Compound, diagnostic marker for gastric cancer, method for detecting enzymatic activity, method for diagnosing gastric cancer, kit comprising compound, use of compound, and method for treating gastric cancer

Abstract

The present invention relates to a novel chemical compound, a diagnostic marker for use in medicine, more particularly in the diagnosis of cancer, in particular gastric cancer. The invention also relates to an in vitro method for detecting enzymatic activity, in particular enzymatic activity derived from gastric cancer cells, present in a bodily fluid of a subject using the compound. The invention also relates to an in vitro method for diagnosing gastric cancer by using the compound, a kit containing the compound, application of the compound to detection of gastric cancer specific enzyme activity and application of the compound to diagnosis of gastric cancer. The invention also relates to a compound used as a gastric cancer diagnosis marker and a method for treating gastric cancer. The method comprises a step of performing the gastric cancer diagnosis method by using the compound.

Description

TECHNICAL FIELD

[0001] The present invention relates to a new compound, a diagnostic marker, for use in medicine, more particularly in the diagnosis of cancer, in particular for the diagnosis of gastric cancer. The present invention also relates to an in vitro method for detecting the presence of an enzyme activity in a body fluid of a subject, in particular an enzyme activity originating from gastric cancer cells, using the compound, an in vitro method for diagnosing gastric cancer using the compound, a kit comprising the compound, the use of the compound for detecting an enzyme activity specific for gastric cancer, the use of the compound for diagnosing gastric cancer, a compound for use as a diagnostic marker for gastric cancer. The present invention also relates to a method for treating gastric cancer comprising the step of carrying out a method for diagnosing gastric cancer as described above. BACKGROUND

[0002] Gastric cancer is the fifth most common cancer worldwide. More than 1.4 million new cases were reported in 2020. Environmental conditions, including diet, such as artificially preserved, pickled and smoked foods and foods that are sources of nitrosamines, play a significant role in the increased risk of developing gastric cancer. As with most cancers, smoking and excessive alcohol consumption, especially of spirits, increase the risk of developing the disease. Substances in tobacco smoke cause failure of mechanisms controlling peristaltic function through direct stimulation of the gastric muscularis and submucosal nerve plexus. This can lead to reflux of duodenal contents into the stomach, causing mucosal gastritis and bile inflammation, which in turn affects the formation of adenocellular neoplastic lesions, including from inflammatory mucosal atrophy to metaplasia, dysplasia and malignant transformation. In addition to the above factors, infections, including bacterial infections, are also of concern. Helicobacter pylori infection is a risk factor for intestinal-type gastric cancer.

[0003] Symptoms caused by gastric cancer are often atypical and can resemble other diseases such as mucositis or peptic ulcer. Thus, patients and physicians often underestimate the worrying symptoms of the disease. The initial symptoms of gastric cancer are non-specific and include abdominal pain, weight loss, vomiting, loss of appetite, early satiety, frequent belching, dysphagia, weakness and indigestion. Abnormalities usually appear late in the physical examination. Symptoms of advanced gastric cancer include: tumors palpable through the abdominal wall, ascites, left supraclavicular fossa (Virchow's) lymph node enlargement, liver enlargement, jaundice, pleural effusion, rectal palpable infiltration. The discovery of these symptoms and the disease usually indicates that gastric cancer has progressed to an advanced stage, which can hamper curative treatment. Gastroscopy is a method that can detect gastric cancer early and distinguish it from precancerous lesions, as it offers the possibility of taking tissue sections for histopathological examination. Endoscopy has a sensitivity of over 90% for gastric cancer. Ultrasound and barium meal X-ray examination are also used for imaging diagnosis of gastric cancer.

[0004] CEA, CA 19-9, and CA 72-4 are the primary cancer markers used to diagnose gastric cancer. CEA is a nonspecific marker. Elevated levels are found in patients with colon, rectal, gastric, pancreatic, cervical, and lung cancers. In contrast, the glycoprotein antigen CA 19-9 is a marker released by proliferating cells of pancreatic, gastric, and colon cancers. The diagnostic usefulness of CEA and CA 19-9 is limited by their low sensitivity in gastric cancer, meaning that normal levels do not exclude the presence of this cancer.

[0005] Attempts are underway to use CA 72-4 as a predictive marker, but due to the low selectivity of this test, this marker is not recommended for diagnostic testing to detect all gastric cancers, including early-stage gastric cancer. The use of CA 72-4 in diagnosis, along with other markers established in gastric cancer patients, namely CEA and CA 19-9, allows for very accurate monitoring and a correct prognosis after curative surgery.

[0006] 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 their smaller fragments. This process enables cancer cells to expand by colonizing new tissues and enhances the process of blood vessel formation (angiogenesis), which enables the efficient delivery of nutrients to the tumor. Furthermore, these enzymes are present due to the death of healthy cells caused by the tumor growth process. All of these processes contribute to the characteristic and specific profile of the enzymatic (proteolytic) activity of cancer cells, which is characteristic of tumors.

[0007] In this field, there are known chromogenic peptide molecules that undergo enzymatic decomposition into smaller fragments, resulting in a change or increase in the color of the test solution. This color-producing effect is the result of the release of a chromophore (such as 4-nitroaniline or 2-aminobenzoic acid) from the chromogenic peptide molecule.

[0008] This type of chromogenic molecule and its use are known, for example, from the publications 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.

[0009] However, the use of this class of compounds in gastric cancer diagnosis has not been described so far.

[0010] Also known in the prior art are methods for obtaining chromogenic peptides, which include 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 away, 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. Subsequently, the solution is separated from the resin during filtration, and the peptide is then precipitated from the solution by a non-polar solvent.

[0011] However, chromogenic peptide compounds suitable for the early diagnosis of specific and gastric cancers or methods for obtaining them are not known in the prior art.

