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

Abstract

The present invention relates to a novel compound-diagnostic marker for use in medicine, more particularly for cancer diagnosis, in particular for the diagnosis of cancer. The invention also relates to an in vitro method for detecting enzymatic activity in a bodily fluid of a subject, in particular derived from ovarian cancer cells, using said compounds. The invention also relates to an in vitro method for diagnosing ovarian cancer by using the compound, a kit containing the compound, application of the compound in detection of ovarian cancer specific enzyme activity and application of the compound in diagnosis of ovarian cancer. The present invention also relates to a compound for use as an ovarian cancer diagnostic marker and a method for treating ovarian cancer, the method comprising a step of performing the ovarian cancer diagnostic method 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, especially for the diagnosis of ovarian cancer. The present invention also relates to a method for using the compound to detect in vitro enzyme activity present in a subject's body fluid, especially enzyme activity derived from ovarian cancer cells, a method for in vitro diagnosis of ovarian cancer using the compound, a kit containing the compound, the use of the compound in detecting ovarian cancer-specific enzyme activity, the use of the compound in the diagnosis of ovarian cancer, and a compound used as a diagnostic marker for ovarian cancer. The present invention also relates to a method for treating ovarian cancer, comprising the steps of implementing the method for diagnosing ovarian cancer as described above. Background Art

[0002] Ovarian cancer is the eighth most common cancer diagnosed in women and the third most common cancer of the female reproductive organs. More than 300,000 new cases were reported in 2020. Worldwide, more than 114,000 women die of ovarian cancer each year. Risk factors for this cancer include childlessness, early or late menstruation, late menopause (after 55 years of age), hormone therapy, and active or passive smoking. In its early stages, ovarian cancer usually does not produce any symptoms; gastrointestinal symptoms appear only when the disease is more advanced. Despite the name, ovarian cancer is not only a disease of the ovaries themselves, in most cases, so-called peritoneal spread can be observed, i.e. the presence of tumors on other abdominal organs. The poor treatment outcome is due to late diagnosis and the fact that the cancer has spread very early throughout the abdomen, to the intestines, liver, spleen and other tissues. This is because the ovaries are organs that are freely located in the abdominal cavity, so cancer cells can easily spread to neighboring organs. In addition, it often metastasizes to the lymph nodes via the lymphatic pathway. Ovarian cancer is characterized by a moderate to poor prognosis, which depends mainly on the stage of the disease and the patient's age and general health. The earlier this cancer is detected, the greater the chances of a permanent cure. The 5-year survival rate for cases detected early is over 90%, while it is only 20% for late-stage cases.

[0003] Symptoms of ovarian cancer are nonspecific and are usually general, such as ascites, lower abdominal pain, and genital tract bleeding. The diagnosis is confirmed by a gynecological examination of the patient, ultrasound examination, and the presence of the CA-125 marker in the blood. CA-125 can be used for diagnosis, monitoring, and prognosis of the course of the disease, as well as to identify its recurrence. In about half of patients with early ovarian cancer, it manifests as an elevated CA-125 level. It is estimated that more than 80% of women with ovarian cancer have elevated levels of the CA-125 marker. The sensitivity and specificity of CA-125 in predicting the occurrence of malignancy and its positive predictive value are low, because before surgery, about 50% of patients with stage I epithelial ovarian cancer have normal CA-125 levels. In addition, an elevated CA-125 level is influenced by many diseases, including malignancies of other abdominal organs and the presence of many benign diseases. Attempts are being made to use CA-125 as a predictive marker, but due to the low selectivity of its detection, this marker is not recommended for the diagnosis of ovarian cancer, including early-stage cancer.

[0004] A new qualitative serum test that helps predict cancer diagnosis in women with pelvic tumors has been released for sale. The test, called OVA1, is based on the evaluation of five biomarkers: transthyretin, apolipoprotein A-1, microglobulin beta 2, transferrin, and CA-125.

[0005] Another recognized biomarker is the measurement of HE4 (human epididymis protein 4 from epithelial cells, human epididymis protein 4). HE4 is a protease inhibitor found in malignant epithelial ovarian cancer cells. It can be detected in the serum of women with ovarian cancer. The combined measurement of HE4 and CA-125 has diagnostic parameters superior to both results interpreted separately. Based on a model that relies on the simultaneous measurement of two circulating cancer markers in serum (CA-125 and HE4), ROMA (Risk of Ovarian Malignancy Algorithm) was created, an algorithm for assessing the risk of the presence of epithelial ovarian cancer and the likelihood that existing small pelvic lesions of unclear status are malignant. Despite the progress, there is no laboratory test that includes cancer markers, or a panel of tests that can diagnose ovarian cancer early and with certainty.

[0006] Basis of the present invention application

[0007] It is well known that the process of initiation, growth and spread of cancer cells involves many factors, including many enzymes, especially hydrolases and especially proteolytic enzymes. Such 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 efficient delivery of nutrients to the tumor. In addition, these enzymes are the result of the death of healthy cells due to the tumor growth process. All these processes form a characteristic and specific profile of the enzymatic (proteolytic) activity of cancer cells, which is characteristic of tumors.

[0008] In the art, it is known that chromogenic peptide molecules can be broken down into smaller fragments by enzymes, causing the color of the solution being tested to change or deepen. This color development effect is the result of the release of chromophores (such as 4-nitroaniline or 2-aminobenzoic acid) from the chromogenic peptide molecules.

