Compound-diagnostic marker for biliary tract cancer, method for detecting enzyme activity, method for diagnosing biliary tract cancer, kit comprising compound, use of compound, and method for treating biliary tract cancer

Abstract

The present invention relates to a novel compound-a diagnostic marker for medicine, more particularly for the diagnosis of cancer, in particular for the diagnosis of biliary tract cancer. The invention also relates to an in vitro method for detecting the activity of an enzyme present in a body fluid of a subject, in particular the activity of an enzyme derived from biliary tract cancer cells, using the compounds. The invention also relates to an in vitro method for diagnosing the biliary tract cancer by using the compound, a kit containing the compound, application of the compound for detecting the specific enzyme activity of the biliary tract cancer and application of the compound for diagnosing the biliary tract cancer. The present invention also relates to a compound for use as a diagnostic marker for biliary tract cancer and a method for treating biliary tract cancer, the method comprising the step of implementing the method for diagnosing biliary tract cancer as defined above using the compound.

Description

[0001] The present invention relates to a novel compound, a diagnostic marker, for use in medicine, more specifically for cancer diagnosis, particularly biliary tract cancer. The present invention also relates to in vitro methods for using the compound to detect enzyme activity present in a subject's body fluid, particularly enzyme activity derived from biliary tract cancer cells, in vitro methods for diagnosing biliary tract cancer using the compound, kits containing the compound, use of the compound for detecting biliary tract cancer-specific enzyme activity, use of the compound in diagnosing biliary tract cancer, and use of the compound as a diagnostic marker for biliary tract cancer. The present invention also relates to a method for treating biliary tract cancer, comprising implementing the steps of the method for diagnosing biliary tract cancer described above. Background Art

[0002] Biliary tract cancer is a relatively rare cancer worldwide. In 2020, 116,000 new cases were reported. The most important disease etiologies that play a role in the pathogenesis of biliary tract cancer include gallstones, chronic inflammation, gallbladder polyps, obesity, toxic exogenous factors, and genetic factors. Biliary tract cancer often develops asymptomatically, and when symptoms do occur, they are atypical and nonspecific. On laboratory testing, elevated serum bilirubin, alkaline phosphatase, gamma-glutamyl transpeptidase, and transaminase activities are observed. Cancer markers in the blood are also frequently elevated, mainly cancer antigen 19-9 and carcinoembryonic antigen. Biliary tract cancer is classified as a cancer with a poor prognosis, especially if the diagnosis is made at an advanced stage.

[0003] If bile tract cancer is suspected, extensive imaging studies are necessary. The primary test, which is important, inexpensive, and safe for the patient, is an abdominal ultrasound to evaluate the gallbladder and bile ducts. Complementary tests include abdominal CT scans, MRI, endoscopic retrograde cholangiopancreatography, and magnetic resonance cholangiopancreatography. Currently, no test is available for the early detection of bile tract cancer. It is well known that the initiation, growth, and spread of cancer cells involve many factors, including numerous enzymes, particularly hydrolases, and especially proteolytic enzymes. These enzymes catalyze the enzymatic cleavage (hydrolysis or proteolysis) of proteins and peptides into smaller fragments. This process enables cancer cells to expand by colonizing new tissues and enhances the process of blood vessel formation (angiogenesis), which allows for the efficient delivery of nutrients to the tumor. Furthermore, these enzymes are activated as a result of the death of healthy cells caused by tumor growth. All of these processes contribute to the characteristic and specific profile of enzymatic (proteolytic) activity in cancer cells, which is characteristic of tumors.

[0004] In this field, it is known that chromogenic peptide molecules can be broken down into smaller fragments by enzymes, resulting in a change or increase in the color of the test solution. This color effect is the result of the release of chromophores (such as 4-nitroaniline or 2-aminobenzoic acid) from the chromogenic peptide molecules.

[0005] For example, Erlanger BF, Kokowsky N, Cohen W. 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. introduce such chromogenic molecules and their uses.

[0006] However, the use of this class of compounds in the diagnosis of cancerous biliary tract has not been described so far.

[0007] Methods for obtaining chromogenic peptides are also known in the prior art, which include connecting individual components under appropriate time and stoichiometric conditions. The connection process includes subsequent steps, in which the individual elements (amino acid derivatives) are connected, the residues are washed off, the protecting groups are removed in sequence and washed again. This cycle is repeated for each amino acid residue. The obtained peptide is separated from the resin by reaction under acidic conditions. Subsequently, the solution is separated from the resin in a filtration process, and the peptide is then precipitated from the solution by a non-polar solvent.

[0008] However, chromogenic peptide compounds suitable for specific and early diagnosis of biliary tract cancer or methods for obtaining them are not known in the prior art.

[0009] Therefore, in this field, there is an urgent need for a "cancer marker" for biliary tract cancer that will enable early, sensitive, and specific diagnosis of biliary tract cancer in a non-invasive and reliable manner, as well as diagnostic methods and treatment methods using such a diagnostic marker.

[0010] The object of the present invention is to provide a new specific diagnostic marker for biliary tract cancer and a diagnostic method using this marker for non-invasive, rapid, sensitive and specific early detection of biliary tract cancer, which is also suitable for screening tests, as well as a therapeutic method using this marker.

[0011] These objects have been achieved by the invention defined in the accompanying patent claims, while preferred variants thereof are defined in the dependent claims. Summary of the Invention

[0012] The present invention provides a compound having formula 1:

[0013] X1 1 -Glu 2 -Arg 3 -Arg 4 -Ala5-X2 6 (Formula 1),

[0014] wherein X1 comprises or consists of the molecule C1, and X2 comprises or consists of the molecule C2,

[0015] The molecular pair C1 and C2 are a pair of fluorescence donor and fluorescence acceptor,

[0016] And wherein the compound is cleaved by an enzyme into fragments X1-Glu-Arg-Arg-Ala-OH (fragment 1) and X2 (fragment 2), and a measurable light signal is generated when molecules C1 and C2 are spatially separated.

[0017] The compounds of the invention are preferably cleaved hydrolytically, more preferably proteolytically.

[0018] Preferably, in the compounds according to the present invention, the molecular pair C1 and C2 is selected from: 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 C1 and C2 pair is (ABZ) / pNA or ABZ / ANB-NH2.

[0019] Preferably, the compound of the present invention is a compound having Formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-Glu-Arg-Arg-Ala-pNA (Formula 3).

[0020] More preferably, the compounds of the present invention undergo hydrolytic cleavage to produce the following fragment 1: ABZ-Glu-Arg-Arg-Ala-OH and fragment 2: ANB-NH2.

