This means that for each of the compounds described herein, i.e. the substrates of the enzymatic reaction tested, any suitable measurement buffer for measuring the absorption can be used, the specific, preferable but non-limiting, measurement buffers for the indicated substrates being given in Table 1 below.

[0107] As used herein, the term substrate or substrates, e.g. substrates 1-7, denotes a compound or compounds having general formula: XI -tetrap eptide-X2 as described above, wherein tetrapeptide means a peptide having a sequence of four amino acids respectively selected from: Gly-Thr-Lys-Asn (SEQ ID NO: 1), Trp-Thr-Glu-Ala (SEQ ID NO: 2), Lys-Pro-Gln- Glu (SEQ ID NO: 3), Glu-Met-Phe-Phe (SEQ ID NO: 4), Thr-Thr-Ala-Arg (SEQ ID NO: 5), Gln-Trp-Phe-Ala (SEQ ID NO: 6) and Glu-Met-Lys-Phe (SEQ ID NO: 7). As described above, XI comprises or consists of a molecule Cl, X2 comprises or consists of a molecule C2, and Cl and C2 are selected from the group consisting of: 2-aminobenzoic acid (ABZ) / 5-amino-2-nitrobenzoic acid (ANB), (ABZ) / pNA, ABZ / ANB-NH2, ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr(3-NO2), more preferably the pair of Cl and C2 is ABZ / pNA or ABZ / ANB-NH2. Preferable substrates 1-7 are, respectively, compounds having formulas 1-7. Also preferable as substrates 1-7 are analogous compounds comprising, instead of the chromophore molecule ANB-NH2, another chromophore molecule, e.g. pNA. Table 1. Reagents for the test: substrates and measurement buffers

[0108] 2. Realisation of the study

[0109] According to the invention, the method can be carried out using a reaction mixture comprising: 20 pl of substrate + 100 pl of measurement buffer+ 80 pl of urine sample to be tested.

[0110] For each urine sample tested, the method is carried out preferably using the combination of 3 of the substrates 1-7, i.e. 3 compounds described herein, preferably selected from the group of compounds having formulas 1-7 (i.e. preferable substrates 1-7).

[0111] As mentioned above, any neutral or alkaline pH measurement buffer, preferably physiological, can be used, preferably using the measurement buffers indicated in Table 1 for the respective substrates to obtain optimal readings.

[0112] Each substrate tested in triplicate with one urine sample, e.g. in a 96-well plate.

[0113] (3) The interpretation of the result obtained by the method according to the invention using combinations of 3 of the substrates, using the exemplary compounds having formulas 1-7, in triplicate for each sample, is shown below. The interpretation of results obtained using other combinations according to the invention in triplicate is carried out in the same way.

[0114] Option I - combination of substrates 1, 2 and 3 Substrate 1 + measurement buffer

[0115] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0116] Substrate 2 + measurement buffer a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signalk,.

[0117] Substrate 3 + measurement buffer

[0118] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0119] Table 2. Interpretation of results for combinations of substrates 1, 2 and 3

[0120] Option II - combination of substrates 1, 3 and 4

[0121] Substrate 1 + measurement buffer:

[0122] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0123] Substrate 3 + measurement buffer:

[0124] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0125] Substrate 4 + measurement buffer:

[0126] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0127] Table 3. Interpretation of results for combinations of substrates 1, 3 and 4

[0128] Option III - combination of substrates 1, 2 and 4

[0129] Substrate 1 + measurement buffer:

[0130] - a positive result when: 2 or 3 signals, while a negative result when: none or 1 '+' signal. Substrate 2 + measurement buffer:

[0131] - a positive result when: 2 or 3 signals, while a negative result when: none or 1 '+' signal. Substrate 4 + measurement buffer:

[0132] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal. Table 4. Interpretation of results for combinations of substrates 1, 2 and 4

[0133] Option IV - combination of substrates 3, 4 and 2

[0134] Substrate 3 + measurement buffer:

[0135] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0136] Substrate 4 + measurement buffer: - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0137] Substrate 2 + measurement buffer:

[0138] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0139] Table 5. Interpretation of results for combinations of substrates 3, 4 and 2

[0140] Option V - combination of substrates 1, 2 and 5

[0141] Substrate 1 + measurement buffer: - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0142] Substrate 2 + measurement buffer:

[0143] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal. Substrate 5 + measurement buffer:

[0144] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0145] Table 6. Interpretation of results for combinations of substrates 1, 2 and 5

[0146] Option VI - combination of substrates 1, 3 and 5

[0147] Substrate 1 + measurement buffer:

[0148] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0149] Substrate 3 + measurement buffer:

[0150] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0151] Substrate 5 + measurement buffer:

[0152] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0153] Table 7. Interpretation of results for combinations of substrates 1, 3 and 5

[0154] VII option - combination of substrates 5, 4 and 2

[0155] Substrate 5 + measurement buffer:

[0156] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0157] Substrate 4 + measurement buffer:

[0158] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0159] Substrate 2 + measurement buffer: - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0160] Table 8. Interpretation of results for combinations of substrates 5, 4 and 2

[0161] Option VIII - - combination of substrates 3, 5 and 2

[0162] Substrate 3 + measurement buffer:

[0163] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal. Substrate 5 + measurement buffer:

[0164] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0165] Substrate 2 + measurement buffer:

[0166] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0167] Table 9. Interpretation of results for combinations of substrates 3, 5 and 2

[0168] Option IX - combination of substrates 3, 4 and 5

[0169] Substrate 3 + measurement buffer:

[0170] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0171] Substrate 4 + measurement buffer:

[0172] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0173] Substrate 5 + measurement buffer:

[0174] - a positive result when: 2 or 3 "+" signals, while a negative result when: none or 1 "+" signal.

[0175] Table 10. Interpretation of results for combinations of substrates 3, 4 and 5

[0176] Option X - combination of substrates 3, 4, and 6

[0177] Substrate 3 + measurement buffer:

[0178] - a positive result when: 2 or 3 “+” signals, while a negative result when: none or 1 “+” signal.

[0179] Substrate 4 + measurement buffer:

[0180] - a positive result when: 2 or 3 “+” signals, while a negative result when: none or 1 signal.

[0181] Substrate 6 + measurement buffer:

[0182] - a positive result when: 2 or 3 “+” signals, while a negative result when: none or 1 “+” signal. Table 11. Interpretation of results for combinations of substrates 3, 4, and 6

[0183] Option XI - combination of substrates 3, 4 and 7

[0184] Substrate 3 + measurement buffer: - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal ,

[0185] Substrate 4 + measurement buffer:

[0186] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal Substrate 7 + measurement buffer:

[0187] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0188] Table 12. Interpretation of results for combinations of substrates 3, 4 and 7 Option XII - combination of substrates 1, 2 and 6

[0189] Substrate 1 + measurement buffer:

[0190] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal ,

[0191] Substrate 2 + measurement buffer:

[0192] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0193] Substrate 6 + measurement buffer:

[0194] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0195] Table 13. Interpretation of results for combinations of substrates 1, 2 and 6

[0196] Option XIII - combination of substrates 1, 2 and 7

[0197] Substrate 1 + measurement buffer:

[0198] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0199] Substrate 2 + measurement buffer:

[0200] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0201] Substrate 7 + measurement buffer:

[0202] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0203] Table 14. Interpretation of results for combinations of substrates 1, 2 and 7

[0204]

[0205] Option XIV - combination of substrates 1, 3 and 6

[0206] Substrate 1 + measurement buffer:

[0207] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal ,

[0208] Substrate 3 + measurement buffer:

[0209] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0210] Substrate 6 + measurement buffer: - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0211] Table 15. Interpretation of results for combinations of substrates 1, 3 and 6 Option XV - combination of substrates 1, 3 and 7

[0212] Substrate 1 + measurement buffer:

[0213] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal ,

[0214] Substrate 3 + measurement buffer:

[0215] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0216] Substrate 7 + measurement buffer:

[0217] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0218] Table 16. Interpretation of results for combinations of substrates 1, 3 and 7

[0219] Option XVI - combination of substrates 1, 4 and 6

[0220] Substrate 1 + measurement buffer:

[0221] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0222] Substrate 4 + measurement buffer:

[0223] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0224] Substrate 6 + measurement buffer:

[0225] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal Table 17. Interpretation of results for combinations of substrates 1, 4 and 6

[0226] Option XVII - combination of substrates 1, 4 and 7

[0227] Substrate 1 + measurement buffer: - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal ,

[0228] Substrate 4 + measurement buffer:

[0229] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal Substrate 7 + measurement buffer:

[0230] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0231] Table 18. Interpretation of results for combinations of substrates 1, 4 and 7 Option XVIII - combination of substrates 1, 5 and 6

[0232] Substrate 1 + measurement buffer:

[0233] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal ,

[0234] Substrate 5 + measurement buffer:

[0235] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0236] Substrate 6 + measurement buffer:

[0237] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0238] Table 19. Interpretation of results for combinations of substrates 1, 5 and 6

[0239] Option XIX - combination of substrates 1, 5 and 7

[0240] Substrate 1 + measurement buffer:

[0241] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0242] Substrate 5 + measurement buffer:

[0243] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0244] Substrate 7 + measurement buffer:

[0245] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0246] Table 20. Interpretation of results for combinations of substrates 1, 5 and 7

[0247]

[0248] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal ,

[0249] Substrate 6 + measurement buffer:

[0250] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0251] Table 21. Interpretation of results for combinations of substrates 2, 3 and 6 Option XXI - combination of substrates 2, 3 and 7

