Novel diagnostic markers for prostate cancer

Abstract

The present invention relates to a compound of formula 1: X1-Trp-Glu-Gly-Asn-X2, The present invention also provides a compound characterized by the formula: wherein cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 results in a detectable signal. The present invention also provides an in vitro method for detecting protease activity in a subject's body fluid, comprising contacting the body fluid with a compound of the present invention and detecting a signal, wherein the body fluid can contain hydrolases derived from prostate cancer cells. The present invention also provides a kit comprising a compound of the present invention and an assay buffer. In addition, the present invention provides the use of a compound of the present invention, an in vitro method, or a kit for detecting prostate cancer or for monitoring a subject suspected of having, at high risk of developing, or having previously had prostate cancer. The present invention also provides the use of a compound of the present invention in a method for treating prostate cancer, comprising carrying out an in vitro method for detecting protease activity in a subject's body fluid and treating prostate cancer in the subject in whom protease activity is detected.

Description

[Technical Field] 【0001】 This invention relates to compounds for use in the diagnosis of prostate cancer. In particular, this invention relates to chromogenic peptides suitable for detecting proteolytic enzymes in a sample. [Background technology] 【0002】 Prostate cancer is the second leading cause of cancer death in men in the Western population, after lung cancer. While the disease is typically diagnosed in men over 65, its impact remains significant, with men who die from prostate cancer experiencing a 9-10 year reduction in average life expectancy. If detected, early-stage prostate cancer can currently be cured surgically in approximately 90% of cases. However, as the tumor spreads outside the glandular region and forms distant metastases, the disease gradually becomes fatal. Therefore, early detection and accurate staging are crucial for the correct selection of treatment. The need for a reliable, rapid, and uncomplicated diagnostic method for detecting prostate cancer remains unmet. Early diagnosis increases the likelihood of surgical intervention and can significantly extend patient survival. 【0003】 The processes of cancer cell initiation, proliferation, and dissemination involve many factors, including several proteolytic enzymes. These proteins can hydrolyze proteins and peptides into smaller fragments. Proteolytic enzymes mediate the degradation of the extracellular matrix, which in turn allows cancer cells to colonize new tissues, facilitates the formation of new blood vessels (angiogenesis), and promotes the efficient delivery of nutrients to the tumor. Furthermore, proteolytic enzymes are present as a result of the death of normal cells during the tumor growth process. All of these processes form a profile of proteolytic enzymes characteristic of tumors. 【0004】 Chromogenic peptide molecules that degrade under the influence of proteolytic enzymes, causing a change or increase in the color of the test solution, have been described previously. This coloration is a result of the release of a chromophore (e.g., 4-nitroanilide or 5-amino-2-nitrobenzoic acid). 【0005】 This type of peptide derivative is known from the following publications: 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. 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. 【0006】 The preferred compound according to the present invention has been developed to detect a signal, for example, an increase in color in the range of 380-440 nm, when the compound is brought into contact with a urine sample from a person with prostate cancer. This effect does not occur when the compound is brought into contact with a urine sample from a healthy person or a patient diagnosed with another type of cancer. 【0007】 The inventors have discovered that such signals, for example, signals from chromogenic compounds or fluorescent signals after separation of fluorescent donor / receptor pairs, can be used to detect the presence of proteolytic enzymes in the urine of subjects with prostate cancer. Enzymatic hydrolysis of the compound results in the generation of a detectable difference, for example, the release of free chromophore molecules that exhibit absorbance at 320–480 nm (ANB-NH2-amide or pNA, i.e., para-nitroaniline, respectively). 【0008】 The compounds of the present invention provide, in particular: (i) a rapid and non-invasive diagnostic method for detecting prostate cancer; (ii) a method suitable for the early detection of prostate cancer; (iii) a method that can be successfully used for screening for prostate cancer; and (iv) a complete diagnostic process in the early stages of cancer development for introducing more effective treatment. [Prior art documents] [Non-patent literature] 【0009】 [Non-Patent Document 1] Arch Biochem Biophys.1961 Nov;95:271-8 [Non-Patent Document 2] Chem Pharm Bull (Tokyo). November 2000; 48(11):1740-4 [Overview of the project] 【0010】 In a first aspect, the present invention relates to a compound characterized by formula 1: X1-Trp-Glu-Gly-Asn-X2 (formula 1), In this regard, cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 yields a detectable signal. The compound contains the tetrapeptide Trp-Glu-Gly-Asn according to SEQ ID NO: 1. 【0011】 In a second aspect, the present invention relates to an in vitro method for detecting protease activity in a subject's body fluid, comprising contacting the body fluid with a compound according to the first aspect of the present invention and detecting a signal, wherein the body fluid can contain hydrolytic enzymes, particularly proteases, derived from prostate cancer cells. 【0012】 In a third aspect, the present invention relates to a kit comprising a compound and a measurement buffer according to the first aspect of the present invention. In a fourth aspect, the present invention relates to the use of compounds according to the first aspect of the present invention, methods according to the second aspect of the present invention, or kits according to the third aspect of the present invention for detecting prostate cancer or for monitoring subjects who are suspected of having prostate cancer, are at high risk of developing prostate cancer, or have had prostate cancer in the past. 【0013】 In a fifth aspect, the present invention relates to the use of a compound according to the first aspect of the present invention in a method for treating prostate cancer, wherein the method comprises (a) the step of carrying out a method according to the second aspect of the present invention, and (b) the step of treating prostate cancer in a subject in which protease activity, in particular increased protease activity, is detected in step (a). [Brief explanation of the drawing] 【0014】 [Figure 1-1] Figure 1 shows the chromatographic analysis (A) and mass spectrometry spectrum (C) of the substrate ABZ-Trp-Glu-Gly-Asn-ANB-NH2, and the chromatographic analysis (B) and mass spectrometry spectrum (D) of ABZ-Trp-Glu-Gly-Asn-pNA. [Figure 1-2] Figure 1 shows the chromatographic analysis (A) and mass spectrometry spectrum (C) of the substrate ABZ-Trp-Glu-Gly-Asn-ANB-NH2, and the chromatographic analysis (B) and mass spectrometry spectrum (D) of ABZ-Trp-Glu-Gly-Asn-pNA. [Figure 2]Figure 2 shows the reverse-phase HPLC analysis of a randomly selected system containing urine from a person diagnosed with prostate cancer. Compounds (1) (ABZ-Trp-Glu-Gly-Asn-ANB-NH2, Figure 2A) and (2) (ABZ-Trp-Glu-Gly-Asn-pNA, Figure 2B) both degrade into the ABZ-Trp-Glu-Gly-Asn-OH peptide fragment and a chromophore (ANB-NH2 or pNA respectively). A: ABZ-Trp-Glu-Gly-Asn-ANB-NH2 substrate in 200 mM Tris-HCl buffer, pH 8.0. B: Hydrolysis of the ABZ-Trp-Glu-Gly-Asn-pNA substrate in the urine of a patient diagnosed with prostate cancer. [Figure 3] Figure 3 shows the hydrolysis rates of substrate (1) ABZ-Trp-Glu-Gly-Asn-ANB-NH2 in urine samples (samples 1 - 25) from patients diagnosed with cancer and urine from healthy individuals (26 - 35). The Arabic numerals indicate the numbers of the selected urine samples. Figure 1 shows that all of samples 1 - 25 disintegrated, but in samples 11, 12, 23, 24, and 25, the degradation of the ABZ-Thr-Thr-Ala-Arg-ANB-NH2 substrate occurred more efficiently than with materials 18 or 22. This result may be due to differences in the activity and amount of enzymes involved in proteolysis. Furthermore, Figure 3 shows that no increase in absorbance occurred when the solution of the compound of formula 2 was incubated with urine samples from healthy individuals (not diagnosed with cancer), and thus hydrolysis of the test compound did not occur. This result indicates that there is no proteolytic enzyme characteristic of prostate cancer. [Figure 4] Figure 4 shows the hydrolysis rates of substrate (2) ABZ-Trp-Glu-Gly-Asn-pNA in urine samples (samples 1 - 25) from patients diagnosed with cancer and urine from healthy individuals (26 - 35). The Arabic numerals indicate the numbers of the selected urine samples. Results similar to those described above for Figure 3 were obtained. [Figure 5]Figure 5 shows the incubation of the substrate (1) ABZ-Trp-Glu-Gly-Asn-ANB-NH2 with urine from various types of malignant tumors (pancreas, liver, intestine, lung, bile duct, stomach, and prostate). An increase in absorbance is observed only for the urine of patients diagnosed with prostate cancer. 【BEST MODE FOR CARRYING OUT THE INVENTION】 【0015】 Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, as these may vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", edited by Leuenberger, H.G.W, Nagel, B., and Koelbl, H. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland). 