RU2802849C2 - New marker for the diagnostics of pancreatic cancer - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to a chromogenic peptide for use in diagnosing pancreatic cancer. The following compound of formula 1 is proposed: X1-Thr-Thr-Ala-Arg-X2, where X1 contains or consists of a C1 component, and X2 contains or consists of a C2 component, while C1 and C2 are a pair of a fluorescence donor and a fluorescence acceptor. Proteolytic cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 results in a detectable signal. Also the following is proposed: a kit for diagnosing pancreatic cancer containing the specified compound, a method of in vitro detection of protease activity in the biological fluid of a subject, which may contain a proteolytic enzyme derived from pancreatic cancer cells, and a method of treating pancreatic cancer.

EFFECT: inventions are applicable to the rapid and non-invasive detection of pancreatic cancer or to the monitoring of a subject suspected of having pancreatic cancer, with increased risk of developing pancreatic cancer, or previously suffering from pancreatic cancer.

14 cl, 4 dwg, 3 tbl, 3 ex

Description

The present invention relates to chemical compounds for use in the diagnosis of pancreatic cancer. In particular, the present invention relates to chromogenic peptides suitable for detecting proteolytic enzymes in a sample.

State of the art

Pancreatic cancer often has a very poor prognosis because it is often diagnosed at an advanced stage. Due to the dynamics of this cancer, survival beyond five years is rare. Approximately 250,000 cases of pancreatic cancer are diagnosed worldwide per year, 3,500 of them in Poland. Unfortunately, the vast majority (more than 80%) of these cases are fatal. There is an urgent need to develop reliable, rapid and uncomplicated diagnostic methods for detecting pancreatic cancer. Earlier diagnosis increases the chances of surgical treatment, which can significantly increase patient survival.

The process of initiation, growth and spread of cancer cells involves many factors, including the participation of a number of proteolytic enzymes. This group of proteins can hydrolyze proteins and peptides into smaller fragments. Proteolytic enzymes mediate the breakdown of the extracellular matrix, which allows cancer cells to colonize new tissues and promotes the formation of new blood vessels (angiogenesis), which facilitates the efficient delivery of nutrients to the tumor. In addition, proteolytic enzymes are formed as a result of the death of healthy cells due to tumor growth. All these processes form a profile of proteolytic enzymes characteristic of a tumor.

Chromogenic peptide molecules, which are destroyed by proteolytic enzymes, leading to a change or increase in the color of the analyzed solution, have been described previously. The chromogenic effect results from the release of a chromophore (eg, 4-nitranilide or 5-amino-2-nitrobenzoic acid).

This type of peptide derivatives 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. 1961 Nov; 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). 2000 Nov; 48(11):1740-4.

Methods for detecting and measuring protease expression have been described previously. The described methods are based on isolated antibodies that specifically bind to specific polypeptides and PCR of polynucleotides encoding specific polypeptides. Demonstration of protease activity is measured by hydrolysis of appropriate synthetic peptide substrates conjugated to chromogenic molecules, said proteases degrading said synthetic substrate, and measuring the uptake of the chromogen released during hydrolysis of the substrate. (CA 2425829).

Preferred compounds of the present invention are formulated such that when the compound is contacted with a urine sample of a person suffering from pancreatic cancer, a signal can be detected, i.e. color enhancement in the range of 380-440 nm. This effect does not occur when the compound comes into contact with a urine sample from a healthy person or a patient diagnosed with another type of cancer.

The present invention shows that such a signal, for example generated by a released chromogenic compound, or a fluorescent signal after separation of a fluorescent donor-acceptor pair, can be used to detect the presence of proteolytic enzymes in the urine of a subject with pancreatic cancer. Enzymatic hydrolysis of the compounds results in a detectable difference, for example the release of free chromophore molecules (ANB-NH ₂ - 5-amino-2-nitrobenzoic acid amide or pNA - para-nitroaniline, respectively), which are characterized by absorption at 320-480 nm.

The compounds of the present invention provide, among other things, other things: (i) a rapid and non-invasive diagnostic method for detecting pancreatic cancer, (ii) a method suitable for the early detection of pancreatic cancer, (iii) a method that can be successfully applied to the screening of pancreatic cancer, (iv) a complete diagnostic process at an early stage of cancer development to increase the effectiveness of treatment.

The essence of the invention

In a first aspect, the present invention relates to a compound characterized by Formula 1:

X1-Thr-Thr-Ala-Arg-X2 (formula 1),

wherein cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 leads to the formation of a detectable signal. The compound contains the tetrapeptide Thr-Thr-Ala-Arg in accordance with SEQ ID NO: 1.

In a second aspect, the present invention relates to a method for detecting protease activity in a biological fluid of a subject in vitro , comprising contacting the biological fluid with a compound according to the first aspect of the present invention and detecting a signal, wherein the biological fluid may contain a hydrolytic enzyme, in particular a protease, derived from pancreatic cancer cells.

In a third aspect, the present invention provides a kit containing a compound according to the first aspect of the present invention and a measurement buffer.

In a fourth aspect, the present invention relates to the use of a compound according to the first aspect of the present invention, a method according to the second aspect of the present invention, or a kit according to the third aspect of the present invention for detecting pancreatic cancer, or for monitoring a subject suspected of having pancreatic cancer at increased risk of developing cancer. pancreas or with pancreatic cancer.