[0252] Substrate 2 + measurement buffer:

[0253] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal ,

[0254] Substrate 3 + measurement buffer:

[0255] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0256] Substrate 7 + measurement buffer:

[0257] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0258] Table 22. Interpretation of results for combinations of substrates 2, 3 and 7

[0259] Option XXII - combination of substrates 2, 4 and 6

[0260] Substrate 2 + measurement buffer:

[0261] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0262] Substrate 4 + measurement buffer:

[0263] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0264] Substrate 6 + measurement buffer:

[0265] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal Table 23. Interpretation of results for combinations of substrates 2, 4 and 6

[0266] Option XXIII - combination of substrates 2, 4 and 7

[0267] Substrate 2 + measurement buffer: - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal ,

[0268] Substrate 4 + measurement buffer:

[0269] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal Substrate 7 + measurement buffer:

[0270] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0271] Table 24. Interpretation of results for combinations of substrates 2, 4 and 7 Option XXIV - combination of substrates 2, 5 and 6

[0272] Substrate 2 + measurement buffer:

[0273] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal ,

[0274] Substrate 5 + measurement buffer:

[0275] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0276] Substrate 6 + measurement buffer:

[0277] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0278] Table 25. Interpretation of results for combinations of substrates 2, 5 and 6

[0279] Option XXV - combination of substrates 2, 5 and 7

[0280] Substrate 2 + measurement buffer:

[0281] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0282] Substrate 5 + measurement buffer:

[0283] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0284] Substrate 7 + measurement buffer:

[0285] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal Table 26. Interpretation of results for combinations of substrates 2, 5 and 7

[0286] Option XXVI - combination of substrates 1, 6 and 7

[0287] Substrate 1 + measurement buffer: - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal ,

[0288] Substrate 6 + measurement buffer:

[0289] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal Substrate 7 + measurement buffer:

[0290] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0291] Table 27. Interpretation of results for combinations of substrates 1, 6 and 7 Option XXVII - combination of substrates 2, 6 and 7

[0292] Substrate 2 + measurement buffer:

[0293] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal ,

[0294] Substrate 6 + measurement buffer:

[0295] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0296] Substrate 7 + measurement buffer:

[0297] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0298] Table 28. Interpretation of results for combinations of substrates 2, 6 and 7

[0299] Option XXVIII - combination of substrates 3, 6 and 7

[0300] Substrate 3 + measurement buffer:

[0301] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0302] Substrate 6 + measurement buffer:

[0303] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0304] Substrate 7 + measurement buffer:

[0305] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal Table 29. Interpretation of results for combinations of substrates 3, 6 and 7

[0306] Option XXIX - combination of substrates 4, 6 and 7

[0307] Substrate 4 + measurement buffer: - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal ,

[0308] Substrate 6 + measurement buffer:

[0309] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal Substrate 7 + measurement buffer:

[0310] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0311] Table 30. Interpretation of results for combinations of substrates 4, 6 and 7 Option XXX - combination of substrates 5, 6 and 7

[0312] Substrate 5 + measurement buffer:

[0313] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal ,

[0314] Substrate 6 + measurement buffer:

[0315] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0316] Substrate 7 + measurement buffer:

[0317] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0318] Table 31. Interpretation of results for combinations of substrates 5, 6 and 7

[0319] Option XXXI - combination of substrates 3, 5 and 6

[0320] Substrate 3 + measurement buffer:

[0321] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0322] Substrate 5 + measurement buffer:

[0323] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0324] Substrate 6 + measurement buffer:

[0325] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal Table 32. Interpretation of results for combinations of substrates 3, 5 and 6

[0326] Option XXXII - combination of substrates 3, 5 and 7

[0327] Substrate 3 + measurement buffer: - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal ,

[0328] Substrate 5 + measurement buffer:

[0329] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal Substrate 7 + measurement buffer:

[0330] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0331] Table 33. Interpretation of results for combinations of substrates 3, 5 and 7 Option XXXIII - combination of substrates 4, 5 and 6

[0332] Substrate 4 + measurement buffer:

[0333] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal ,

[0334] Substrate 5 + measurement buffer:

[0335] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0336] Substrate 6 + measurement buffer:

[0337] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0338] Table 34. Interpretation of results for combinations of substrates 4, 5 and 6

[0339] Option XXXIV - combination of substrates 4, 5 and 7

[0340] Substrate 4 + measurement buffer:

[0341] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0342] Substrate 5 + measurement buffer:

[0343] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal

[0344] Substrate 7 + measurement buffer:

[0345] - a positive result when: 2 or 3 „+” signals, while a negative result when: none or 1 „+” signal Table 35. Interpretation of results for combinations of substrates 4, 5 and 7

[0346] In summary, above there are embodiment examples according to the invention for the diagnosis of pancreatic cancer of all possible combinations according to the invention (options I-XXXIV), i.e. combinations of 3 compounds among the substrates 1-7 being the compounds described herein, based on an example of 3 among the compounds having formulas 1-7, as well as rules for the interpretation of the results obtained according to the invention, both the interpretation of positive and negative results being indicated (Tables 2- 35).

[0347] The method for the diagnosis of pancreatic cancer carried out in this way allows for reliable, non-invasive, rapid, highly sensitive and specific detection and diagnosis of pancreatic cancer, especially at an early stage, while also effectively differentiating pancreatic cancer from other pancreatic diseases such as pancreatitis, eliminating the risk of false positive results.

[0348] In a fifth aspect of the present invention, there is provided a kit comprising at least one compound according to the invention as defined above or the combination according to the invention as defined above and a measurement buffer.

[0349] Preferably, such a kit according to the invention comprises the combination according to the invention comprising 3 compounds selected from the following compounds: ABZ-Gly-Thr- Lys-Asn-ANB-NH2 (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NH2(formula 2), ABZ-Lys- Pro-Gln-Glu-ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ- Thr-Thr-Ala-Arg-ANB-NH2(formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NH2(formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NH2(formula 7), more preferably the combination according to the invention consisting of 3 of the said compounds. Such combinations are particularly suitable for use in the methods according to the invention and in the uses according to the invention.

[0350] In a sixth aspect of the present invention, there is provided use of the compound according to the invention as defined above or the combination according to the invention as defined above for detecting enzymatic activity specific for pancreatic cancer.

[0351] In a seventh aspect of the present invention there is provided the compound according to the invention as defined above for use in a method for the diagnosis of pancreatic cancer.

[0352] In such aspects of the present invention, the compound according to the invention selected from the following compounds according to the invention: ABZ-Gly-Thr-Lys-Asn-ANB- NH2(formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NHz (formula 2), ABZ-Lys-Pro-Gln-Glu- ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Gln-Trp-Phe- Ala-ANB-NHz (formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NHz (formula 7) is used.

[0353] In an eighth aspect of the present invention there is provided the combination according to the invention as defined above for use in a method for the diagnosis of pancreatic cancer. Preferably, in such an aspect of the invention, the combination comprising 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NHz (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NHz (formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NHz (formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Thr-Thr-Ala-Arg-ANB-NH2(formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NH2(formula 6) and ABZ-Glu-Met-Lys-Phe- ANB-NH2(formula 7), more preferably a combination consisting of 3 of these compounds, is used.

[0354] In a ninth aspect of the present invention, there is provided use of the compound according to the invention as defined above, preferably selected from: ABZ-Gly-Thr-Lys-Asn-ANB- NH2(formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NH2(formula 2), ABZ-Lys-Pro-Gln-Glu- ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Gln-Trp-Phe- Ala-ANB-NH2(formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NH2(formula 7), in a method of in vitro diagnosis of pancreatic cancer.

[0355] In a tenth aspect of the present invention, there is provided use of the combination according to the invention as defined above, more preferably comprising 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NH2(formula 1), ABZ-Trp-Thr- Glu-Ala-ANB-NH2(formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NH2(formula 3), ABZ-Glu- Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Thr-Thr-Ala-Arg-ANB-NH2(formula 5), ABZ- Gln-Trp-Phe-Ala-ANB-NH2(formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NH2(formula 7), even more preferably consisting of 3 such compounds, in an in vitro method for the diagnosis of pancreatic cancer.

[0356] In the above aspects of the present invention, the diagnosis of pancreatic cancer preferably comprises detecting primary pancreatic cancer, detecting residual disease after surgical resection of pancreatic cancer and / or detecting recurrence of pancreatic cancer.

[0357] In a further aspect of the present invention, a method for the treatment of pancreatic cancer is provided, wherein

[0358] (a) the presence of enzyme activity specific for pancreatic cancer is detected by the method for the detection as defined above in a sample of body fluid, preferably urine, from an examined individual, preferably human, and

[0359] (b) when the said enzymatic activity is found in said sample, treatment of pancreatic cancer is applied to the individual.

[0360] Treatment of pancreatic cancer undertaken in step b) of the method for the treatment according to the invention is carried out in accordance with the assessment of the attending physician, in accordance with schemes known in the art.

[0361] Preferably, after completion of the treatment according to point b), monitoring of the said enzymatic activity specific for pancreatic cancer is carried out at defined time intervals to detect possible residual disease or recurrence.

[0362] Preferably, in step a) of the method for the treatment of pancreatic cancer, the compound according to the invention selected from: ABZ-Gly-Thr-Lys-Asn-ANB-NHz (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NHz (formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NHz (formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Gln-Trp-Phe-Ala-ANB-NH2(formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NH2(formula 7), or the combination comprising 3 of the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NH2(formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NH2(formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Thr-Thr-Ala-Arg-ANB-NH2(formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NH2(formula 6) and ABZ-Glu-Met-Lys-Phe- ANB-NH2(formula 7), more preferably consisting of 3 of the said compounds, is used. The use of such compounds according to the invention, in particular such combination according to the invention, in the method according to the discussed aspect of the present invention allows a highly sensitive and specific detection of enzymatic activity specific for pancreatic cancer, allowing an increase in the efficiency of pancreatic cancer treatment.