【0016】 Throughout the text of this specification, several documents are cited. Each document cited in this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether above or below, is hereby incorporated by reference in its entirety. Nothing in this specification should be construed as an admission that the present invention has no right to antedate such disclosure based on prior invention. 【0017】 To carry out the present invention, unless otherwise specified, conventional chemical, biochemical, and recombinant DNA techniques described in the literature of the art (see, for example, Molecular Cloning: A Laboratory Manual, 2nd edition, edited by J. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989) are used. 【0018】 Throughout this specification and the accompanying claims, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to mean that they encompass the integer or stage or group of integers or stages presented, but not exclude any other integer or stage or group of integers or stages. Where used herein and in the accompanying claims, the singular forms “a,” “an,” and “the” encompass multiple referents unless the content explicitly indicates otherwise. 【0019】 The elements of the present invention are described below. These elements are listed together with specific embodiments, but it should be understood that they may be combined in any way and in any number to create additional embodiments. The various examples and preferred embodiments described should not be construed as limiting the invention to only the embodiments explicitly described. This description should be understood as supporting and encompassing embodiments that combine the explicitly described embodiments with any number of disclosed elements and / or preferred elements. Furthermore, any rearrangement and combination of all elements described in this application should be considered disclosed by this description unless the context indicates otherwise. 【0020】 The terms and abbreviations used in the patent specification and claims should be understood as follows: In the context of this invention, the term "chromophore" is used to refer to a compound having "color-developing properties." The term "color-developing properties" refers to the ability of a compound to form a colored product. 【0021】 In the context of this invention, the term "fluorophore" is used to refer to a compound having "fluorescence properties." The term "fluorescence properties" refers to the ability of a compound to form a fluorescent product. 【0022】 In the context of this invention, NMP refers to N-methylpyrrolidone. In the context of this invention, DMF refers to dimethylformamide. In the context of this invention, DCM refers to methylene chloride. 【0023】 In the context of this invention, pNA refers to 4-nitroaniline, which is sometimes also called para-nitroaniline. In the context of this invention, ABZ refers to 2-aminobenzoic acid. 【0024】 In the context of the present invention, ANB-NH2 refers to the amide of 5-amino-2-nitrobenzoic acid. In the context of this invention, AFC refers to 7-amide-4-trifluoromethylcoumarin. 【0025】 In the context of this invention, Boc refers to a tert-butyloxycarbonyl group. In the context of the present invention, Fmoc refers to a 9-fluorenylmethoxycarbonyl group. In the context of this invention, TFA refers to trifluoroacetic acid. 【0026】 In the context of this invention, the term “prostate cancer” is used in its broadest sense and refers to all cancers that originate in the prostate. This includes adenocarcinoma, sarcoma, small cell carcinoma, neuroendocrine tumors (other than small cell carcinoma), and subtypes of transitional cell carcinoma. It also includes the following stages (defined by the corresponding TNM classification (1 or greater) in parentheses): Stage I (T1, N0, M0, Gleason score ≤6, PSA <10; or T2a, N0, M0, Gleason score ≤6, PSA <10), Stage IIA (T1, N0, M0, Gleason score ≤7, PSA <20; T1, N0, M0, Gleason score ≤6, PSA at least 10 but less than 20; or T2a or T2b, N0, M0, Gleason score ≤7, PSA <20), Stage IIB (T Stage 2c, N0, M0, any Gleason score, any PSA; T1 or T2, N0, M0, any Gleason score, PSA ≥ 20; or T1 or T2, N0, M0, Gleason score ≥ 8, any PSA), Stage III (T3, N0, M0, any Gleason score, any PSA) and Stage IV (T4, N0, M0, any Gleason score, any PSA; any T, N1, M0, any Gleason score, any PSA; or any T, any N, M1, any Gleason score, any PSA). 【0027】 Peptides according to the present invention, preferably chromogenic or fluorescent peptides, can be obtained by carrying out peptide synthesis on a solid support, as is known in the art. The solid support may be in the form of a resin having Fmoc groups that are removed during the reaction process. The resin used to carry out this process should be properly prepared. The preparation of this resin involves increasing its volume by repeatedly washing it with a hydrophobic solvent. 【0028】 The Fmoc protecting group must be removed from the resin by washing with a 20% solvent solution. Known processes for obtaining chromogenic peptides involve the attachment of individual components under appropriate time and stoichiometric conditions. This attachment process consists of subsequent steps: attaching individual elements (amino acid derivatives), washing and removing residues, removing protecting groups, and washing again. This cycle is repeated for each amino acid residue. The resulting peptides are separated from the resin by a reaction under acidic conditions. Subsequently, the solution is separated from the resin by filtration, and the peptides are precipitated from the resulting solution using a nonpolar solvent. The peptide precipitate is then centrifuged. 【0029】 Known methods for obtaining chromogenic peptides are based on processes carried out on a solid support, partially in a buffer solution. Amide resins are used as the solid support, and a mixture of hydrophobic solvents is used as the solution. 【0030】 The compounds according to the present invention were prepared using the method described in Hojo et al., Chem Pharm Bull (Tokyo), 2000. A detailed description of the synthesis can be found in the Examples section below. 【0031】 In a first aspect, the present invention provides a compound characterized by formula 1:X1-Trp-Glu-Gly-Asn-X2 (formula 1), wherein the cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 yields a detectable signal. 【0032】 In a preferred embodiment, the sequence Trp-Glu-Gly-Asn is accessible to hydrolases, in particular to hydrolases that cleave the compound into X1-Trp-Glu-Gly-Asn-OH (fragment 1) and NH2-X2 (fragment 2). 【0033】 Hydrolytic enzymes can be proteases or combinations of several proteases. The detectable signal resulting from the cleavage of the compound can be selected from a variety of suitable signals known to those skilled in the art. In a preferred embodiment, the detectable signal is an optical signal. 【0034】 In a preferred embodiment, X1 contains or consists of component C1, and X2 contains or consists of component C2, and the detectable signal is brought about by the spatial separation of C1 and C2, i.e., by the hydrolytic cleavage of the peptide Trp-Glu-Gly-Asn. 【0035】 In addition to C1, X1 may contain, for example, one or more amino acids on either side of C1. In addition to C2, X2 may contain, for example, one or more amino acids on either side of C2. Therefore, before cleavage, C1 and C2 can be separated by an amino acid sequence containing Trp-Glu-Gly-Asn or by an amino acid sequence of 4 to 20 amino acids or 5 to 10 amino acids consisting of Trp-Glu-Gly-Asn. In a preferred embodiment, C1 and C2 are separated by 10 or fewer amino acids. 【0036】 In a preferred embodiment, the detectable signal is a change in absorption or fluorescence. The change may be an increase or a decrease. In a preferred embodiment, the detectable signal is an increase in absorbance intensity at 300–500 nm, particularly at 380–430 nm. 【0037】 In a preferred embodiment, one of C1 and C2 is a chromophore having an absorption maximum 1 (AM1) at wavelength 1, and the compound has an absorption maximum 2 (AM2) at wavelength 2, which is different from wavelength 1. Therefore, when the absorption is measured at wavelength 2 before and after the cleavage of the compound, an increase in absorption is detected. 【0038】 It is particularly preferable that the chromophore is released in a free form upon cleavage. Upon cleavage, fragment 1 consists of X1-Trp-Glu-Gly-Asn, and fragment 2 consists solely of NH2-X2. Therefore, in a preferred embodiment, the chromophore is C2 rather than C1. If C2 is the chromophore, it is preferable that X2 consists of C2 or substantially C2. On the other hand, X1 may contain additional amino acids on either side of C1, and as a result, C1 and C2 may be separated by an amino acid sequence longer than Trp-Glu-Gly-Asn before cleavage. 【0039】 In a preferred embodiment, the chromophore is selected from para-nitroaniline (pNA), 5-amino-2-nitrobenzoic acid amide (ANB-NH2), 7-amido-4-trifluoromethylcoumarin (AFC), and 3-nitro-L-tyrosine (Tyr3-NO2). In a preferred embodiment, the chromophore is para-nitroaniline (pNA). In a preferred embodiment, the chromophore is 5-amino-2-nitrobenzoic acid amide (ANB-NH2). 【0040】 In a preferred embodiment, C1 and C2 are a pair of a fluorescent donor and a fluorescent acceptor. When C1 and C2 are a pair of a fluorescent donor and acceptor, and the compound is used in a method for measuring the change in fluorescence due to the cleavage of the compound, it is preferable that C1 and C2 are separated by 10 or fewer amino acids to ensure efficient quenching of the fluorescent donor by the fluorescent acceptor. Those skilled in the art know that the distance between the fluorescent donor and the fluorescent acceptor is an important parameter. Therefore, if the amino acid sequence separating C1 and C2 is folded into a condensed or twisted secondary structure, resulting in a closer proximity of C1 and C2 than in the case of a linear linker, a longer spacer between C1 and C2 may be acceptable. 