[0021] The present invention also provides an in vitro method for detecting enzyme activity present in a subject's body fluid, particularly enzyme activity derived from biliary cancer cells, comprising:

[0022] a) contacting a body fluid sample with a compound of formula 1:

[0023] X1 1 -Glu 2 -Arg 3 -Arg 4 -Ala 5 -X2 6 (Formula 1),

[0024] wherein X1 comprises or consists of the molecule C1, and X2 comprises or consists of the molecule C2,

[0025] The pair of molecules C1 and C2 are a pair of fluorescence donor and fluorescence acceptor,

[0026] and wherein the compound is cleaved by an enzyme into fragments X1-Glu-Arg-Arg-Ala-OH (fragment 1) and X2 (fragment 2), and

[0027] b) A measurable light signal is generated when the detection molecules C1 and C2 are spatially separated.

[0028] In the method for detecting enzyme activity according to the present invention, the enzyme activity is preferably hydrolytic activity, more preferably proteolytic activity.

[0029] In the method for detecting enzyme activity according to the present invention, it is preferred to use a compound of Formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 (Formula 2) or a compound of Formula 3: ABZ-Glu-Arg-Arg-Ala-pNA (Formula 3) as the compound.

[0030] In the method for detecting enzyme activity according to the present invention, urine is preferably used as the body fluid, and human urine is more preferably used.

[0031] The present invention also relates to an in vitro method for diagnosing biliary tract cancer, wherein the presence or absence of biliary tract cancer in a subject is detected by measuring the activity of a biliary tract cancer-specific enzyme in a body fluid sample from the subject being examined, and wherein the absence of the enzyme activity indicates the absence of biliary tract cancer, while the presence of the enzyme activity indicates the presence of biliary tract cancer.

[0032] In the method for detecting / diagnosing biliary tract cancer according to the present invention, the detection of enzyme activity is performed by the enzyme activity detection method defined above.

[0033] In the method for detecting / diagnosing biliary tract cancer according to the present invention, the enzyme activity is measured using a compound having Formula 1:

[0034] X1 1 -Glu 2 -Arg 3 -Arg4 -Ala 5 -X2 6 (Formula 1),

[0035] wherein X1 comprises or consists of the molecule C1, and X2 comprises or consists of the molecule C2,

[0036] The molecular pair C1 and C2 are a pair of fluorescence donor and fluorescence acceptor,

[0037] And wherein the compound undergoes enzymatic cleavage into fragments X1-Glu-Arg-Arg-Ala-OH (fragment 1) and X2 (fragment 2), and a measurable light signal is generated when molecules C1 and C2 are spatially separated.

[0038] In the detection / diagnosis method of biliary tract cancer according to the present invention, the body fluid sample and the compound are preferably incubated in a measurement buffer having a neutral or alkaline pH, more preferably a physiological pH, in a ratio of sample to measurement buffer in the range of 1:2 to 1:10, preferably 1:5.

[0039] In the method for detecting / diagnosing biliary tract cancer according to the present invention, the compound is preferably used at a concentration of 0.1-10 mg / mL, particularly 0.25-7.5 mg / mL.

[0040] In the detection / diagnosis method of biliary tract cancer according to the present invention, as the compound, it is preferred to use a compound having formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 (Formula 2) or a compound having formula 3: ABZ-Glu-Arg-Arg-Ala-pNA (Formula 3).

[0041] In the method for detecting / diagnosing biliary tract cancer according to the present invention, as the sample, preferably a urine sample is used, more preferably human urine is used.

[0042] In the method for detecting / diagnosing biliary tract cancer according to the present invention, the measurement of the enzyme activity preferably includes measuring the absorption intensity in the range of 300-500nm, more preferably 380-430nm, and in particular 405nm at a temperature in the range of 25-40°C, more preferably 36-38°C during 40-60 minutes.

[0043] The present invention also provides a kit comprising any compound of the present invention as described above and a measurement buffer.

[0044] In the kit of the present invention, the compound is preferably a compound having Formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 or a compound having Formula 3: ABZ-Glu-Arg-Arg-Ala-pNA.

[0045] The present invention also provides the use of any compound of the present invention as defined above for detecting biliary tract cancer-specific enzyme activity.

[0046] The present invention also provides the use of any compound of the invention as defined above for the diagnosis of biliary tract cancer.

[0047] Preferably, in this use, the diagnosis of biliary tract cancer includes the detection of primary biliary tract cancer, the detection of minimal residual disease after surgical resection of cancer and / or the detection of recurrence of biliary tract cancer.

[0048] Preferably, the compound for use according to the present invention is a compound having formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 or a compound having formula 3: ABZ-Glu-Arg-Arg-Ala-pNA.

[0049] The present invention also provides any compound of the present invention as defined above for use as a diagnostic marker for detecting biliary tract cancer.

[0050] Preferably, the compound used as a diagnostic marker of the present invention is a compound having Formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 or a compound having Formula 3: ABZ-Glu-Arg-Arg-Ala-pNA.

[0051] The present invention also provides a method for treating biliary tract cancer, wherein

[0052] a) detecting the presence of biliary tract cancer-specific enzyme activity in a body fluid sample from the subject by any of the methods defined above, and

[0053] b) administering a biliary tract cancer treatment to the subject if the presence of said enzyme activity is found in said sample.

[0054] Preferably, in the treatment method according to the present invention, the biliary tract cancer-specific enzyme activity is monitored at predetermined time intervals after the end of the treatment according to point b).

[0055] Preferably, in the method of treatment according to the invention, a urine sample, preferably human urine, is used as sample.

[0056] Preferably, in the treatment method of the present invention, a compound of Formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 or a compound of Formula 3: ABZ-Glu-Arg-Arg-Ala-pNA is used as the compound. DETAILED DESCRIPTION

[0057] It should be understood that the present invention is defined in the appended claims. This specification describes various non-limiting embodiments and examples of the present invention. Unless otherwise indicated, the present invention is not limited to any specific method, protocol or reagent for practicing the present invention. The terms and scientific and technical expressions used herein have the meanings commonly known and used by those skilled in the art of the present invention. However, for the sake of clarity, the following expressions / terms and abbreviations used in the patent should be understood as follows:

[0058] A chromophoric compound or chromophoric molecule is a compound that has the property of chromophoricity. Chromophoricity refers to the ability of a compound to form a colored product.

[0059] Fluorescent compounds or fluorescent molecules are compounds that have the property of fluorescence. Fluorescence refers to the ability of a compound to form products that emit fluorescence.

[0060] NMP represents N-methylpyrrolidone; DMF represents dimethylformamide; DCM represents methylene chloride or dichloromethane; pNA represents 4-nitroaniline or p-nitroaniline; ABZ represents 2-aminobenzoic acid, and ANB-NH2 represents the amide of 5-amino-2-nitrobenzoic acid; Boc represents tert-butyloxycarbonyl; Fmoc represents 9-fluorenyloxycarbonyl; and TFA represents trifluoroacetic acid.

