FRET enzyme substrates and their use in lung cancer

Abstract

The present invention relates to a novel compound, diagnostic marker, for use in medical treatment, more particularly in cancer diagnosis, particularly in lung cancer diagnosis.The present invention also relates to an in vitro method for detecting enzyme activity present in a subject's body fluid, particularly from lung cancer cells, using the compound.The present invention further relates to an in vitro method for diagnosing lung cancer using the compound, a kit containing the compound, and the use of the compound for detecting enzyme activity specific to lung cancer, as well as the use of the compound for diagnosing lung cancer.The present invention also relates to the compound for use as a diagnostic marker for lung cancer, and to a method for treating lung cancer, comprising the procedure of carrying out the above-mentioned method for diagnosing lung cancer using the compound.

Description

[Technical field]

[0001] The present invention relates to a novel compound, diagnostic marker, for use in medical treatment, more particularly in cancer diagnosis, particularly in lung cancer diagnosis.The present invention also relates to an in vitro detection method for detecting the enzyme activity present in the body fluid of a subject, particularly from lung cancer cells, using the compound, an in vitro diagnosis method for lung cancer using the compound, a kit containing the compound, the use of the compound for detecting the enzyme activity specific to lung cancer, the use of the compound for diagnosing lung cancer, and the compound for use as a diagnostic marker for lung cancer.The present invention also relates to a method for treating lung cancer, comprising the procedure of carrying out the above-mentioned lung cancer diagnosis method. [Background technology]

[0002] Lung cancer is the second most common malignant neoplasm in the world, with more than 2.2 million patients affected in 2020. Lung cancer is characterized by late onset symptoms, very rapid disease progression, and high mortality. Approximately 1.8 million patients died from lung cancer in 2020. Lung cancer will remain the most lethal cancer until 2030. In the course of lung cancer, early and precancerous lesions do not cause symptoms. For this reason, early detection of the disease is difficult and rarely occurs. In the majority of affected patients, the cancer is found incidentally as a localized lesion during an X-ray examination performed intentionally or for other reasons. In cases of a definite (positive) diagnosis, the 5-year survival rate is at a level of less than 20%. Currently, patients are diagnosed at a late stage of the disease, when treatment options are very limited. Early detection is associated with a good prognosis. Surgical treatment of lung cancer is the only effective method that can lead to the removal of lung cancer. Unfortunately, only about 30% of patients diagnosed with lung cancer are candidates for surgical treatment. Patients who undergo definitive surgical treatment have a 50% 5-year survival rate. There are no clinical tests, "cancer markers" or test sets that allow early and reliable diagnosis of lung cancer. Imaging modalities such as X-ray, CT scan, MRI or positron emission tomography (PET) are used for effective diagnosis, followed by a tumor biopsy. A small proportion of patients (5-10%) show altered expression of certain genes, making this population amenable to genetic testing.

[0003] It is known that the processes of initiation, proliferation and dissemination of cancer cells involve many factors, including a large number of enzymes, especially hydrolases, especially proteolytic enzymes. Such enzymes catalyze the process of enzymatic (hydrolytic or proteolytic) cleavage of proteins and peptides into their smaller fragments. This process allows cancer cells to colonize new tissues and to grow and spread by enhancing the process of blood vessel formation (angiogenesis) that allows an effective delivery of nutrients to the tumor. Moreover, these enzymes are present as a result of the death of healthy cells due to the tumor proliferation process. All these processes form a distinctive and specific aspect of the enzymatic (proteolytic) activity of cancer cells, characteristic of tumors.

[0004] Chromogenic peptide molecules are known in the art that change or increase the color of a test solution upon enzymatic degradation into smaller fragments, the coloring effect being the result of the release of a chromophore (e.g., 4-anilide or 2-aminobenzoic acid) from the chromogenic peptide molecule. Chromogenic molecules of this kind and their uses are known, for example, from the publications 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.

[0005] However, there have been no reports to date on the use of this class of compounds in the diagnosis of lung cancer. Methods for coupling the individual components under suitable time and stoichiometric conditions to obtain chromogenic peptides are also known in the prior art. The coupling method consists of successive steps of coupling the individual building blocks (amino acid derivatives), washing off the residues, sequentially removing the protecting groups and washing again. This cycle is repeated for each amino acid residue. The resulting peptide is separated from the resin by reaction under acidic conditions. The solution is then separated from the resin in a filtration step, after which the peptide is precipitated from the solution by a non-polar solvent.

[0006] However, chromogenic peptide compounds suitable for specific and early diagnosis of lung cancer and methods for obtaining them are not known in the prior art. Thus, there is an urgent need in the art for "cancer markers" for lung cancer that allow for early, sensitive and specific diagnosis of lung cancer in a non-invasive and reliable manner, and for diagnostic and therapeutic methods using such diagnostic markers.

[0007] The object of the present invention is to provide novel and specific diagnostic markers for lung cancer and diagnostic methods for non-invasive, rapid, sensitive and specific early detection of lung cancer using such markers (which may also be suitable as screening tests), as well as therapeutic methods using such markers.

[0008] These objects are achieved by the invention as defined in the appended claims, while preferred variants thereof are defined in the dependent claims. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Erlanger BF, Kokowsky N, Cohen W., “The preparation and properties of two new chromogenic substrates of trypsin”, Arch Biochem Biophys., November 1961; 95:271-8 [Non-Patent Document 2] Hojo K, Maeda M, Iguchi S, Smith T, Okamoto H, Kawasaki K. Amino acids and peptides. XXXV. “Facile preparation of p-nitroanilide analogs by the solid-phase method”, Chem Pharm Bull (Tokyo), November 2000; 48(11):1740-4 Summary of the Invention

[0010] The present invention relates to a compound of formula 1: X1 1 -Tyr 2 -Ile 3 -Phe 4 -Arg 5 -X2 6 (Formula 1) The present invention provides a compound having the formula:

[0011] In the formula, X1 comprises or consists of a molecule C1, and X2 comprises or consists of a molecule C2; Here, the pair of molecules C1 and C2 is a pair of a fluorescent donor and a fluorescent acceptor, The compound undergoes enzymatic cleavage into fragments X1-Tyr-Ile-Phe-Arg-OH (fragment 1) and X2 (fragment 2) and generates a measurable light signal due to the spatial separation of molecules C1 and C2.