[0363] The advantages of the present invention lie in providing new compounds having properties that make them suitable for use for highly sensitive and specific detection of enzymatic activity specific for pancreatic cancer, for use as a diagnostic biomarker for the detection of pancreatic cancer, as well as for use for rapid, non-invasive diagnosis of pancreatic cancer, while allowing detection of pancreatic cancer at an early stage of its development, as well as for effective differentiation of pancreatic cancer from other pancreatic diseases, including pancreatitis. The combinations of compounds described herein show a synergistic effect in increasing the specificity of detection of pancreatic cancer, allowing extremely effective differentiation of pancreatic cancer from other pancreatic diseases, including pancreatitis. Another advantage is that the diagnostic methods according to the invention are non-invasive and can be successfully used for screening tests. This offers the possibility of a complete diagnosis at an early stage of cancer development and consequently more effective treatment. Early diagnosis offers the possibility of surgical treatment, which significantly prolongs patient survival. It is also important for monitoring the effectiveness of the surgical and / or chemotherapeutic treatment given to pancreatic cancer, thanks to the possibility of detecting residual disease or possible recurrence.

[0364] The present invention will now be illustrated in the following figures and examples, which, however, are not intended to limit in any way the scope of the invention as defined in the patent claims.

[0365] Brief description of the figures

[0366] Fig. 1 A-H , panels I, show exemplary results of chromatographic analysis (HPLC) confirming the preparation of herein described compounds, and Fig. 1 A-H, panels II, show results of chromatographic analysis (HPLC) of the cleavage of the above-mentioned compounds in a urine sample from an individual with pancreatic cancer.

[0367] Fig. 2 A-K show a hydrolysis of the substrates, i.e. compounds having, respectively, formulas 1-4 and 6-7 according to the invention (Fig. 2 A-D and F-G, respectively) and compounds having formulas 5 and 8-11 (Fig. 2 E and G-K, respectively) in urine samples from individuals diagnosed with pancreatic cancer (samples 1-20), urine samples from healthy persons (samples Z1-Z10) and urine samples from persons with pancreatitis (samples inflammation 1 to inflammation 10). Arabic numerals on the X axis indicate the number of the urine sample selected.

[0368] Fig. 3 shows the hydrolysis selectivity of compounds having formulas 1-4 and 6-7 according to the invention, (Fig. 3 A-D and F-G, respectively) and compound having formula 5 (Fig. 3 E) in urine samples from persons diagnosed with pancreatic cancer and urine samples from persons diagnosed with another cancer (stomach, large intestine, kidney, prostate, bladder, lung, ovary and liver). The samples tested for each cancer type were derived from 20 different patients for each of the cancers tested. Results represent mean values for each cancer type. These results demonstrate the selectivity of the cleavage of the tested compounds towards urine samples from patients with pancreatic cancer relative to urine samples from patients with other cancers.

[0369] Fig. 4 shows the dependence of the degree of hydrolysis of compounds having formulas 1- 4 and 6-7 according to the invention, (Fig. 4 A-D and F-G, respectively) and compound having formula 5 (Fig. 4 E) on the pH environment.

[0370] Examples

[0371] The invention is illustrated by the following examples, which do not constitute a limitation thereof. Unless otherwise indicated, the following examples use known and / or commercially available apparatus, methods, reaction conditions, reagents and kits, such as are commonly used in the field to which the present invention belongs and as recommended by the manufacturers of the respective reagents and kits.

[0372] Example 1: Synthesis of compounds according to the invention

[0373] Example 1 A: Synthesis of compounds comprising the ANB-NH2 molecule as a chromophore

[0374] This example shows the synthesis of a representative compound according to the present invention, namely compound: ABZ1-Lys2-Pro3-Gln4-Glu5-ANB6-NH2, i.e. the compound having formula 3. The remaining compounds according to the invention can be synthesized in an analogous way, i.e. using corresponding amino acid residues and chromophore molecules. The numerals in superscript indicate subsequent positions of residues in the compound according to the invention. The compounds according to the invention can be alternatively represented by an analogous formula without the indication of residue positions, which does not change the sequence of residues in the compounds according to the invention, as it remains unchanged.

[0375] 1. Obtaining a chromogenic peptide a) The first step of the synthesis was to obtain the chromogenic peptide, which was obtained by solid phase synthesis, on a solid support, using Fmoc / tBu chemistry, i.e. with the use of protection.

[0376] The compound having formula 3: ABZ1-Lys2-Pro3-Gln4-Glu5-ANB6-NH2, where ABZ stands for 2-aminobenzoic acid and ANB-NH2 stands for 5-amino-2 -benzoic acid amide and ANB is 5-amino-2 -benzoic acid, was obtained by solid-phase chemical synthesis using the following amino acid derivatives.

[0377] Boc-ABZ, Fmoc-Lys(Boc), Fmoc-Pro, Fmoc-Gln(Trt), Fmoc-Glu(OtBu). The synthesis of the compound according to the invention, which can be used as a diagnostic marker for the detection of pancreatic cancer, especially in a combination with other compounds herein described, was carried out on a solid support enabling the conversion of 5-amino-2-beznoic acid into ANB-NH2 amide, namely amide resin TentaGel S RAM from RAPP Polymere (Germany), with a substitution of 0.23 mmol / g. However, it is possible to use any other amide resin, e.g. Rink Amide (Germany).

[0378] The synthesis of the compound was carried out manually using a laboratory shaker. In most of the steps a 25 mL sintered syringe for solid phase synthesis was used as a reactor.

[0379] All the obtained final compounds contained 2-aminobenzoic acid (ABZ) at the position 1 of their sequence, i.e. at the N-terminus, and a 5-amino-2-nitrobenzoic acid (ANB) molecule at the position 6, i.e. at the C-terminus. ABZ acts here as a fluorescence donor, while ANB- 5- amino-2-nitrobenzoic acid - acts as a fluorescence quencher and simultaneously a chromophore. The peptides contained at least and preferably one reactive site in their sequence, located at the amino acid residue Ile-ANB-NH2, i.e. at the position 5 of the compound. The synthesis consisting in attaching amino acid derivatives is carried out from residue 6 to 1, i.e. from the C- to N-terminus. b) Attachment of ANB to TentaGel S RAM resin:

[0380] The synthesis of peptides was performed on TentaGel S RAM resin from Rapp Polymere with a substitution of 0.23 mmol / g. In the first step, the resin was prepared, including swelling it by the wash cycle. Subsequently, the protection of the Fmoc amino group was removed from the solid support with the 20% solution of piperidine in NMP. Then, the solvent washing cycle was carried out. In order to confirm the presence of free amino groups, a chloranil test was performed.

[0381] Solvent wash cycle:

[0382] DMF 1 x 10 minutes; IsOH 1 x 10 minutes; DCM 1 x 10 minutes.

[0383] Removal of Fmoc protection:

[0384] DMF 1 x 5 minutes; 20% piperidine in NMP 1 x 3 minutes; 20% piperidine in NMP 1 x 8 minutes.

[0385] Solvent wash cycle:

[0386] DMF 3 x 2 minutes; IsOH 3 x 2 minutes; DCM 3 x 2 minutes. c) Chloranil test:

[0387] The chloranil test consisted in transferring, by means of a spatula, several grains of resin from the reactor - a syringe, into a glass ampule, to which subsequently 100 pL of saturated solution of p-chloranil in toluene and 50 pL of fresh acetaldehyde were added. After 10 minutes, the control of grains colour was carried out.

[0388] At that stage, after performing the test, a green colour of the grains was obtained, which evidenced the presence of free amino groups. After confirming the removal of 9-fluorenylmethoxy carbonyl protection from the resin, it was possible to proceed to the next step, the attachment of the ANB derivative (5-amino-2-nitrobenzoic acid). d) Substitution of 5-amino-2-nitrobenzoic acid on solid support:

[0389] The first step in the synthesis of the peptide was substitution of ANB on 1 g of resin. Before attaching the chromophore, the resin used for the reaction was washed with the following solvents: DMF, DCM and again DMF, after which the Fmoc- protection was removed from the functional group of the solid support. One cycle of the Fmoc- protection removal comprised the following steps: Removal of Fmoc- protection:

[0390] 20% piperidine in NMP 1 x 3 minutes; 20% piperidine in NMP 1 x 8 minutes. e) Washing:

[0391] DMF 3 x 2 minutes; IsOH 3 x 2 minutes; DCM 3 x 2 minutes. f) Chloranil test:

[0392] The resin with a free amino group was washed with 5% solution of A-methylmorpholine (NMM) in DMF, and then DMF. The procedure of removing the Fmoc- protection and the wash cycle were carried out in a Merrifield vessel. In a separate flask, ANB was dissolved in DMF, and TBTU, DMAP and finally diisopropylethylamine (DIPEA) were subsequently added in the following excess relative to polymer substitution: ANB / TBTU / DMAP / DIPEA, 3:3:2:6 N / N / N / N. The mixture prepared in this way was added to the resin and was stirred for 3 hours. The resin was filtered off under reduced pressure, washed with DMF, DCM and isopropanol, and the entire acylation procedure was repeated twice. To carry out subsequent reactions of attaching ANB to the resin, hexafluorophosphate-O-(7-azabenzotriazol-l-yl)- N,N,N',N'-tetramethyluronium (HATU) and then hexafluorophosphate-O-(benzotriazol-l- yl)-N,N,N',N'-tetramethyluronium (HATU) were used. In the last step, the resin was washed successively with DMF, DCM and isopropanol, and was air dried.