【0041】 In a preferred embodiment, the C1 and C2 pair is selected from the group consisting of 2-aminobenzoic acid (ABZ) / pNA, ABZ / ANB-NH2, ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, and ABZ / Tyr(3-NO2), and in particular, the C1 and C2 pair is selected from ABZ / pNA and ABZ / ANB-NH2. It is preferable that C1 and C2 have molecular weights of less than 500 g / mol, particularly less than 400 g / mol, more specifically less than 300 g / mol, and even more specifically 100 to 200 g / mol. 【0042】 The C1 and C2 pair may also be a pair of protein fluorescent donors and receptors selected from the group consisting of BFP / GFP, BFP / CFP, BFP / YFP, BFP / DsRed, CFP / GFP, CFP / YFP, CFP / mVenus, CeFP / YFP, CeFP / mVenus, CeFP / mCitrine, CFP / dsRed, CFP / mCherry, mTurquoise / mVenus, GFP / YFP, GFP / DsRed, GFP / RFP, Clover / mRuby, Cy3 / C5, Alexa 488 / Alexa 555, and FITC / TRITC. Those skilled in the art will know how to select an appropriate pair of protein fluorescent donors and receptors based on their emission and absorbance spectra. 【0043】 If C1 and C2 are a pair of protein fluorescence donors and receptors, it must be ensured that the relatively large size of the protein does not hinder the cleavage of the compound, and that the sequence Trp-Glu-Gly-Asn is accessible for cleavage by hydrolytic enzymes. This can be achieved, for example, by increasing the length of the amino acid sequence containing the peptide according to SEQ ID NO: 1. 【0044】 In a preferred embodiment, the compound is characterized by formula 2: ABZ-Trp-Glu-Gly-Asn-ANB-NH2 (Formula 2). In a preferred embodiment, the compound is characterized by formula 3: ABZ-Trp-Glu-Gly-Asn-pNA (Formula 3). 【0045】 Cleavage of the compound characterized by Equation 2 or 3 releases a free chromophore molecule (ANB-NH2 or pNA, respectively). Therefore, an increase in absorbance intensity can be detected at 380–430 nm. In addition, cleavage results in the spatial separation of the fluorescence donor (ABZ) from the fluorescence receptor (ANB-NH2 or pNA, respectively). Thus, the fluorescence emitted from the ABZ is no longer quenched, and an increase in fluorescence intensity can be detected at 420 nm. 【0046】 In a second aspect, the present invention provides an in vitro method for detecting protease activity in a subject's body fluid, comprising contacting the body fluid with a compound according to the first aspect of the present invention and detecting a signal, wherein the body fluid can contain hydrolytic enzymes, particularly proteases, derived from prostate cancer cells. 【0047】 In a preferred embodiment, the presence of protease activity in body fluids indicates the presence of prostate cancer, and the absence of protease activity in body fluids indicates the absence of prostate cancer. In a preferred embodiment, the present invention provides a method for diagnosing prostate cancer. 【0048】 In a preferred embodiment, the body fluid is selected from blood or urine. In a preferred embodiment, the body fluid is urine. Surprisingly, the inventors found that compounds according to the first aspect of the present invention can detect hydrolytic enzyme activity in urine samples. In subjects diagnosed with prostate cancer, hydrolytic enzyme activity is significantly elevated compared to healthy subjects (Figures 1 and 2). 【0049】 In a preferred embodiment, the subject is at high risk of developing prostate cancer, suspected of having prostate cancer, has had prostate cancer in the past, or has prostate cancer. 【0050】 In a preferred embodiment, the compound is provided in a measurement buffer having a neutral or alkaline pH, preferably pH 6.8 to 8.5, more preferably physiological pH, at a concentration of 0.1 to 10 mg / mL, particularly 0.25 to 7.5 mg / mL, more specifically 0.5 to 5 mg / mL, more specifically 0.75 to 2 mg / mL, and even more specifically about 1 mg / mL, and the body fluid sample is added to the compound in a ratio of 1:2 to 1:10, particularly 1:3 to 1:8, more specifically 1:4 to 1:6, and even more specifically about 1:5. 【0051】 In the context of this specification, the term "neutral pH" refers to a pH of approximately 7.0. In the context of this specification, the term "physiological pH" refers to a pH of approximately 7.4. In a preferred embodiment, detecting the signal includes measuring absorbance or fluorescence. 【0052】 In a preferred embodiment, signal detection involves measuring the absorbance intensity at 300-500 nm, particularly 380-430 nm, at 25-40°C, particularly 36-38°C, preferably for 40-60 minutes. 【0053】 In a preferred embodiment, an increase in absorbance indicates the presence of hydrolytic enzyme activity. In a third aspect, the present invention provides a kit comprising a compound and a measurement buffer according to the first aspect of the present invention. 【0054】 In a fourth aspect, the present invention provides the use of a compound according to the first aspect, a method according to the second aspect, or a kit according to the third aspect for detecting prostate cancer or for monitoring subjects who are at high risk of developing prostate cancer, suspected of having prostate cancer, or have had prostate cancer in the past. 【0055】 Depending on the intended use of the method in the second perspective, the term “subject” may have different limitations. For example, if the method is used to detect or screen subjects for prostate cancer, the subject may not be known to have prostate cancer; that is, they may or may not have prostate cancer. In this example, the subject is preferably at high risk of developing prostate cancer, suspected of having prostate cancer, or has had prostate cancer in the past (i.e., a detectable prostate cancer has been cured). “High risk” means that one or more risk factors for cancer in general or for prostate cancer may be attributable to the subject, preferably as defined by the American Cancer Society for cancer in general or for prostate cancer. Examples of risk factors for prostate cancer include: age 50 or older (especially 65 or older), male sex, ethnic background (especially African American and Caribbean men of African descent), family history of prostate cancer (especially first-degree relatives), genetic syndrome or predisposition (such as mutations in the BRCA1 or BRCA2 gene, or Lynch syndrome), diet (especially one rich in red meat or high-fat dairy products), obesity, tobacco consumption (especially smoking), occupation (especially firefighters), prostate inflammation, sexually transmitted infections (especially gonorrhea or chlamydia), and vasectomy. 【0056】 Prostate cancer may be of any subtype and stage defined above; that is, the presence or absence of any subtype and / or stage can be detected. In a preferred embodiment, the presence of a significant amount of protease activity, or an amount of protease activity greater than that of the control, indicates the presence of prostate cancer, and the absence of a significant amount of protease activity, or an amount of protease activity less than or equal to that of the control, indicates the absence of prostate cancer. 【0057】 In certain embodiments, the method of the second aspect further includes confirming the detection of prostate cancer by using one or more further means for detecting prostate cancer. The further means may be cancer markers (or "biomarkers") or conventional (non-marker) detection means. Cancer markers may be, for example, DNA methylation markers, mutation markers (e.g., SNPs), antigen markers, protein markers, miRNA markers, cancer-specific metabolites, or expression markers. Conventional means may be, for example, biopsies (e.g., visual biopsies with or without staining methods for proteins or expression markers), imaging techniques, or physical examinations, such as tactile examinations. In a preferred embodiment, the further means for detecting prostate cancer are selected from the group consisting of PSA testing, DRE testing, PCA3 testing, ultrasound (especially transrectal ultrasound), biopsy, MRI, MRI-fused biopsy, and CT scans. 【0058】 As used herein, the terms “indicative for” or “indicate” refer to the act of identifying or specifying the indicated object. As those skilled in the art will understand, such an assessment is preferably accurate, but may not be accurate for 100% of the subject. However, the term requires that an accurate representation of a statistically significant portion of the subject can be made. Those skilled in the art can easily determine whether a portion is statistically significant using several well-known statistical assessment tools, such as confidence interval calculation, p-value calculation, Student's t-test, and Mann-Whitney test. Details are provided in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95%. P-values ​​are preferably 0.05, 0.01, or 0.005. 【0059】 When used herein in relation to prostate cancer, the phrase "method for detecting the presence or absence of cancer" refers to the determination of whether or not a subject has cancer. As those skilled in the art will understand, such an assessment is preferably accurate, but may not be accurate for 100% of subjects. However, this term requires that an accurate representation be made for a statistically significant portion of the subjects. For explanations of statistical significance and appropriate confidence intervals and p-values, see above. 【0060】 As used herein, the term “diagnosis” refers to the determination of whether or not a subject has cancer. Diagnosis by protease activity analysis, as described herein, may be supplemented by further means, as described herein, to confirm the cancer detected by protease activity analysis. As those skilled in the art will understand, while a diagnosis is preferably accurate, it may not be accurate for 100% of subjects. However, this term requires that an accurate diagnosis can be made for a statistically significant portion of the subjects. For explanations of statistical significance and appropriate confidence intervals and p-values, see above. 