[0012] The compounds according to the invention are preferably subject to hydrolytic cleavage, more preferably proteolytic cleavage. Preferably, in the compound according to the invention, the pair of molecules C1 and C2 is 2-aminobenzoic acid (ABZ) / 5-amino-2-nitrobenzoic acid (ANB), (ABZ) / pNA, ABZ / ANB-NH 2 , ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr(3-NO 2), more preferably, the pair of C1 and C2 is selected from the group consisting of (ABZ) / pNA or ABZ / ANB-NH 2 It is.

[0013] Preferably, the compound according to the invention has formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2 (formula 2) or a compound having the formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA (formula 3).

[0014] More preferably, the compounds according to the invention undergo hydrolytic cleavage to give the following fragment 1: ABZ-Tyr-Ile-Phe-Arg-OH and fragment 2: ANB-NH 2 Generate.

[0015] The present invention further provides an in vitro method for detecting an enzymatic activity, particularly from lung cancer cells, present in a body fluid of a subject, the method comprising: a) A body fluid sample was subjected to a reaction with a solvent according to Equation 1: X1 1 -Tyr 2 -Ile 3 -Phe 4 -Arg 5 -X2 6 (Formula 1) contacting the compound having the formula [wherein X1 comprises or consists of a molecule C1, and X2 comprises or consists of a molecule C2; Here, the pair of molecules C1 and C2 is a pair of a fluorescent donor and a fluorescent acceptor, the compound undergoes enzymatic cleavage to give the fragments X1-Tyr-Ile-Phe-Arg-OH (fragment 1) and X2 (fragment 2); and b) Detecting a measurable light signal generated by the spatial separation of molecules C1 and C2 This includes:

[0016] In the method for detecting an enzymatic activity according to the present invention, the enzymatic activity is preferably a hydrolytic activity, more preferably a proteolytic activity. In the method for detecting an enzyme activity according to the present invention, the compound is preferably a compound represented by formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2 Either a compound having the formula 2 or a compound having the formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA (formula 3) is used.

[0017] In the method for detecting enzyme activity according to the present invention, urine, preferably human urine, is used as said body fluid. The present invention also relates to an in vitro method for diagnosing lung cancer, which detects the presence or absence of lung cancer in a subject by measuring a lung cancer-specific enzyme activity in a body fluid sample of the subject, the absence of said enzyme activity indicating the absence of lung cancer, whereas the presence of said enzyme activity indicates the presence of lung cancer.

[0018] In the method for detecting / diagnosing lung cancer according to the present invention, the detection of the enzyme activity is carried out by the method for detecting the enzyme activity defined above. In the method for detecting / diagnosing lung cancer according to the present invention, the measurement of the enzyme activity is carried out by using a method according to Formula 1: X1 1 -Tyr 2 -Ile 3 -Phe 4 -Arg 5 -X2 6 (Formula 1) The reaction is carried out using a compound having the formula:

[0019] In the above formula, X1 comprises or consists of a molecule C1, and X2 comprises or consists of a molecule C2; Here, the pair of molecules C1 and C2 is a pair of a fluorescent donor and a fluorescent acceptor, The compound undergoes enzymatic cleavage into fragments X1-Tyr-Ile-Phe-Arg-OH (fragment 1) and X2 (fragment 2) and generates a measurable light signal due to the spatial separation of molecules C1 and C2.

[0020] In the lung cancer detection / diagnosis method 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, preferably physiological pH, in a sample to assay buffer ratio ranging from 1:2 to 1:10, preferably 1:5.

[0021] In the method for detecting / diagnosing lung cancer according to the present invention, the compound is preferably used at a concentration of 0.1 to 10 mg / mL, particularly 0.25 to 7.5 mg / mL. In the method for detecting / diagnosing lung cancer according to the present invention, the compound is preferably a compound represented by formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2 Either a compound having the formula 2 or a compound having the formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA (formula 3) is used.

[0022] In the method for detecting / diagnosing lung cancer according to the present invention, preferably a urine sample, more preferably human urine, is used as the sample. In the method for detecting / diagnosing lung cancer according to the present invention, the measurement of the enzyme activity preferably comprises measuring the absorbance intensity within the range of 300 to 500 nm, more preferably 380 to 430 nm, particularly 405 nm, for 40 to 60 minutes at a temperature within the range of 25 to 40°C, more preferably 36 to 38°C.

[0023] The present invention further provides a kit comprising any compound according to the invention as defined above and an assay buffer. In the kit according to the invention, the compound preferably has the formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2 or a compound having formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA.

[0024] The present invention also provides the use of any compound according to the invention as defined above for detecting an enzyme activity specific for lung cancer. The present invention also provides the use of any compound according to the invention as defined above for the diagnosis of lung cancer.

[0025] Preferably, in such uses, diagnosing lung cancer includes detecting primary lung cancer, detecting minimal residual disease following surgical resection of lung cancer, and / or detecting recurrence of lung cancer. Preferably, the compound in the use according to the invention has formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2 or a compound having formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA.

[0026] The present invention further provides any compound according to the invention as defined above for use as a diagnostic marker for the detection of lung cancer. Preferably, the compound for use as a diagnostic marker according to the present invention has the formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2 or a compound having formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA.

[0027] The present invention further comprises: a) detecting the presence of a lung cancer-specific enzyme activity in a body fluid sample to be tested by any of the methods defined above, and b) administering to the subject a treatment for lung cancer if the presence of said enzymatic activity is found in said sample. It also offers treatment for lung cancer.

[0028] Preferably, in the method of treatment according to the invention, after the end of the treatment according to point b), said enzymatic activity specific for lung cancer is monitored at defined time intervals. Preferably, in the therapeutic method according to the invention a urine sample, preferably human urine, is used as sample.