[0012] Therefore, in this field, there is an urgent need for "cancer markers" for gastric cancer, which will enable early, sensitive, and specific diagnosis of cancer in a non-invasive and reliable manner, as well as diagnostic methods and therapeutic methods using such diagnostic markers.

[0013] The object of the present invention is to provide a novel, specific cancer diagnostic marker and a diagnostic method using this marker for the non-invasive, rapid, sensitive and specific early detection of gastric cancer, which is also suitable for screening tests, as well as a therapeutic method using this marker.

[0014] 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

[0015] The present invention provides a compound having formula 1:

[0016] X1 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -X2 6 (Formula 1),

[0017] wherein X1 comprises or consists of the molecule C1, and X2 comprises or consists of the molecule C2,

[0018] The molecules C1 and C2 are the fluorescence donor and fluorescence acceptor pair,

[0019] And wherein the compound is enzymatically cleaved into fragments X1-Gly-Thr-Thr-Gly-OH (fragment 1) and X2 (fragment 2), generating a measurable light signal upon spatial separation of molecules C1 and C2.

[0020] The compounds according to the invention preferably undergo hydrolytic cleavage, more preferably proteolysis.

[0021] Preferably, in the compounds according to the present invention, the pair of molecules 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 pair of C1 and C2 is (ABZ) / pNA or ABZ / ANB-NH2.

[0022] Preferably, the compound according to the present invention is a compound having Formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3).

[0023] More preferably, the compounds according to the invention undergo hydrolytic cleavage, generating the following fragment 1: ABZ-Gly-Thr-Thr-Gly-OH and fragment 2: ANB-NH2.

[0024] 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 gastric cancer cells, comprising:

[0025] a) contacting a body fluid sample with a compound having formula 1:

[0026] X1 1-Gly 2 -Thr 3 -Thr 4 -Gly 5 -X2 6 (Formula 1),

[0027] wherein X1 comprises or consists of the molecule C1, and X2 comprises or consists of the molecule C2,

[0028] The molecules C1 and C2 are the fluorescence donor and fluorescence acceptor pair,

[0029] and wherein the compound is enzymatically cleaved into fragments X1-Gly-Thr-Thr-Gly-OH (fragment 1) and X2 (fragment 2), and

[0030] b) A measurable light signal is generated when the detection molecules C1 and C2 are spatially separated.

[0031] In the method for detecting enzyme activity according to the present invention, the enzyme activity is preferably hydrolytic activity, more preferably proteolytic activity.

[0032] In the method for detecting enzyme activity according to the present invention, as the compound, it is preferred to use a compound having formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 (Formula 2) or a compound having formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3).

[0033] In the method for detecting enzyme activity according to the present invention, the body fluid is preferably urine, more preferably human urine.

[0034] The present invention also relates to an in vitro method for diagnosing gastric cancer, wherein the presence of gastric cancer in a subject is detected by measuring the activity of a gastric 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 gastric cancer, while the presence of the enzyme activity indicates the presence of gastric cancer.

[0035] In the method for detecting / diagnosing gastric cancer according to the present invention, detection of enzyme activity is performed by the method for detecting enzyme activity as described above.

[0036] In the method for detecting / diagnosing gastric cancer according to the present invention, the measurement of the enzyme activity is performed using a compound having Formula 1:

[0037] X1 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -X2 6 (Formula 1),

[0038] wherein X1 comprises or consists of the molecule C1, and X2 comprises or consists of the molecule C2,

[0039] The molecules C1 and C2 are the fluorescence donor and fluorescence acceptor pair,

[0040] And wherein the compound is enzymatically cleaved into fragments X1-Gly-Thr-Thr-Gly-OH (fragment 1) and X2 (fragment 2), generating a measurable light signal upon spatial separation of molecules C1 and C2.

[0041] In the method for detecting / diagnosing gastric cancer according to the present invention, the body fluid sample is preferably incubated with the compound in a measurement buffer having a neutral or alkaline pH (more preferably physiological), and the ratio of the sample to the measurement buffer is 1:2 to 1:10, preferably 1:5.

[0042] In the method for detecting / diagnosing gastric 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.

[0043] In the method for detecting / diagnosing gastric cancer according to the present invention, as the compound, it is preferred to use a compound having formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 (Formula 2) or a compound having formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3).

[0044] In the method for detecting / diagnosing gastric cancer according to the present invention, a urine sample, more preferably human urine, is preferably used as the sample.

[0045] In the method for detecting / diagnosing gastric cancer according to the present invention, the measurement of the enzyme activity preferably includes measuring the absorbance 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, within 40-60 minutes.

[0046] The present invention also provides a kit comprising any compound according to the present invention as described above and a measurement buffer.

[0047] In the kit according to the present invention, the compound is preferably a compound having Formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 or a compound having Formula 3: ABZ-Gly-Thr-Thr-Gly-pNA.

[0048] The present invention also provides use of any compound according to the present invention as defined above for detecting gastric cancer-specific enzyme activity.

[0049] The present invention also provides the use of any compound according to the invention as defined above in the diagnosis of gastric cancer.

[0050] Preferably, in this use, the diagnosis of gastric cancer includes detecting primary gastric cancer, detecting minimal residual lesions after surgical resection of gastric cancer and / or detecting gastric cancer recurrence.

[0051] Preferably, the compound for use according to the present invention is a compound of formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 or a compound of formula 3: ABZ-Gly-Thr-Thr-Gly-pNA.

[0052] The present invention further provides any compound according to the present invention as defined above for use as a diagnostic marker for detecting gastric cancer.

[0053] Preferably, the compound used as a diagnostic marker according to the present invention is a compound having formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 or a compound having formula 3: ABZ-Gly-Thr-Thr-Gly-pNA.

[0054] The present invention also provides a method for treating gastric cancer, wherein

[0055] a) detecting the presence of gastric cancer-specific enzyme activity in a body fluid sample of the subject by any of the methods described above, and

[0056] b) if the enzyme activity is found in the sample, treating the subject for gastric cancer.