[0393] (g) Attachment of the C-terminal amino acid residue (Fmoc-Glu(OtBu)-OH) to ANB:

[0394] The corresponding amino acid derivative (9-fold molar excess relative to resin substitution) was dissolved in pyridine and was transferred to the flask comprising the resin with deposited ANB. The whole was cooled until the temperature of -15°C was reached (ice bath: 1 part by weight of NH4CI, 1 part by weight of NaNCh, 1 part by weight of ice). After the desired temperate was reached, POCE was added (in 1 : 1 ratio to the amount of amino acid derivative used) and the whole was stirred on a magnetic stirrer: for 20 minutes at -15°C, 30 minutes at room temperature and 6 hours at 40°C (oil bath). When the reaction was completed, the resin was filtered off under reduced pressure, washed with DMF and MeOH and left to dry.

[0395] In the next stage, the residue was attached in the P2 position (Fmoc-Gln(Trt)).

[0396] Every attachment of amino acid residue was preceded by washing the resin with DMF for 5 minutes. Diisopropylcarbodiimide was used as a coupling agent in subsequent attachments. The procedure was repeated twice.

[0397] After each acylation, a resin wash cycle was started and then the chloranil test was performed in order to monitor the attachment of the amino acid derivative to free amino acid groups of the resin.

[0398] Solvent wash cycle:

[0399] DMF 3 x 2 minutes; IsOH 3 x 2 minutes; DCM 3 x 2 minutes.

[0400] Chloranil test:

[0401] As a result of the performed tests, after the first two coupling procedures, the colour of the grains was first green and then grey, so it was necessary to perform another acylation, as a result of which the resin grains tested by the chloranil test were colourless. This evidenced the attachment of ANB to the TentaGel S RAM resin, and thus it was possible to proceed to the next step of peptide synthesis. h) Attachment of subsequent protected amino acid residues:

[0402] The resin together with the attached fragment ANB-Glu-Gln, located in the reactor, was washed with DMF, which was followed by deprotection of the Fmoc from the amino group in order to attach the protected amino acid derivative Pro.

[0403] Removal of Fmoc protection:

[0404] DMF 1 x 5 minutes; 20% piperidine in NMP 1 x 3 minutes; 20% piperidine in NMP 1 x 8 minutes.

[0405] Solvent wash cycle:

[0406] DMF 3 x 2 minutes; IsOH 3 x 2 minutes; DCM 3 x 2 minutes.

[0407] Chloranil test:

[0408] The chloranil test produced a positive result, which was evidenced by the green colour of the resin grains. Thus, it was possible to proceed to the next step - attachment of the amino acid residue Fmoc-Pro-OH.

[0409] Attachment of the amino acid derivative The process of coupling was preceded by washing the resin with DMF. The composition of the coupling mixture remained unchanged when attaching the protected serine residue.

[0410] After the end of each acylation, a solvent wash cycle was performed according to the specified procedure and then the chloranil test for the presence of free amino acid groups in the solution was performed.

[0411] Solvent wash cycle

[0412] DMF 3 x 2 minutes; IsOH 3 x 2 minutes; DCM 3 x 2 minutes.

[0413] Chloranil test:

[0414] During the test performed after the second acylation the resin grains were colourless, and thus it was possible to proceed to the next step of the synthesis i.e. the introduction of another protected amino acid derivative - Fmoc-Lys(Boc) and 2-aminobenzoic acid molecule. The coupling processes were performed according to the procedure discussed earlier.

[0415] Tests carried out after attaching the above-mentioned residues showed positive results - the resin grains were colourless.

[0416] 2. Removal of the peptide from the solid support

[0417] After the synthesis, the amide of ABZ- Lys-Pro-Gln-Glu-ANB-NFF peptide was removed from the solid support and simultaneously the side protection was removed using the mixture: TF A: phenol: water: TIPS (88:5:5:2, v / v / v / v) in a round-bottom flask on a magnetic stirrer.

[0418] After 3 hours, the content of the flask was filtered off under reduced pressure in sintered (Schott) funnels and washed with diethyl ether. The obtained sediment was centrifuged on a SIGMA 2K30 centrifuge (Laboratory Centrifuges) for 20 minutes. The precipitate obtained after centrifugation was dissolved in water by means of ultrasound and then it was subjected to lyophilisation. The remaining compounds according to the invention can be obtained in an analogous way.

[0419] The identity / characteristics of the novel compound according to the invention were confirmed using the HPLC analysis. The conditions of the HPLC analysis were as follows: RP Bio Wide Pore Supelco C8 column, 250 mm 4 mm, a phase system A 0.1% TFA in water, B: 80% acetonitrile in A), flow rate 1 mL / min, UV detection at 226 nm.

[0420] The analysis carried out confirmed that the compound according to the invention was obtained.

[0421] Confirmation of the identity / characteristics of the new compound according to the invention was carried out using HPLC analysis. The conditions for this HPLC analysis were as follows: RP Bio Wide Pore Supelco C8 column, 250 mm 4 mm, phase system A 0.1% TFA in water, B: 80% acetonitrile in A), flow rate 1 ml / min, UV detection at 226 nm.

[0422] The analysis performed confirmed the obtaining of the compound according to the invention. The results of the chromatographic analysis for the exemplary compounds described herein, as well as remaining compounds herein described are shown in Fig. 1 (panels I).

[0423] Example IB: Synthesis of compounds comprising pNA molecule as chromophore

[0424] In this example, the synthesis of another representative compound according to the present invention is presented, namely the compound: ABZ1-Lys2-Pro3-Gln4-Glu5-pNA6, i.e. a compound comprising an alternative chromophore molecule to the chromophore molecule used above in the form of the ANB-NFE molecule - as in the representative compound according to the invention - compound having formula 3. The remaining compounds according to the present invention can be synthesised in an analogous manner using the corresponding amino acid residues and chromophore molecules. The numerals in superscript indicate subsequent positions of residues in the compound according to the invention. The compounds according to the invention can be alternatively represented by an analogous formula without the indication of residue positions, which does not change the sequence of residues in the compounds according to the invention, as it remains unchanged.

[0425] 1. Obtaining a chromogenic peptide a) The first step of the synthesis was to obtain the chromogenic peptide, which was obtained by solid phase synthesis, on a solid support, using Fmoc / tBu chemistry, i.e. with the use of protections.

[0426] The compound having formula 3: ABZ1-Lys2-Pro3-Gln4-Glu5-pNA6, where ABZ stands for 2-aminobenzoic acid and pNA stands for para-nitroanilide, in this case Glu-pNA, which can be obtained by solid-phase chemical synthesis using the following amino acid derivatives. Boc-ABZ, Fmoc-Lys(Boc), Fmoc-Pro, Fmoc-Gln(Trt), Glu-pNA.

[0427] The synthesis of a compound that can be used as a diagnostic marker for the detection of pancreatic cancer, especially in combination with the other compounds described herein, can be carried out on a solid support, namely 2-chlorochlorotrityl resin.

[0428] The synthesis of the compound was carried out manually using a laboratory shaker. In most of the steps a 25 mL sintered syringe for solid phase synthesis was used as a reactor.

[0429] All the final compounds obtained contain in their sequence at position 1, i.e. at the N- terminus, 2-aminobenzoic acid (ABZ), and at position 6, i.e. at the C-terminus, para nitroanilide (pNA). The synthesis involving the attachment of amino acid derivatives is carried out from residue 6 to 1, i.e. from the C-end to the N-end. (b) Substitution of Fmoc-Gln(Trt) on 2-chlorochlorotrityl resin:

[0430] The synthesis of peptides can be performed on Rapp Polymere's 2-chlorochlorotrityl resin. In the first step, the resin is prepared, including resin swelling through the wash cycle, peptide Solvent wash cycle:

[0431] DMF 1 x 10 minutes; IsOH 1 x 10 minutes; DCM 1 x 10 minutes.

[0432] Removal of Fmoc protection:

[0433] DMF 1 x 5 minutes; 20% piperidine in NMP 1 x 3 minutes; 20% piperidine in NMP 1 x 8 minutes.

[0434] Solvent wash cycle:

[0435] DMF 3 x 2 minutes; IsOH 3 x 2 minutes; DCM 3 x 2 minutes.

[0436] (c) Chloranil test:

[0437] The chloranil test involves transferring, using a spatula, a few grains of resin from a reactor - a syringe - into a glass ampoule, to which 100 pl of a saturated solution of p-chloranil in toluene and 50 pl of fresh acetaldehyde are then added. After 10 minutes, a check on the colour of the grains is carried out.

[0438] At that stage, after performing the test, a green colour of the grains is obtained, which evidences the presence of free amino groups. After confirming the removal of 9-fluorenylmethoxycarbonyl protection from the resin, it is possible to proceed to the next step, the attachment of the Fmoc-Gln(Trt) derivative.

[0439] (d) Substitution of Fmoc-Gln(Trt) on a solid support:

[0440] The first step in the synthesis of the peptide is the substitution of Fmoc-Gln(Trt) on 1 g of resin. Before attachment of the amino acid derivative, the resin used for the reaction must be washed with the following solvents: DMF, DCM and again DMF, followed by removal of the Fmoc- protection from the support functional group. One cycle of Fmoc- protection removal includes the following steps:

[0441] Removal of the Fmoc-protection:

[0442] 20% piperidine in NMP 1 x 3 minutes; 20% piperidine in NMP 1 x 8 minutes.