[0029] Preferably, in the treatment method according to the invention, a compound of formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2or a compound having the formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA is used as said compound. [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 shows the results of chromatographic analysis of the substrate cleavage, i.e., ABZ-Tyr-Ile-Phe-Arg-ANB-NH2, in urine samples of subjects with lung cancer. [Diagram 2] Figure 2 shows the hydrolysis rate of the substrate ABZ-Tyr-Ile-Phe-Arg-ANB-NH2 in urine samples from subjects diagnosed with lung cancer (samples 1–50) and urine collected from healthy subjects (samples 1Z–25Z). Arabic numerals indicate the numbers of the selected urine samples. [Diagram 3] FIG. 3 shows the selectivity of hydrolysis of the substrate ABZ1-Tyr2-Ile3-Phe4-Arg5-ANB6-NH2 (i.e., compound of formula 2) in a urine sample from a subject diagnosed with lung cancer (sample 1) and urine samples taken from subjects diagnosed with different neoplastic diseases (cancer) (samples 2-9). Arabic numerals indicate the number of a given cancer type. Samples tested for each type of cancer were obtained from 20 different patients for each cancer tested. Results are average values ​​for a given cancer type. Results show the selectivity of substrate cleavage in the case of urine from lung cancer patients compared to urine samples from other cancer patients. [Figure 4] FIG. 4 shows the dependence of the hydrolysis level of the substrate ABZ1-Tyr2-Ile3-Phe4-Arg5-ANB6-NH2 on pH conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] It should be understood that the present invention is defined in the appended claims. In this description, various non-limiting aspects and embodiments of the present invention are illustrated. The present invention is not limited to any particular methodology, protocol, or reagents used to carry it out, unless otherwise specified. 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 acronyms used in this patent shall be understood as follows:

[0032] Chromogenic compound or molecule refers to a compound that has chromogenic properties. Chromogenic properties refers to the ability of a compound to form a colored product. A fluorescent compound or molecule refers to a compound that has fluorescent properties, which refers to the ability of a compound to form products that fluoresce.

[0033] NMP stands for N-methylpyrrolidone; DMF stands for dimethylformamide; DCM stands for methylene chloride or dichloromethane; pNA stands for 4-nitroaniline or para-nitroaniline; ABZ stands for 2-aminobenzoic acid, and ANB-NH 2 represents 5-amino-2-nitrobenzoic acid; Boc represents a tert-butyloxycarbonyl group; Fmoc represents a 9-fluorenylmethoxycarbonyl group; and TFA represents trifluoroacetic acid.

[0034] In the context of the present invention, the term lung cancer shall be understood to mean primary lung cancer (malignant neoplasm) that originates from tissue located in the lung.The most frequent lung cancer is non-small cell lung cancer (about 90%); less frequent is small cell lung cancer.The term lung cancer used herein therefore includes all malignant lung neoplasms that originate from tissue located in the lung.

[0035] In the context of the present invention, the term diagnosis of lung cancer shall be understood to mean the identification of the disease, especially at an early stage when other diagnostic methods are not sufficiently sensitive and / or specific. In this specification, diagnosis of lung cancer also includes the detection of minimal residual disease (MRD) after surgical resection of lung cancer, and the detection of recurrence of lung cancer after previously completed lung cancer treatment.

[0036] In the context of the present invention, the term treatment of lung cancer shall be understood to mean a treatment at an early stage in the progression of the disease, making it possible to significantly extend the survival time and improve the quality of life of affected individuals.

[0037] In the context of the present invention, the term monitoring shall be understood to mean diagnosing the presence of minimal residual disease (MRD), i.e. a small number of surviving cancer cells in the body (during treatment or remission) in amounts that cannot be detected by standard diagnostic methods.

[0038] In the context of the present invention, the term subject is to be understood to mean a human subject or mammal suspected of having lung cancer or belonging to a group at high risk of lung cancer, or after resection of lung cancer or after completion of treatment for lung cancer. The subject is preferably a human subject.

[0039] The compounds according to the invention have color-producing and fluorescent properties due to the presence of a chromophore, i.e. they contain a fluorescent donor and a fluorescent acceptor molecule. Due to their structure, which has been developed in such a way that as a result of contact with a test body fluid sample of a subject, particularly having lung cancer, an increase in color is observed in the wavelength range of 380-440 nm, while such an effect is not observed in reaction with a body fluid sample of a healthy subject or a subject diagnosed with another type of cancer, these compounds make it possible to detect an enzyme activity specific for lung cancer and in particular to diagnose lung cancer with specificity and high sensitivity, even at an early stage of the progression of this cancer. The subject to be examined is preferably a human subject. The body fluid is preferably urine, more preferably human urine.

[0040] In a first aspect of the present invention, there is provided a novel compound having the formula 1: X1 1 -Tyr 2 -Ile 3 -Phe 4 -Arg 5 -X2 6 (Formula 1) where 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, and the pair of molecules C1 and C2 is a fluorescent donor and fluorescent acceptor pair. The superscripts indicate the position of the subsequent residues in the compound according to the invention and the order of attachment of the residues during synthesis. According to the invention, in this context, formula 1 can alternatively be written without showing the numbering of the residues. The core of all compounds according to the invention is a tetrapeptide with the indicated four amino acid sequence (Tyr-Ile-Phe-Arg), which is also shown in the sequence listing as SEQ ID NO: 1.

[0041] The compound according to the invention undergoes enzymatic cleavage into fragments: X1-Tyr-Ile-Phe-Arg-OH (fragment 1) and X2 (fragment 2), generating a measurable light signal due to the spatial separation of molecules C1 and C2. The measurable light signal is measured by a method for measuring the change in absorbance / fluorescence after enzymatic cleavage of the compound. Preferably, molecules C1 and C2 are separated from each other by 10 or fewer amino acid residues to ensure efficient quenching of the fluorescent donor by the fluorescent acceptor. It is clear to those skilled in the art that the key factor is the distance between the fluorescent donor and the acceptor. Thus, the distance between molecules C1 and C2 may be more than 10 amino acid residues, if the amino acid sequence separating them is folded into a twisted or condensed secondary structure, resulting in a closer proximity of molecules C1 and C2 compared to the primary structure.

[0042] Due to their chromogenic properties and the presence of a reactive site at position 5 allowing enzymatic (preferably proteolytic) cleavage into smaller fragments, the compounds are particularly suitable for use as diagnostic markers, in particular specific diagnostic biomarkers for lung cancer, in particular for the early diagnosis of lung cancer.

[0043] In a preferred embodiment, the compounds according to the invention undergo hydrolytic cleavage, preferably proteolytic cleavage. In a preferred embodiment, the pair of molecules C1 and C2 is 2-aminobenzoic acid (ABZ) / 5-amino-2-nitrobenzoic acid (ANB), (ABZ) / pNA, ABZ / ANB-NH 2 , ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr(3-NO 2 ), more preferably the pair of molecules C1 and C2 is selected from the group consisting of ABZ / pNA or ABZ / ANB-NH 2 It is.