【0061】 Where used herein in relation to cancer, the phrase “screen a subject population” means applying the methods of the first aspect to a sample of the subject population. Preferably, subjects are at high risk of cancer, suspected of having cancer, or have had cancer in the past. In particular, one or more risk factors listed herein may be attributable to subjects in a population. In certain embodiments, the same one or more risk factors may be attributable to all subjects in a population. For example, a population may consist of subjects characterized by heavy alcohol use and / or tobacco consumption. The term “screening” should be understood to mean the above-described diagnosis of subjects in a population, which is preferably confirmed by further means as described herein. As will be understood by those skilled in the art, screening results are preferably accurate, but may not usually be accurate for 100% of subjects. However, the term requires that accurate screening results be obtained for a statistically significant portion of the subjects. See above for an explanation of statistical significance and appropriate confidence intervals and p-values. 【0062】 As used herein, the term “monitoring” refers to the accompaniment of cancer diagnosed during a treatment procedure or over a specific period, typically at least one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, one year, two years, three years, five years, ten years, or any other period. The term “accompaniment” means that the state of cancer, in particular changes in these states of cancer, can be detected over a treatment cycle that may be up to one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fifteen times per month (no more than one determination per day), based on the amount of protease activity, in particular changes in the amount in any type of periodic time interval determined, for example, daily or one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fifteen times per month (no more than one determination per day). The amount or change in amount may also be determined by treatment-specific events, e.g., before and / or after each treatment cycle or drug / treatment application. One cycle is the time between one round of treatment and the start of the next round. Cancer treatment is usually a course of treatment, not a single treatment. A course usually takes 3 to 6 months, but may be longer or shorter. During a course of treatment, usually 4 to 8 cycles of treatment are performed. Typically, one treatment cycle includes a break in treatment to allow the body to recover. As those skilled in the art will understand, monitoring results are preferably accurate, but may not usually be accurate for 100% of the subject. However, this term requires that accurate monitoring results can be obtained for a statistically significant portion of the subject. See above for an explanation of statistical significance and appropriate confidence intervals and p-values. 【0063】 In a fifth aspect, the present invention provides the use of a compound according to the first aspect of the present invention in a method for treating prostate cancer, wherein the method comprises the steps of carrying out a method according to the second aspect of the present invention, and treating prostate cancer in a subject in which protease activity, particularly increased protease activity, is detected. 【0064】 In other words, the present invention provides a method for treating prostate cancer, comprising carrying out a method according to a second aspect of the present invention, and treating prostate cancer in a subject in which protease activity, in particular increased protease activity, is detected. 【0065】 As used herein, the terms “treatment” or “treating” in relation to cancer refer to therapeutic treatment, the goal of which is to reduce the progression of cancer. Beneficial or desirable clinical outcomes include, but are not limited to, release of symptom, reduction of disease duration, stabilization of the pathological condition (specifically, no worsening), slowing of disease progression, improvement of the pathological condition and / or remission (both partial and total), which are preferably detectable. Success of treatment does not necessarily mean a cure, but may mean an extension of survival compared to the survival expected without treatment. In a preferred embodiment, the treatment is a primary treatment, i.e., the cancer has not been treated previously. Treatment of cancer encompasses a treatment regimen. 【0066】 As used herein, the term “treatment regimen” refers to a method of treating a subject, taking into account the disease and available procedures and medications. Non-limiting examples of cancer treatment regimens include chemotherapy, surgery and / or radiation, or combinations thereof. The early detection of cancer made possible by the present invention enables surgical intervention, particularly curative resection. In particular, the term “treatment regimen” refers to the administration of one or more anticancer agents or anticancer treatments as defined below. As used herein, the term “anticancer agent or anticancer treatment” refers to chemical, physical or biological agents or treatments, or surgical procedures, or combinations thereof, that have antiproliferative, anti-cancer and / or anti-cancer properties. 【0067】 Chemical anticancer agents or treatments can be selected from the group consisting of alkylating agents, antimetabolites, plant alkaloids and terpenoids, and topoisomerase inhibitors. Preferably, the alkylating agent is a platinum-based compound. In one embodiment, the platinum-based compound is selected from the group consisting of cisplatin, oxaliplatin, eptaplatin, lovaplatin, nedaplatin, carboplatin, iproplatin, tetraplatin, lovaplatin, DCP, PLD-147, JM18, JM216, JM335, and satraplatin. 【0068】 Physical anticancer agents or treatments may be selected from a group consisting of radiotherapy (e.g., curative radiotherapy, adjuvant radiotherapy, palliative radiotherapy, remote radiotherapy, brachytherapy, or metabolic radiotherapy), phototherapy (e.g., using hematoporphoryn or Photofrin II), and hyperthermia. 【0069】 Surgical procedures can be curative resection, palliative surgery, prophylactic surgery, or tumor reduction surgery. Typically, it includes resection, such as intracapsular resection, marginal resection, extensive resection, or radical resection, as described by Baron and Valin (Rec. Med. Vet, Special Canc. 1990; 11(166):999-1007). 【0070】 Biological anticancer agents or therapies include: antibodies (e.g., antibodies that stimulate an immune response to destroy cancer cells, e.g., retuximab or alemtuzubab; antibodies that stimulate an immune response by binding to receptors on immune cells and inhibiting signals that prevent immune cells from attacking their own cells, e.g., ipilimumab; antibodies that interfere with the action of proteins necessary for tumor growth, e.g., bevacizumab, cetuximab or panitumumab; or antibodies that conjugate to cytotoxic substances such as drugs, preferably toxins, chemotherapeutic molecules or radioactive molecules, e.g., Y-ibritumomab tiuxetan, I-tocitumomab or ad-trastuzumab emtansine); cytokines (e.g., interferons or interleukins, e.g., INF-alpha and IL-2); vaccines (e.g., vaccines containing cancer-related antigens, e.g., cypro The following can be selected from the group consisting of: Icel-T); oncolytic viruses (e.g., naturally occurring oncolytic viruses such as reovirus, Newcastle disease virus, or mumps virus, or genetically modified viruses such as measles virus, adenovirus, vaccinia virus, or herpesvirus that preferentially target cells with cancer-associated antigens); gene therapies (e.g., DNA or RNA that replaces altered tumor suppressors, blocks the expression of oncogenes, improves the subject's immune system, makes cancer cells more sensitive to chemotherapy, radiotherapy, or other treatments, induces cell suicide, or produces anti-angiogenic effects); and adoptive T cells (e.g., tumor-infiltrating T cells taken from the subject selected for antitumor activity, or T cells taken from the subject that have been genetically modified to recognize cancer-associated antigens). 【0071】 In one embodiment, one or more anticancer agents include abiraterone acetate, ABVD, ABVE, ABVE-PC, AC, AC-T, ADE, ad-trastuzumab emtacin, afatinib dimaleate, aldesleukin, alemtuzumab, aminolevulinic acid, anastrozole, aprepitant, arsenic trioxide, asparaginase erwinia chrysanthemum, axitinib, azacitidine, BEACOPP, bellinostat, bendamustine hydrochloride, BEP, bevacizumab, bexarotene, bicalutamide, bleomycin, bortezomib, bosutinib, and b Lentuximab vedotin, busulfan, cabazitaxel, cabozantinib-S-malate, CAF capecitabine, CAPOX, carboplatin, carboplatin-taxol, carfilzomib, carmustine, carmustine implant, ceritinib, cetuximab, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, clofarabine, CMF, COPP, COPP-ABV, crizotinib, CVP, cyclophosphamide, cytarabine, cytarabine, liposome, dabrafenib, dacarbazine, dactinomycin, dasatinib, Daunorubicin hydrochloride, decitabine, degarelix, deniroukin difutitox, denosumab, dexrazoxane hydrochloride, docetaxel, doxorubicin hydrochloride, doxorubicin hydrochloride liposome, eltrombopagolamine, enzalutamide, epirubicin hydrochloride, EPOCH, eribulin mesylate, erlotinib hydrochloride, etoposide phosphate, everolimus, exemestane, FEC, filgrastim, fludarabine phosphate, fluorouracil, FU-LV, fulvestrant, gefitinib, gemcitabine hydrochloride, gemcitabine-cisp Latin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, glucarpidase, goserelin acetate, HPV bivalent vaccine, recombinant HPV quadrivalent vaccine, Hyper-CVAD, ibritumomab tiuxetan, ibrutinib, ICE, idelalisib, ifosfamide, imatinib, mesylate, imiquimod, iodine-131 tositumomab and tositumomab, ipilimumab, irinotecan hydrochloride, ixabepyrone, lapatinib nitosylate, lenalidomide, letrozole, leucovorin calcium, leuprolide acetate, liposomal cytarabine,Lomustine, mechloretamine hydrochloride, megestrol acetate, mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone hydrochloride, MOPP, nelarabine, nilotinib, obinutuzumab, ofatumumab, omasetaxin mepesuccinate, OEPA, OFF, OPPA, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palifermin, palonosetron hydrochloride, pamidronate disodium, panitumumab, pazopanib hydrochloride, pegaspargaze, pegylated interferon alpha-2b, pem Brolizumab, pemetrexed disodium, pertuzumab, prelixafor, pomalidomide, ponatinib hydrochloride, pralatrexate, prednisone, procarbazine hydrochloride, radium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant HPV bivalent vaccine, recombinant HPV quadrivalent vaccine, recombinant interferon alpha-2b, regorafenib, rituximab, romidepsin, romiplostim, ruxolitinibulinate, siltuximab, ciproisel-T, sorafenib tosylate, STANFORD Selected from the group consisting of V, sunitibralate, TAC, talc, tamoxifen citrate, temozolomide, temsirolimus, thalidomide, topotecan hydrochloride, toremifene, tositumomab, and I131 iodine tositumomab, TPF, trametinib, trastuzumab, vandetanib, VAMP, VeIP, vemurafenib, vinblastine sulfate, vincristine sulfate, vincristine sulfate liposomes, vinorelbine tartrate, bismodegib, vorinostat, XELOX, Ziv-aflibercept, and zoledronic acid, or salts thereof. 