[0443] (e) Washing

[0444] DMF 3 x 2 minutes; IsOH 3 x 2 minutes; DCM 3 x 2 minutes.

[0445] (f) Chloranil test:

[0446] All attachments of amino acid residues were preceded by washing the resin with DMF for 5 minutes. In subsequent attachments diisopropylcarbodiimide is used as a coupling agent. The procedure is repeated twice. After each acylation, a cycle of resin washes is initiated, followed by a chloranil test to monitor the attachment of the amino acid derivative to the free amino groups of the resin. Solvent wash cycle:

[0447] DMF 3 x 2 minutes; IsOH 3 x 2 minutes; DCM 3 x 2 minutes.

[0448] Chloranil test:

[0449] As a result of the tests, after the first two couplings, the colour of the grains may be green and then grey, so that a further acylation is then necessary, resulting in colourless grains of the resin tested by the chloranil test. This indicates the attachment of Fmoc-Gln(Trt) to the 2-chloro-chlorotrityl resin, allowing the next step of peptide synthesis to proceed.

[0450] (h) Attachment of further protected amino acid residues:

[0451] The resin with the attached amino acid Fmoc-Gln(Trt) - located in the reactor is washed with DMF and then proceeds to deprotect the Fmoc protection from the amino group in order to attach the protected amino acid derivative Pro.

[0452] Removal of Fmoc protection:

[0453] DMF 1 x 5 minutes; 20% piperidine in NMP 1 x 3 minutes; 20% piperidine in NMP 1 x 8 minutes.

[0454] Solvent wash cycle:

[0455] DMF 3 x 2 minutes; IsOH 3 x 2 minutes; DCM 3 x 2 minutes.

[0456] Chloranil test:

[0457] As a result of the chloranil test, obtaining an positive result, as indicated by the green colouration of the resin grains, allows the next step - the attachment of the amino acid residue Fmoc-Pro-OH - to begin.

[0458] Attachment of an amino acid derivative:

[0459] The couplings performed are preceded by washing the resin in DMF. During attachment of the protected proline residue, the composition of the coupling mixture remained unchanged. After the completion of each acylation, a cycle of solvent washes is applied according to the procedure given, followed by the chloranil test for the presence of free amine groups in the solution.

[0460] Solvent wash cycle:

[0461] DMF 3 x 2 minutes; IsOH 3 x 2 minutes; DCM 3 x 2 minutes.

[0462] Chloranil test:

[0463] The resin grains when tested after the second acylation if colourless, we can proceed to the next step of the synthesis, which will be the introduction of another protected amino acid derivative, Fmoc-Lys(Boc), and a 2-aminobenzoic acid molecule. The coupling processes followed the procedure discussed earlier.

[0464] 2. Removal of the peptide from the with preservation of the side group protection

[0465] After completion of the synthesis, the ABZ-Lys(Boc)-Pro-Gln(Trt)-OH peptide is removed from the support without removing the side protection using a mixture of: acetic acid:trifluoroethanol:dichloromethane 2:2:6 (v / v / v / v / v) in a round-bottomed flask on a magnetic stirrer.

[0466] After 2 hours, the contents of the flask are filtered off under reduced pressure on Schott sintered funnels, the filtrate obtained is washed several times with hexane using a vacuum evaporator. The product obtained is lyophilised with tert-butanol.

[0467] 3. Coupling of para-nitroanilide to the protected peptide (ABZ-Lys(Boc)-Pro-Gln(Trt)-OH+Glu-pNA)

[0468] The coupling of the protected peptide ABZ-Lys(Boc)-Pro-Gln(Trt)-OH to para-nitroanilide- Glu is carried out in solution using the coupling agents DEBPT and DIPEA in ratios of protected peptide:Glu-pNa:DEBPT:DIPEA 1 : 1,1 :2:3 (w:w:w:v). The reaction is carried out in DMF at 27°C. At the end of the process, the solvent is evaporated under reduced pressure and the product obtained is treated with a mixture of TFA : phenol : water : TIPS, in a ratio of 88 : 5 : 5 : 2, N / N / N / N, to remove the side group protections. After detachment of the peptide, the precipitate obtained is filtered off, centrifuged, dissolved in water by means of ultrasound and then lyophilisation. Confirmation of the identity / characteri sties of the new compound is carried out using HPLC analysis. The conditions for this HPLC analysis are as follows: RP Bio Wide Pore Supelco C8 column, 250 mm 4 mm, phase system A 0.1% TFA in water, B: 80% acetonitrile in A), flow rate 1 ml / min, UV detection at 226 nm.

[0469] The analysis performed confirms that the compound identified here was obtained (ABZ1- Lys2-Pro3-Gln4-Glu5-pNA6)-pNA.

[0470] Example 2: Testing the properties of compounds and combinations according to the invention as cancer biomarkers

[0471] Testing the activity of individual representative substrates 1-7, i.e. the following compounds: 1) ABZ-Gly-Thr-Lys-Asn-ANB-NH2, 2) AZB-Trp-Thr-Glu-Ala-ANB-NH2, 3) AZB- Lys- Pro-Gln-Glu-ANB-NH2, 4) AZB-Glu-Met-Phe-Phe-ANB-NH2, 5) AZB-Thr-Thr-Ala-Arg- ANB-NH26) ABZ-Gln-Trp-Phe-Ala-ANB-NH2and 7) ABZ-Glu-Met-Lys-Phe-ANB-NH2, was performed as described above on a group of 20 patients diagnosed with pancreatic cancer individually and in combination, including compounds having formulas 8-11. Compounds having formulas 8-11 have an analogous structure to compounds of formulas 1- 7, following the general formula Xl-tetrapeptide-X2, differing only in the sequence of the tetrapeptide, namely compound having formula 8 comprises the 4-amino acid sequence Ile- Ile-Lys-Ile (SEQ ID NO: 8 according to the sequence listing), the compound having formula 9 comprises the 4-amino acid sequence Met-Ala-Trp-His (SEQ ID NO: 9 according to the sequence listing), the compound having formula 10 comprises the 4-amino acid sequence Gln-Glu-Gln-Leu (SEQ ID NO: 10 according to the sequence listing), and the compound having formula 11 comprises the 4-amino acid sequence Ala-Phe-Val-Val (SEQ ID NO: 11 according to the sequence listing). The mechanism of action of the compounds according to the invention, including the representative compounds according to the invention having formulas 1-7, as well as the compounds having formulas 8-11, consists in a specific enzymatic cleavage, more precisely an enzymatic hydrolysis, at such a site that leads to the release of free chromophore molecules, e.g. ANB-NEE (5-amino-2-nitrobenzoic acid amide), or pNA (para-nitroanilide) in the case of compounds comprising para-nitroanilide (pNA) as the C2 molecule, which show absorbance at 320-480 nm, especially 380-430 nm, in particular 410 nm. For this purpose, representative compounds according to the invention (compounds having formulas 1-7 as defined herein) and compounds having formulas 8-11 as defined herein were dissolved in dimethyl sulfoxide (at a concentration of 0.5 mg / mL) and then 20 pL of such solution was mixed with 100 pL of a suitable measurement buffer (see Table 36 below) and 80 pL of urine from a pancreatic cancer patient. The measurement was performed in a 96-well plate designed for absorbance measurement, and each sample was analysed in triplicate at 37 °C. The measurement duration was 120 minutes. During the measurement, the wavelength characteristic of the releasing chromophore (ANB-NFE ) was monitored at 410 nm (range 380-430 nm). Measurements with other compounds described herein, i.e. comprising other chromophore molecules, are carried out in the same way. The results are shown in Fig. 1 A-E (panels II). As shown in Fig. 1 A-E (panels II), the RP HPLC analysis of randomly selected samples comprising urine from a person diagnosed with pancreatic cancer and the test substrate indicates that compounds having formulas 1-7 dissociate, i.e. hydrolyse, into the corresponding peptide fragment and chromophore group of the compound (in this case ANB-NFE).

[0472] As a result of the measurement, an increase in solution colour over time was observed in the majority of urine samples from persons diagnosed with pancreatic cancer when one of substrates 1-7 was used as described above. A summary of the diagnostic parameters for the individual substrates is shown in Tables 37-43, with Tables marked as A showing a summary of the results obtained for the test substrate (the compound described herein) and Tables marked as B showing the values obtained and specificities for the test substrate (the compound described herein). The results obtained for the individual substrates are also shown in Fig. 2 A-K.

[0473] The use of any of the substrates 1-4 and 6-7, results in high specificity values (100%) against both pancreatic cancer and inflammation of this organ. Unfortunately, sensitivity reaches a range of values from 55 to 90%, which is not satisfactory. For substrate 5, such as the compound having formula 5, the situation is the opposite. It is characterised by a low specificity, as we obtain an positive result also for most samples with pancreatitis, resulting in a specificity of only 55%. Its sensitivity in the tested material is 95% and is the highest of all the compounds tested.

[0474] Given the results obtained for the above individual substrates (diagnostic biomarkers), studies of their properties in combinations, such as those comprising the 3 substrates described herein, were also carried out using representative compounds having formulas 1- 11, in order to verify whether their use in combination would allow to increase specificity of the detection of pancreatic cancer, as well as to effectively differentiate from other pancreatic diseases such as inflammation.