[0044] In a preferred embodiment, the compound according to the invention is Formula 2: ABZ 1 -Tyr 2 -Ile 3 -Phe 4 -Arg 5 -ANB 6 -NH 2 (Formula 2) or Formula 3: ABZ 1 -Tyr 2 -Ile 3 -Phe 4 -Arg 5 -pNA 6 (Formula 3) In the formula, ABZ represents 2-aminobenzoic acid, and ANB-NH 2 stands for 5-amino-2-nitrobenzoic acid, and pNA stands for 4-nitroaniline.

[0045] The compounds undergo hydrolytic cleavage to give the following fragment 1: ABZ-Tyr-Ile-Phe-Arg-OH and fragment 2: ANB-NH for compounds having formula 2: 2 On the other hand, in the case of a compound having formula 3, the following fragment 1: ABZ-Tyr-Ile-Phe-Arg-OH and fragment 2: pNA are generated. Thus, fragment 2 is a free chromophore.

[0046] Upon enzymatic cleavage of the compound according to the invention, the molecules C1 and C2 are spatially separated, resulting in the generation of a measurable optical signal, since the fluorescence emitted by the fluorescent donor is no longer quenched by the fluorescent acceptor. Such a measurable optical signal can be detected at a wavelength preferably between 300 and 500 nm, more preferably between 380 and 430 nm.

[0047] The compounds according to the invention can be obtained by known methods. For example, they can be obtained using a method for obtaining chromogenic peptides, in which the process is carried out on a solid support in the form of a resin bearing an Fmoc group (which is removed in the course of 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 method should be appropriately prepared. The preparation of the resin consists in increasing its volume by repeated washing with a hydrophobic solvent. Preferably, a resin with a loading of 0.23 mmol / g is used. The Fmoc protecting group must be removed from the resin by washing with a 20% solvent solution.

[0048] The known methods for obtaining chromogenic peptides then involve coupling the individual components under suitable time and stoichiometric conditions. The coupling method consists of successive steps of coupling the individual building blocks (amino acid derivatives), washing off the residues, sequentially removing the protecting groups and washing again. This cycle is repeated for each amino acid residue. The resulting peptide is separated from the resin by reaction under acidic conditions. The peptide is then precipitated from the resulting solution by a non-polar solvent after the solution has been separated from the resin in a filtration step. The peptide precipitate thus obtained is centrifuged.

[0049] Exemplary detailed, but non-limiting, syntheses of compounds according to the invention are described below and in Example 1 below. The synthesis of the compounds according to the invention is such that the process is carried out on a solid support, preferably in the form of a resin carrying an Fmoc group. Before starting the process, the solid support is prepared by increasing its volume by repeated washing with a hydrophobic solvent, preferably dimethylformamide, methylene chloride or N-methylpyrrolidone, and removing the Fmoc protecting group, preferably by washing with a 10-30% piperidine solution in a solvent such as dimethylformamide, methylene chloride or N-methylpyrrolidone.

[0050] The method is then carried out in the following steps: a) Prior to attachment of 5-amino-2-nitrobenzoic acid (ANB) (or another chromophore suitable for use according to the invention as defined in the claims) onto the resin, the solid support is washed with a 3-6% solution of N-methylmorpholine (NMM) in DMF, then with DMF, after which a solution of ANB in ​​DMF is prepared, to which is added in the following order: TBTU, DMAP and finally diisopropylethylamine (DIPEA) in the following excess relative to polymer attachment: ANB / TBTU / DMAP / DIPEA 3:3:2:6. The mixture thus prepared is added to the resin and mixed until homogeneous. The resin is then filtered under reduced pressure and washed with solvents such as DMF, DCM and isopropanol, after which the conjugation of ANB to the resin is continued with an excess of hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HATU) followed by hexafluorophosphate-O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HBTU). After completion, the solid support is washed successively with DMF, DCM and isopropanol and gently dried.

[0051] b) The attachment of the amino acid residue to the ANB is carried out by reaction with an amino acid derivative, namely Fmoc-Arg(OtBu)-OH. Here, at least a 5-fold excess of the amino acid derivative relative to the resin is dissolved in anhydrous pyridine and contacted with the resin with the ANB attached. After this, the whole is cooled to a temperature not below -20 °C and then POCl 3 is added in a ratio of 1:1 relative to the amount of amino acid derivative used, the whole is mixed, then the mixing steps are carried out at room temperature and then at elevated temperature, once the reaction is complete the resin is filtered under reduced pressure, washed with DMF and MeOH and gently dried, after which the intermediate compound obtained is subjected to an acylation step followed by coupling of the Tyr-Ile-Phe fragment.

[0052] c) Acylation of the intermediate compound obtained is carried out with an amino acid derivative, preferably Fmoc-Phe-OH, followed by Fmoc-Ile-OH, then Fmoc-Tyr(tBu)-OH and, in the final stage of the synthesis, Boc-Abz-OH. The acylation is carried out stepwise from residue 6 to 1, using diisopropylcarbodiimide as coupling agent (used in excess). After each step, the resin is washed with DMF and preferably subjected to the chloranil test (test for the presence of a free amino group) to monitor the binding of the amino acid derivative.

[0053] d) Removal of the Fmoc protecting group is carried out by washing with a 10-30% piperidine solution in DMF, followed by washing with each of the solvents, namely DMF, isopropanol and methylene chloride.

[0054] e) Separation of the peptide from the resin is carried out using a mixture, namely TFA:phenol:water:TIPS, maintaining the ratio of 88:5:5:2 v / v / v / v respectively. The mixture is stirred for at least 1 hour, preferably 3 hours, the resulting precipitate is filtered under reduced pressure, then washed with diethyl ether and the resulting peptide is centrifuged.

[0055] f) The preparation of the final product is carried out by dissolving the peptide in water by ultrasound and then subjecting it to lyophilization. In a second aspect of the present invention, there is provided an in vitro detection method for detecting an enzymatic activity, preferably a proteolytic activity, present in a body fluid of a subject, in particular from lung cancer cells, comprising a) contacting a body fluid sample with a compound according to the present invention, and b) detecting a measurable optical signal generated by the spatial separation of molecules C1 and C2 present in the compound according to the present invention. In a preferred embodiment of this aspect, the subject examined in this case is a human subject. In another preferred embodiment of this aspect, the body fluid is urine, in particular human urine.

[0056] In a preferred embodiment of this aspect, the compound of formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2or a compound having the formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA is used.