[0009] Chromogenic molecules of this type and their uses are known, for example, from Erlanger BF, Kokowsky N, Cohen W., "The preparation and properties of two new chromogenic substrate softrypsin", 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.

[0010] However, the use of this class of compounds in the diagnosis of ovarian cancer has not been described so far.

[0011] Methods for obtaining chromogenic peptides are also known in the prior art and 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, the residues are washed off, the protecting groups are removed in turn 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.

[0012] However, a chromogenic peptide compound suitable for specific and early diagnosis of ovarian cancer or a method for obtaining the same is unknown in the prior art.

[0013] Therefore, in this field, there is an urgent need for a "cancer marker" for ovarian cancer that can perform early, sensitive and specific diagnosis of ovarian cancer in a non-invasive and reliable manner, as well as a diagnostic method and a therapeutic method using such a diagnostic marker.

[0014] The object of the present invention is to provide a novel, specific diagnostic marker for ovarian cancer and a diagnostic method using such a marker for non-invasive, rapid, sensitive and specific early detection of ovarian cancer, which is also suitable for screening tests, as well as a therapeutic method using such a marker.

[0015] These objects have been achieved by the invention defined in the attached patent claims, while preferred variants thereof are defined in the dependent claims. Summary of the invention

[0016] The present invention provides a compound having formula 1:

[0017] X1 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -X2 6 (Formula 1),

[0018] wherein X1 comprises or consists of molecule C1, X2 comprises or consists of molecule C2,

[0019] The pair of molecules C1 and C2 are a pair of fluorescence donor and fluorescence acceptor,

[0020] And wherein the compound undergoes enzymatic cleavage into fragments X1-Asp-Thr-Phe-Ile-OH (fragment 1) and X2 (fragment 2) upon spatial separation of molecules C1 and C2, and generates a measurable light signal.

[0021] The compounds of the invention preferably undergo hydrolytic cleavage, more preferably proteolytic cleavage.

[0022] 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.

[0023] Preferably, the compound of the present invention is a compound having Formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (Formula 3).

[0024] More preferably, the compounds of the invention undergo hydrolytic cleavage to produce the following fragment 1: ABZ-Asp-Thr-Phe-Ile-OH and fragment 2: ANB-NH2.

[0025] The present invention also provides a method for in vitro detection of enzyme activity present in a subject's body fluid, particularly enzyme activity derived from ovarian cancer cells, comprising:

[0026] a) contacting a body fluid sample with a compound of formula 1:

[0027] X1 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -X2 6 (Formula 1),

[0028] wherein X1 comprises or consists of molecule C1, X2 comprises or consists of molecule C2,

[0029] The pair of molecules C1 and C2 are a pair of fluorescence donor and fluorescence acceptor,

[0030] And wherein the compound is cleaved by enzyme into fragments X1-Asp-Thr-Phe-Ile-OH (fragment 1) and X2 (fragment 2), and

[0031] b) A measurable light signal is generated when the detection molecules C1 and C2 are spatially separated.

[0032] In the method for detecting enzyme activity according to the present invention, the enzyme activity is preferably hydrolytic activity, more preferably proteolytic activity.

[0033] In the method for detecting enzyme activity according to the present invention, it is preferred to use a compound of Formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 (Formula 2) or a compound of Formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (Formula 3) as the compound.

[0034] 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.

[0035] The present invention also relates to an in vitro method for diagnosing ovarian cancer, wherein the presence or absence of ovarian cancer in a subject is detected by measuring the activity of an ovarian cancer-specific enzyme in a body fluid sample from the subject, and wherein the absence of the enzyme activity indicates the absence of ovarian cancer, while the presence of the enzyme activity indicates the presence of ovarian cancer.

[0036] In the method for detecting / diagnosing ovarian cancer according to the present invention, the detection of enzyme activity is performed by the method for detecting enzyme activity as defined above.

[0037] In the method for detecting / diagnosing ovarian cancer according to the present invention, the enzyme activity is measured using a compound having Formula 1:

[0038] X1 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -X2 6 (Formula 1),

[0039] wherein X1 comprises or consists of molecule C1, X2 comprises or consists of molecule C2,

[0040] The pair of molecules C1 and C2 are a pair of fluorescence donor and fluorescence acceptor,

[0041] And wherein the compound undergoes enzymatic cleavage into fragments X1-Asp-Thr-Phe-Ile-OH (fragment 1) and X2 (fragment 2) when molecules C1 and C2 are spatially separated, and a measurable light signal is generated.

[0042] In the method for detecting / diagnosing ovarian cancer according to the present invention, the body fluid sample is preferably incubated with the compound in an assay buffer having a neutral or alkaline pH (more preferably a physiological pH), in a sample to assay buffer ratio ranging from 1:2 to 1:10, preferably 1:5.

[0043] In the method for detecting / diagnosing ovarian 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.

[0044] In the method for detecting / diagnosing ovarian cancer according to the present invention, as the compound, it is preferred to use a compound having Formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (Formula 3).

[0045] In the method for detecting / diagnosing ovarian cancer according to the present invention, as the sample, preferably a urine sample, more preferably human urine is used.

[0046] In the method for detecting / diagnosing ovarian cancer according to the present invention, the determination of the enzyme activity preferably includes determining the absorption intensity in the range of 300-500nm, more preferably 380-430nm, and especially 405nm, within a temperature range of 25-40°C, more preferably 36-38°C, during 40-60 minutes.

[0047] The present invention also provides a kit comprising any compound of the present invention as described above and an assay buffer.

[0048] In the kit of the present invention, the compound is preferably a compound having Formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 or a compound having Formula 3: ABZ-Asp-Thr-Phe-Ile-pNA.