[0475] The results of the activity tests carried out on combinations of compounds, using representative compounds (substrates) described herein, namely compounds according to the invention having formulas 1-4 and compounds having formulas 5-11 (combinations including 3 of these compounds), show that certain of these combinations, i.e. combinations of compounds comprising as the tetrapeptide core 4-amino acid sequences having SEQ ID NOS: 1-7 (according to the sequence listing), show a synergistic effect and allow for an increased sensitivity and specificity for the detection of pancreatic cancer relative to single biomarkers. Combinations with compounds having formulas 8-11 did not show such an effect, and their specificity was found to be strongly unsatisfactory. A summary of the diagnostic parameters for the substrate combinations tested is presented in Tables 44-76, with Tables marked as A showing a summary of the results obtained for the substrate combination tested, and Tables marked as B showing the values and specificities obtained for the substrate combination tested.

[0476] As indicated in Tables 44-76, the use of any combination of the three substrates 1-7 described herein, preferably compounds having formulas 1-7 (e.g. combinations of compounds having formulas 1, 2 and 3) in independent measurements of the same urine sample, and assuming that a positive result is one where for the three substrates tested for two an increase in colour is observed, all urine samples of persons diagnosed with pancreatic cancer were diagnosed as positive. When a combination of substrates 1-7, preferably compounds having formulas 1-7, is used, a synergistic effect is observed if any three of them can be used in the analysis process. For example, 1, 2, 4 or 3, 5, 1. Such combinations provide a sensitivity of such a test of 95-100% and a specificity of 100% (Tables 44-76, in particular Tables marked as B). The observed increase in absorbance over time is different for each of the samples tested. A different effect was obtained for 10 samples from healthy persons, as no increase in absorbance in the diagnostic range was observed in any of 10 urine samples tested. A similar effect was observed when measuring samples from persons diagnosed with pancreatitis. Only individual substrates were efficiently cleaved (e.g. substrate 3 or 5). The other two substrates did not hydrolyse (substrate 1, 2 or 4). In contrast, the use of any of the compounds having formulas 8-11 (which are examples of compounds unsuitable for the diagnosis of pancreatic cancer according to the present invention): 8: ABZ-Ile-Ile-Lys-Ile-ANB-NH2, 9: ABZ-Met-Ala-Trp-His-ANB-NH2, 10: ABZ-Gln-Glu-Gln-Leu-ANB-NH2, 11 : ABZ-Ala- Phe-Val-Val-ANB-NH2in combination with two compounds from the group of compounds having formulas 1-7 results in an increase in colour being observed for only one of the substrates and consequently the result obtained is NEGATIVE, confirming that the effect obtained with combinations of substrates 1-7, such as compounds having formulas 1-7, is not additive and that compounds having formulas 8-11 are not suitable for use according to the invention.

[0477] The studies carried out indicate that the majority of samples 1-20 from pancreatic cancer patients resulted in the cleavage of the substrates tested, i.e. substrate 1 (specificity 80%), substrate 2 (specificity 85%), substrate 3 (specificity 55%), substrate 4 (specificity 90%), substrate 5 (specificity 95%), substrate 6 (specificity 90%), substrate 7 (specificity 90%), substrate 8 (specificity 15%), substrate 9 (specificity 50%), substrate 10 (specificity 18%) and substrate 11 (specificity 30%) (Tables 36-76, especially Tables marked B). This means that the compounds having formulas 8-11 are not suitable for use in the combinations according to the invention, as their specificity is insufficient to effectively diagnose pancreatic cancer according to the present invention. For samples from healthy persons, no increase in absorbance over time was observed for any of the analysed substrates. Urine samples from persons diagnosed with pancreatitis resulted in a cleavage of compounds and a consequent increase in absorbance for substrate 5 (60%), and compounds 8 (20%), 9 (20%), 10 (20%) and 11 (30%) being examples of compounds unsuitable for the combination of the three substrates according to the invention.

[0478] The scheme of the study was as follows: 1. Reagents

[0479] Table 36. Test reagents used: substrates and measurement buffers

[0480] 2. Realisation of the study The following reaction mixture was used in the study: 20 pl of substrate + 100 pl of measurement buffer+ 80 pl of urine sample. The test was carried out in a standard 96-well plate in triplicate for each urine sample and using different combinations of 3 substrates, using representative compounds having formulas 1-5 as substrates, respectively.

[0481] In the case of the use of any combination of 3 substrates from among those described herein, as shown for representative substrates 1-7, i.e. compounds having formulas 1-7, a synergistic effect is observed (for example the combination of compounds having formulas 1, 2 and 4 or 3, 5 and 1). Combinations of any 3 compounds of compounds having formulas 1-7 provide a sensitivity of such a test of 95-100% and a specificity of 100% (See Tables 44-76). Combinations of the compounds according to the invention with compounds having formulas 8-11 do not result in satisfactory sensitivity and specificity, and are therefore not suitable for use as diagnostic biomarkers, more specifically for the diagnosis of pancreatic cancer (See Tables 77-80). The following tables show the results of the test carried out by the method according to the invention using samples from persons with pancreatic cancer (samples 1-20), from healthy persons (samples Z1-Z10) and from persons with pancreatitis (inflammation 1 - inflammation 10) and individual compounds having formulas 1-7, wherein the tables marked as A show the results obtained for the test substrate (compound described herein) and B: the obtained values and specificities for the test substrate (compound described herein). means that the parameter is not read - we do not expect false positive or true negative results in the group of patients with pancreatic cancer, and we do not expect true positive or false negative results in the group of patients without pancreatic cancer. Table 37. Test results for a representative substrate 1 according to the invention - compound having formula 1

[0482] A

[0483]

[0484] B Table 38. Test results for representative substrate 2 - compound having formula 2

[0485] A

[0486]

[0487] B

[0488] Table 39. Test results for representative substrate 3 - compound having formula 3

[0489] A

[0490]

[0491] Table 40. Test results for representative substrate 4 - compound having formula 4

[0492] A

[0493]

[0494] B Table 41. Test results for representative substrate 5 - compound having formula 5

[0495] A

[0496]

[0497] B

[0498] Table 42. Test results for representative substrate 6 - compound having formula 6

[0499] A B

[0500] Table 43. Test results for representative substrate 7 - compound having formula 7

[0501] A

[0502]

[0503] B

[0504] The following Tables 44-76 show the results obtained for combinations of test compounds having formulas 1-7 using samples from persons with pancreatic cancer (samples 1-20), from healthy persons (samples Zl-Z 10) and from persons with pancreatitis (inflammation 1 - inflammation 10), with Tables marked as A showing the results obtained for the substrate combination tested, and B: the values obtained and specificities for the substrate combination tested. Table 44. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 1, 2 and 3

[0505] A

[0506] B

[0507] Table 45. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 1, 3 and 4 A

[0508] B

[0509] Table 46. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 1, 2 and 4 A

[0510]

[0511] B

[0512] Table 47. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 3, 4 and 2:

[0513] A

[0514]

[0515] B Table 48. Results obtained for a representative combination according to the invention -the combination of compounds having formulas 1, 2 and 5

[0516] A

[0517] B

[0518] Table 49. Results obtained for a representative combination according to the invention - a combination of compounds having formulas 1, 3 and 5 A

[0519] Table 50. Results obtained for a representative combination according to the invention - a combination of compounds having formulas 5, 4 and 2 A

[0520]

[0521] B Table 51. Results obtained for a representative combination according to the invention - a combination of compounds having formulas 3, 5 and 2

[0522] A

[0523] B

[0524] Table 52. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 3, 4 and 5 A B

[0525] Table 53. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 3, 4 and 6 A

[0526]

[0527] B Table 54. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 3, 4 and 7

[0528] A

[0529] B

[0530] Table 55. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 1, 2 and 6 A

[0531] Table 56. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 1, 2 and 7 A

[0532]

[0533] B Table 57. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 1, 3 and 6

[0534] A

[0535] B

[0536] Table 58. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 1, 3 and 7 A

[0537] B

[0538] Table 59. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 1, 4 and 6 A

[0539]

[0540] B Table 60. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 1, 4 and 7

[0541] A

[0542] B

[0543] Table 61. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 1, 5 and 6 A

[0544] B

[0545] Table 62. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 1, 5 and 7 A B

[0546] Table 63. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 2, 3 and 6 A

[0547]

[0548] B Table 64. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 2, 3 and 7

[0549] A

[0550] B

[0551] Table 65. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 2, 4 and 6 A

[0552] Table 66. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 2, 4 and 7 A

[0553]

[0554] B Table 67. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 2, 5 and 6

[0555] A

[0556] B

[0557] Table 68. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 2, 5 and 7 A

[0558] Table 69. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 1, 6 and 7 A

[0559]

[0560] B Table 70. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 3, 6 and 7

[0561] A

[0562] B

[0563] Table 71. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 4, 6 and 7 A

[0564] B

[0565] Table 72. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 2, 6 and 7

[0566] A

[0567]

[0568] B Table 73. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 3, 5 and 6

[0569] A

[0570] B

[0571] Table 74. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 3, 5 and 7 A

[0572] Table 75. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 4, 5 and 6 A

[0573]

[0574] B Table 76. Results obtained for a representative combination according to the invention - the combination of compounds having formulas 4, 5 and 7

[0575] A

[0576] B

[0577] Tables 77-80 show the results obtained for combinations of compounds according to the invention with compounds having formulas 8-11. The results obtained for these combinations show that compounds having formulas 8-11 are not suitable, either individually or in combination, for use in accordance with the present invention.

[0578] Table 77. Results obtained for the combination of compounds having formulas 3, 4 and 8

[0579] A B

[0580] Table 78. Results obtained for the combination of compounds having formulas 3, 4 and 9.