[0057] In a third aspect of the present invention, there is provided an in vitro method for the diagnosis of lung cancer, which detects the presence or absence of lung cancer in a subject by measuring a lung cancer specific enzymatic activity in a body fluid sample of the subject, the absence of said enzymatic activity indicating the absence of lung cancer, whereas the presence of said enzymatic activity indicates the presence of lung cancer. The detection of such enzymatic activity is preferably carried out using the method for detecting enzymatic activity described above. In a preferred embodiment of this aspect, the subject is a human subject. In a preferred embodiment of this aspect, the body fluid is urine, in particular human urine. In a preferred embodiment of this aspect, the lung cancer specific enzymatic activity is a proteolytic activity. In a preferred embodiment of this aspect, a compound of formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2 or a compound having the formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA is used.

[0058] Furthermore, in a preferred embodiment of this aspect, the measurement of the enzyme activity in the method according to the present invention comprises measuring the absorbance intensity at 300 to 500 nm, preferably at 380 to 430 nm, particularly at 405 nm, for 40 to 60 minutes at a temperature in the range of 25 to 40° C., preferably at 36 to 38° C. This makes it possible to obtain the strongest possible measurable optical signal resulting from an increase in absorbance or fluorescence.

[0059] Furthermore, in a preferred embodiment of the method according to the invention, the measurement of the enzyme activity is carried out with a compound according to the invention at a concentration in the range of 0.1-10 mg / mL, more preferably at a concentration of 1 mg / mL. In a preferred embodiment of the method according to the invention, the test sample is incubated with the compound according to the invention in an assay buffer having a neutral or alkaline pH, preferably physiological pH. The body fluid sample is preferably human urine, the ratio of sample (e.g. urine sample) to assay buffer being in the range of 1:2-1:10, preferably 1:5. The sample is preferably taken from a subject referred for a diagnosis of lung cancer. Preferably, the absorbance intensity is measured in the range of 300-500 nm, preferably 380-430 nm, in particular 405 nm, for 40-60 minutes, at a temperature in the range of 25-40° C., preferably 36-38° C. Under the above conditions, a maximally intense measurable light signal is obtained as a result of the increase in absorbance or fluorescence.

[0060] In a fourth aspect, the present invention provides a kit comprising any of the compounds according to the present invention and an assay buffer. Assay buffers are known in the art, and a suitable buffer for use in the kit according to the present invention is, for example, but not limited to, Tris-HCl buffer. In a preferred embodiment, in the kit according to the present invention, the compound is represented by the formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2 or a compound having formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA.

[0061] In a fifth aspect, the present invention provides the use of a compound according to the present invention for detecting an enzyme activity specific for lung cancer. In a sixth aspect, the present invention provides the use of a compound according to the present invention for diagnosing lung cancer. Preferably, diagnosing lung cancer according to the present invention includes detecting primary lung cancer, detecting minimal residual disease after surgical resection of lung cancer, and / or detecting recurrence of lung cancer after previously completed lung cancer treatment.

[0062] In a seventh aspect, the present invention provides a compound according to the present invention for use as a diagnostic marker for detecting lung cancer. In a preferred embodiment of this aspect, the compound has the formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2 or a compound having formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA.

[0063] In an eighth aspect, the present invention provides a method for producing a composition comprising: a) detecting the presence of an enzymatic activity specific for lung cancer in a body fluid sample to be examined by any of the methods according to the invention as defined above, and b) administering to the subject a treatment for lung cancer if said enzymatic activity is found to be present in said sample; Provides a treatment for lung cancer.

[0064] In a preferred embodiment of the method, after completion of the treatment according to point b), the activity of said enzyme specific for lung cancer is monitored at defined time intervals as known in the art, for example, every week, every few weeks, every month, every few months, every year, or any other interval that the skilled person considers appropriate, in order to detect minimal residual disease or recurrence after surgical resection of lung cancer. Furthermore, in a preferred embodiment of the method, a urine sample, preferably human urine, is used as the test sample. In a preferred embodiment of the method, a compound of formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2 A compound having the formula 2 or a compound having the formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA (formula 3) is used as the compound.

[0065] The advantage of the present invention is to provide novel compounds with suitable properties for use in the specific and sensitive detection of enzyme activity specific for lung cancer, for use as diagnostic biomarkers for the detection of lung cancer, for use in the rapid and non-invasive diagnosis of lung cancer, while allowing lung cancer to be detected at an early stage of its progression. Another advantage is that the diagnostic method according to the present invention can also be successfully used as a screening test. This allows a complete diagnosis at an early stage of the cancer's progression and, as a result, a more effective treatment. Early diagnosis allows for a surgical treatment that significantly extends the patient's survival time. It is also important when monitoring the effectiveness of the applied surgical and / or chemotherapy treatment of lung cancer, since it allows the detection of any minimal residual disease or recurrence.

[0066] The invention will now be described in the following figures and examples which are not intended in any way to limit the scope of the invention as defined in the claims. EXAMPLES

[0067] The present invention is illustrated by the following non-limiting examples. Unless otherwise indicated, the following examples use known and / or commercially available equipment, methods, reaction conditions, reactants and kits that are commonly used in the field to which the present invention pertains and that are recommended by the manufacturers of the respective reactants and kits.

[0068] Example 1: Synthesis of compounds according to the invention In this example, one representative compound according to the present invention, namely compound ABZ 1 -Tyr 2 -Ile 3 -Phe 4 -Arg 5 -ANB 6 -NH 2The remaining peptides according to the invention can be synthesized similarly. The superscripts indicate the position of the subsequent residues in the compounds according to the invention and the order of attachment of the residues during synthesis. The compounds according to the invention can alternatively be represented by a similar formula without indicating the positions of the residues. This does not change the sequence of the residues in the compounds according to the invention, which remains unchanged.

[0069] 1. Obtaining Chromogenic Peptides a) The first step of the synthesis was to obtain the chromogenic peptide, which was obtained by solid phase synthesis on a solid support using Fmoc / tBu chemistry, i.e. with protection.

[0070] Array ABZ 1 -Tyr 2 -Ile 3 -Phe 4 -Arg 5 -ANB 6 -NH 2 wherein ABZ is 2-aminobenzoic acid and ANB-NH 2 is an amide of 5-amino-2-benzoic acid, and ANB is 5-amino-2-benzoic acid] was obtained by the method of solid phase chemical synthesis using the following amino acid derivatives: Boc-ABZ, Fmoc-Tyr(tBU), Fmoc-Ile, Fmoc-Phe, Fmoc-Arg(Pbf).