[0049] The present invention also provides the use of any compound of the present invention as defined above for detecting ovarian cancer specific enzyme activity.

[0050] The present invention also provides the use of any compound of the present invention as defined above for the diagnosis of ovarian cancer.

[0051] Preferably, in this use, the diagnosis of ovarian cancer comprises detecting primary ovarian cancer, detecting minimal residual disease after surgical resection of cancer and / or detecting recurrence of ovarian cancer.

[0052] Preferably, the compound used in the present invention is a compound of Formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 or a compound of Formula 3: ABZ-Asp-Thr-Phe-Ile-pNA.

[0053] The present invention also provides any compound of the present invention as defined above for use as a diagnostic marker for detecting ovarian cancer.

[0054] Preferably, the compound used as a diagnostic marker of the present invention is a compound having Formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 or a compound having Formula 3: ABZ-Asp-Thr-Phe-Ile-pNA.

[0055] The present invention also provides a method for treating ovarian cancer, wherein

[0056] a) detecting the presence of ovarian cancer-specific enzyme activity in a body fluid sample from a subject by any of the methods defined above, and

[0057] b) treating the subject for ovarian cancer if the presence of said enzyme activity is found in said sample.

[0058] Preferably, in the treatment method according to the present invention, the ovarian cancer-specific enzyme activity is monitored at predetermined time intervals after the treatment according to point b) is completed.

[0059] Preferably, in the method of treatment according to the invention a urine sample, preferably human urine, is used as sample.

[0060] Preferably, in the treatment method of the present invention, a compound of Formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 or a compound of Formula 3: ABZ-Asp-Thr-Phe-Ile-pNA is used as the compound. DETAILED DESCRIPTION

[0061] It should be understood that the invention is defined in the appended claims. This specification describes various non-limiting embodiments and examples of the invention. Unless otherwise specified, the invention is not limited to any specific method, protocol or reagent for practicing the 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 invention. However, for the sake of clarity, the following expressions / terms and abbreviations used in the patent should be understood as follows:

[0062] A chromogenic compound or chromogenic molecule is a compound that has the property of chromogenicity. Chromogenicity refers to the ability of a compound to form a colored product.

[0063] A fluorescent compound or fluorescent molecule is a compound that has the property of fluorescence. The fluorescent property refers to the ability of a compound to form products that emit fluorescence.

[0064] 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, ANB-NH2 represents 5-amino-2-nitrobenzamide; Boc represents tert-butyloxycarbonyl; Fmoc represents 9-fluorenyloxycarbonyl; TFA represents trifluoroacetic acid.

[0065] In the context of the present invention, the term ovarian cancer is understood to refer to a primary cancer (malignant tumor) of the ovary that develops from tissue located in the ovary. Ovarian cancer is most commonly an endometrial adenocarcinoma (about 90%) and less commonly a serous clear cell carcinoma. The term ovarian cancer as used herein therefore includes all malignant ovarian tumors that develop from tissue located in the ovary.

[0066] In the context of the present invention, the term "diagnosis of ovarian cancer" should be understood to refer to the identification of the disease, especially in its early stages, when other diagnostic methods are not sensitive and / or specific enough. As used herein, the diagnosis of ovarian cancer also includes the detection of minimal residual disease (MRD) after surgical resection of ovarian cancer and the detection of ovarian cancer recurrence after previously completed ovarian cancer treatment.

[0067] In the context of the present invention, the term "treatment of ovarian cancer" is understood to mean treatment at an early stage of disease progression, which can significantly prolong patient survival time and improve the patient's quality of life.

[0068] In the context of the present invention, the term: monitoring is to be understood as referring to the diagnosis of minimal residual disease (MRD), i.e. the presence of a small number of viable cancer cells in the organism (during treatment or remission), in an amount that cannot be detected by standard diagnostic methods.

[0069] In the context of the present invention, the term "subject" should be understood to refer to a human subject or mammal suspected of having ovarian cancer, or alternatively, a human subject or mammal belonging to a group with increased risk of ovarian cancer, or a human subject or mammal after ovarian cancer resection or after completion of treatment for ovarian cancer. The subject is preferably a human subject.

[0070] Due to the presence of chromophores, the compounds of the invention have both chromogenic and fluorescent properties, i.e. they contain molecules of fluorescent donors and acceptors. Since the structures of the compounds of the invention are such that, in particular due to contact with a test body fluid sample of a subject suffering from ovarian cancer, an increase in color is observed in the wavelength range of 380-440 nm, while this effect is not observed in reactions with body fluid samples from healthy subjects or subjects diagnosed with another type of cancer, these compounds make it possible to detect enzyme activities specific to ovarian cancer, in particular to diagnose ovarian cancer in a specific and sensitive manner, also at an early stage of the progression of ovarian cancer. The subject is preferably a human subject. The body fluid is preferably urine, more preferably human urine.

[0071] In the first aspect of the present invention, a novel compound is provided, which has formula 1:

[0072] X1 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -X2 6 (Formula 1),

[0073] 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 residue in the compound of the present invention and the order in which the residues are connected during the synthesis process. According to the present invention, in this case, Chemical Formula 1 can be written alternatively without indicating the number of the residue. The core of all compounds of the present invention is a tetrapeptide having a specified sequence of 4 amino acids, i.e., Asp-Thr-Phe-Ile (the symbol in the three-letter amino acid abbreviation is equivalent to the symbol in the one-letter amino acid abbreviation: DTFI), which is also shown as sequence number 1 in the sequence table.