[0057] Preferably, in the treatment method according to the present invention, the gastric cancer-specific enzyme activity is monitored at predetermined time intervals after the treatment according to point b) is completed.

[0058] Preferably, in the method of treatment according to the present invention, a urine sample (preferably human urine) is used as a sample.

[0059] Preferably, in the treatment method according to the present invention, a compound having formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 or a compound having formula 3: ABZ-Gly-Thr-Thr-Gly-pNA is used as the compound. DETAILED DESCRIPTION

[0060] 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 specified, the present invention is not limited to any particular method, protocol or reagent for achieving it. 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 this patent should be understood as follows:

[0061] A chromogenic compound or chromogenic molecule is a compound that has the property of producing color. Chromogenicity refers to the ability of a compound to form a colored product.

[0062] 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.

[0063] NMP stands for N-methylpyrrolidone; DMF stands for dimethylformamide; DCM stands for methylene chloride or dichloromethane; pNA stands for 4-nitroaniline or p-nitroaniline; ABZ stands for 2-aminobenzoic acid, and ANB-NH2 stands for 5-amino-2-nitrobenzamide; Boc stands for tert-butyloxycarbonyl; Fmoc stands for 9-fluorenylmethoxycarbonyl; and TFA stands for trifluoroacetic acid.

[0064] In the context of the present invention, the term gastric cancer should be understood as a primary gastric cancer (malignant tumor) that originates from the tissue lining the stomach. The most common type of gastric cancer is endometrial adenocarcinoma (approximately 90%); others include undifferentiated carcinoma, adenosquamous carcinoma, and other rare variants. Therefore, the term gastric cancer as used herein includes any gastric malignancy that develops from the tissue lining the stomach.

[0065] In the context of the present invention, the term gastric cancer treatment is understood to mean treatment at an early stage of disease progression, which makes it possible to significantly prolong survival time and improve the quality of life of the affected subject.

[0066] In the context of the present invention, the term monitoring is understood as the diagnosis of minimal residual disease (MRD) - 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.

[0067] In the context of the present invention, the term "subject" should be understood to refer to a human subject or mammal suspected of having gastric cancer, or a human subject or mammal belonging to an increased risk group for gastric cancer, or a human subject or mammal after gastric cancer resection or completion of gastric cancer treatment. The subject is preferably a human subject.

[0068] The compounds according to the present invention have color development properties due to the presence of chromophores and have fluorescent properties, i.e., they contain molecules of fluorescent donors and acceptors. Due to their structure, color deepening is observed in the wavelength range of 380-440nm, particularly when contacted with test fluid samples of subjects being examined who suffer from gastric cancer, while this effect is not observed in reactions with body fluid samples of healthy subjects or subjects diagnosed with another type of cancer. These compounds make it possible to detect gastric cancer-specific enzymatic activity, particularly in the early stages of such cancers, in a specific and sensitive manner to diagnose gastric cancer. The subjects being examined are preferably human subjects. The body fluid is preferably urine, more preferably human urine.

[0069] In a first aspect of the present invention, a novel compound is provided, which has the formula 1:

[0070] X1 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -X2 6 (Formula 1),

[0071] Wherein X1 is an amino acid derivative or a peptide fragment comprising molecule C1, or X1 consists of such molecule C1, X2 is an amino acid derivative or a peptide fragment comprising molecule C2, or X2 consists of such molecule C2, wherein molecules C1 and C2 are fluorescent donor and fluorescent acceptor pairs. The superscript represents the subsequent position of the residue in the compound according to 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 to not indicate the residue number. The core of all compounds according to the present invention is a tetrapeptide with a specified sequence of 4 amino acids, i.e. Gly-Thr-Thr-Gly (the symbol of the three-letter amino acid abbreviation format is equivalent to the symbol of the single-letter amino acid abbreviation format: GTTG), which is also presented in the sequence list as sequence No. 1.

[0072] The compound according to the present invention is enzymatically cleaved into fragments: X1-Gly-Thr-Thr-Gly-OH (fragment 1) and X2 (fragment 2), which generate a measurable light signal upon spatial separation of molecules C1 and C2. 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 fluorescence donor by the fluorescence acceptor. It is obvious to the skilled person that the key factor is the distance between the fluorescence donor and the acceptor. Therefore, if the amino acid sequences of the isolated molecules C1 and C2 are folded into a distorted 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.

[0073] Due to its colorimetric properties and the presence of a reactive site at position 5, the compound can be enzymatically (preferably proteolytically) cleaved into smaller fragments, making it particularly suitable for use as a diagnostic marker, especially a specific diagnostic biomarker for gastric cancer, especially for the early diagnosis of gastric cancer.

[0074] In a preferred embodiment, the compounds according to the invention undergo hydrolytic cleavage, more preferably proteolytic cleavage.

[0075] In a preferred embodiment, the pair of molecules 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 pair of molecules C1 and C2 is ABZ / pNA or ABZ / ANB-NH2.

[0076] In a preferred embodiment, the compounds according to the invention are:

[0077] Compound of formula 2:

[0078] ABZ 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -ANB 6 -NH2 (Formula 2) or

[0079] Compound of formula 3:

[0080] ABZ 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -pNA6 (Formula 3),

[0081] Wherein ABZ represents 2-aminobenzoic acid, ANB-NH2 represents 5-amino-2-nitrobenzamide, and pNA represents 4-nitroaniline.

[0082] In the case of the compound having Formula 2, the compound undergoes hydrolytic cleavage to produce the following fragment 1: ABZ-Gly-Thr-Thr-Gly-OH and fragment 2: ANB-NH2, while in the case of the compound having Formula 3, the following fragment 1: ABZ-Gly-Thr-Thr-Gly-OH and fragment 2: pNA are produced. Therefore, fragment 2 is a free chromophore.