[0061] In the context of the present invention, the term "diagnosis of biliary tract cancer" should be understood to refer to the identification of the disease, particularly in its early stages, when other diagnostic methods are not sensitive and / or specific enough. As used herein, diagnosis of biliary tract cancer also includes the detection of minimal residual disease (MRD) after surgical resection of biliary tract cancer and the detection of biliary tract cancer recurrence after previous completion of biliary tract cancer treatment.

[0062] In the context of the present invention, the term "treatment of biliary tract cancer" is understood as a treatment at an early stage of disease progression, which allows to significantly prolong survival time and improve the quality of life of the affected subject.

[0063] In the context of the present invention, the term "monitoring" should be understood as referring to the diagnosis of minimal residual disease (MRD), i.e. the presence of a small number of cancer cells surviving in the organism (during treatment or remission) in an amount that cannot be detected by standard diagnostic methods.

[0064] In the context of the present invention, the term "subject" is understood to refer to a human subject or mammal suspected of having biliary tract cancer, or alternatively, a human subject or mammal belonging to a population at increased risk for biliary tract cancer, or a human subject or mammal after resection of biliary tract cancer or after completion of biliary tract cancer treatment. The subject is preferably a human subject.

[0065] Due to the presence of chromophores, the compounds of the present invention have both chromogenic and fluorescent properties, i.e., they contain molecules that are fluorescent donors and acceptors. Due to their structure, an increase in color is observed in the wavelength range of 380-440 nm, particularly upon contact with test body fluid samples from subjects with cancer, while this effect is not observed in reactions with body fluid samples from healthy subjects or subjects with another type of cancer. These compounds enable the detection of cancer-specific enzymatic activity, particularly for the specific and sensitive diagnosis of biliary tract cancer in the early stages of such cancer progression. The subject is preferably a human subject. The body fluid is preferably urine, more preferably human urine.

[0066] In the first aspect of the present invention, a novel compound is provided, which has the formula 1:

[0067] X1 1 -Glu 2 -Arg 3 -Arg 4 -Ala 5 -X2 6 (Formula 1),

[0068] Wherein X1 is an amino acid derivative or peptide fragment comprising molecule C1, or X1 consists of such molecule C1, X2 is an amino acid derivative or peptide fragment comprising molecule C2, or X2 consists of such molecule C2, wherein the pair of molecules C1 and C2 is a fluorescent donor and a fluorescent acceptor pair. The superscript indicates the subsequent position of the residues in the compound of the present invention and the order in which the residues are connected during synthesis. According to the present invention, in this case, Chemical Formula 1 can be written alternatively without indicating the numbering of the residues. The core of all compounds of the present invention is a tetrapeptide having a specified sequence of 4 amino acids, i.e., Glu-Arg-Arg-Ala (the symbol in the three-letter amino acid abbreviation format is equivalent to the symbol in the one-letter amino acid abbreviation format: DTFI), which is also represented as SEQ ID NO.1 in the sequence table.

[0069] The compound of the present invention is enzymatically cleaved into fragments: X1-Glu-Arg-Arg-Ala-OH (fragment 1) and X2 (fragment 2), generating a measurable light signal when molecules C1 and C2 are spatially separated. The measurable light signal is measured by measuring the absorbance / fluorescence change after enzymatic cleavage of the compound. Preferably, molecules C1 and C2 are separated from each other by no more than 10 amino acid residues, which ensures effective quenching of the fluorescent donor by the fluorescent acceptor. For those skilled in the art, the key factor is obviously the distance between the fluorescent donor and the acceptor. Therefore, when the amino acid sequence separating molecules C1 and C2 folds into a twisted or condensed secondary structure, resulting in the proximity of molecules C1 and C2 relative to the primary structure, the distance between molecules C1 and C2 can be greater than 10 amino acid residues.

[0070] This compound, due to its chromogenic properties and the presence of a reactive site at position 5, enables enzymatic (preferably proteolytic) cleavage into smaller fragments, and is particularly suitable for use as a diagnostic marker, in particular a specific diagnostic biomarker for biliary tract cancer, especially for the early diagnosis of biliary tract cancer.

[0071] In a preferred embodiment, the compounds of the invention are cleaved hydrolytically, more preferably proteolytically.

[0072] In a preferred embodiment, the molecular pair C1 and C2 are selected from: 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 molecular pair C1 and C2 are ABZ / pNA or ABZ / ANB-NH2.

[0073] In a preferred embodiment, the compound of the present invention is:

[0074] A compound having formula 2:

[0075] ABZ 1 -Glu 2 -Arg 3 -Arg 4 -Ala 5 -ANB 6 -NH2 (Formula 2) or

[0076] Compounds having formula 3:

[0077] ABZ 1 -Glu 2 -Arg 3 -Arg 4 -Ala 5 -pNA 6 (Formula 3),

[0078] Wherein ABZ represents 2-aminobenzoic acid, ANB-NH2 represents the amide of 5-amino-2-nitrobenzoic acid, and pNA represents 4-nitroaniline.

[0079] In the case of the compound of formula 2, the compound is hydrolytically cleaved to produce the following fragment 1: ABZ-Glu-Arg-Arg-Ala-OH and fragment 2: ANB-NH2, while in the case of the compound of formula 3, the following fragment 1: ABZ-Glu-Arg-Arg-Ala-OH and fragment 2: pNA are produced. Therefore, fragment 2 is a free chromophore.

[0080] The spatial separation of molecules C1 and C2 is the result of enzymatic cleavage of the compound of the present invention, which results in the generation of a measurable light signal because the fluorescence emitted by the fluorescence donor is no longer quenched by the fluorescence acceptor. This measurable light signal can be detected preferably at a wavelength of 300-500 nm, more preferably at 380-430 nm.

[0081] The compounds of the invention can be obtained by known methods. For example, they can be obtained using a method for obtaining chromogenic peptides, which involves carrying out the process on a solid support in the form of a resin having an Fmoc group, which resin is removed during the reaction. For example, it can be an amide resin such as Teenage S RAM or RinkAmide, but any other commercially available resin can also be used. The resin used to carry out this process should be appropriately prepared. The preparation of the resin consists in increasing its volume by repeated washing with a hydrophobic solvent. Preferably, a resin with a deposit of 0.23 mmol / g is used. The resin must be washed with a 20% solvent solution to remove the Fmoc protecting group.

[0082] Then, the known method of obtaining chromogenic peptides comprises connecting individual components under appropriate time and stoichiometric conditions. The connection process includes subsequent steps, in which individual elements (amino acid derivatives) are connected, residues are washed off, protective groups are removed in sequence and washed again. This cycle is repeated for each amino acid residue. The peptide obtained is separated from the resin by reaction under acidic conditions. Then, the solution is separated from the resin during filtration, and the peptide is then precipitated from the solution obtained by a non-polar solvent. The peptide precipitate obtained in this way is centrifuged.