[0074] The compound according to the present invention undergoes enzymatic cleavage into fragments: X1-Asp-Thr-Phe-Ile-OH (fragment 1) and X2 (fragment 2), generating a measurable light signal when the molecules C1 and C2 are spatially separated. The measurable light signal is determined by a method of measuring the absorbance / fluorescence change after the enzymatic cleavage of the compound. Preferably, the 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 the molecules C1 and C2 is folded into a twisted or condensed secondary structure, resulting in the proximity of the molecules C1 and C1 relative to the primary structure, the distance between the molecules C1 and C2 can be greater than 10 amino acid residues.

[0075] This compound, due to its chromogenic properties and the presence of a reactive site at position 5 enabling enzymatic (preferably proteolytic) cleavage into smaller fragments, is particularly suitable for use as a diagnostic marker, in particular a specific diagnostic biomarker for ovarian cancer, in particular for the early diagnosis of ovarian cancer.

[0076] In a preferred embodiment, the compounds of the invention undergo hydrolytic cleavage, more preferably proteolytic cleavage.

[0077] 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 C1 and C2 pair is (ABZ) / pNA or ABZ / ANB-NH2.

[0078] In a preferred embodiment, the compounds of the present invention are:

[0079] The compound has Formula 2:

[0080] ABZ 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -ANB 6 -NH2 (Formula 2) or

[0081] The compound has Formula 3:

[0082] ABZ 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -pNA 6 (Formula 3),

[0083] Wherein ABZ represents 2-aminobenzoic acid, ANB-NH2 represents 5-amino-2-nitrobenzamide, and pNA represents 4-nitroaniline.

[0084] In the case of the compound of formula 2, the compound undergoes hydrolytic cleavage to produce the following fragment 1: ABZ-Asp-Thr-Phe-Ile-OH and fragment 2: ANB-NH2, while in the case of the compound of formula 3, the following fragment 1: ABZ-Asp-Thr-Phe-Ile-OH and fragment 2: pNA are produced. Therefore, fragment 2 is a free chromophore.

[0085] The spatial separation of molecules C1 and C2 is the result of the 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 preferably detected at a wavelength of 300-500nm, more preferably at 380-430nm.

[0086] The compounds of the present invention can be obtained by known methods. For example, the compounds of the present invention can be obtained using a method for obtaining chromogenic peptides, which comprises 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 to carry out the process should be appropriately prepared. The preparation of the resin consists in increasing its volume by repeated washing with a hydrophobic solvent. It is preferred to use a resin with a deposition amount of 0.23 mmol / g. The resin must be washed with a 20% solvent solution to remove the Fmoc protecting group.

[0087] Then, the known method of obtaining chromogenic peptides consists in connecting the 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 protective groups are removed in turn 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 in a filtering process, and the peptide is then precipitated from the solution obtained by a non-polar solvent. The peptide precipitate obtained in this way is centrifuged.

[0088] Exemplary, detailed but non-limiting synthetic methods for compounds of the invention are described below and in Example 1 below.

[0089] 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 with an Fmoc group, wherein before the method starts, the solid support is prepared by repeatedly washing with a hydrophobic solvent (preferably dimethylformamide, dichloromethane or N-methylpyrrolidone) to increase its volume and removing the Fmoc protecting group by washing in a solvent (such as dimethylformamide, dichloromethane or N-methylpyrrolidone) preferably with a 10-30% piperidine solution.

[0090] Then, execute the method in the following steps:

[0091] a) before depositing 5-amino-2-nitrobenzoic acid ANB (or other chromophores suitable for the present invention as defined in the claims) on the resin, washing the solid support with a 3-6% solution of N-methylmorpholine (NMM) in DMF, then washing with DMF, thereafter preparing a DMF solution of ANB to which TBTU, DMAP and finally diisopropylethylamine (DIPEA) are added in order in excess relative to the polymer deposition: ANB / TBTU / DMAP / DIPEA, 3:3:2:6; The mixture prepared in this way is added to the resin and mixed until homogeneous, then the resin is filtered under reduced pressure and washed with solvents such as DMF, DCM and isopropanol, then O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) is used, followed by an excess of benzotriazole-N,N,N'N'-tetramethyluronium hexafluorophosphate (HBTU), and ANB is continued to be attached to the resin. After completion, the solid support is washed with DMF, DCM and isopropanol in sequence and gently dried;

[0092] b) the connection of the amino acid residue to ANB is carried out by reaction with an amino acid derivative - Fmoc-Ile-OH, wherein at least five times molar excess of the amino acid derivative relative to the resin is dissolved in anhydrous pyridine and brought into contact with the deposited ANB, and then the whole is cooled to a temperature not lower 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, and 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 an acylation process, and the Asp-Thr-Phe fragments are sequentially connected;

[0093] c) acylation of the intermediate compound obtained with an amino acid derivative, preferably Fmoc-Phe-OH, followed by Fmoc-Thr(tBu)-OH, then Fmoc-Asp(OtBu)-OH and in the last step of the synthesis with Boc-Abz-OH, the acylation being carried out in steps from residues 6 to 1, using diisopropylcarbodiimide used in excess as coupling agent, washing the resin with DMF after each step, preferably carrying out a chlorobenzoquinone test (a test for the presence of free amino groups), wherein the attachment of the amino acid derivative is monitored;

[0094] d) removing the Fmoc protecting group by washing with 10-30% piperidine in DMF, followed by washing with each of DMF, isopropanol and dichloromethane;

[0095] e) using a mixture: 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 obtained precipitate under reduced pressure, then washing with diethyl ether, and centrifuging the obtained peptide to separate the peptide from the resin;

[0096] f) The peptide is dissolved in water by ultrasonication and then freeze-dried to prepare a finished product.