[0581] A

[0582]

[0583] B

[0584] Table 79. Results obtained for the combination of compounds having formulas 1, 3 and 10

[0585] A B

[0586] Table 80. Results obtained for the combination of compounds having formulas 1, 2 and 11

[0587] A

[0588] B

[0589] As results from Tables 77-80 above, the results obtained for the combinations of compounds according to the invention with compounds having formulas 8-11, having an analogous structure according to the general formula (Xl-tetrapeptide-X2), differing only in the specific sequence of the tetrapeptide (sequences numbered 8-11, respectively), show a lack of satisfactory sensitivity (50-70%).

[0590] Table 81 below summarises the results obtained regarding sensitivity and specificity in the sample groups tested. Table 81. Summary of results obtained regarding sensitivity and specificity of individual compounds as well as their combinations

[0591] In summary, it has been shown that the compounds according to the invention allow an extremely high specificity for samples from patients who may be suspected of having pancreatic cancer, e.g., patients with inflammation of pancreas (pancreatitis), and the combinations according to the invention show a synergistic action in the detection of enzymatic activity specific for pancreatic cancer, allowing their use in a method for the detection of specific activity for pancreatic cancer and allowing a very efficient, highly sensitive and specific diagnosis of pancreatic cancer, as well as effective differentiation from other pancreatic diseases, such as pancreatitis, eliminating the risks of false positives that can occur with biomarkers known from the state of the art.

[0592] Example 3: Study of the selectivity of the cleavage of compounds according to the invention depending on the type of cancer

[0593] This example describes the performed tests for selectivity of the compounds according to the invention, using exemplary, representative compounds having formulas 1-7, depending on the type of cancer tested. The results of the performed tests are shown in Fig. 3 and they indicate that the tested compounds having formulas 1-7: 1) ABZ-Gly-Thr-Lys-Asn-ANB- NH2, 2) AZB-Trp-Thr-Glu-Ala-ANB-NH2, 3) AZB- Lys-Pro-Gln-Glu-ANB-NH2, 4) AZB-Glu-Met-Phe-Phe-ANB-NH2, 5) AZB-Thr-Thr-Ala-Arg-ANB-NH2, 6) ABZ-Gln-Trp- Phe-Ala-ANB-NH2and 7) ABZ-Glu-Met-Lys-Phe-ANB-NH2incubated with samples from patients diagnosed with cancers of specific organs: stomach, large intestine, kidney, prostate, bladder, lung, ovary, liver do not break down and do not cause an increase in absorbance in the indicated range. This means that the tested compounds allow a sensitive and specific detection of activity specific for pancreatic cancer, and thus specific diagnosis of pancreatic cancer, and an effective and selective detection of pancreatic cancer against other cancers. The samples tested in each case were a mixture of 20 samples obtained for each of the cancers tested. This indicates the selectivity of the cleavage of the compounds according to the invention, which makes them suitable for the specific detection of enzymatic activity specific for pancreatic cancer and the specific diagnosis of pancreatic cancer according to the present invention.

[0594] Example 4: Study of the dependence of the proteolytic activity of the compounds according to the invention on the reaction environment

[0595] This example describes the measurements carried out on the dependence of the proteolytic activity of the tested compounds (substrates of the enzymatic reaction analysed according to the invention), using exemplary, representative compounds having formulas 1-7, on the reaction environment. As a result of this experiment, it was found that there is at least one enzyme in the tested material showing maximum activity at alkaline pH (Fig. 4), and so the optimum pH value for substrate 1 is 8, for substrate 2 is 7.5, for substrate 3 is 8, for substrate 4 is 8, for substrate 5 is 7.5, for substrate 6 is 7.5, and for substrate 7 is 7.5. In conclusion, the studies and analyses carried out confirmed the usefulness of the compounds according to the invention and the combinations according to the invention in the sensitive and specific detection of enzymatic activity specific for pancreatic cancer, and thus their usefulness also for the diagnosis of pancreatic cancer, and also as a diagnostic marker for pancreatic cancer. It is noteworthy that the compounds according to the invention, and in particular the combinations according to the invention, allow not only an extremely sensitive and but also a highly specific diagnosis of pancreatic cancer, as well as allowing effective differentiation from other pancreatic diseases, and eliminating false positives that may occur with the compounds according to the state of the art for other pancreatic diseases, such as pancreatitis. Combinations of compounds according to the invention allow for a synergistic effect in this respect with respect to individual compounds. The mechanism of action of all compounds according to the invention consists in their specific enzymatic cleavage in such a position that leads to the release of free chromophore molecules, resulting in a measurable optical signal that can be used for diagnostic purposes, in particular for the detection of activity specific for pancreatic cancer, and in the diagnosis of pancreatic cancer according to the present invention.

[0596] SEQUENCE LISTING

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[0608] <InventionTitle languageCode="pl">Nowe zwiqzki i kombinacje zwiqzkow oraz ich zastosowania jako biomarkery diagnostyczne, zwlaszcza do wykrywania raka trzustki, sposob wykrywania aktywnosci enzymatycznej, sposob diagnozowania raka trzustki, zestawy zawierajqce takie zwiqzki i kombinacje, zastosowania takich zwiqzkow i kombinacji w diagnostyce i leczeniu raka trzustki < / InventionTitle>

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[0874] 10

Claims

Claims1. A compound having general formula:XI -tetrapeptide-X2 whereinXI comprises or consists of a molecule Cl, tetrapeptide means a peptide having a sequence of four amino acids selected from: Gly- Thr-Lys-Asn (SEQ ID NO: 1), Trp-Thr-Glu-Ala (SEQ ID NO: 2), Lys-Pro-Gln-Glu (SEQ ID NO: 3), Glu-Met-Phe-Phe (SEQ ID NO: 4), Gln-Trp-Phe-Ala (SEQ ID NO: 6) and Glu-Met-Lys-Phe (SEQ ID NO: 7),X2 comprises or consists of a molecule C2, wherein a pair of molecules Cl and C2 is a pair of a fluorescence donor and a fluorescence acceptor, and wherein the compound undergoes enzymatic cleavage into fragments 1 and 2: XI- tetrapeptide-OH (fragment 1) and X2 (fragment 2), respectively, with a generation of a measurable optical signal upon spatial separation of molecules Cl and C2.

2. The compound according to claim 1, wherein the compound undergoes hydrolytic cleavage, preferably proteolytic cleavage.

3. The compound according to claim 1 or 2, wherein the pair of molecules Cl and C2 is selected from the group consisting of: 2-aminobenzoic acid (ABZ) / 5-amino-2- nitrobenzoic acid (ANB), (ABZ) / pNA, ABZ / ANB-NH?, ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr(3-NO2), preferably the pair of Cl and C2 is ABZ / pNA or ABZ / ANB-NH2.

4. The compound according to any one of claims 1 to 3, wherein the compound is selected from the group consisting of: ABZ-Gly-Thr-Lys-Asn-ANB-NH2(formula 1), ABZ- Trp-Thr-Glu-Ala-ANB-NH2(formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), Gln-Trp-Phe-Ala (formula 6) and Glu-Met- Lys-Phe (formula 7).

5. The compound according to claim 4, wherein the compound undergoes hydrolytic cleavage with the generation of fragment 1 selected from: ABZ-Gly-Thr-Lys-Asn-OH, ABZ-Trp-Thr-Glu-Ala-OH, ABZ-Lys-Pro-Gln-Glu-OH, ABZ-Glu-Met-Phe-Phe-OH, ABZ-Gln-Trp-Phe-Ala-OH and ABZ-Glu-Met-Lys-Phe-OH, and fragment 2: ANB-NH2.

6. A combination of compounds, which combination comprises three compounds having general formula:XI -tetrapeptide-X2 whereinXI comprises or consists of a molecule Cl, tetrapeptide is a peptide having a sequence of four amino acids selected from: Gly-Thr- Lys-Asn (SEQ ID NO: 1), Trp-Thr-Glu-Ala (SEQ ID NO: 2), Lys-Pro-Gln-Glu (SEQ ID NO: 3), Glu-Met-Phe-Phe (SEQ ID NO: 4), Thr-Thr-Ala-Arg (SEQ ID NO: 5), Gln-Trp- Phe-Ala (SEQ ID NO: 6) and Glu-Met-Lys-Phe (SEQ ID NO: 7),X2 comprises or consists of a molecule C2, wherein a pair of molecules Cl and C2 is a pair of a fluorescence donor and a fluorescence acceptor, and wherein the compound undergoes enzymatic cleavage into fragments 1 and 2: XI- tetrapeptide-OH (fragment 1) and X2 (fragment 2), respectively, with a generation of a measurable optical signal upon spatial separation of molecules Cl and C2.

7. The combination according to claim 6, wherein the compounds undergo hydrolytic cleavage, preferably proteolytic cleavage.

8. The combination according to claim 6 or 7, wherein the pair of molecules Cl and C2 is selected from the group consisting of: 2-aminobenzoic acid (ABZ) / 5-amino-2-nitrobenzoic acid (ANB), (ABZ) / pNA, ABZ / ANB-NH2, ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr(3-NO2), preferably the pair of Cl and C2 is ABZ / pNA or ABZ / ANB-NH2.

9. The biomarker combination according to any one of claims 6-8, wherein the combination comprises 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn- ANB-NH2 (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NH2(formula 2), ABZ-Lys-Pro-Gln- G1U-ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Thr-Thr- Thr-Ala-Arg-ANB-NEE (formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NH2(formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NH2(formula 7).