[0083] Exemplary, detailed, but non-limiting, syntheses of the compounds of the present invention are described below and in Example 1 below.

[0084] The synthesis method of the compound of the present invention is that the method is carried out on a solid support in the form of a resin (preferably having an Fmoc group), wherein before the start of the method, the solid support is prepared by repeatedly washing with a hydrophobic solvent (preferably dimethylformamide, dichloromethane or N-methylpyrrolidone) to increase its volume and remove the Fmoc protecting group, preferably washing with a 10-30% piperidine solution in a solvent (such as dimethylformamide, methylene chloride or N-methylpyrrolidone).

[0085] The method then proceeds in the following steps:

[0086] a) before depositing 5-amino-2-nitrobenzoic acid ANB (or another chromophore suitable for the present invention as defined in the claims) on the resin, the solid support is washed with a 3-6% solution of N-methylmorpholine (NMM) in DMF, then with DMF, after which a solution of ANB in DMF is prepared, to which TBTU, DMAP and finally diisopropylethylamine (DIPEA) are added in the following excess relative to the polymer deposited: ANB / TBTU / DMAP / DIPEA, 3:3:2:6; the 5-amino-2-nitrobenzoic acid ANB prepared in this way is added to the resin. The mixture was added to the resin and mixed until uniform, and then the resin was filtered under reduced pressure and washed with solvents such as DMF, DCM and isopropanol, and ANB was continued to be connected to the resin using hexafluorophosphate-O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium salt (HATU) and then an excess of hexafluorophosphate-O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium salt (HATU). After completion, the solid support was washed with DMF, DCM and isopropanol in sequence and gently dried;

[0087] b) the connection of the amino acid residue to ANB is carried out by reaction with an amino acid derivative -Fmoc-Ala-OH, wherein at least a five-fold molar excess of the amino acid derivative relative to the resin is dissolved in anhydrous pyridine and contacted with the deposited ANB, and then the whole is cooled to a temperature of not less than -20°C, and then POCl3 is added in a ratio of 1:1 relative to the amount of the amino acid derivative used and the whole is mixed, and then the mixing process is carried out at room temperature and then at an elevated temperature. After the reaction is completed, the resin is filtered under reduced pressure, washed with DMF and MeOH and gently dried, and then the intermediate compound obtained is subjected to acylation treatment, and the Glu-Arg-Arg fragment is connected in sequence;

[0088] c) using an amino acid derivative, preferably Fmoc-Arg(Pbf)-OH, followed by Fmoc-Arg(Pbf)-OH, then Fmoc-Glu(tBu-OH, and in the last step of the synthesis using Boc-Abz-OH, acylation is carried out in steps from residue 6 to 1, using diisopropylcarbodiimide used in excess as coupling agent, washing the resin with DMF after each step, preferably performing a chloranil test (a test for the presence of free amino groups), wherein the attachment of the amino acid derivative is monitored;

[0089] d) Removal of the Fmoc protecting group was performed by washing with 10-30% piperidine in DMF, followed by washing with each of DMF, isopropanol, and dichloromethane;

[0090] e) separating the peptide from the resin using a mixture of: TFA:phenol:water:TIPS, while maintaining a ratio of 88:5:5:2 v / v / v / v, respectively, stirring the mixture for at least one hour, preferably three hours, filtering off the precipitate obtained under reduced pressure, then washing with diethyl ether, and centrifuging the peptide obtained;

[0091] f) The peptide is dissolved in water by ultrasonication and then freeze-dried to prepare a finished product.

[0092] A second aspect of the present invention provides an in vitro method for detecting enzyme activity, preferably proteolytic activity, present in a body fluid of a subject, in particular an enzyme activity derived from biliary tract cancer cells, the method comprising a) contacting a body fluid sample with a compound of the present invention, and b) detecting a measurable light signal generated upon spatial separation of molecules C1 and C2 present in the compound of the present invention. In a preferred embodiment of this aspect, in this case, the subject being examined is a human subject. In another preferred embodiment of this aspect, the body fluid is urine, in particular human urine.

[0093] In a preferred embodiment of this aspect, a compound having formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 or a compound having formula 3: ABZ-Glu-Arg-Arg-Ala-pNA is used.

[0094] The third aspect of the present invention provides an in vitro method for diagnosing biliary tract cancer, wherein the presence or absence of biliary tract cancer in a subject is detected by measuring the biliary tract cancer-specific enzyme activity in a body fluid sample from the subject being examined, wherein the absence of the enzyme activity indicates the absence of biliary tract cancer, and the presence of the enzyme activity indicates the presence of biliary tract cancer. The detection of this enzyme activity is preferably carried out using the method for detecting enzyme activity as described above. In a preferred embodiment of this aspect, the subject is a human subject. In a preferred embodiment of this aspect, the body fluid is urine, in particular human urine. In a preferred embodiment of this aspect, the enzyme activity specific for biliary tract cancer is proteolytic activity. In a preferred embodiment of this aspect, a compound having formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 or a compound having formula 3: ABZ-Glu-Arg-Arg-Ala-pNA is used.

[0095] Furthermore, in a preferred embodiment of this aspect, the measurement of the enzyme activity in the method of the present invention comprises measuring the absorbance intensity in the range of 300-500 nm, preferably 380-430 nm, in particular 405 nm, at a temperature of 25-40° C., preferably 36-38° C., within 40-60 minutes. This enables a measurable light signal of maximum intensity caused by an increase in absorbance or fluorescence to be obtained.

[0096] Furthermore, in a preferred embodiment of the method of the present invention, the measurement of the enzyme activity is performed using a compound of the present invention at a concentration in the range of 0.1-10 mg / mL, more preferably at a concentration of 1 mg / mL. In a preferred embodiment of the method of the present invention, the test sample is incubated with the compound of the present invention in a measurement buffer having a neutral or alkaline pH (preferably physiological pH) and mixed with a body fluid sample (preferably human urine) in a ratio of sample to measurement buffer of 1:2 to 1:10, preferably 1:5. The sample is preferably collected from a patient referred for a diagnosis of biliary tract cancer. Preferably, the absorbance intensity is measured at a temperature of 25-40°C (preferably 36-38°C) within 40-60 minutes in the range of 300-500 nm, in particular 380-430 nm, and especially 405 nm. Under these conditions, a measurable optical signal of maximum intensity can be obtained due to an increase in absorbance or fluorescence.

[0097] In a fourth aspect, the present invention provides a kit comprising any compound of the present invention and a measurement buffer. Measurement buffers are known in the art, and suitable buffers for the kits of the present invention include, but are not limited to, Tris-HCl buffer. In a preferred embodiment, in the kits according to the present invention, the compound is a compound having Formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 or a compound having Formula 3: ABZ-Glu-Arg-Arg-Ala-pNA.