[0097] A second aspect of the invention provides an in vitro method for detecting enzyme activity present in a subject's body fluid, preferably proteolytic activity, in particular enzyme activity derived from ovarian cancer cells, the method comprising a) contacting a body fluid sample with a compound of the invention and b) detecting a measurable light signal generated upon spatial separation of the molecules C1 and C2 present in the compound of the 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.

[0098] In a preferred embodiment of this aspect, the compound has Formula 2:

[0099] ABZ-Asp-Thr-Phe-Ile-ANB-NH2 or a compound having Formula 3:

[0100] ABZ-Asp-Thr-Phe-Ile-pNA was used.

[0101] The third aspect of the present invention provides an in vitro method for diagnosing ovarian cancer, wherein the presence or absence of ovarian cancer in a subject is detected by measuring the ovarian cancer-specific enzyme activity in a body fluid sample from the subject, wherein the lack of the enzyme activity indicates the absence of ovarian cancer, and the presence of the enzyme activity indicates the presence of ovarian 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, particularly human urine. In a preferred embodiment of this aspect, the ovarian cancer-specific enzyme activity is proteolytic activity. In a preferred embodiment of this aspect, a compound having formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 or a compound having formula 3: ABZ-Asp-Thr-Phe-Ile-pNA is used.

[0102] Furthermore, in a preferred embodiment of this aspect, the determination of the enzyme activity in the method of the present invention comprises measuring the absorbance intensity within 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 the maximum intensity of a measurable light signal caused by an increase in absorbance or fluorescence to be obtained.

[0103] In addition, in a preferred embodiment of the method of the present invention, the enzyme activity is measured using a compound of the present invention in a concentration 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 a compound according to the present invention in a measurement buffer having a neutral or alkaline pH (preferably physiological pH) with a body fluid sample (preferably human urine), and the ratio of the sample (e.g., urine) to the measurement buffer is 1:2 to 1:10, preferably 1:5. The sample is preferably taken from a subject with a referral for diagnosis of ovarian cancer. Preferably, the absorbance intensity is measured within 40-60 minutes at a temperature of 25-40°C, preferably 36-38°C, within a range of 300-500nm, preferably 380-430nm, and particularly 405nm. Under the above conditions, a maximum intensity of a measurable light signal is obtained due to an increase in absorbance or fluorescence.

[0104] In a fourth aspect, the present invention provides a kit comprising any compound of the present invention and an assay buffer. Assay buffers are known in the art, and buffers suitable for the kit of the present invention are, for example, but 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-Asp-Thr-Phe-Ile-ANB-NH2 or a compound having Formula 3: ABZ-Asp-Thr-Phe-Ile-pNA.

[0105] In a fifth aspect, the present invention provides the use of a compound according to the present invention in detecting ovarian cancer specific enzyme activity. In a sixth aspect, the present invention provides the use of a compound according to the present invention in diagnosing ovarian cancer. Preferably, according to the present invention, the diagnosis of ovarian cancer includes detecting primary ovarian cancer, detecting minimal residual disease after surgical resection of ovarian cancer, and / or detecting recurrence of ovarian cancer after previously completing ovarian cancer treatment.

[0106] In a seventh aspect, the present invention provides a compound of the present invention for use as a diagnostic marker for detecting ovarian cancer. In a preferred embodiment of this aspect, the compound is a compound having Formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 or a compound having Formula 3: ABZ-Asp-Thr-Phe-Ile-pNA.

[0107] In an eighth aspect, the present invention provides a method for treating ovarian cancer, wherein

[0108] a) detecting the presence of ovarian cancer-specific enzyme activity in a body fluid sample from a subject by any of the methods of the invention as defined above, and

[0109] b) treating ovarian cancer in the subject when the enzyme activity is found to be present in the sample.

[0110] In a preferred embodiment of the method of treatment, after completion of the treatment according to point b), the ovarian 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 ovarian 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-Asp-Thr-Phe-Ile-ANB-NH2 (Formula 2) or a compound of Formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (Formula 3) is used as the compound.

[0111] The present invention has the advantage of providing a new compound having an enzymatic activity that makes it suitable for specific and sensitive detection of ovarian cancer, for use as a diagnostic biomarker for detecting ovarian cancer, for rapid, non-invasive diagnosis of ovarian cancer, while being able to detect ovarian cancer at an early stage of its development. Another advantage is that the diagnostic method according to the present invention can be successfully used in screening tests. This enables comprehensive diagnosis at an early stage 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 ovarian cancer, because minimal residual disease or recurrence (if any) can be detected.

[0112] The present invention will now be described in the following figures and examples which, however, are not intended in any way to limit the scope of the invention defined in the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0113] Figure 1 Shown are the results of chromatographic analysis of substrate cleavage, i.e., expression of ABZ-Asp-Thr-Phe-Ile-ANB-NH2 in urine samples from subjects with ovarian cancer.

[0114] Figure 2 The hydrolysis rates of the substrate -ABZ-Asp-Thr-Phe-Ile-ANB-NH2- in urine samples from subjects diagnosed with ovarian cancer (samples 1-20) and urine samples from healthy subjects (samples Z21-Z40) are shown. The Arabic numerals indicate the number of the selected urine sample.