[0071] The compound according to the present invention, i.e., a diagnostic marker for detecting lung cancer, can be synthesized by the synthesis of 5-amino-2-benzoic acid as ANB-NH 2 The reaction was carried out on a solid support that allows the conversion to an amide, namely the amide resin TentaGel S RAM from RAPP Polymere (Germany) (with a loading of 0.23 mmol / g), however, it is also possible to use any other amide resin, for example Rink Amide (Germany).

[0072] The synthesis of the compounds was carried out manually on a laboratory shaker. For most steps, 25 mL sintered syringes for solid-phase synthesis were used as reactors. All the final compounds obtained contained a 2-aminobenzoic acid (ABZ) molecule at position 1, i.e., the N-terminus, and a 5-amino-2-nitrobenzoic acid (ANB) molecule at position 6, i.e., the C-terminus, of their sequence. ABZ acts here as a fluorescent donor, whereas ANB (5-amino-2-benzoic acid) acts as a fluorescent quencher and simultaneously as a chromophore.

[0073] The peptides consist of the amino acid residues Arg-ANB-NH 2 i.e., position 5 of the compound). The synthesis of the attachment of the amino acid derivative is carried out from residue 6 to 1, i.e., from the C-terminus to the N-terminus.

[0074] b) Attachment of ANB to TentaGel S RAM resin: The synthesis of peptides was carried out on TentaGel S RAM from Rapp Polymere with a loading of 0.23 mmol / g. In the first step the resin was prepared (including loosening the resin by washing cycles). Afterwards the Fmoc amino protection was removed from the solid support with a 20% piperidine solution in NMP. Solvent washing cycles were then performed. A chloranil test was performed to confirm the presence of free amino groups.

[0075] Solvent wash cycle: DMF 1x10min; IsOH 1x10min; DCM 1x10min Removal of Fmoc protecting group: DMF 1x5 min; 20% piperidine in NMP 1x3 min; 20% piperidine in NMP 1x8 min Solvent wash cycle: DMF 3x2min; IsOH 3x2min; DCM 3x2min c) Chloranil test: The chloranil test consisted in transferring a few grains of resin with a spatula from the reactor (syringe) to a glass ampoule, after which 100 μL of a saturated solution of p-chloranil in toluene and 50 μL of fresh acetaldehyde were added to it. After 10 min, a control of the color of the grains was performed.

[0076] At this stage (after carrying out the test), green particles were obtained, which is evidence of the presence of free amino groups. After confirming the removal of the 9-fluorenylmethoxycarbonyl protecting group from the resin, it was possible to proceed to the next step, i.e. the coupling of the ANB derivative (5-amino-2-nitrobenzoic acid).

[0077] d) Attachment of 5-amino-2-nitrobenzoic acid to a solid support The first step in the synthesis of the peptide library (mixture of peptides) was the attachment of ANB onto 1 g of resin. Prior to the attachment of the chromophore, the resin used in the reaction was washed with the following solvents: DMF, DCM, and again with DMF, after which the Fmoc protecting group was removed from the functional groups of the solid support. One cycle of Fmoc protecting group removal included the following steps:

[0078] Removal of Fmoc protecting group: 20% piperidine in NMP 1x3 min; 20% piperidine in NMP 1x8 min e) Washing: DMF 3x2min; IsOH 3x2min; DCM 3x2min f) Chloranil test The resin with free amino groups was washed with a 5% solution of N-methylmorpholine (NMM) in DMF and then with DMF. The procedure for removal of the Fmoc protecting group and the washing cycles were carried out in a Merrifield vessel. In a separate flask, ANB was dissolved in DMF, followed by TBTU, DMAP and finally diisopropylethylamine (DIPEA) in the following excess amounts relative to the polymer attachment: ANB / TBTU / DMAP / DIPEA 3:3:2:6 v / v / v / v. The mixture thus prepared 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 procedure was repeated twice. To carry out the subsequent coupling reaction of ANB to the resin, hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HATU) was used, followed by hexafluorophosphate-O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HBTU). In the last step, the resin was washed successively with DMF, DCM and isopropanol and air-dried.

[0079] g) Binding of the C-terminal amino acid residue (Fmoc-Arg(OtBu)) to ANB The corresponding amino acid derivative (9-fold molar excess relative to resin attachment) was dissolved in pyridine and transferred to the flask containing the ANB-attached resin. The whole was cooled (ice bath: 1 part by weight of NH 4 Cl, 1 part by weight of NaNO 3 , 1 part by weight of ice). When the desired temperature is reached, add POCl 3 (1:1 ratio with respect to the amount of amino acid derivative used) was added and the whole was stirred on a magnetic stirrer for 20 min at -15°C, 30 min at room temperature and 6 h at 40°C (oil bath). Once the reaction was complete, the resin was filtered under reduced pressure, washed with DMF and MeOH and left to dry.

[0080] In the next step, a residue was attached at the P2 position (Fmoc-Phe). After each coupling of amino acid residues, the resin was first washed with DMF for 5 min. Diisopropylcarbodiimide was used as a coupling agent for the subsequent couplings. The procedure was repeated twice.

[0081] After each acylation, a resin washing cycle was initiated and then a chloranil test was performed to monitor the binding of the amino acid derivatives to the free amino acid groups of the resin. Solvent wash cycle: DMF 3x2min; IsOH 3x2min; DCM 3x2min Chloranil Test: After the first two coupling steps, a test was performed and the color of the particles was initially green and then gray, so another acylation had to be performed. As a result, the resin particles tested by the chloranil test became colorless, which was evidence of the binding of ANB to the TentaGel S RAM resin, and it was possible to proceed to the next peptide synthesis step.

[0082] h) Coupling of subsequent protected amino acid residues: After washing both the resin and the binding fragment ANB-Arg(Pbf) in the reaction vessel with DMF, the amino group was deprotected with Fmoc in order to couple the protected amino acid derivative Phe.

[0083] Removal of Fmoc protecting group: DMF 1x5 min; 20% piperidine in NMP 1x3 min; 20% piperidine in NMP 1x8 min Solvent wash cycle: DMF 3x2min; IsOH 3x2min; DCM 3x2min Chloranil Test: The chloranil test was successful, as evidenced by the green colour of the resin particles, making it possible to proceed to the next step, namely the coupling of the amino acid residue Fmoc-Ile-OH.

[0084] Binding of amino acid derivatives The resin was washed with DMF prior to the coupling step. The composition of the coupling mixture remained unchanged when attaching the protected serine residue.