[0083] The spatial separation of molecules C1 and C2 is the result of enzymatic cleavage of the compound according to the invention, resulting in the generation of a measurable light signal, since the fluorescence emitted from the fluorescence donor is no longer quenched by the fluorescence acceptor. This measurable light signal can preferably be detected at a wavelength of 300-500 nm, more preferably 380-430 nm.

[0084] The compounds according to the invention can be obtained by known methods. For example, they can be obtained using the 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 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 for this process should be properly prepared. The preparation of the resin involves increasing its volume by repeated washing with a hydrophobic solvent. Preferably, a resin with a degree of substitution of 0.23 mmol / g is used. The resin must be washed with a 20% solvent solution to remove the Fmoc protecting group.

[0085] Then, the known method for obtaining chromogenic peptides is included in the connection of individual components under appropriate time and stoichiometric conditions. The connection process includes a subsequent step in which individual elements (amino acid derivatives) are connected, residues are washed off, blocking 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, in a filtering process, the solution is separated from the resin, and then the peptide is precipitated from the resulting solution by a non-polar solvent. The peptide precipitate obtained in this way is centrifuged.

[0086] Exemplary, detailed but non-limiting methods for synthesizing compounds according to the present invention are described below and in Example 1 below.

[0087] The method for synthesizing the compound according to the present invention comprises carrying out the method 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 increasing its volume by washing with a hydrophobic solvent (preferably dimethylformamide, dichloromethane or N-methylpyrrolidone) and preferably removing the Fmoc protecting group by washing in a solvent (e.g. dimethylformamide, dichloromethane or N-methylpyrrolidone) containing 10-30% piperidine solution.

[0088] Then, execute the method in the following steps:

[0089] a) before attaching 5-amino-2-benzoic acid, ANB (or another chromophore suitable for the present invention as defined in the claims) to the resin, the solid support is washed with a DMF solution containing 3-6% N-methylmorpholine (NMM), then washed with DMF, and then a DMF solution containing ANB is prepared, to which TBTU, DMAP and finally diisopropylethylamine (DIPEA) are added in sequence in the following excess relative to the polymer substitution: ANB / TBTU / DMAP / DIPEA, 3:3:2:6; The mixture prepared in this way is added to the resin and mixed until homogeneous, and then the resin is filtered under reduced pressure and washed with solvents such as DMF, DCM and isopropanol. Then, hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea (HATU) is used, followed by an excess of hexafluorophosphate-O-(benzotriazol-1-yl)-N,N,N'N'-tetramethylurea (HBTU) to attach ANB to the resin. After completion, the solid support is washed with DMF, DCM and isopropanol in sequence and gently dried.

[0090] b) the connection of the amino acid residue to ANB is carried out by reaction with an amino acid derivative -Fmoc-Gly-OH, wherein an amino acid derivative in at least five-fold molar excess relative to the resin is dissolved in anhydrous pyridine and contacted with the resin on which the ANB is deposited, 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 the reaction is carried out 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 resulting intermediate compound is subjected to an acylation process, and the Gly-Thr-Thr fragment is sequentially connected;

[0091] c) acylation of the intermediate compound obtained is carried out using an amino acid derivative, preferably using Fmoc-Thr(tBu)-OH, subsequently using Fmoc-Thr(tBu)-OH, then using Fmoc-Gly-OH, and in the last step of the synthesis using Boc-Abz-OH, using diisopropylcarbodiimide as a coupling agent, which is used in excess, and the acylation is carried out stepwise from residue 6 to 1, and after each step, the resin is washed with DMF, preferably performing a chloranil test (a test for the presence of free amino groups), wherein the attachment of the amino acid derivative is monitored;

[0092] d) removing the Fmoc protecting group by washing with DMF containing 10-30% piperidine solution and then washing with each solvent (DMF, isopropanol and dichloromethane);

[0093] 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 / v, respectively, stirring the mixture for at least one hour, preferably three hours, and filtering the resulting precipitate under reduced pressure, followed by washing with diethyl ether and centrifuging the resulting peptide;

[0094] f) The finished product was prepared by dissolving the peptide in water by ultrasonication and then lyophilizing.

[0095] A second aspect of the present invention provides an in vitro method for detecting enzymatic activity, preferably proteolytic activity, in a body fluid of a subject, in particular activity derived from gastric cancer cells, the method comprising a) contacting a sample of the body fluid with a compound according to the invention, and b) detecting a measurable light signal generated upon spatial separation of molecules C1 and C2 present in the compound according to the invention. In a preferred embodiment of this aspect, the subject being examined is a human subject. In another preferred embodiment of this aspect, the body fluid is urine, in particular human urine.

[0096] In a preferred embodiment of this aspect, a compound having formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 or a compound having formula 3: ABZ-Gly-Thr-Thr-Gly-pNA is used.

[0097] A third aspect of the present invention provides an in vitro method for diagnosing gastric cancer, wherein the presence of gastric cancer in a subject is detected by measuring the activity of an enzyme specific for gastric cancer in a body fluid sample from the subject being examined, wherein the absence of the enzyme activity indicates the absence of gastric cancer, and the presence of the enzyme activity indicates the presence of gastric cancer. This detection of 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, particularly human urine. In a preferred embodiment of this aspect, the enzyme activity specific for gastric cancer is proteolytic activity. In a preferred embodiment of this aspect, a compound having formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 or a compound having formula 3: ABZ-Gly-Thr-Thr-Gly-pNA is used.

[0098] Furthermore, in a preferred embodiment of this aspect, the measurement of the enzyme activity in the method according to the invention comprises measuring the absorbance intensity in the range of 300-500 nm, preferably 380-430 nm, in particular 405 nm, at a temperature in the range 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.