[0115] Figure 3 The substrate-ABZ extracted from urine samples of a subject diagnosed with ovarian cancer (sample 1) and from subjects diagnosed with another neoplastic disease (samples 2-9) is shown. 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -ANB 6 -NH2 (i.e., compound of Formula 2) hydrolysis selectivity. Arabic numerals represent the number of a specific cancer type. The test samples for each cancer were from 20 different patients for each tested cancer. The results are averages for a given cancer type. The results show that urine from ovarian cancer patients has selectivity for substrate cleavage compared to urine samples from patients with other tumors.

[0116] Figure 4 The substrate-ABZ is shown 1 -Asp 2 -Thr 3 -Phe4 -Ile 5 Dependence of the hydrolysis level of -NH2 on pH conditions.

[0117] Example

[0118] The present invention is illustrated by the following non-limiting examples. Unless otherwise specified, the following examples use known and / or commercially available devices, methods, reaction conditions, reactants and kits, which are commonly used in the art to which the present invention belongs and are recommended by the manufacturers of the corresponding reactants and kits.

[0119] Example 1: Synthesis of the compounds of the present invention

[0120] This example describes the synthesis of a representative compound of the present invention, namely compound: ABZ 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -NH2. The remaining peptides of the present invention can be synthesized in a similar manner. The 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 the synthesis. The compounds of the present invention can alternatively be represented by a similar formula that does not indicate the position of the residues, which does not change the order of the residues in the compounds of the present invention because it remains unchanged.

[0121] 1. Obtaining Chromogenic Peptides

[0122] 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 with protection.

[0123] In the solid phase chemical synthesis process, the following amino acid derivatives were used: 5-amino-2-nitrobenzoic acid Boc-ABZ, Fmoc-Asp(OtBu), Fmoc-Thr(tBu), Fmoc-Phe, Fmoc-Ile, and the sequence ABZ was obtained. 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -NH2 compound, wherein ABZ is 2-aminobenzoic acid, ANB-NH2 is 5-amino-2-nitrobenzamide, and ANB is 5-amino-2-nitrobenzoic acid.

[0124] The synthesis of the compounds useful as diagnostic markers for the detection of ovarian cancer according to the invention was carried out on a solid support capable of converting 5-amino-2-benzoic acid into ANB-NH2 amide, namely the amide resin TentaGel S RAM from RAPP Polymere (Germany) with a deposition of 0.23 mmol / g. However, any other amide resin may be used, for example RinkAmide (Germany).

[0125] 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.

[0126] All final compounds obtained contain 2-aminobenzoic acid (ABZ) 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 Ile-ANB-NH2, i.e. position 5 of the compound. The synthesis consisting of the linked amino acid derivatives is carried out from residues 6 to 1, i.e. from the C- to the N-terminus.

[0127] b) Deposition of ANB on TentaGel S RAM resin:

[0128] The peptide synthesis was performed on TentaGel S RAM resin from Rapp Polymere with a deposit of 0.23 mmol / g. In the first step, the resin was prepared, including loosening it by washing cycles. Subsequently, the Fmoc-amino group was deprotected from the solid support with 20% piperidine in NMP. Then, solvent washing cycles were performed. To confirm the presence of free amino groups, a chloranil test was performed.

[0129] Solvent cleaning cycle:

[0130] DMF 1x 10 min; IsOH 1x 10 min; DCM 1x 10 min.

[0131] Removal of Fmoc protection:

[0132] DMF 1 x 5 min; 20% piperidine in NMP 1 x 3 min; 20% piperidine in NMP 1 x 8 min.

[0133] Solvent cleaning cycle:

[0134] DMF 3x 2 min; IsOH 3x 2 min; DCM 3x 2 min.

[0135] c) Chloranil test:

[0136] The chloranil test consists in transferring a few pellets of resin from the reactor (syringe) into a glass ampoule with a spatula, to which 100 μL of a saturated toluene solution of chloranil and 50 μL of fresh acetaldehyde are then added. After 10 minutes, a control of the pellet color is performed.

[0137] 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).

[0138] d) Deposition of 5-amino-2-nitrobenzoic acid on a solid support:

[0139] The first step in peptide synthesis is to deposit ANB on 1 g 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 protection is removed from the functional groups of the solid support. One cycle of removing the Fmoc protection includes the following steps:

[0140] Removal of Fmoc protection:

[0141] 1 x 3 min in 20% piperidine in NMP; 1 x 8 min in 20% piperidine in NMP.

[0142] e) Washing:

[0143] DMF 3x 2 min; IsOH 3x 2 min; DCM 3x 2 min.

[0144] f) Chloranil test:

[0145] The resin with free amino groups was washed with 5% DMF solution of N-methylmorpholine (NMM), and then washed with DMF. The procedure and washing cycle for removing Fmoc protection were carried out in a Merrifield container. In another flask, ANB was dissolved in DMF, and then the following TBTU, DMAP and the last diisopropylethylamine (DIPEA) were added in excess relative to polymer deposition: ANB / TBTU / DMAP / DIPEA, 3:3:2:6v / 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. In order to carry out the subsequent reaction in which ANB was subsequently connected to the resin, O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) was used, followed by benzotriazole-N,N,N'N'-tetramethyluronium hexafluorophosphate (HBTU). In the last step, the resin was washed sequentially with DMF, DCM and isopropanol and air-dried.