[0098] In a fifth aspect, the present invention provides the use of a compound according to the present invention for detecting biliary tract cancer-specific enzyme activity. In a sixth aspect, the present invention provides the use of a compound according to the present invention for diagnosing biliary tract cancer. Preferably, according to the present invention, the diagnosis of biliary tract cancer includes the detection of primary biliary tract cancer, the detection of minimal residual disease after surgical resection of the cancer, and / or the detection of biliary tract cancer recurrence after previously completed biliary tract cancer treatment.

[0099] In a seventh aspect, the present invention provides a compound of the present invention for use as a diagnostic marker for the detection of biliary tract cancer. In a preferred embodiment of this aspect, the compound is a compound having Formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 or a compound having Formula 3: ABZ-GluArg-Arg-Ala-pNA.

[0100] In an eighth aspect, the present invention provides a method for treating biliary tract cancer, wherein

[0101] a) detecting the presence of biliary tract cancer-specific enzyme activity in a body fluid sample from the subject being examined by any of the methods according to the invention as defined above, and

[0102] b) administering a biliary tract cancer treatment to the subject when the enzyme activity is found to be present in the sample.

[0103] In a preferred embodiment of the method for treatment, after completion of the treatment according to point b), the biliary tract cancer-specific enzyme activity is monitored at predetermined time intervals known in the art, such as weekly, weekly, monthly, monthly, annually, or any other interval deemed appropriate by a person skilled in the art to detect minimal residual disease after surgical resection or recurrence of biliary tract cancer. In addition, in a preferred embodiment of the method, a urine sample, preferably human urine, is used as a test sample. In a preferred embodiment of the method of treatment, a compound of Formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 (Formula 2) or a compound of Formula 3: ABZ-Glu-Arg-Arg-Ala-pNA (Formula 3) is used as the compound.

[0104] The advantage of the present invention is that it provides a new compound having an enzyme activity that makes it suitable for specific and sensitive detection of biliary tract cancer, for use as a diagnostic biomarker for detecting biliary tract cancer, for rapid and non-invasive diagnosis of biliary tract cancer, while being able to detect biliary tract cancer in the early stages of its development. Another advantage is that the diagnostic method according to the present invention can be successfully used for screening tests. This enables comprehensive diagnosis at the early stages of cancer progression, thereby obtaining more effective treatment. Early diagnosis allows surgical treatment, which significantly prolongs the patient's survival time. This is also important when monitoring the effectiveness of surgical treatment and / or chemotherapy for cancer, because minimal residual disease or recurrence (if any) can be detected.

[0105] The invention will now be illustrated in the following figures and examples which, however, are not intended in any way to limit the scope of the invention as defined in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 Shown are the results of chromatographic analysis of the cleavage of the substrate, ABZ-Glu-Arg-Arg-Ala-ANB-NH2, in a urine sample from a subject with biliary tract cancer.

[0107] Figure 2 Shown are the hydrolysis rates of the substrate ABZ-Glu-Arg-Arg-Ala-ANB-NH2 in urine samples from subjects diagnosed with biliary tract cancer (samples 1-13) and healthy subjects (samples 14-23). Arabic numerals indicate the number of the selected urine sample.

[0108] Figure 3Shown are the substrate ABZ in urine samples from subjects diagnosed with biliary tract cancer and urine samples from subjects diagnosed with another neoplastic disease. 1 -Glu 2 -Arg 3 -Arg 4 -Ala 5 Selectivity of hydrolysis of -NH2 (i.e., compound of Formula 2). Test samples for each cancer were obtained from 20 different patients with each cancer tested. Results are averaged for a given cancer type. The results show selectivity of substrate cleavage in urine from patients with biliary tract cancer compared to urine samples from patients with other tumors.

[0109] Figure 4 The dependence of the hydrolysis level of the substrate -ABZ1-Glu2-Arg3-Arg4-Ala5-NH2 on pH conditions is shown.

[0110] Example

[0111] The present invention is illustrated by the following non-limiting examples. Unless otherwise indicated, the following examples utilize known and / or commercially available devices, methods, reaction conditions, reactants, and kits that are commonly used in the art to which the present invention pertains and are recommended by the manufacturers of the corresponding reactants and kits.

[0112] Example 1: Synthesis of the compounds of the present invention

[0113] This example describes the synthesis of a representative compound of the present invention, namely compound: ABZ 1 -Glu 2 -Arg 3 -Arg 4 -Ala 5 -NH2. The remaining peptides of the present invention can be synthesized in a similar manner. Superscripts indicate the subsequent position of the residues in the compounds of the present invention and the order in which the residues are connected during synthesis. The compounds of the present invention can alternatively be represented by similar formulas that do not indicate the position of the residues. This does not change the order of the residues in the compounds of the present invention, as it remains unchanged.

[0114] 1. Obtaining Chromogenic Peptides

[0115] a) The first step of the synthesis is to obtain the chromogenic peptide obtained by solid phase synthesis on a solid support using Fmoc / tBu chemistry, ie using protection.

[0116] The amino acid derivatives having the sequence ABZ were obtained during solid phase chemical synthesis using the following 1 -Glu 2 -Arg 3 -Arg 4 -Ala5 -NH2 compounds, wherein ABZ is 2-aminobenzoic acid, ANB-NH2 is the amide of 5-amino-2-benzoic acid, and ANB is 5-amino-2-benzoic acid:

[0117] Boc-ABZ, Fmoc-Glu(OtBu), Fmoc-Arg(Pbf), Fdoc-Arg(Pbf), Fmoc-Ala.

[0118] The synthesis of the compounds of the present invention, which can be used as diagnostic markers for the detection of biliary tract cancer, was carried out on a solid support capable of converting 5-amino-2-benzoic acid to ANB-NH2 amide, namely the amide resin TentaGel S RAM from RAPP Polymer (Germany), with a deposition capacity of 0.23 mmol / g. However, any other amide resin, such as Rink amide (Germany), can also be used.

[0119] The synthesis of this compound was performed manually using a laboratory shaker. In most steps, a 25 mL fritted syringe for solid phase synthesis was used as the reactor.

[0120] All final compounds obtained contain a 2-aminobenzoic acid (ABZ) molecule at position 1, i.e., the N-terminus, of their sequence, and a 5-amino-2-nitrobenzoic acid (ANB) molecule at position 6, i.e., the C-terminus. ABZ acts as a fluorescence donor, while ANB-5-amino-2-nitrobenzoic acid acts as a fluorescence quencher and also as a chromophore. The peptide contains at least one, and preferably one, reactive site in its sequence, located between the amino acid residues Ala-ANB-NH2, i.e., position 5 of the compound. The synthesis, including the step of attaching the amino acid derivatives, proceeds from residues 6 to 1, i.e., from the C-terminus to the N-terminus.