[0099] In addition, in a preferred embodiment of the method according to the present invention, the measurement of the enzyme activity is performed using the compound according to the present invention in a concentration range of 0.1-10 mg / mL, more preferably in a concentration range of 1 mg / mL. In a preferred embodiment of the method according to the present invention, the test sample is incubated with the compound according to the present invention in a measurement buffer having a neutral or alkaline pH (preferably physiological) together with a body fluid sample (preferably human urine), and the ratio of the sample (e.g., urine) to the measurement buffer is in the range of 1:2 to 1:10, preferably 1:5. The sample is preferably taken from a subject referred for a diagnosis of gastric cancer. Preferably, the absorbance intensity is measured within 40-60 minutes at a temperature in the range of 25-40°C, preferably 36-38°C, within a range of 300-500 nm, preferably 380-430 nm, in particular 405 nm. Under the above conditions, a measurable light signal of maximum intensity is obtained due to an increase in absorbance or fluorescence.

[0100] In a fourth aspect, the present invention provides a kit comprising any compound according to the present invention and a measurement buffer. Measurement buffers are known in the art, and suitable buffers for use in the kit according to the present invention include, but are not limited to, Tris-HCl buffer. In a preferred embodiment, in the kit according to the present invention, the compound is a compound having Formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 or a compound having Formula 3: ABZ-Gly-Thr-Thr-Gly-pNA.

[0101] In a fifth aspect, the present invention provides the use of a compound according to the present invention for detecting gastric cancer-specific enzyme activity. In a sixth aspect, the present invention provides the use of a compound according to the present invention for diagnosing gastric cancer. Preferably, according to the present invention, the diagnosis of gastric cancer includes detecting primary gastric cancer, detecting minimal residual lesions after surgical resection of gastric cancer, and / or detecting gastric cancer recurrence after previously completed gastric cancer treatment.

[0102] In a seventh aspect, the present invention provides a compound according to the present invention for use as a diagnostic marker for detecting gastric cancer. In a preferred embodiment of this aspect, the compound is a compound having formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 or a compound having formula 3: ABZ-Gly-Thr-Thr-Gly-pNA.

[0103] In an eighth aspect, the present invention provides a method for treating gastric cancer, wherein

[0104] a) detecting the presence of gastric cancer-specific enzyme activity in a body fluid sample from a subject to be examined by any of the methods of the present invention as defined above, and

[0105] b) when the enzyme activity is found in the sample, treating the subject for gastric cancer.

[0106] In a preferred embodiment of the method of treatment, after the treatment according to point b) is completed, the gastric cancer-specific enzyme activity is monitored at predetermined time intervals known in the art (e.g., weekly, every few weeks, monthly, every few months, annually, or any other interval deemed appropriate by those skilled in the art) to detect minimal residual lesions or recurrence after surgical resection of gastric 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 treatment method, a compound having Formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3) is used as the compound.

[0107] The advantage of the present invention is to provide a new compound, the properties of which make it suitable for the specificity and sensitivity of the detection of the gastric cancer-specific enzyme activity, for use as a diagnostic biomarker for the detection of gastric cancer, for the rapid, non-invasive diagnosis of gastric cancer, while being able to detect gastric cancer in its early stages of progression. Another advantage is that the diagnostic method according to the present invention can be successfully used for screening tests. This enables a comprehensive diagnosis in the early stages of cancer progression, thus obtaining a more effective treatment. Early diagnosis can be treated surgically, thus significantly prolonging the survival time of the patient. This is also important when monitoring the effectiveness of the application of surgical treatment and / or chemotherapy for gastric cancer, as it enables the detection of minimal residual lesions or recurrence, if any.

[0108] The following figures and the following examples will now illustrate the present invention, however, these figures and examples are not intended to limit the scope of the invention as defined in the claims in any way. BRIEF DESCRIPTION OF DRAWINGS

[0109] Figure 1 The chromatographic analysis results of the substrate cleavage, i.e. ABZ-Gly-Thr-Thr-Gly-ANB-NH2, in urine samples from gastric cancer patients are shown.

[0110] Figure 2 The hydrolysis rates of the substrate ABZ-Gly-Thr-Thr-Gly-ANB-NH2 in urine samples of subjects diagnosed with gastric cancer (samples 1-20) and healthy subjects (samples 21-40) are shown. The Arabic numerals indicate the number of selected urine samples.

[0111] Figure 3 The selectivity of the hydrolysis of the substrate ABZ-Gly-Thr-Thr-Gly-ANB-NH2 (i.e. compound of formula 2) extracted from urine samples taken from a subject diagnosed with gastric cancer (sample 1) and from subjects diagnosed with another tumor disease (samples 2-9) is shown. The Arabic numerals indicate the number of individual cancer types. The samples tested for each type of cancer come from 20 different patients for each tested cancer. The results are the average values for a given cancer type. The results show the selectivity of substrate cleavage in the urine of gastric cancer patients compared to urine samples of patients with other tumors. 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -NH2. The Arabic numerals indicate the number of individual cancer types. The samples tested for each type of cancer come from 20 different patients for each tested cancer. The results are the average values for a given cancer type. The results show the selectivity of substrate cleavage in the urine of gastric cancer patients compared to urine samples of patients with other tumors.

[0112] Figure 4 The selectivity of the hydrolysis of the substrate ABZ-Gly-Thr-Thr-Gly-ANB-NH2 (i.e. compound of formula 2) extracted from urine samples taken from a subject diagnosed with gastric cancer (sample 1) and from subjects diagnosed with another tumor disease (samples 2-9) is shown. The Arabic numerals indicate the number of individual cancer types. The samples tested for each type of cancer come from 20 different patients for each tested cancer. The results are the average values for a given cancer type. The results show the selectivity of substrate cleavage in the urine of gastric cancer patients compared to urine samples of patients with other tumors. 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5The level of hydrolysis of -NH2 as a function of the pH conditions. The values displayed on the X axis represent the pH of the environment.

[0113] Example

[0114] The following non-limiting examples illustrate the application. Unless otherwise indicated, the following examples use known and / or commercially available devices, methods, reaction conditions, reagents and components that are generally employed in the field to which the application pertains and are recommended by the manufacturers of the respective reagents and kits.