[0146] g) Linking of the C-terminal amino acid residue (Fmoc-Ile-OH) to ANB:

[0147] The corresponding amino acid derivative (9-fold molar excess relative to the resin deposition) was dissolved in pyridine and transferred to the flask containing the resin deposited with ANB. The whole was cooled to 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 (in a 1:1 ratio with the amount of amino acid derivative used) was added and stirred on a magnetic stirrer: 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.

[0148] In the next stage, the residue was attached at the P2 position (Fmoc-Phe(tBu)).

[0149] Before each connection of amino acid residues, the resin was washed with DMF for 5 minutes. Diisopropylcarbodiimide was used as a coupling agent in subsequent connections. This process was repeated twice.

[0150] 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.

[0151] Solvent cleaning cycle:

[0152] DMF 3x 2 min; IsOH 3x 2 min; DCM 3x 2 min.

[0153] Chloranil test:

[0154] As a result of the tests performed, after the first two coupling steps, the color of the particles was first green and then gray, so it was necessary to perform another acylation, resulting in the resin particles being colorless when tested with chloranil. This proved that the ANB was attached to the TentaGel S RAM resin and that the next step of peptide synthesis could be performed.

[0155] h) Subsequent attachment of protected amino acid residues:

[0156] The resin was washed with DMF together with the linker fragment ANB-Ile in the reactor, and then Fmoc was deprotected from the amino group to link the protected amino acid derivative Phe.

[0157] Removal of Fmoc protection:

[0158] DMF 1 x 5 min; 20% piperidine in NMP 1 x 3 min; 20% piperidine in NMP 1 x 8 min.

[0159] Solvent cleaning cycle:

[0160] DMF 3x 2 min; IsOH 3x 2 min; DCM 3x 2 min.

[0161] Chloranil test:

[0162] The chloranil test yielded a positive result, as evidenced by the green color of the resin particles, so the next step - attachment of the amino acid residue Fmoc-Thr(tBu)-OH - was possible.

[0163] Linking of amino acid derivatives

[0164] Prior to the coupling process, the resin was washed with DMF. When attaching protected serine residues, the composition of the coupling mixture remained unchanged.

[0165] After each acylation, solvent washing cycles were performed according to the specified procedure, followed by a chloranil test for the presence of free amino acid groups in the solution.

[0166] Solvent Wash Cycle

[0167] DMF 3x 2 min; IsOH 3x 2 min; DCM 3x 2 min.

[0168] Chloranil test:

[0169] In tests performed after the second acylation, the resin particles were colorless and thus the next step of the synthesis could be carried out, which was the introduction of another protected amino acid derivative, Fmoc-Asp(OtBu), and the 2-aminobenzoic acid molecule. The coupling process was carried out as previously discussed.

[0170] Tests conducted after connecting the above residues showed positive results - the resin particles were colorless.

[0171] 2. Removal of peptides from solid supports

[0172] After synthesis, the amide of the ABZ-Asp-Thr-Phe-Ile-ANB-NH2 peptide was removed from the solid support and the side protection was removed simultaneously using a mixture of TFA:phenol:water:TIPS (88:5:5:2, v / v / v / v) on a magnetic stirrer.

[0173] After 3 hours, the contents in the flask were filtered under reduced pressure in a Schott funnel and washed with ether. The precipitate obtained was centrifuged for 20 minutes on a SIGMA2K30 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.

[0174] The identity / characteristics of the novel compounds of the present invention were confirmed by HPLC analysis. The conditions of HPLC analysis were as follows: RPBio Wide Pore Supelco C8 column, 250mm 4mm, phase system A: 0.1% TFA aqueous solution, B: 80% acetonitrile A solution, flow rate 1mL / min, UV detection wavelength 226nm.

[0175] The analyses performed confirmed that the compound of the present invention was obtained.

[0176] Example 2: Testing the properties of the compounds of the invention as cancer markers

[0177] The activity of the novel compounds of the present invention was studied in 20 subjects diagnosed with ovarian cancer using representative compounds of the present invention. The mechanism of action of the compounds of the present invention, including the representative compound of formula 2, is that a specific enzymatic cleavage, more specifically enzymatic hydrolysis, occurs at the position leading to the release of the free molecule of the respective chromophore: ANB-NH2 (amide of 5-amino-2-nitrobenzoic acid) in the case of the compound of formula 2 and pNA (p-nitroaniline) in the case of the compound of formula 3, 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 of the present invention, ABZ 1 -Asp2 -Thr 3 -Phe 4 -Ile 5 -NH2 was dissolved in dimethyl sulfoxide (concentration of 0.5 mg / mL), and then 50 μL of the solution was mixed with 120 μL of buffer (200 mM Tris-HCl, pH 8.0) and 80 μL of urine from a subject with ovarian cancer. The assay was performed in a 96-well plate designed for absorbance determination, and each sample was analyzed three times at a temperature of 37°C. The duration of the assay was 60 minutes. During the assay, the wavelength characteristics of the released chromophore (ANB-NH2) were monitored at a wavelength of 405 nm (range 380-430 nm).

[0178] like Figure 1 As shown, RP HPLC analysis of a randomly selected system containing urine from a person diagnosed with ovarian cancer showed that the compound according to the invention was cleaved into the peptide fragment ABZ-Asp-Thr-Phe-Ile-OH and the chromophore of the compound (ANB-NH2).

[0179] The results of the assay showed that the color intensity of the solution increased over time in all urine samples collected from people diagnosed with cancer. The magnitude of the observed increase in absorbance over time was different for each sample examined. Different effects were obtained for the 20 samples from healthy subjects, as no increase in absorbance within the diagnostic range was observed in any of the 20 urine samples tested.