[0121] b) Deposition of ANB on TentaGel S RAM resin:

[0122] Peptide synthesis was performed on Rapp Polymer's TentaGel S RAM resin at a deposition rate of 0.23 mmol / g. In the first step, the resin was prepared, including loosening through wash cycles. Subsequently, the Fmoc-amino group was deprotected from the solid support using a 20% piperidine solution in NMP. Solvent wash cycles were then performed. A chloranil test was performed to confirm the presence of free amino groups.

[0123] Solvent Wash Cycle:

[0124] DMF 1 x 10 min; IsOH 1 x 10 min; DCM 1 x 10 min.

[0125] Removal of Fmoc protection:

[0126] DMF 1 x 5 min; 20% piperidine in NMP 1 x 3 min; 20% piperidine in NMP 1 x 8 min.

[0127] Solvent Wash Cycle:

[0128] DMF 3x2 min; IsOH 3x2 min; DCM 3x2 min.

[0129] c) Chloranil test:

[0130] The chloranil test involves transferring a few pellets of resin from the reactor (syringe) into a glass ampoule with a spatula, followed by the addition of 100 μL of a saturated toluene solution of chloranil and 50 μL of fresh acetaldehyde. After 10 minutes, a color control of the pellets is performed.

[0131] At this stage, after testing, green particles were obtained, which confirmed the presence of free amino groups. After confirming the removal of the 9-Fmoc protection from the resin, the next step can be carried out, namely the attachment of the ANB derivative (5-amino-2-nitrobenzoic acid).

[0132] d) Deposition of 5-amino-2-nitrobenzoic acid on a solid support:

[0133] The first step in peptide synthesis is to deposit ANB onto 1 g of resin. Prior to attaching the chromophore, the resin used for the reaction is washed with the following solvents: DMF, DCM, and DMF, followed by removal of the Fmoc protection from the functional groups on the solid support. One cycle of Fmoc removal involves the following steps:

[0134] Removal of Fmoc protection:

[0135] 20% piperidine in NMP 1 x 3 min; 20% piperidine in NMP 1 x 8 min.

[0136] e) Washing:

[0137] DMF 3x2 min; IsOH 3x2 min; DCM 3x2 min.

[0138] f) Chloranil test:

[0139] The resin with free amino groups was washed with 5% N-methylmorpholine (NMM) in DMF and then washed with DMF. The procedure for removing the Fmoc protection and the washing cycle were carried out in a Merrifield container. In another flask, ANB was dissolved in DMF and then TBTU, DMAP and finally diisopropylethylamine (DIPEA) were added in excess relative to the polymer deposition: ANB / TBTU / DMAP / DIPEA, 3:3:2:6 v / v / v / v. The mixture prepared in this way was added to the resin and stirred for 3 hours. The resin was filtered under reduced pressure, washed with DMF, DCM and isopropanol, and the whole acylation process was repeated twice. For the subsequent reaction to attach ANB to the resin, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) was used, followed by O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU). In the final step, the resin was washed sequentially with DMF, DCM, and isopropanol and air-dried.

[0140] g) Linkage of the C-terminal amino acid residue (Fmoc-Ala-OH) to ANB:

[0141] The corresponding amino acid derivative (9-fold molar excess relative to the resin deposit) was dissolved in pyridine and transferred to the flask containing the ANB-deposited resin. The mixture was cooled to -15°C (ice bath: 1 part by weight NH4Cl, 1 part by weight NaNO3, 1% by weight ice). Once the desired temperature was reached, POCl3 (1:1 ratio to the amount of amino acid derivative used) was added and stirred on a magnetic stirrer for 20 minutes at -15°C, 30 minutes at room temperature, and 6 hours at 40°C (oil bath). After the reaction was complete, the resin was filtered under reduced pressure, washed with DMF and MeOH, and dried.

[0142] In the next stage, the residue was linked at the P2 position (Fmoc-Arg(Pbf)).

[0143] Before each amino acid residue attachment, the resin was washed with DMF for 5 minutes. Diisopropylcarbodiimide was used as a coupling agent in subsequent attachments. This procedure was repeated twice.

[0144] After each acylation, a resin wash cycle was initiated followed by a chloranil test to monitor the attachment of the amino acid derivative to the free amino acid groups on the resin.

[0145] Solvent Wash Cycle:

[0146] DMF 3x2 min; IsOH 3x2 min; DCM 3x2 min.

[0147] Chloranil test:

[0148] As a result of the tests performed, after the first two coupling procedures, the color of the particles was first green and then gray, necessitating another acylation step, resulting in colorless resin particles after the chloranil test. This confirmed that the ANB was attached to the TentaGel S RAM resin, and the next step of peptide synthesis could be carried out.

[0149] h) Attachment of subsequent protected amino acid residues:

[0150] The resin was washed with DMF together with the linker fragment ANB-Ala in the reactor, and then Fmoc was deprotected from the amino group to link the protected amino acid derivative Arg.

[0151] Removal of Fmoc protection:

[0152] DMF 1 x 5 min; 20% piperidine in NMP 1 x 3 min; 20% piperidine in NMP 1 x 8 min.

[0153] Solvent Wash Cycle:

[0154] DMF 3x2 min; IsOH 3x2 min; DCM 3x2 min.

[0155] Chloranil test:

[0156] The chloranil test gave a positive result, as evidenced by the green color of the resin particles, and thus the next step—attachment of the amino acid residue Fmoc-Arg(Pbf)-OH—was possible.

[0157] Linking of amino acid derivatives

[0158] Before the coupling process, the resin was washed with DMF. When attaching protected serine residues, the composition of the coupling mixture remained unchanged.

[0159] After each acylation, solvent wash cycles were performed according to the specified procedure, followed by a chloranil test for the presence of free amino acid groups in the solution.

[0160] Solvent wash cycle

[0161] DMF 3x2 min; IsOH 3x2 min; DCM 3x2 min.

[0162] Chloranil test:

[0163] In tests performed after the second acylation, the resin particles were colorless, so the next step in the synthesis could be carried out, which was to introduce another protected amino acid derivative, Fmoc-Glu(OtBu), and the 2-aminobenzoic acid molecule. The coupling process was carried out according to the procedures discussed previously.

[0164] Tests conducted after connecting the above residues showed positive results - the resin particles were colorless.

[0165] 2. Removal of peptides from the solid support

[0166] After synthesis, the amide of the ABZ-Glu-Arg-Arg-Ala-ANB-NH2 peptide was removed from the solid support and the side chain protection was simultaneously removed using a mixture of TFA:phenol:water:TIPS (88:5:5:2, v / v / v / v) in a round-bottom flask on a magnetic stirrer.