[0115] Example 1: Synthesis of compounds according to the present invention

[0116] This example describes the synthesis of a representative compound according to the application, namely the compound: ABZ 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -NH2. The remaining peptides according to the application can be synthesized in a similar manner. The superscripts indicate the subsequent position of the residues in the compounds according to the application and the order of the connection of the residues during the synthesis. The compounds according to the application can alternatively be represented with a similar formula without indicating the residue positions, which does not change the sequence of the residues in the compounds according to the application, as it remains unchanged.

[0117] 1. Obtaining the chromogenic peptide

[0118] a) The first step of the synthesis is to obtain the chromogenic peptide obtained by solid-phase synthesis using Fmoc / tBu chemistry, i.e. using protecting agents, on a solid support.

[0119] The following amino acid derivatives (Boc-ABZ, Fmoc-Gly, Fmoc-Thr(tBu), Fmoc-Thr(tBu), Fmoc-Gly) were used to obtain the compound having the sequence ABZ 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -NH2, wherein ABZ is 2-aminobenzoic acid, ANB-NH2 is 5-amino-2-benzamide and ANB is 5-amino-2-benzoic acid, during the solid-phase chemical synthesis.

[0120] The synthesis of the compounds according to the invention, which can be used as diagnostic markers for the detection of gastric cancer, was carried out on a solid support capable of converting 5-amino-2-nitrobenzoic acid to ANB-NH2 amide, namely the amide resin TentaGel SRAM from RAPP Polymere (Germany) with a degree of substitution of 0.23 mmol / g. However, any other amide resin, such as Rink amide (Germany), can be used.

[0121] The synthesis of the compounds was performed manually using a laboratory shaker. In most steps, a 25 mL fritted syringe for solid phase synthesis was used as the reactor.

[0122] All final compounds obtained contain a 2-aminobenzoic acid (ABZ) molecule at position 1 (i.e., N-terminus) and a 5-amino-2-nitrobenzoic acid (ANB) molecule at position 6 (i.e., C-terminus) of their sequence. ABZ acts as a fluorescence donor, while ANB—5-amino-2-nitrobenzoic acid—acts as a fluorescence quencher and also acts as a chromophore. The peptides contain at least, and preferably one, reactive site in their sequence, located between the amino acid residues Gly-ANB-NH2, i.e., at position 5 of the compound. Synthesis consisting of linked amino acid derivatives is performed from residues 6 to 1, i.e., from the C-terminus to the N-terminus.

[0123] b) Attaching ANB to TentaGel S RAM resin:

[0124] Peptide synthesis was performed on Rapp Polymere's TentaGel S RAM resin with a degree of substitution of 0.23 mmol / g. The first step involved preparing the resin, which involved loosening it through wash cycles. Subsequently, the Fmoc amino protecting group was removed from the solid support using a 20% piperidine solution in NMP. Solvent wash cycles were then performed. A chlorobenzoquinone test was performed to confirm the presence of free amino groups.

[0125] Solvent cleaning cycle:

[0126] DMF 1x 10 min; IsOH 1x 10 min; DCM 1x 10 min.

[0127] Removal of Fmoc protection:

[0128] DMF 1 x 5 min; NMP containing 20% ​​piperidine 1 x 3 min; NMP containing 20% ​​piperidine 1 x 8 min.

[0129] Solvent cleaning cycle:

[0130] DMF 3x 2 min; IsOH 3x 2 min; DCM 3x 2 min.

[0131] c) Chlorobenzoquinone test:

[0132] 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 chloranil solution in toluene and 50 μL of fresh acetaldehyde. After 10 minutes, the pellets are checked for color.

[0133] At this stage, after testing, green particles were obtained, which confirmed the presence of free amino groups. After confirming that the 9-fluorenylmethoxycarbonyl protecting group was detached from the resin, the next step could be carried out, namely the attachment of the ANB derivative (5-amino-2-nitrobenzoic acid).

[0134] d) Connecting 5-amino-2-nitrobenzoic acid to a solid support:

[0135] The first step in peptide synthesis is to displace ANB onto 1 gram of resin. Before attaching the chromophore, the resin used for the reaction is washed with the following solvents: DMF, DCM, and DMF, and then the Fmoc protecting group is removed from the functional group of the solid support. A cycle of Fmoc removal includes the following steps:

[0136] Removal of Fmoc-protecting group:

[0137] 20% piperidine in NMP 1 x 3 min; 20% piperidine in NMP 1 x 8 min.

[0138] e) Washing:

[0139] DMF 3x 2 min; IsOH 3x 2 min; DCM 3x 2 min.

[0140] f) Chlorobenzoquinone test:

[0141] 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 washing cycles was carried out in a Merrifield vessel. In a separate flask, ANB was dissolved in DMF, followed by the addition of TBTU, DMAP, and finally diisopropylethylamine (DIPEA), and the amount added was as follows relative to the polymer substitution excess: 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 entire acylation process was repeated twice. For the subsequent reaction to attach ANB to the resin, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) was used, followed by O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU). In the final step, the resin was washed sequentially with DMF, DCM, and isopropanol and air-dried.

[0142] g) Linkage of the C-terminal amino acid residue (Fmoc-Gly-OH) to ANB:

[0143] The corresponding amino acid derivative (9-fold molar excess relative to the resin substitution) was dissolved in pyridine and transferred to the flask containing the ANB-deposited resin. The whole was cooled until it reached a temperature of -15°C (ice bath: 1 part by weight of NH4Cl, 1 part by weight of NaNO3, 1 part by weight of ice). After reaching the desired temperature, POCl3 was added (in a 1:1 ratio relative to the amount of amino acid derivative used), and the whole was stirred on a magnetic stirrer: at -15°C for 20 minutes, at room temperature for 30 minutes, and at 40°C for 6 hours (oil bath). After the reaction was complete, the resin was filtered under reduced pressure, washed with DMF and MeOH, and dried.