[0180] The tests performed showed that all samples 1-20 from humans with ovarian cancer underwent cleavage, but in the case of samples 3 and 17, cleavage of the substrate (i.e., ABZ-Asp-Thr-Phe-Ile-ANB-NH2) was not as efficient as in the case of samples 1 or 13 (Table 1, Figure 2 ). Such results may be due to differences in the activity and quantity of enzymes responsible for enzymatic cleavage (proteolysis). In addition, the results shown in Table 1 below indicate that incubation of the substrate solution - the compound of the present invention - with urine samples taken from healthy people (no cancer diagnosis, labeled with Arabic numbers from Z21 to Z40) does not result in an increase in absorbance, and therefore the test compound does not undergo hydrolysis. The results indicate the lack of proteolytic enzymes specific / characteristic to ovarian cancer.

[0181] Table 1 Absorbance analysis results

[0182]

[0183]

[0184] Furthermore, the dependence of the substrate, i.e. the cleavage selectivity of the compounds according to the invention, on the type of cancer being tested was investigated. Figure 3 As shown, it shows that the detected substrate, ABZ 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -NH2 was incubated with samples extracted from patients diagnosed with the following cancers: colorectal cancer, kidney cancer, prostate cancer, pancreatic cancer, stomach cancer, lung cancer, uterine body cancer and liver cancer, and no cleavage occurred within a specific range and no increase in absorbance was caused. In each case, the sample tested was a mixture of 20 samples from each cancer studied. This shows the cleavage selectivity of the compound of the present invention, which makes it suitable for the specific detection of ovarian cancer-specific enzyme activity and the specific diagnosis of ovarian cancer.

[0185] Table 2 below shows the results of the three replicates for each sample.

[0186] Table 2 Pyrolysis selectivity analysis results

[0187]

[0188]

[0189] In addition, the dependence of the proteolytic activity of representative compounds of the invention on the reaction pH was determined. The experiments showed that at least one enzyme of the studied materials exhibited maximum activity at alkaline pH values ​​( Figure 4 ).

[0190] The analysis performed demonstrates the suitability of the compounds of the invention for the sensitive and specific detection of ovarian cancer-specific enzymatic activity and, by this analysis, for the specific diagnosis of ovarian cancer and as diagnostic markers for ovarian cancer. The mechanism of action of the compounds of the invention is their specific enzymatic cleavage at the 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 ovarian cancer according to the invention.

[0191]

[0192]

[0193]

Claims

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

2. The compound according to claim 1, which has been subjected to hydrolytic cleavage, preferably proteolytic cleavage.

3. The compound according to claim 1 or 2, wherein 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), 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, which is a compound having Formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (Formula 3).

5. The compound according to claim 4, which undergoes hydrolytic cleavage to produce the following fragment 1: ABZ-Asp-Thr-Phe-Ile-OH and fragment 2: ANB-NH2.

6. An in vitro method for detecting enzyme activity present in a subject's body fluid, in particular enzyme activity derived from ovarian cancer cells, comprising: a) contacting a body fluid sample with a compound of formula 1: X1 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -X2 6 (Formula 1), wherein X1 comprises or consists of molecule C1, X2 comprises or consists of molecule C2, The pair of molecules C1 and C2 are a pair of fluorescence donor and fluorescence acceptor, And wherein the compound is cleaved by enzyme into fragments X1-Asp-Thr-Phe-Ile-OH (fragment 1) and X2 (fragment 2), and b) Detecting the measurable light signal generated when 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 a compound of Formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 (Formula 2) or a compound of Formula 3: ABZ-Asp-Thr-Phe-Ile-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 ovarian cancer, wherein the presence or absence of ovarian cancer in a subject is detected by measuring the activity of an ovarian cancer-specific enzyme in a body fluid sample from the subject being examined, and wherein the absence of said enzyme activity indicates the absence of ovarian cancer, while the presence of said enzyme activity indicates the presence of ovarian cancer.

11. The method according to claim 10, wherein the detection of 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 enzyme activity is measured using a compound of formula 1: X1 1 -Asp 2 -Thr 3 -Phe 4 -Ile 5 -X2 6 (Formula 1), Where X1 contains or consists of C1 molecules, and X2 contains or consists of C2 molecules: The pair of molecules C1 and C2 are a pair of fluorescence donor and fluorescence acceptor, And wherein the compound undergoes enzymatic cleavage into fragments X1-Asp-Thr-Phe-Ile-OH (fragment 1) and X2 (fragment 2), generating a measurable light signal 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 an assay buffer having a neutral or alkaline pH, preferably a physiological pH, in a sample to assay buffer ratio ranging from 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 claims 10 to 14, wherein a compound of Formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 (Formula 2) or a compound of Formula 3: ABZ-Asp-Thr-Phe-Ile-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 determination of the enzyme activity comprises measuring the absorbance intensity within 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.

18. A kit comprising the compound of any one of claims 1 to 5 and an assay buffer.

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

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

21. Use of the compound according to any one of claims 1 to 5 in diagnosing ovarian cancer.

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

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

24. A compound according to any one of claims 1 to 5 for use as a diagnostic marker for detecting ovarian cancer.

25. The compound according to claim 24, which is a compound of Formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 (Formula 2) or a compound of Formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (Formula 3).

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

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

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: As the compound, a compound having Formula 2: ABZ-Asp-Thr-Phe-Ile-ANB-NH2 (Formula 2) or a compound having Formula 3: ABZ-Asp-Thr-Phe-Ile-pNA (Formula 3) is used.

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