【0072】 In another respect, the present invention provides a method for producing compounds according to the first aspect of the present invention. In a preferred embodiment, the compound characterized by the formula ABZ1-Trp2-Glu3-Gly4-Asn5-ANB-NH26 [wherein ABZ is 2-aminobenzoic acid and ANB-NH2 is an amide of 5-amino-2-nitrobenzoic acid] is produced by a process preferably carried out on a resin support having an Fmoc group. Before starting the process, the support is prepared: the volume of the support is increased by repeatedly washing with a hydrophobic solvent, preferably dimethylformamide, methylene chloride, or N-methylpyrrolidone, and removing the Fmoc protecting group by washing with a 10-30% solution of piperidine in a solvent such as dimethylformamide, methylene chloride, or N-methylpyrrolidone. Next, the process in the subsequent steps is carried out: 【0073】 a) Before depositing 5-amino-2-nitrobenzoic acid ANB onto the resin, the support is washed with a 3-6% solution of N-methylmorpholine (NMM) in DMF, followed by DMF. Next, a solution of ANB in ​​DMF is prepared, to which TBTU, DMAP, and finally diisopropylethylamine (DIPEA) are added in excess of the polymer deposit in the following ratio: ANB / TBTU / DMAP / DIPEA, 3:3:2:6. The resulting mixture is added to the resin and mixed until homogeneous, after which the resin is filtered under reduced pressure and washed with solvents such as DMF, DCM, and isopropanol. Next, excess hexafluorophosphate-O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HATU), followed by excess hexafluorophosphate-O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (HBTU), is used to continue the binding of ANB to the resin. After completion, the support is successively washed with DMF, DCM, and isopropanol, and gently dried. 【0074】 b) The attachment of amino acid residues to ANB is carried out by the reaction of an amino acid derivative of Fmoc-Asn(Trt)-OH, in which at least 5 molar excess of the amino acid derivative relative to the resin is dissolved in pyridine anhydride and brought into contact with the resin on which ANB is deposited. Next, the whole is cooled to a temperature not below 20°C, and POCl3 is added in a 1:1 ratio relative to the amount of amino acid derivative used and mixed. The mixing process is carried out at room temperature and then at high temperature; after the reaction is complete, the resin is filtered under reduced pressure, washed with DMF and MeOH, and gently dried; the resulting intermediate compound is subjected to an acylation process. 【0075】 c) The obtained intermediate compound is acylated using an amino acid derivative, preferably Fmoc-Gly-OH, then Fmoc-Glu(OtBu)-OH, then Fmoc-Trp(Trt)-OH, and finally Boz-Abz-OH. The acylation is carried out in steps 6 through 1 using diisopropylcarbodiimide as the coupling agent, with an excess of the coupling agent used. At the end of each step, the resin is washed with DMF and then subjected to a chloroanyl test to monitor the adhesion of the amino acid derivative (preferably). 【0076】 d) The Fmoc protecting group is removed by washing with a 10-30% piperidine solution in DMF, followed by washing with DMF, isopropanol, and methylene chloride solvents, respectively. 【0077】 e) Separation of peptides from the resin is carried out using a mixture of TFA, phenol, water, and TIPS, maintaining a v / v / v / v ratio of 88:5:5:2 for each component; the mixture is stirred for at least 1 hour, preferably 3 hours, the resulting precipitate is filtered under reduced pressure and washed with diethyl ether, and the resulting peptides are centrifuged. 【0078】 f) The final product is prepared by dissolving the peptide in water using ultrasound and then freeze-drying it. 【0079】 In a preferred embodiment, the compound characterized by ABZ1-Trp2-Glu3-Gly4-Asn5-pNA6 [wherein ABZ is 2-aminobenzoic acid and pNA is p-nitroanilide] is produced by a process preferably carried out on a resin support having an Fmoc group. Before starting the process, the support is prepared: the support volume is increased by repeatedly washing with a hydrophobic solvent, preferably dimethylformamide, methylene chloride, or N-methylpyrrolidone, and removing the Fmoc protecting group by washing with a 10-30% solution of piperidine in a solvent such as dimethylformamide, methylene chloride, or N-methylpyrrolidone; then the process in the subsequent steps is carried out: 【0080】 a) The third amino acid residue (Fmoc-Gly) is attached to the resin using a suitable amino acid derivative in a 9-fold molar excess relative to the resin deposit dissolved in anhydrous methylene chloride. The mixture is then stirred at room temperature for at least 2 hours; after the reaction is complete, the resin is filtered under reduced pressure, washed with DMF and MeOH, and then dried. 【0081】 b) In the next step, amino acid residues are attached, hereafter referred to as acylation, using derivatives of Fmoc-Glu(OtBu)-OH, followed by Fmoc-Trp(Trt)-OH and Boc-ABZ-OH; before each step, the resin is washed with DMF, preferably for 5 minutes; and for the subsequent attachment, a coupling agent, preferably diisopropylcarbodiimide, is used, preferably in excess. This procedure is repeated twice, and after each step, the resin is washed with DMF and preferably subjected to a chloroanyl test to monitor the attachment of the amino acid derivatives. 【0082】 c) The Fmoc protecting group is removed by washing with a 10-30% piperidine solution in DMF, followed by washing with DMF, isopropanol, and methylene chloride solvents, respectively. 【0083】 d) Repeat steps b) to c) until compound (ABZ1-Trp(Trt)2-Glu(OtBu)3-Gly4-OH) is obtained; after synthesis from residues 6 to 3, the obtained compound is separated from the support using a mixture of TFA:phenol:water:TIPS in a ratio of 88:5:5:2, v / v / v / v, while stirring. After at least 2 hours, the contents of the flask are filtered under reduced pressure and the precipitate is washed with diethyl ether. The precipitate is then preferably centrifuged for 20 minutes, dissolved in water by sonication, and subsequently freeze-dried. 【0084】 e) The Fmoc-Asn(Trt)-pNA synthesis is carried out stepwise; in the first step, 2 mmol of Fmoc-Asn(Trt) is dissolved in anhydrous tetrahydrofuran (THF) in the presence of 2 mmol of N-methylmorpholine (NMM), and the carboxyl group of the amino acid derivative is activated with 2 mmol of isobutyl chloride. After activation for 10 minutes, 3 mmol of p-nitroaniline is added, and the reaction is carried out at a temperature of (preferably) -15°C for (preferably) 2 hours, followed by 1 day at room temperature. Once the reaction is complete, the solvent is evaporated, and the dried residue is dissolved in ethyl acetate; the resulting solution is then washed sequentially with saturated aqueous NaCl, 10% citric acid, and 5% sodium bicarbonate, and dried over anhydrous sodium sulfate; the ethyl acetate is distilled under reduced pressure, and the dried residue is dried. 【0085】 f) Combining the protective peptide ABZ1-Trp(Trt)2-Glu(OtBu)3-Gly4-OH with the paranitroanilide Asn(Trt) is based on the following process: Dissolve the protective peptide ABZ1-Trp(Trt)2-Glu(OtBu)3-Gly4-OH in a small amount of DCM, then activate with TFFH (tetramethylfluoroformamide) preferably at a temperature of 0°C for 30 minutes, and then add catalytic amounts of DMAP and Fmoc-Asn(Trt)5-pNA6. The reaction is preferably carried out at room temperature for 24 hours, after which the solvent is evaporated, and the resulting solution is poured in with a mixture to remove side group protection: TFA:phenol:water:TIPS (88:5:5:2, v / v / v / v), and preferably mixed for 3 hours. 【0086】 g) After dissolving the peptide in water using ultrasonic waves and subjecting it to lyophilization, the final product is prepared. 【Example】 【0087】 The present invention is illustrated by the following non-limiting examples. Example 1: Compound ABZ 1 -Trp 2 -Glu 3 -Gly 4 -Asn 5 -ANB-NH 2 6 synthesis 1. Preparation of the chromogenic peptide a) The first step of the synthesis was to obtain the chromogenic peptide by solid-phase synthesis on a solid support using Fmoc / tBu chemistry, i.e., using protection. 【0088】 Sequence ABZ 1 -Trp 2 -Glu 3 -Gly 4 -Asn 5 -ANB-NH2 6 [where ABZ is 2-aminobenzoic acid, ANB-NH2 is the amide of 5-amino-2-benzoic acid, and ANB is 5-amino-2-benzoic acid] The compound having the formula was obtained by a solid-phase chemical synthesis process using the following amino acid derivatives. 【0089】 Boc-ABZ, Fmoc-Trp(Trt), Fmoc-Glu(OtBu), Fmoc Gly Fmoc-Asn(Trt), ANB The synthesis of a compound, i.e., a diagnostic marker for prostate cancer detection in which the diagnosis is linked to the hydrolysis of this compound under the influence of proteolytic enzymes, was carried out on a solid carrier that allows the conversion of 5-amino-2-benzoic acid to ANB-NH2 amide: e.g., an amide resin, e.g., TentaGel S RAM from RAPP Polymere (Germany), with deposition of e.g., 0.23 mmol / g. 