[0144] In the next stage, the residue was attached at the P2 position (Fmoc-Thr(tBu)).

[0145] Before each amino acid residue connection, the resin was washed with DMF for 5 minutes. Diisopropylcarbodiimide was used as a coupling agent in subsequent connections. This process was repeated twice.

[0146] After each acylation, a resin wash cycle was initiated followed by a chlorobenzoquinone test to monitor the attachment of the amino acid derivative to the free amino acid groups on the resin.

[0147] Solvent cleaning cycle:

[0148] DMF 3x 2 min; IsOH 3x 2 min; DCM 3x 2 min.

[0149] Chloranil test:

[0150] 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, resulting in colorless resin particles tested with tetrachlorobenzoquinone. This confirmed the attachment of ANB to the TentaGel S RAM resin, allowing for the next step in peptide synthesis.

[0151] h) Attachment of subsequent protected amino acid residues:

[0152] The resin was washed with DMF together with the attached fragment ANB-Gly in the reactor, and then Fmoc was deprotected from the amino group to attach the protected amino acid derivative Thr.

[0153] Removal of Fmoc protecting group:

[0154] DMF 1 x 5 min; NMP containing 20% ​​piperidine 1 x 3 min; NMP containing 20% ​​piperidine 1 x 8 min.

[0155] Solvent cleaning cycle:

[0156] DMF 3x 2 min; IsOH 3x 2 min; DCM 3x 2 min.

[0157] Chloranil test:

[0158] The chloranil test gave a positive result, as evidenced by the green color of the resin particles. Therefore, the next step - attachment of the amino acid residue Fmoc-Thr(tBu)-OH - was possible.

[0159] Linking of amino acid derivatives

[0160] The resin was washed with DMF before the coupling process. When attaching protected serine residues, the composition of the coupling mixture remained unchanged.

[0161] After each acylation, solvent wash cycles were performed according to the specified procedure, followed by a chloranil test to verify the presence of free amino acid groups in the solution.

[0162] Solvent cleaning cycle

[0163] DMF 3x 2 min; IsOH 3x 2 min; DCM 3x 2 min.

[0164] Chloranil test:

[0165] After the second acylation, the resin particles were colorless, allowing the next step of the synthesis to be carried out, which was to introduce another protected amino acid derivative, Fmoc-Gly, and a 2-aminobenzoic acid molecule. The coupling process was carried out according to the procedures discussed previously.

[0166] The test after connecting the above residue showed a positive result - the resin pellets were colorless.

[0167] 2. Removal of peptides from solid supports

[0168] After synthesis, the amide of the ABZ-Gly-Thr-Thr-Gly-ANB-NH2 peptide was cleaved from the solid support and the side chain protecting groups were 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.

[0169] After 3 hours, the contents of the flask were filtered under reduced pressure in a Schott funnel and washed with diethyl ether. The precipitate obtained was centrifuged for 20 minutes in a SIGMA 2K30 centrifuge (laboratory centrifuge). The precipitate obtained after centrifugation was dissolved in water by ultrasound and then freeze-dried. The remaining compounds according to the present invention can be obtained in a similar manner.

[0170] The identity / characterization of the novel compounds according to the present invention was confirmed using HPLC analysis. HPLC conditions were as follows: RP Bio Wide Pore Supelco C8 column, 250 mm x 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.

[0171] The analyses carried out confirmed that the compound according to the invention was obtained.

[0172] Example 2: Testing the properties of compounds according to the invention as cancer markers

[0173] The activity of the novel compound according to the present invention was studied in a group of 20 subjects diagnosed with gastric cancer using a representative compound according to the present invention. The mechanism of action of the compound according to the present invention (including the representative compound with formula 2) includes a specific enzymatic cleavage at the position of the free molecule that leads to the release of each chromophore, more specifically enzymatic hydrolysis, the chromophore being: ANB-NH2 (5-amino-2-nitrobenzamide) in the case of the compound with formula 2, and pNA (p-nitroaniline) in the case of the compound with formula 3, which exhibits absorbance at a wavelength of 320-480nm, particularly 380-430nm, particularly 405nm. The remaining compounds according to the present invention are characterized by a similar mechanism of action. For this reason, the representative compound ABZ according to the present invention is 1 -Gly 2 -Thr 3 -Thr 4 -Gly 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 gastric cancer. The measurement was performed on a 96-well plate designed for absorbance measurement, and each sample was analyzed in triplicate at a temperature of 37°C. The duration of the measurement 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).

[0174] A Figure 1 As shown, RP HPLC analysis of a randomly selected system including urine collected from a human diagnosed with gastric cancer showed that the compound according to the present invention was cleaved into the peptide fragment ABZ-Gly-Thr-Thr-Gly-OH and the chromophore of the compound (ANB-NH2).

[0175] Measurements showed that the color intensity of the solution increased over time in all urine samples collected from people diagnosed with gastric cancer. The magnitude of the observed increase in absorbance over time varied for each sample examined. A different effect was observed for 20 samples from healthy subjects, as no increase in absorbance within the diagnostic range was observed in any of the 20 urine samples tested.

[0176] The tests performed showed that all samples 1-20 from humans with gastric cancer underwent cleavage, but in the case of samples 4 and 9, the cleavage efficiency of the substrate, i.e., ABZ-Gly-Thr-Thr-Gly-ANB-NH2, was lower than that of samples 6 or 9 (Table 1 Figure 2). This result may be due to differences in the activity and amount of enzymes responsible for enzymatic cleavage (proteolysis). Furthermore, the results shown in Table 1 below indicate that incubation of the substrate solution (compounds according to the present invention) with urine samples taken from healthy individuals (without a cancer diagnosis, labeled sequentially with Arabic numerals 21 to 40) did not result in an increase in absorbance, and therefore, no hydrolysis of the test compound occurred. These results suggest the lack of proteolytic enzymes specific / characteristic of gastric cancer.