[0085] After each acylation, a solvent washing cycle was performed according to a specific procedure, followed by a chloranil test to check for the presence of free amino acid groups in the solution. Solvent wash cycle: DMF 3x2min; IsOH 3x2min; DCM 3x2min Chloranil Test: During the test carried out after the second acylation, the resin particles were colorless, so it was possible to proceed to the next step of the synthesis, i.e. the introduction of another protected amino acid derivative Fmoc-Tyr(tBu) and a 2-aminobenzoic acid molecule. The coupling step was carried out according to the procedure discussed previously.

[0086] Tests carried out after coupling of the aforementioned residues showed good results, i.e. the resin particles were colourless. 2. Removal of peptides from solid support After synthesis, ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2 The peptide amide was removed from the solid support and simultaneously the side chain protecting groups were removed using a mixture: TFA:phenol:water:TIPS (88:5:5:2, v / v / v / v) in a round bottom flask on a magnetic stirrer.

[0087] After 3 hours, the contents of the flask were filtered under reduced pressure through a Schott funnel and washed with diethyl ether. The precipitate obtained was centrifuged for 20 minutes in a SIGMA 2K30 Laboratory Centrifuges. The precipitate obtained after centrifugation was dissolved in water by ultrasound and then subjected to freeze-drying. The remaining compounds according to the invention can be obtained in the same manner.

[0088] The identity / characterization of the novel compounds according to the present invention was confirmed using HPLC analysis. The conditions of the HPLC analysis 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: 226 nm.

[0089] The analyses carried out confirm that the compound according to the invention was obtained. Example 2: Testing the properties of peptides according to the invention as cancer markers The activity of the novel compounds according to the present invention was examined in a group of 20 subjects diagnosed with lung cancer using a representative compound according to the present invention. The mechanism of action of the compounds according to the present invention (including the representative compound having formula 2) is as follows: In the case of the compound having formula 2, ANB-NH 2 In the case of the compounds having formula 3, it is the specific enzymatic cleavage, more particularly enzymatic hydrolysis, that occurs at a position which results in the release of a free molecule of the respective chromophore, pNA (para-nitroaniline), which exhibits an absorbance at a wavelength between 320 and 480 nm, in particular between 380 and 430 nm, and in particular at 405 nm. The remaining compounds according to the invention are also characterized by a similar mechanism of action. To this end, the representative compound according to the invention, ABZ 1 -Tyr 2 -Ile 3 -Phe 4 -Arg 5 -ANB 6 -NH 2 was dissolved in dimethyl sulfoxide (at a concentration of 0.5 mg / mL) and 50 μL of this solution was mixed with 120 μL of buffer (200 mM Tris-HCl, pH 8.0) and 80 μL of urine from a subject suffering from lung cancer. The measurements were carried out on a 96-well plate designed for absorbance measurements, each sample was analyzed in triplicate at a temperature of 37 °C. The measurement time was 60 min. During the measurement, the released chromophore (ANB-NH 2 ) was monitored at a wavelength of 405 nm (range 380-430 nm).

[0090] As shown in FIG. 1, RP HPLC analysis of a randomly selected system containing urine samples from individuals diagnosed with lung cancer demonstrated that the compounds of the present invention are characterized by a peptide fragment ABZ-Tyr-Ile-Phe-Arg-OH and a chromophore group (ANB-NH 2 ) was disconnected.

[0091] Measurements showed that the color intensity of the solution increased over time in all urine samples taken from people diagnosed with lung cancer. The magnitude of the observed increase in absorbance over time varied for each tested sample. Different results were obtained for the 20 samples taken from healthy subjects, since no increase in absorbance was observed within the diagnostic range in any of the 20 tested urine samples.

[0092] According to the performed studies, all samples 1-50 from lung cancer patients underwent cleavage, but in the case of samples 7 and 16, the substrate, i.e., ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2 It is shown that the cleavage of the test compound proceeded less efficiently than in the case of samples 50 or 42 (Figure 2). Such results may be due to the difference in activity and amount of the enzyme responsible for the enzymatic cleavage (proteolysis). Furthermore, the results shown in Table 1 below show that when the substrate solution (compound according to the present invention) is incubated with urine samples collected from healthy subjects (not diagnosed with cancer, marked with Arabic numerals 1Z to 25Z in order), there is no increase in absorbance, and therefore no hydrolysis of the test compound occurs. This result indicates the absence of proteolytic enzymes specific / characteristic of lung cancer.

[0093] Table 1. Results of absorbance analysis

[0094] [Table 1-1]

[0095] [Table 1-2]

[0096] [Table 1-3]

[0097] Furthermore, it was also investigated whether the cleavage selectivity of the substrate, i.e. the compound according to the invention, depends on the cancer being examined. The results of the carried out tests are shown in Figure 3. According to it, the test substrate, i.e. ABZ, incubated with samples taken from patients diagnosed with the following cancers, i.e. testicular, intestinal, renal, prostate, pancreatic, bile duct, lung, ovarian, and rectal cancers, was examined. 1 -Tyr 2 -Ile 3 -Phe 4 -Arg 5 -ANB 6 -NH 2 It has been shown that the cleavage of the 20 samples of the present invention does not occur and does not cause an increase in absorbance within a certain range. The test samples were in each case a mixture of 20 samples from each cancer examined. This demonstrates the cleavage selectivity of the compounds according to the invention, making them suitable for the specific detection of the enzyme activity specific for lung cancer and for the specific diagnosis of lung cancer.

[0098] Table 2 below shows the results of three measurements taken for each sample. Table 2. Analysis results of cleavage selectivity

[0099] [Table 2]

[0100] Additionally, the dependence of the proteolytic activity of representative compounds according to the invention on reaction pH was also determined, and experiments showed that the studied materials possess at least one enzyme that exhibits maximum activity at alkaline pH (Figure 4).

[0101] The analyses carried out confirmed that the compounds according to the invention are suitable for the sensitive and specific detection of enzyme activity specific for lung cancer, as well as for the specific diagnosis of lung cancer and as diagnostic markers for lung cancer. The mechanism of action of the compounds according to the invention consists in their specific enzymatic cleavage at a position which leads to the release of free chromophore molecules, which generate a measurable light signal which can be used for diagnostic purposes, in particular for the diagnosis of lung cancer according to the invention.