【0090】 It is also possible to use other commercially available amide resins, such as Rink Amide (Germany). The compounds were synthesized manually using a laboratory shaker. For most steps, a 25 mL sintered syringe for solid-phase synthesis was used as the reactor. 【0091】 All of the final compounds obtained contained ABZ2-aminobenzoic acid at position 1 (i.e., the N-terminus) and an ANB5-amino-2-nitrobenzoic acid molecule at position 6 (the C-terminus). ABZ acts as a fluorescence donor, while ANB(5-amino-2-benzoic acid) acts as a fluorescence quencher and chromophore. The peptide contained at least (and preferably) one reactive site in its sequence between the amino acid residues Arg-ANB-NH2 at position 5 of the compound. The synthesis involving the attachment of the amino acid derivative was carried out from residue 6 to 1, i.e., from the C-terminus to the N-terminus. 【0092】 b) Deposition of ANB on TentaGel S RAM resin: Peptide synthesis was performed on TentaGel S RAM resin (Rapp Polymere) with a deposition rate of 0.23 mmol / g. In the first step, the resin was prepared, including loosening by a washing cycle. Subsequently, the Fmoc amino group protection was removed from the support using a 20% solution of piperidine in NMP, and a solvent washing cycle was performed. A chloranil test was performed to confirm the presence of free amino groups. Solvent washing cycle: DMF 1 x 10 minutes IsOH 1×10 min DCM 1×10 minutes Removal of Fmoc protection: DMF 1 x 5 minutes 20% piperidine in NMP, 1 x 3 minutes 20% piperidine in NMP, 1 x 8 minutes Solvent washing cycle: DMF 3 x 2 minutes IsOH 3 x 2 min DCM 3×2 minutes 【0093】 c) Chloranil test: The chloranil test involved transferring several resin particles from the reactor (i.e., syringe) to a glass ampoule (using a spatula), to which 100 μL of a saturated solution of p-chloranil in toluene and 50 μL of unused acetaldehyde were added. After 10 minutes, the color of the particles was controlled. 【0094】 In this step, after testing, green particles were obtained, indicating the presence of free amino groups. After confirming the removal of the 9-fluorenylmethoxycarbonyl protection from the resin, it was possible to proceed to the next step, the attachment of the ANB derivative (5-amino-2-nitrobenzoic acid). 【0095】 d) Deposition of 5-amino-2-nitrobenzoic acid on a solid support The first step in the synthesis of the peptide library, i.e., the peptide mixture, was ANB deposition on 1 g of resin. Before attaching the chromophore, the resin used for the reaction was washed with the following solvents: DMF, DCM, and again with DMF, and then the Fmoc protection was removed from the functional groups of the support. One cycle of removing the Fmoc protection included the following steps: Removal of Fmoc protection: 20% piperidine in NMP, 1 x 3 minutes 20% piperidine in NMP, 1 x 8 minutes e) Cleaning DMF 3 x 2 minutes IsOH 3 x 2 min DCM 3×2 minutes f) Chloranil test for the presence of free amino groups 【0096】 The resin containing free amino groups was washed with a 5% solution of N-methylmorpholine (NMM) in DMF, followed by washing with DMF. The procedure for removing Fmoc protection and the washing cycle were performed 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 deposit: ANB / TBTU / DMA / DIPEA, 3:3:2:6. The mixture thus prepared was added to the resin and stirred for 3 hours. The resin was filtered under reduced pressure and washed with DMF, DCM, and isopropanol, and the entire acylation procedure was repeated twice. The subsequent reaction for ANB adhesion to the resin was carried out using 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). In the final step, the resin was successively washed with DMF, DCM, and isopropanol and air-dried. 【0097】 g) Attachment of C-terminal amino acid residues to ANB The corresponding amino acid derivative (in a 9-fold molar excess relative to the resin deposit) was dissolved in pyridine and transferred to a flask containing the resin with ANB deposition. The entire mixture was cooled to -15°C (ice bath: 1 part by weight of NH4Cl, 1 part by weight of NaNO3, 1 part by weight of ice). After reaching the desired temperature, POCl3 was added (in a 1:1 ratio relative to the amount of amino acid derivative used), and the mixture was stirred on a magnetic stirrer at -15°C for 20 minutes, at room temperature for 30 minutes, and at 40°C (oil bath) for 6 hours. After the reaction was complete, the resin was filtered under reduced pressure, washed with DMF and MeOH, and allowed to stand to dry. 【0098】 In the next step, the residue (alanine) was attached to the P2 position. Before attaching amino acid residues, the resin was washed with DMF for 5 minutes each time. Diisopropylcarbodiimide was used as a coupling agent for subsequent attachment. This procedure was repeated twice. 【0099】 After each acylation, a resin washing cycle was initiated, followed by a chloranil test to monitor the adhesion of amino acid derivatives to the free amino groups of the resin. Solvent washing cycle: DMF 3 x 2 minutes IsOH 3 x 2 min DCM 3×2 minutes 【0100】 Chloranil test: The test results showed that after the first two couplings, the particle color was initially green, then gray. Therefore, another acylation was necessary, and as a result, the resin particles tested in the chloranil test were colorless. This indicated the adhesion of ANB to the TentaGel S RAM resin, which allowed for the transition to the next step in peptide synthesis. 【0101】 h) Attachment of further protective amino acid residues: After washing the resin in the reactor with attached ANB residues using DMF, the Fmoc was deprotected from the amino group, and a protected amino acid alanine derivative was attached. Removal of Fmoc protection: DMF 1 x 5 minutes 20% piperidine in NMP, 1 x 3 minutes 20% piperidine in NMP, 1 x 8 minutes Solvent washing cycle: DMF 3 x 2 minutes IsOH 3 x 2 min DCM 3×2 minutes 【0102】 Chloranil test: The chloranil test yielded positive results, as evidenced by the green color of the resin particles. This allowed us to proceed to the next step, namely the attachment of the Fmoc-Thr(tBu)-OH amino acid residue. 【0103】 Adhesion of amino acid derivatives: Prior to the coupling process, the resin was washed in DMF. The composition of the coupling mixture remained unchanged even after the protective glutamate residues were attached. 【0104】 At the end of each acylation, a solvent washing cycle was performed according to a predetermined procedure, followed by a chloranil test to check for the presence of free amino groups in the solution. Solvent washing cycle: DMF 3 x 2 minutes IsOH 3 x 2 min DCM 3×2 minutes 【0105】 Chloranil test: The resin particles tested after the second acylation were colorless, allowing us to proceed to the next synthesis step, namely the introduction of other protective amino acid derivatives, threonine and 2-aminobenzoic acid molecules. The coupling process followed the procedure discussed earlier. Tests conducted after attaching the above residues showed positive results: the resin particles were colorless. 【0106】 2. Removal of peptides from the carrier After synthesis, the amide of the ABZ-Thr-Thr-Ala-Arg-ANB-NH2 peptide was removed from the support, and simultaneously, the side group protection was removed in a round-bottom flask on a magnetic stirrer using a mixture of:TFA:phenol:water:TIPS (88:5:5:2, v / v / v / v). 【0107】 After 3 hours, the contents of the flask were filtered under reduced pressure using a sintered (Schott) funnel and washed with diethyl ether. The resulting precipitate was centrifuged for 20 minutes using a SIGMA 2K30 laboratory centrifuge. The precipitate obtained after centrifugation was dissolved in water using ultrasound and freeze-dried. 【0108】 Identification / characterization of novel compounds - HPLC analysis, MS HPLC conditions: RP Bio Wide Pore Supelco C8 250mm 4mm column, A phase system: 0.1% TFA in water, B phase: 80% acetonitrile in A phase, flow rate 1 mL / min, UV detection at 226 nm. We confirmed that the compound was obtained. 【0109】 Example 2: Preparation of a compound having the formula:ABZ1-Thr2-Thr3-Ala4-Arg5-pNA6 This process is carried out in the same manner as described in Example 1, except that the corresponding amino acid derivatives and additional substituents are used. This process is carried out partly in solution and partly on a solid support. 【0110】 Preparation of p-nitroanilide Ala a) The first step of the synthesis was to obtain the protected peptide by solid-phase synthesis using Fmoc / tBu chemistry. ABZ 1 -Thr(tBu) 2 -Thr(tBu) 3 -Ala 4 -OH compounds [wherein ABZ is 2-aminobenzoic acid] were obtained by solid-phase chemical synthesis using the following amino acid derivatives: Boc-ABZ, Fmoc-Thr(tBu), Fmoc-Ala. 【0111】 The compound was synthesized by deposition of 1.6 mmol Cl / g groups on a solid support, e.g., 2-chloro-chlorotriyl resin from Iris BIOTECH GMBH (Germany). The compounds were synthesized manually using a laboratory shaker. A 25 mL sintered syringe for solid-phase synthesis was used as the reactor throughout the entire process. 【0112】 Peptide synthesis was carried out by deposition of 1.6 mmol Cl / g groups on a support: for example, a 2-chloro-chlorotrityl resin from Iris BIOTECH GMBH (Germany). In the first step, the resin was relaxed by a washing cycle. Subsequently, the Fmoc amino group protection was removed from the support using a 20% solution of piperidine in NMP. A solvent washing cycle was then performed. A chloranil test was performed to confirm the presence of free amino groups. Solvent washing cycle: DMF 1 x 10 minutes IsOH 1×10 min DCM 1×10 minutes Removal of Fmoc protection: DMF 1 x 5 minutes 20% piperidine in NMP, 1 x 3 minutes 20% piperidine in NMP, 1 x 8 minutes Solvent washing cycle: DMF 3 x 2 minutes IsOH 3 x 2 min DCM 3×2 minutes 【0113】 b) Chloranil test: The chloranil test involved transferring several resin particles from the reactor (i.e., syringe) to a glass ampoule (using a spatula), and then adding 100 μL of a saturated solution of p-chloranil in toluene and 50 μL of unused acetaldehyde. After 10 minutes, the color of the particles was controlled. 