[0177] Table 1: Absorbance analysis results

[0178]

[0179]

[0180] Furthermore, the dependence of the cleavage selectivity of the substrates (ie the compounds according to the invention) on the type of cancer examined was investigated. Figure 3 As shown, they indicate that the substrate tested, namely ABZ 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -ANB 6 -NH2, incubated with samples collected from patients diagnosed with the following cancers: renal cancer, lung cancer, intestinal cancer, prostate cancer, stomach cancer, pancreatic cancer, uterine corpus cancer and liver cancer, was not affected by lysis and did not lead to an increase in absorbance within the specific 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, which makes them suitable for the specific detection of gastric cancer-specific enzymatic activity and the specific diagnosis of gastric cancer.

[0181] Table 2 below lists the measurement results obtained in triplicate for each sample.

[0182] Table 2: Pyrolysis selectivity analysis results

[0183] Stomach 1 0.15416 0.16476 0.1487 Kidney 2 0 0 0 Lung 3 0 0 0 Intestine 4 0 0 0 Prostate 5 0 0 0 Stomach 6 0 0 0 Pancreas 7 0 0 0 Uterine body 8 0 0 0 Liver 9 0 0 0

[0184] Furthermore, the dependence of the proteolytic activity of representative compounds according to the invention on the reaction pH was measured. The experiments showed that the investigated materials have at least one enzyme that exhibits maximum activity at alkaline pH ( Figure 4 ).

[0185] The analysis performed confirmed the suitability of the compounds according to the application for sensitive and specific detection of the enzyme activity specific for gastric cancer, and by the same method, they are also suitable for specific diagnosis of gastric cancer and as diagnostic markers of gastric cancer. The mechanism of action of the compounds according to the application lies in their specific enzymatic cleavage at the site resulting in the release of the free chromophore molecule, which generates a measurable light signal, which can be used for diagnostic purposes, in particular for the diagnosis of gastric cancer according to the application.

[0186] SEQUENCE LISTING in accordance with WIPO Standard ST.25 format

[0187] <110> ELTE Ltd.

[0188] <120> Novel diagnostic markers for gastric cancer

[0189] <130> 17P51411P100

[0190] <160> 1

[0191] <170> BiSSAP 1.3.6

[0192] <210> 1

[0193] <211> 4

[0194] <212> PRT

[0195] <213> Homo sapiens

[0196] <400> 1

[0197] Gly Thr Thr Gly 1

[0199]

[0200]

[0201]

[0202]

Claims

1. A compound having formula 1: X1 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -X2 6 (Formula 1) wherein X1 comprises or consists of the molecule C1, and X2 comprises or consists of the molecule C2, The molecules C1 and C2 are the fluorescence donor and fluorescence acceptor pair, And wherein the compound is enzymatically cleaved into fragments X1-Gly-Thr-Thr-Gly-OH (fragment 1) and X2 (fragment 2), generating a measurable light signal upon spatial separation of molecules C1 and C2.

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

3. The compound according to claim 1 or 2, wherein the molecules 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), preferably the pair of C1 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 a compound having Formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3).

5. The compound according to claim 4, wherein the compound undergoes hydrolytic cleavage to produce the following fragment 1: ABZ-Gly-Thr-Thr-Gly-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 gastric cancer cells, comprising: a) contacting a body fluid sample with a compound having formula 1: X1 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -X2 6 (Formula 1) wherein X1 comprises or consists of the molecule C1, and X2 comprises or consists of the molecule C2, The molecules C1 and C2 are the fluorescence donor and fluorescence acceptor pair, wherein the compound is enzymatically cleaved into fragments X1-Gly-Thr-Thr-Gly-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 used is a compound having Formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3).

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 gastric cancer, wherein the presence or absence of gastric cancer in a subject is detected by measuring the activity of a gastric cancer-specific enzyme in a body fluid sample from the subject being tested, and wherein the absence of the enzyme activity indicates the absence of gastric cancer, while the presence of the enzyme activity indicates the presence of gastric 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 having formula 1: X1 1 -Gly 2 -Thr 3 -Thr 4 -Gly 5 -X2 6 (Formula 1) wherein X1 comprises or consists of the molecule C1, and X2 comprises or consists of the molecule C2, The molecules C1 and C2 are the fluorescence donor and fluorescence acceptor pair, And wherein the compound undergoes enzymatic cleavage into fragments X1-Gly-Thr-Thr-Gly-OH (fragment 1) and X2 (fragment 2), generating a measurable light signal upon spatial separation of molecules C1 and C2.

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, and the ratio of the sample to the measurement buffer is 1:2 to 1:10, preferably 1:

5.

14. The method according to any one of claims 10 to 13, wherein the compound is used in a concentration of 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 used is a compound having Formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3).

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 in the range of 25-40°C, preferably 36-38°C, within 40-60 minutes.

18. A kit comprising the compound according to any one of claims 1 to 5 and a measurement buffer.

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

20. Use of the compound according to any one of claims 1 to 5 for detecting gastric cancer-specific enzyme activity.

21. Use of the compound according to any one of claims 1 to 5 in the diagnosis of gastric cancer.

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

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

24. A compound as defined in any one of claims 1 to 5 for use as a diagnostic marker for detecting gastric cancer.

25. The compound for use according to claim 24, wherein the compound is a compound having Formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3).

26. A method for treating gastric cancer, wherein a) detecting the presence of gastric cancer-specific enzyme activity in a body fluid sample from a subject to be examined by the method according to any one of claims 6 to 9, and, b) if the enzyme activity is found in the sample, treating the subject for gastric cancer.

27. The method according to claim 26, wherein the gastric 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 used is a compound having Formula 2: ABZ-Gly-Thr-Thr-Gly-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-Gly-Thr-Thr-Gly-pNA (Formula 3).

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