Claims

1. Formula 1: X1 １ -Tyr ２ -Ile ３ -Phe ４ -Arg ５ -X2 ６ (Formula 1) A compound having the formula: wherein X1 comprises or consists of a molecule C1, and X2 comprises or consists of a molecule C2; Here, the pair of molecules C1 and C2 is a pair of a fluorescent donor and a fluorescent acceptor, The compound undergoes enzymatic cleavage into fragments X1-Tyr-Ile-Phe-Arg-OH (fragment 1) and X2 (fragment 2), generating a measurable optical signal due to the spatial separation of molecules C1 and C2. compound.

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

3. In the compound, the pair of molecules C1 and C2 is 2-aminobenzoic acid (ABZ) / 5-amino-2-nitrobenzoic acid (ANB), (ABZ) / pNA, ABZ / ANB-NH ２ , ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr (3-NO ２ ) Preferably, the pair of C1 and C2 is selected from the group consisting of ABZ / pNA or ABZ / ANB-NH ２ 2. The compound of claim 1, wherein:

4. The compound has the formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH ２ 3. The compound according to claim 1, which is a compound having the formula: (Formula 2) or a compound having the formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA (Formula 3).

5. The compound undergoes hydrolytic cleavage to give the following fragment 1: ABZ-Tyr-Ile-Phe-Arg-OH and fragment 2: ANB-NH ２ The compound of claim 4, which produces

6. 1. An in vitro method for detecting an enzyme activity, particularly derived from lung cancer cells, present in a body fluid of a subject, comprising: a) A body fluid sample is subjected to a quantification of the following: X1 １ -Tyr ２ -Ile ３ -Phe ４ -Arg ５ -X2 ６ (Formula 1) contacting the compound having the formula: [wherein X1 comprises or consists of a molecule C1, and X2 comprises or consists of a molecule C2; Here, the pair of molecules C1 and C2 is a pair of a fluorescent donor and a fluorescent acceptor, the compound undergoes enzymatic cleavage to give fragments X1-Tyr-Ile-Phe-Arg-OH (fragment 1) and X2 (fragment 2), and b) detecting a measurable light signal generated by the spatial separation of molecules C1 and C2; An in vitro method comprising:

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

8. The compound may be a compound represented by Formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH ２ 8. The in vitro method according to claim 6 or 7, wherein a compound having the formula 2 or a compound having the formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA (formula 3) is used.

9. 8. The method according to claim 6 or 7, wherein urine, preferably human urine, is used as the body fluid.

10. 1. An in vitro method for diagnosing lung cancer, wherein the presence or absence of lung cancer in a subject is detected by measuring a lung cancer-specific enzyme activity in a body fluid sample of the test subject, wherein the absence of said enzyme activity indicates the absence of lung cancer, while the presence of said enzyme activity indicates the presence of lung cancer, wherein said measuring of the enzyme activity is performed using a method according to Formula 1: X1 1 -Tyr 2 -Ile 3 -Phe 4 -Arg 5 -X2 6 (Formula 1) The method is carried out using a compound according to claim 1 having the formula In the above formula, X1 comprises or consists of a molecule C1, and X2 comprises or consists of a molecule C2; Here, the pair of molecules C1 and C2 is a pair of a fluorescent donor and a fluorescent acceptor, The compound undergoes enzymatic cleavage into fragments X1-Tyr-Ile-Phe-Arg-OH (fragment 1) and X2 (fragment 2), generating a measurable optical signal due to the spatial separation of molecules C1 and C2.

11. 11. The method according to claim 10, wherein the detection of the enzymatic activity is carried out by the method defined in claim 6.

12. A method according to claim 10 or 11, wherein the body fluid sample is incubated with the compound in a measurement buffer having a neutral or alkaline pH, preferably a physiological pH, at a sample to measurement buffer ratio in the range of 1:2 to 1:10, preferably 1:

5.

13. The method described in claim 10 or 11, wherein the compound is used at a concentration of 0.1 to 10 mg / mL, in particular 0.25 to 7.5 mg / mL.

14. The method described in claim 10 or 11, wherein the compound used is a compound having the formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2 (formula 2) or a compound having the formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA (formula 3).

15. A method according to claim 10 or 11, wherein a urine sample, preferably human urine, is used as the sample.

16. The method of claim 10 or 11, wherein the measurement of the enzyme activity comprises measuring the absorbance intensity in the range of 300 to 500 nm, preferably 380 to 430 nm, in particular 405 nm, for 40 to 60 minutes at a temperature in the range of 25 to 40°C, preferably 36 to 38°C.

17. A kit comprising a compound defined in claim 1 or 2 and an assay buffer.

18. The kit of claim 17, wherein the compound is a compound having the formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2 (formula 2) or a compound having the formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA (formula 3).

19. Use of a compound defined in claim 1 or 2 for detecting enzyme activity specific to lung cancer.

20. A kit as defined in claim 17 for use in diagnosing lung cancer.

21. A kit for use as described in claim 20, wherein the diagnosis of lung cancer includes detection of primary lung cancer, detection of minimal residual disease after surgical resection of lung cancer, and / or detection of recurrence of lung cancer.

22. The kit for use according to claim 20, wherein the compound is a compound having the formula 2: ABZ-Tyr-Ile-Phe-Arg-ANB-NH 2 (formula 2) or a compound having the formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA (formula 3).

23. A kit as defined in claim 17 for use as a diagnostic marker for detecting lung cancer.

24. The kit for use according to claim 23, wherein the compound is a compound having the formula 2: ABZ-Tyr-Ile-Phe-Arg ANB-NH 2 (formula 2) or a compound having the formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA (formula 3).

25. A kit as defined in claim 17 for use in a method of treating lung cancer, the method comprising: a) detecting the presence of a lung cancer-specific enzyme activity in a body fluid sample to be examined by the method defined in claim 6; and b) if the presence of said enzyme activity is found in said sample, then administering to the subject a treatment for lung cancer; Including the kit.

26. A kit for use as described in claim 25, wherein after the end of treatment according to point b), the activity of the enzyme specific for lung cancer is monitored at predetermined time intervals.

27. A kit for use according to claim 25 or 26, characterized in that as sample a urine sample, preferably human urine, is used.

28. The kit for use according to claim 25 or 26, wherein the compound used is a compound having the formula 2: ABZ-Tyr-Ile-Phe-Arg ANB-NH 2 (formula 2) or a compound having the formula 3: ABZ-Tyr-Ile-Phe-Arg-pNA (formula 3).

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