【0114】 In this process, after testing, green particles were obtained, indicating the presence of free amino groups. After confirming the removal of the 9-fluorenylmethoxycarbonyl protection from the resin, the attachment of the Fmoc-Ala derivative was initiated. 【0115】 c) Embedding of Fmoc-Ala on a solid support The first step in the synthesis of the peptide library was the deposition of Fmoc-Ala on 1 g of resin. Before attaching the amino acid derivatives, the resin used for the reaction was washed with the following solvents: DMF (dimethylformamide), DCM (methylene chloride), and again with DMF, and then the Fmoc protection was removed from the functional groups of the support. One cycle of removing the Fmoc protection included the following steps: Removal of Fmoc protection: 20% piperidine in NMP, 1 x 3 minutes 20% piperidine in NMP, 1 x 8 minutes Cleaning DMF 3 x 2 minutes IsOH 3 x 2 min DCM 3×2 minutes Chloranil test for the presence of free amino groups. 【0116】 The resin containing free amino groups was washed with DMF. In a separate flask, Fmoc-Ala was dissolved in DMF. Then, TBTU, DMAP, and finally diisopropylethylamine (DIPEA) were added in the following excess amounts relative to the polymer deposit: Fmoc-Pro / TBTU / DMA / DIPEA, 3:3:2:6. The resulting mixture was added to the resin and stirred for 3 hours. The resin was filtered under reduced pressure and washed with DMF, DCM, and isopropanol, and the entire acylation procedure was repeated twice. Subsequent reactions for the attachment of Fmoc-Pro to the resin were carried out using hexafluorophosphate-O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium (HATU), followed by hexafluorophosphate-O-(benzotriazole-1-yl)-N,N,N',N'-tetramethyluronium (HBTU). In the final stage, the resin was sequentially washed with DMF, DCM, and isopropanol, and then air-dried. 【0117】 c. Attachment of further protective amino acid residues: After washing the resin in the reactor with the attached Fmoc-Ala residues using DMF, the Fmoc was deprotected from the amino group, and a protected threonine derivative was attached. Removal of Fmoc protection: DMF 1 x 5 minutes 20% piperidine in NMP, 1 x 3 minutes 20% piperidine in NMP, 1 x 8 minutes Solvent washing cycle: DMF 3 x 2 minutes IsOH 3 x 2 min DCM 3×2 minutes 【0118】 Chloranil test: The chloranil test yielded positive results, as evidenced by the green color of the resin particles. This allowed us to proceed to the next step, namely the attachment of the Fmoc-Thr(tBu)-OH amino acid residue. 【0119】 Adhesion of amino acid derivatives: Prior to the coupling process, the resin was washed in DMF. The composition of the coupling mixture remained unchanged even after the protective glutamate residues were attached. 【0120】 At the end of each acylation, a solvent washing cycle was performed according to a predetermined procedure, followed by a chloranil test to check for the presence of free amino groups in the solution. Solvent washing cycle: DMF 3 x 2 minutes IsOH 3 x 2 min DCM 3×2 minutes 【0121】 Chloranil test: The resin particles tested after the second acylation were colorless, allowing us to proceed to the next synthesis step, namely the introduction of other protective amino acid derivatives, threonine and 2-aminobenzoic acid molecules. The coupling process followed the procedure discussed earlier. Tests conducted after attaching the above residues showed positive results: the resin particles were colorless. 【0122】 d) Removal of peptides from the carrier while maintaining side group protection. After synthesis was complete, the protected ABZ-Thr(tBu)-Th(tBu)-Ala-OH peptide was removed from the support, and the side group protection was maintained in a round-bottom flask on a magnetic stirrer using a mixture of acetic acid, TFE (trifluoroethanol), and DCM (2:2:6, v / v / v). 【0123】 After 2 hours, the contents of the flask were filtered under reduced pressure using a sintered (Schott) funnel and washed with an astringent mixture. The solution was washed with hexane (1:10, v / v), evaporated under reduced pressure, and then freeze-dried. 【0124】 e) Chemical synthesis of Arg derivatives of p-nitroanilide Fmoc-Arg(Pbf)-pNA was synthesized using a mixed anhydrous method. In the first step, 2 mmol of Fmoc-Arg(Pbf) was dissolved in anhydrous tetrahydrofuran (THF) in the presence of 2 mmol of N-methylmorpholine (NMM). The carboxyl group of the amino acid derivative was activated with 2 mmol of isobutyl chloride. After 10 minutes of activation, 3 mmol of p-nitroaniline was added. The reaction was carried out at -15°C for 2 hours, followed by 24 hours at room temperature. After the reaction was complete, the solvent was evaporated, and the dried residue was dissolved in ethyl acetate. The resulting solution was successively washed with saturated aqueous NaCl, 10% citric acid, and 5% sodium bicarbonate. The resulting solution was dried over anhydrous sodium sulfate, ethyl acetate was removed under reduced pressure, and the dried residue was dried over P2O5 and KOH in a vacuum desiccator. 【0125】 f) Coupling of protective peptide with p-nitroanilide Arg (Pbf) protection ABZ 1 -Thr(tBu) 2 -Thr(tBu) 3 -Ala 4-OH peptide was dissolved in a small amount of DCM and then activated with TFFH (tetramethylfluoroformamide) at 0°C for 30 minutes. Catalytic amounts of DMAP and Arg(Pbf)-pNA were then added. The reaction was carried out at room temperature for 24 hours, after which the solvent was evaporated. The resulting solution was poured with a mixture to remove side group protection: TFA:phenol:water:TIPS (88:5:5:2, v / v / v / v), and mixed in a round-bottom flask on a magnetic stirrer for 3 hours. 【0126】 After this time, cold diethyl ether was added to the flask, and the resulting precipitate was centrifuged at 5000 rpm for 20 minutes using a high-speed centrifuge. The precipitate obtained after centrifugation was dissolved in water using ultrasound, and then freeze-dried. 【0127】 Identification / characterization of novel compounds - HPLC analysis, MS HPLC conditions: RP Bio Wide Pore Supelco C8 250mm 4mm column, A phase system (0.1% TFA in water, B phase (80% acetonitrile in A)), flow rate 1 mL / min, UV detection at 226 nm. The compound was confirmed to have been obtained. 【0128】 Assay conditions A study on the application of a novel compound was conducted in a group of 25 patients diagnosed with prostate cancer. For this purpose, a compound having the formula: ABZ-Trp-Glu-Gly-Asn-ANB-NH2 or ABZ-Trp-Glu-Gly-Asn-pNA was dissolved in dimethyl sulfoxide (concentration 0.5 mg / mL); 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 prostate cancer patients. Measurements were performed using a 96-well plate designed for absorbance measurement, and each sample was analyzed three times at 37°C. The measurement time was 60 minutes. During measurement, the wavelength characteristic of the emitted chromophore (ANB-NH2 or pNA) was monitored at 405 nm (range 380-430 nm). 【0129】 The measurements revealed that the solution color increased over time in all urine samples from patients diagnosed with prostate cancer. The increase in absorbance observed over time differed for each test sample. Different results were obtained for 15 samples from healthy individuals, none of which showed an increase in absorbance within the diagnostic range. 【0130】 Analysis confirmed the use of the compounds according to this embodiment in the diagnosis of prostate cancer. The mechanism of action of the novel compounds is based on enzymatic hydrolysis at sites that result in the release of a free chromophore molecule, ANB-NH2, i.e., an amide of 5-amino-2-nitrobenzoic acid. This specification encompasses the following disclosures of the invention. [1] Compounds characterized by formula 1: X1-Trp-Glu-Gly-Asn-X2 (formula 1), In this regard, the cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 yields a detectable signal. [2] The compound according to [1], wherein the sequence Trp-Glu-Gly-Asn is accessible to hydrolases, in particular hydrolases that cleave the compound into X1-Trp-Glu-Gly-Asn-OH (fragment 1) and NH2-X2 (fragment 2). [3] The compound according to [1] or [2], wherein X1 contains or consists of component C1, and X2 contains or consists of component C2, and a detectable signal is obtained by spatial separation of C1 and C2. [4] A compound according to any one of [1] to [3], wherein one of C1 and C2, in particular C2, is a chromophore having an absorption maximum 1 (AM1) at wavelength 1, and the compound has an absorption maximum 2 (AM2) at wavelength 2 different from wavelength 1. [5] A compound according to any of [1] to [4], wherein C1 and C2 are a pair of fluorescent donor and fluorescent acceptor. [6] A compound according to any one of [1] to [5], wherein the C1 and C2 pair is selected from the group consisting of 2-aminobenzoic acid (ABZ) / pNA, ABZ / ANB-NH2, ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, and ABZ / Tyr(3-NO2), and in particular, the C1 and C2 pair is selected from ABZ / pNA and ABZ / ANB-NH2. [7] An in vitro method for detecting protease activity in a body fluid of a subject, comprising contacting the body fluid with a compound described in any of [1] to [6] and detecting a signal, wherein the body fluid can contain hydrolytic enzymes, particularly proteases, derived from prostate cancer cells. [8] An in vitro method according to claim 7 for detecting the presence or absence of prostate cancer in a subject, wherein the presence of protease activity in a body fluid indicates the presence of prostate cancer, and the absence of protease activity in a body fluid indicates the absence of prostate cancer. [9] In vitro methods described in [7] or [8] for the diagnosis of prostate cancer.