[0167] After 3 hours, the contents of the flask were filtered under reduced pressure in a Schott funnel and washed with ether. The obtained precipitate was centrifuged for 20 minutes on a SIGMA 2K30 centrifuge (laboratory centrifuge). The precipitate obtained after centrifugation was dissolved in water by ultrasound and then freeze-dried. The remaining compounds of the present invention can be obtained in a similar manner.

[0168] The identity / characteristics of the novel compounds of the present invention were confirmed by HPLC analysis. HPLC analysis conditions were as follows: RPBio Wide Pore Supelco C8 column, 250 mm x 4 mm, phase system A: 0.1% TFA in water, phase system B: 80% acetonitrile in water, flow rate 1 mL / min, UV detection wavelength 226 nm.

[0169] The analyses performed confirmed that the compound of the present invention was obtained.

[0170] Example 2: Testing the properties of the compounds of the present invention as cancer markers

[0171] The activity of the novel compounds of the present invention was studied in 20 subjects diagnosed with biliary tract cancer using representative compounds of the present invention. The mechanism of action of the compounds of the present invention, including the representative compounds of formula 2, is that a specific enzymatic cleavage, more specifically enzymatic hydrolysis, occurs at a position leading to the release of the free molecule of the respective chromophore: in the case of the compound of formula 2, it is ANB-NH2 (amide of 5-amino-2-nitrobenzoic acid), in the case of the compound of formula 3, it is pNA (p-nitroaniline), which exhibits absorbance at a wavelength of 320-480 nm, in particular 380-430 nm, in particular 405 nm. The remaining compounds of the present invention are characterized by a similar mechanism of action. For this purpose, the representative compound ABZ of the present invention was used. 1 -Glu2 -Arg 3 -Arg 4 -Ala 5 -NH2 was dissolved in dimethyl sulfoxide (at a concentration of 0.5 mg / mL), and 50 μL of the solution was then mixed with 120 μL of buffer (200 mM Tris-HCl, pH 8.0) and 80 μL of urine from a subject with cancer. The measurements were performed on a 96-well plate designed for absorbance measurement, and each sample was analyzed three times at a temperature of 37°C. The measurement duration was 60 minutes. During the measurement, the wavelength characteristics of the released chromophore (ANB-NH2) were monitored at a wavelength of 405 nm (range 380-430 nm).

[0172] like Figure 1 As shown, RP HPLC analysis of a randomly selected system containing urine extracted from a person diagnosed with biliary tract cancer showed that the compound according to the invention was cleaved into the peptide fragment ABZ-Glu-Arg-Arg-Ala-OH and the chromophore of the compound (ANB-NH2).

[0173] The measurements showed that the color intensity of the solution increased over time in all urine samples from patients diagnosed with biliary tract cancer. The magnitude of the observed absorbance increase over time varied for each sample examined. A different effect was observed for the 10 samples from healthy subjects, as no increase in absorbance within the diagnostic range was observed in any of the 10 urine samples tested.

[0174] The tests performed showed that all samples 1-13 from humans with biliary tract cancer were cleaved, but in the case of samples 1, 6, and 12, cleavage of the substrate (i.e., ABZ-Glu-Arg-Arg-Ala-ANB-NH2) was not as efficient as in the case of samples 7 or 8 (Table 1 Figure 2 ). Such results may be due to differences in the activity and quantity of the enzymes responsible for enzymatic cleavage (proteolysis). In addition, the results shown in Table 1 below show that incubation of the substrate solution - the compound according to the present invention - with urine samples extracted from healthy individuals (without a cancer diagnosis, labeled sequentially with Arabic numerals 14 to 23) does not result in an increase in absorbance, and therefore no hydrolysis of the test compound occurs. The results indicate the absence of proteolytic enzymes specific / characteristic of cancer biliary tract.

[0175] Table 1. Absorbance analysis results

[0176] 1 0.004 0.002 0.003 2 0.0068 0.0034 0.0053 3 0.0035 0.005 0.0048 4 0.004 0.0067 0.0051 5 0.0474 0.0461 0.0042 6 0.002 0.0021 0.0026 7 0.084 0.092 0.087 8 0.0746 0.0948 0.084 9 0.053 0.0458 0.0497 10 0.0436 0.035 0.041 11 0.0051 0.0041 0.0048 12 0.0025 0.0028 0.0032 13 0.0046 0.005 0.0047 14 0.000000 0.000000 0.000000 15 0.000000 0.000000 0.000000 16 0.000000 0.000000 0.000000 17 0.000000 0.000000 0.000000 18 0.000000 0.000000 0.000000 19 0.000000 0.000000 0.000000 20 0.000000 0.000000 0.000000 21 0.000000 0.000000 0.000000 22 0.000000 0.000000 0.000000 23 0.000000 0.000000 0.000000

[0177] Furthermore, the dependence of the substrate, i.e. the cleavage selectivity of the compounds according to the invention, on the type of cancer tested was investigated. The results of the tests performed are shown in FIG. Figure 3 As shown, they indicate that the substrate tested, namely ABZ 1 -Glu 2 -Arg 3 -Arg 4 -Ala 5 -NH2, incubated with samples extracted from patients with the following cancers: testicular cancer, colorectal cancer, kidney cancer, prostate cancer, pancreatic cancer, liver cancer, lung cancer, ovarian cancer, and rectal cancer, was not cleaved and did not cause an increase in absorbance within the specified range. In each case, the sample tested was a mixture of 20 samples from each cancer studied. This demonstrates the cleavage selectivity of the compounds according to the invention, making them suitable for the specific detection of biliary tract cancer-specific enzymatic activity and the specific diagnosis of biliary tract cancer.

[0178] Table 2 below shows the results of triplicate measurements for each sample.

[0179] Table 2. Cleavage selectivity analysis results

[0180] testis 0 0 0 the large intestine 0 0 0 kidney 0 0 0 prostate 0 0 0 pancreas 0 0 0 liver 0 0 0 lung 0 0 0 ovaries 0 0 0 rectum 0 0 0 bile duct 0.0075 0.0084 0.0071

[0181] In addition, the dependence of the proteolytic activity of representative compounds of the present invention on the reaction pH was measured. The experiments showed that at least one enzyme of the materials studied exhibited maximum activity at alkaline pH ( Figure 4 ).