10. The biomarker combination according to any one of claims 6-9, wherein the combination consists of 3 compounds selected from the following: ABZ-Gly-Thr-Lys-Asn-ANB-NH2(formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NEE (formula 2), ABZ-Lys-Pro-Gln-Glu-ANB- NH2 (formula 3), ABZ -Glu-Met-Phe-Phe- ANB -NH2 (formula 4), ABZ-Thr-Thr-Thr-Ala- Arg-ANB-NH2(formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NH2(formula 6) and ABZ-Glu- Met-Lys-Phe-ANB-NEE (formula 7).

11. An in vitro method for the detection of enzymatic activity present in an individual’ s body fluid sample, in particular deriving from pancreatic cancer cells, comprising: a) contacting the body fluid sample with the compound as defined in any one of the claims 1-5, or the combination of compounds as defined in one of the claims 6-10, and(b) detecting a measurable optical signal, which is generated upon spatial separation of molecules Cl and C2.

12. The in vitro method according to claim 11, wherein the enzymatic activity is hydrolytic activity, preferably proteolytic activity.

13. The in vitro method according to claim 11 or 12, wherein the combination comprising 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NH2 (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NJL (formula 2), ABZ-Lys-Pro-Gln-Glu-ANB- NH2 (formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NBL (formula 4), ABZ-Thr-Thr-Ala-Arg- ANB-NH2 (formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NTL (formula 6) and ABZ-Glu-Met- Lys-Phe-ANB-NH2 (formula 7), preferably the combination consisting of 3 of these compounds, is used.

14. The in vitro method according to any one of claims 11 to 13, wherein as the said body fluid urine, preferably human urine, is used.

15. The in vitro method according to any one of claims 11 to 14, wherein as the said measurement buffer a Tris buffer, or a buffer comprising Tris, NaCl and DMSO, is used.

16. An in vitro method for the diagnosis of pancreatic cancer, wherein the presence or absence of pancreatic cancer in an individual is detected by measuring enzymatic activity specific for pancreatic cancer in a body fluid sample of an examined individual using a compound as defined in one of claims 1 to 5 or a combination of compounds as defined in one of claims 6 to 10, wherein the absence of the said enzymatic activity indicates the absence of pancreatic cancer and the presence of the said enzymatic activity indicates the presence of pancreatic cancer.

17. The in vitro method according to claim 16, wherein the detection of enzymatic activity is carried out by the method as defined in any one of claims 11 to 15.

18. The in vitro method according to claim 16 or 17, wherein the said enzymatic activity is measured using the combination comprising 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NBL (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB- NH2 (formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NBL (formula 3), ABZ-Glu-Met-Phe-Phe- ANB-NH2 (formula 4), ABZ-Thr-Thr-Ala-Arg-ANB-NTB (formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NH2 (formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-Nfb (formula 7), preferably the combination consisting of 3 of these compounds.

19. The in vitro method according to any one of claims 16 to 18, wherein the said body fluid sample is incubated with the said compound in a measurement buffer having neutral pH or alkaline pH, preferably physiological pH, within the range of sample-to-measurement buffer ratio of 1 :2 to 1 : 10, preferably 1 :5.

20. The in vitro method according to any one of claims 16 to 19, wherein the said compound is used at a concentration of 0.1-10 mg / mL, in particular 0.25-7.5 mg / mL.

21. The in vitro method according to any one of claims 16 to 20, wherein as the said sample a urine sample, preferably human urine, is used.

22. The in vitro method according to any one of claims 16 to 21, wherein the measurement of the said enzyme activity comprises measuring the absorbance intensity in the range SOO- SOO nm, preferably 380-430 nm, in particular 410 nm, for a time period of 40-60 minutes, at a temperature in the range 25-40° C, preferably 36-38° C.

23. A kit comprising at least one compound as defined in any one of claims 1 to 5 or the combination as defined in any one of the claims 6 to 10 and a measurement buffer.

24. The kit according to claim 23, wherein the kit comprises the combination comprising 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NH2 (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NH2 (formula 2), ABZ-Lys-Pro-Gln-Glu-ANB- NH2 (formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2 (formula 4), ABZ-Thr-Thr-Ala-Arg- ANB-NH2 (formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NH2 (formula 6) and ABZ-Glu-Met- Lys-Phe-ANB-NH2 (formula 7), preferably the combination consisting of 3 of these compounds.

25. Use of the compound as defined in any one of the claims 1 to 5 or the combination as defined in any one of claims 6 to 10 for the detection of enzymatic activity specific for pancreatic cancer.

26. The compound as defined in any one of claims 1-5 for use in a method for the diagnosis of pancreatic cancer.

27. The use of the compound according to claim 25 or the compound for use according to claim 26, wherein as the said compound the compound selected from ABZ-Gly-Thr-Lys- Asn-ANB-NH2 (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NH2 (formula 2), ABZ-Lys-Pro- Gln-Glu-ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Gln- Trp-Phe-Ala-ANB-NH2 (formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NH2 (formula 7), is used.

28. The combination as defined in any one of claims 6-10 for use in a method for the diagnosis of pancreatic cancer.

29. The combination for use according to claim 28, wherein as the said combination the combination comprising 3 compounds selected from the following compounds: ABZ-Gly- Thr-Lys-Asn-ANB-NH2 (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-Nff (formula 2), ABZ- Lys-Pro-Gln-Glu-ANB-NH2 (formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NIU (formula 4), ABZ-Thr-Thr-Ala-Arg-ANB-NH2 (formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NIU (formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NTB (formula 7), preferably the combination consisting of 3 of these compounds, is used.

30. Use of the compound as defined in any one of claims 1-5 in an in vitro method for the diagnosis of pancreatic cancer.

31. The use according to claim 30, wherein as the said compound the compound selected from ABZ-Gly-Thr-Lys-Asn-ANB-NH2 (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NIU (formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NBU (formula 3), ABZ-Glu-Met-Phe-Phe-ANB- NH2 (formula 4), ABZ-Gln-Trp-Phe-Ala-ANB-NTU (formula 6) and ABZ-Glu-Met-Lys- Phe-ANB-NH2 (formula 7), is used.

32. Use of the combination as defined in any one of claims 6-10 in an in vitro method for the diagnosis of pancreatic cancer.

33. The use according to claim 32, wherein as the said combination the combination comprising 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn- ANB-NH2 (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NIU (formula 2), ABZ-Lys-Pro-Gln- Glu-ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Thr-Thr- Ala-Arg-ANB-NH2 (formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NIU (formula 6) and ABZ- Glu-Met-Lys-Phe-ANB-NH2 (formula 7), preferably the combination consisting of 3 of these compounds, is used.

34. The compound for use according to claim 26 or 27, the combination for use according to claim 28 or 29, the use of the compound according to claim 30 or 31, or use of the combination according to claim 32 or 33, wherein the diagnosis of pancreatic cancer comprises detection of primary pancreatic cancer, detection of residual disease after surgical resection of pancreatic cancer and / or detection of recurrence of pancreatic cancer.

35. The compound for use, the combination for use, the use of the compound, or the use of the combination according to claim 34, wherein the combination for use comprises 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NTU (formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NIU (formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Thr-Thr-Ala-Arg- ANB-NH2 (formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NH2(formula 6) and ABZ-Glu-Met- Lys-Phe-ANB-NH2(formula 7), preferably the combination consists of 3 of these compounds.

36. The compound as defined in any one of claims 1 to 5 or the combination as defined in any one of claims 6 to 10 for use as a diagnostic marker for the detection of pancreatic cancer.

37. The compound or combination for use according to claim 36, wherein the said combination is a combination comprising 3 compounds selected from the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NH2(formula 1), ABZ-Trp-Thr-Glu-Ala-ANB- NH2(formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe- ANB-NH2(formula 4), ABZ-Thr-Thr-Ala-Arg-ANB-NH2(formula 5), ABZ-Gln-Trp-Phe- Ala-ANB-NH2(formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NH2(formula 7), preferably the combination consisting of 3 of these compounds.

38. A method for the treatment of pancreatic cancer, wherein(a) the presence of enzymatic activity specific for pancreatic cancer as defined in any one of claims 11-15 is detected in a body fluid sample from an examined individual, and(b) when the said enzymatic activity is found in the said sample, treatment of pancreatic cancer is applied to the individual.

39. The method according to claim 38, wherein after completion of the treatment in accordance with point b), monitoring of the said enzymatic activity specific for pancreatic cancer is carried out at predetermined time intervals.

40. The method according to claim 38 or 39, characterised in that as the said sample a urine sample, preferably human urine, is used.

41. The method according to any one of claims 38-40, wherein in step (a) the compound selected from the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NH2(formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NH2(formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NH2(formula 3) and ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Gln-Trp-Phe-Ala-ANB-NH2(formula 6) and ABZ-Glu-Met-Lys-Phe-ANB-NH2(formula 7), or the combination comprising 3 of the following compounds: ABZ-Gly-Thr-Lys-Asn-ANB-NH2(formula 1), ABZ-Trp-Thr-Glu-Ala-ANB-NH2(formula 2), ABZ-Lys-Pro-Gln-Glu-ANB-NH2(formula 3), ABZ-Glu-Met-Phe-Phe-ANB-NH2(formula 4), ABZ-Thr-Thr-Ala-Arg-ANB-NH2(formula 5), ABZ-Gln-Trp-Phe-Ala-ANB-NH2(formula 6) and ABZ-Glu-Met-Lys-Phe- ANB-NH2(formula 7), preferably the combination consisting of 3 of these compounds, is used.

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