[10] An in vitro method described in any of [7] to [9], wherein the body fluid is urine.

[11] The method according to any one of [7] to

[10] , wherein the compound is provided in a measurement buffer having a neutral or alkaline pH, preferably a physiological pH, at a concentration of 0.1 to 10 mg / mL, particularly 0.25 to 7.5 mg / mL, more specifically 0.5 to 5 mg / mL, more specifically 0.75 to 2 mg / mL, and even more specifically about 1 mg / mL, and a body fluid sample is added to the compound in a ratio of 1:2 to 1:10, particularly 1:3 to 1:8, more specifically 1:4 to 1:6, and even more specifically about 1:5.

[12] The method according to any one of [7] to

[11] , wherein detecting a signal is comprising measuring absorbance or fluorescence, in particular measuring absorbance intensity at 300 to 500 nm, more specifically 380 to 430 nm, at 25 to 40°C, particularly 36 to 38°C, for 40 to 60 minutes. A kit comprising the compound and measurement buffer described in any of

[13] [1] to [6].

[14] Use of any of the compounds described in [1] to [6], any of the methods described in [7] to

[12] , or any of the kits described in

[13] for detecting prostate cancer or for monitoring subjects suspected of having prostate cancer, at high risk of developing prostate cancer, or who have had prostate cancer in the past.

[15] A compound according to any of [1] to [6] for use in a method for treating prostate cancer, wherein the method is a. The step of carrying out any of the methods described in [7] to

[12] , b. In subjects in which protease activity, particularly increased protease activity, was detected in step a, the stage of treating prostate cancer. The compound comprising the above.

Claims

[Claim 1] Formula 1: X1-Trp-Glu-Gly-Asn-X2 (formula 1), A compound characterized by, Here, the cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 yields a detectable signal. X1 consists of component C1, X2 consists of component C2, the detectable signal is an optical signal, and A detectable signal is obtained by the spatial separation of C1 and C2 due to hydrolytic cleavage of the peptide Trp-Glu-Gly-Asn, where C1 and C2 are a fluorescent donor and fluorescent acceptor pair, and the C1 and C2 pair is 2-aminobenzoic acid (ABZ) / pNA, ABZ / ANB-NH ２ , ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr (3-NO ２ Selected from the group consisting of ) The aforementioned compound. [Claim 2] The compound according to claim 1, wherein the sequence Trp-Glu-Gly-Asn is accessible to a hydrolytic enzyme. [Claim 3] The hydrolase breaks the compound into X1-Trp-Glu-Gly-Asn-OH (fragment 1) and NH ２ The compound according to claim 2, which is a hydrolytic enzyme that cleaves into -X2 (fragment 2). [Claim 4] The compound according to any one of claims 1 to 3, wherein one of C1 and C2 is a chromophore having an absorption maximum 1 (AM1) at wavelength 1, and the compound has an absorption maximum 2 (AM2) at wavelength 2 different from wavelength 1. [Claim 5] The compound according to claim 4, wherein C2 is a chromophore having an absorption maximum 1 (AM1) at wavelength 1, and the compound has an absorption maximum 2 (AM2) at wavelength 2 different from wavelength 1. [Claim 6] The pair of C1 and C2 is ABZ / pNA and ABZ / ANB-NH ２ A compound according to any one of claims 1 to 5, selected from the above. [Claim 7] A method for detecting protease activity in a subject's body fluid in vitro, comprising contacting the body fluid with a compound according to any one of claims 1 to 4 and detecting a signal, wherein the body fluid can contain a hydrolase derived from prostate cancer cells. [Claim 8] The method according to claim 7, wherein the enzyme is a protease. [Claim 9] A method according to claim 7 or 8, used to detect the presence or absence of prostate cancer in a subject, wherein the presence of protease activity in a body fluid indicates the presence of prostate cancer, and the absence of protease activity in a body fluid indicates the absence of prostate cancer. [Claim 10] The method according to any one of claims 7 to 9 for the diagnosis of prostate cancer. [Claim 11] The method according to any one of claims 7 to 10, wherein the bodily fluid is urine. [Claim 12] The method according to any one of claims 7 to 11, wherein the compound is provided in a pH measuring buffer having a neutral or alkaline concentration at a concentration of 0.1 to 10 mg / mL, and the body fluid sample is added to the compound in a compound:body fluid sample ratio of 1:2 to 1:10 v / v. [Claim 13] The method according to claim 12, wherein the compound is provided at a concentration of 0.25 to 7.5 mg / mL. [Claim 14] The method according to claim 13, wherein the compound is provided at a concentration of 0.5 to 5 mg / mL. [Claim 15] The method according to claim 14, wherein the compound is provided at a concentration of 0.75 to 2 mg / mL. [Claim 16] The method according to claim 15, wherein the compound is provided at a concentration of 1 mg / mL. [Claim 17] The method according to any one of claims 12 to 16, wherein the measurement buffer has a physiological pH. [Claim 18] The method according to any one of claims 12 to 17, wherein the body fluid sample is added to the compound in a compound:body fluid sample ratio of 1:3 to 1:8 v / v. [Claim 19] The method according to claim 18, wherein the ratio of compound to body fluid sample is 1:4 to 1:6 v / v. [Claim 20] The method according to claim 19, wherein the ratio of compound to body fluid sample is 1:5 v / v. [Claim 21] The method according to any one of claims 7 to 20, wherein detecting a signal includes measuring absorbance or fluorescence. [Claim 22] The method according to claim 21, wherein the measurement of absorbance is the measurement of absorbance intensity at 300 to 500 nm. [Claim 23] The method according to claim 22, wherein the measurement of absorbance is the measurement of absorbance intensity at 380 to 430 nm. [Claim 24] The method according to any one of claims 21 to 23, wherein detecting a signal includes measuring absorbance or fluorescence at 25 to 40°C for 40 to 60 minutes. [Claim 25] The method according to claim 24, wherein detecting a signal includes measuring absorbance or fluorescence at 36 to 38°C. [Claim 26] The method according to claim 25, wherein detecting a signal is done by measuring absorbance at 36 to 38°C. [Claim 27] The method according to any one of claims 12 to 26 for the detection of prostate cancer, or for monitoring subjects suspected of having prostate cancer, at high risk of developing prostate cancer, or who have had prostate cancer in the past. [Claim 28] A kit comprising the compound and measurement buffer according to any one of claims 1 to 6. [Claim 29] To assist in the detection of prostate cancer, or to assist in monitoring subjects suspected of having prostate cancer, at high risk of developing prostate cancer, or who have had prostate cancer in the past, Compounds characterized by formula 1: X1-Trp-Glu-Gly-Asn-X2 (formula 1), [In the formula, The cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 yields a detectable signal. X1 consists of component C1, X2 consists of component C2, the detectable signal is an optical signal, and A detectable signal is obtained by the spatial separation of C1 and C2 due to hydrolytic cleavage of the peptide Trp-Glu-Gly-Asn, and, C1 and C2 are a pair of fluorescent donor and fluorescent acceptor, where the pair C1 and C2 is 2-aminobenzoic acid (ABZ) / pNA, ABZ / ANB-NH ２ , ABZ / DNP, ABZ / EDDNP, EDANS / DABCYL, TAM / DANSYL, ABZ / Tyr (3-NO ２ Selected from the group consisting of ) the compound Use. [Claim 30] A method to assist in identifying subjects requiring treatment for prostate cancer, a. The step of carrying out the method according to any one of claims 7 to 27, and b. The step of selecting subjects in whom protease activity was detected in step a. The method, including the method described above. [Claim 31] The method according to claim 30, wherein the protease activity at step b is increased protease activity.

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