[0182] The assays performed demonstrate the suitability of the compounds according to the invention for the sensitive and specific detection of biliary tract cancer-specific enzyme activities and, by virtue of this assay, their suitability for the specific diagnosis of biliary tract cancer and as diagnostic markers for biliary tract cancer. The mechanism of action of the compounds according to the invention resides in their specific enzymatic cleavage at a site leading to the release of free chromophore molecules, which generates a measurable optical signal that can be used for diagnostic purposes, in particular for the diagnosis of biliary tract cancer according to the invention. Sequence listing in WIPO standard ST.25 format <110> Ertest

[0183] <120> New diagnostic markers for biliary tract cancer

[0184] <130> 17P51015PL00

[0185] <160> 1

[0186] <170> BiSSAP 1.3.6

[0187] <210> 1

[0188] <211> 4

[0189] <212> PRT

[0190] <213> Homo sapiens

[0191] <400> 1GluArgArgAla 1Sequence listing in WIPO standard ST.26 format

[0192]

[0193]

Claims

1. A compound having formula 1: X1 1 -Glu 2 -Arg 3 -Arg 4 -Ala 5 -X2 6 (Formula 1) wherein X1 comprises or consists of the molecule C1, and X2 comprises or consists of the molecule C2, The molecular pair C1 and C2 are a pair of fluorescence donor and fluorescence acceptor, And wherein the compound is cleaved by an enzyme into fragments X1-Glu-Arg-Arg-Ala-OH (fragment 1) and X2 (fragment 2) and generates a measurable light signal when molecules C1 and C2 are spatially separated.

2. The compound according to claim 1, which is hydrolytically cleaved, preferably proteolytically cleaved.

3. The compound according to claim 1 or 2, wherein the molecular pair C1 and C2 is selected from: 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 molecular pair C1 and C2 is ABZ / pNA or ABZ / ANB-NH2.

4. The compound according to any one of claims 1 to 3, which is a compound having Formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-Glu-Arg-Arg-Ala-pNA (Formula 3).

5. The compound according to claim 4, which is hydrolytically cleaved to produce the following fragment 1: ABZ-Glu-Arg-Arg-Ala-OH and fragment 2: ANB-NH2.

6. An in vitro method for detecting enzyme activity present in a subject's body fluid, particularly enzyme activity derived from biliary cancer cells, comprising: a) contacting a body fluid sample with a compound of formula 1: X1 1 -Glu 2 -Arg 3 -Arg 4 -Ala 5 -X2 6 (Formula 1) wherein X1 comprises or consists of the molecule C1, and X2 comprises or consists of the molecule C2, The C1 and C2 molecular pairs are a pair of fluorescence donor and fluorescence acceptor, and wherein the compound is cleaved by an enzyme into fragments X1 Glu-Arg-Arg-Ala-OH (fragment 1) and X2 (fragment 2), and b) A measurable light signal is generated when the detection molecules C1 and C2 are spatially separated.

7. The in vitro method according to claim 6, wherein the enzymatic activity is a hydrolytic activity, preferably a proteolytic activity.

8. The in vitro method according to claim 6 or 7, wherein the compound of formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 (Formula 2) or the compound of formula 3: ABZ-Glu-Arg-Arg-Ala-pNA (Formula 3) is used as the compound.

9. The method according to any one of claims 6 to 8, wherein urine, preferably human urine, is used as the body fluid.

10. An in vitro method for diagnosing biliary tract cancer, wherein the presence or absence of biliary tract cancer in a subject is detected by measuring the activity of an enzyme specific for biliary tract cancer in a body fluid sample from the subject being examined, and wherein the absence of the enzyme activity indicates the absence of biliary tract cancer, while the presence of the enzyme activity indicates the presence of biliary tract cancer.

11. The method according to claim 10, wherein the detection of the enzyme activity is performed by the method according to any one of claims 6 to 9.

12. The method according to claim 10 or 11, wherein the measurement of the enzyme activity is performed using a compound of formula 1: X1 1 -Glu 2 -Arg 3 -Arg 4 -Ala 5 -X2 6 (Formula 1) wherein X1 comprises or consists of the molecule C1, and X2 comprises or consists of the molecule C2, The molecular pair C1 and C2 are a pair of fluorescence donor and fluorescence acceptor. And wherein the compound is cleaved by an enzyme into fragments X1 Glu-Arg-Arg-Ala-OH (fragment 1) and X2 (fragment 2), a measurable light signal is generated when molecules C1 and C2 are spatially separated.

13. The method according to any one of claims 10 to 12, wherein the body fluid sample is incubated with the compound in a measurement buffer having a neutral or alkaline pH, preferably a physiological pH, in a sample to measurement buffer ratio in the range of 1:2 to 1:10, preferably 1:

5.

14. The method according to any one of claims 10 to 13, wherein the concentration of the compound is 0.1-10 mg / mL, in particular 0.25-7.5 mg / mL.

15. The method according to any one of claims 10 to 14, wherein the compound of Formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 (Formula 2) or the compound of Formula 3: ABZ-Glu-Arg-Arg-Ala-pNA (Formula 3) is used as the compound.

16. The method according to any one of claims 10 to 15, wherein a urine sample, preferably human urine, is used as the sample.

17. The method according to any one of claims 10 to 16, wherein the measurement of the enzyme activity comprises measuring the absorbance intensity in the range of 300-500 nm, preferably 380-430 nm, in particular 405 nm at a temperature of 25-40°C, preferably 36-38°C, for 40-60 minutes. 18 . A kit comprising the compound according to claim 1 and a measurement buffer.

19. The kit according to claim 18, wherein the compound is a compound having Formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-Glu-Arg-Arg-Ala-pNA (Formula 3).

20. Use of a compound as defined in any one of claims 1 to 5 for detecting biliary tract cancer-specific enzyme activity.

21. Use of a compound as defined in any one of claims 1 to 5 for the diagnosis of biliary tract cancer.

22. The use according to claim 21, wherein the diagnosis of biliary tract cancer comprises detecting primary biliary tract cancer, detecting minimal residual disease after surgical resection of cancerous biliary tract cancer and / or detecting recurrence of biliary tract cancer.

23. The use according to any one of claims 21 to 22, wherein the compound is a compound having Formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-Glu-Arg-Arg-Ala-pNA (Formula 3).

24. A compound as defined in any one of claims 1 to 5 for use as a diagnostic marker for the detection of biliary tract cancer.

25. The compound for use according to claim 24, wherein the compound is a compound of Formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 (Formula 2) or a compound of Formula 3: ABZ-Glu-Ag-Arg-AlapNA (Formula 3).

26. A method for treating biliary tract cancer, wherein a) detecting the presence of biliary tract cancer-specific enzyme activity in a body fluid sample from the subject by the method defined in any one of claims 6 to 9, and b) applying a treatment for biliary tract cancer in said subject if the presence of said enzyme activity is found in said sample.

27. The method according to claim 26, wherein the biliary tract cancer-specific enzyme activity is monitored at predetermined time intervals after completion of the treatment according to point b).

28. The method according to claim 26 or 27, characterized in that A urine sample, preferably human urine, is used as the sample.

29. The method according to any one of claims 26 to 28, wherein the compound of Formula 2: ABZ-Glu-Arg-Arg-Ala-ANB-NH2 (Formula 2) or the compound of Formula 3: ABZ-Glu-Arg-Arg-Ala-pNA (Formula 3) is used as the compound.

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