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 pancreatic cancer, said method comprising the steps of (a) performing the method according to the second aspect of the present invention and (b) treating pancreatic cancer in a subject who has Step (a) detects protease activity, in particular increased protease activity.

Detailed Description of the Invention

Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as they are subject to change. Moreover, it should be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not to be construed as limiting the scope of the present invention, which is to be limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. Preferably, the terms used herein correspond to the definitions described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, H.G.W, Nagel, B. and Kölbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Several documents are mentioned throughout the text of this description. Each of the documents referred to in this application (including all patents, patent applications, patent publications, scientific publications, manufacturer's specifications, instructions, etc.) above or below is incorporated herein by reference in its entirety. Nothing herein should be interpreted as an admission that the present invention cannot or cannot precede such disclosure by virtue of an earlier invention.

To practice the present invention, unless otherwise indicated, conventional methods used in the field of chemistry, biochemistry and recombinant DNA technology are used, as explained in the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual , ^2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989) .

It should be understood that in this specification and the following claims, the word “comprise” and its derivatives, for example, “comprises” and “comprising” are intended to include the specified element or step or group of elements or steps and not to exclude any other element or step or group elements or steps unless the context otherwise requires. In the present specification and the accompanying claims, the singular forms include the plural unless the meaning of the specification clearly indicates otherwise.

Elements of the present invention are described below. These elements are listed with specific embodiments, but it should be understood that they can be combined in any manner and in any quantity to create additional embodiments. The various examples and preferred embodiments described should not be construed as limiting the present invention to those embodiments expressly described. It should be understood that this description is provided to confirm and cover embodiments combining the explicitly described embodiments with any number of described and/or preferred elements. In addition, any permutations and combinations of all elements described in this application should be considered to be included in the description in this application unless the context otherwise requires.

The terms and abbreviations used in the patent description and claims should be understood as follows:

In the context of the present invention, "detectable signal" refers to any signal, for example, generated by any label, including a magnetic label, a fluorescent group, an enzyme, a chemiluminescent probe, a metal particle, a non-metallic colloidal particle, a polymer dye particle, a pigment molecule, a pigment particle, an electrochemically active compound, semiconductor nanocrystal, or other nanoparticle, including quantum dots or gold particles. The label may be, for example, chromophores, fluorophores, quantum dots or radioactive labels.

In the context of the present invention, the expression “chromophore” is used to refer to a compound having “chromogenic properties”. The expression “chromogenic properties” refers to the ability of a compound to form a colored product.

In the context of the present invention, the expression “fluorophore” is used to refer to a compound having “fluorogenic properties”. The expression “fluorogenic properties” refers to the ability of a compound to form a product that emits fluorescence.

Fluorescent dyes of the present invention include the following classes of dyes: xanthenes (eg, fluorescein), acridines (eg, acridine yellow), oxazines (eg, oxazine 1), cyanines (eg, Cy7/Cy3), styryl dyes (eg, Dye-28 ), coumarins (e.g. Alexa Fluor 350), porphins (e.g. Chlorophyll B), metal ligand complexes (e.g. PtOEPK), fluorescent proteins (e.g. APC, R-phycoerythrin), nanocrystals (e.g. QuantumDot 705), perylenes (e.g. , Lumogen Red F300) and phthalocyanines (e.g. IRDYE™700DX), as well as conjugates and combinations of dyes of these classes.

A quantum dot is a semiconductor consisting of atoms of elements of groups II-VI or III-V of the periodic table of elements (for example, CdSe, CdTe, InP). Other alternatives include any two-dye system.

In the context of the present invention, NMP refers to N- methylpyrrolidone.

In the context of the present invention, DMF refers to dimethylformamide.

In the context of the present invention, DCM refers to dichloromethane.

In the context of the present invention, pNA refers to 4-nitroaniline, which may also be referred to as para-nitroaniline.

In the context of the present invention, ABZ refers to 2-aminobenzoic acid.

In the context of the present invention, ANB-NH2 refers to 5-amino-2-nitrobenzoic acid amide.

In the context of the present invention, AFC refers to 7-amido-4-trifluoromethylcoumarin.

In the context of the present 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 the present invention, TFA refers to trifluoroacetic acid.

In the context of the present invention, the term "pancreatic cancer" is used in its broadest possible sense and refers to all types of cancer that begin in the pancreas. It includes the subtypes of exocrine malignancies, endocrine malignancies, pancreoblastoma, pancreatic sarcomas and lymphoma. Exocrine malignancies include adenocarcinomas, particularly ductal adenocarcinomas, as well as cystic tumors and acinar cell malignancies. Endocrine malignancies include gastrinomas, insulinomas, somatostatinomas, VIPomas and glucagonomas. It also includes the following stages (the definitions of which according to the respective TNM classifications are indicated in parentheses): stage 0 (Tis, N0, M0), stage IA (T1, N0, M0), stage IB (T2, N0, M0), stage IIA ( T3, N0, M0), stage IIB (T1-3, N1, M0), stage III (T4, any N, M0) and stage IV (any T, any N, M1).

The peptides of the present invention, preferably chromogenic or fluorogenic peptides, can be obtained by performing solid-support peptide synthesis known in the art. The solid support may be in the form of a resin containing an Fmoc group which is removed during the reaction. The resin used to perform this process must be properly prepared. Preparing the resin involves increasing its volume by repeated washing with hydrophobic solvents.

The Fmoc protecting group should be removed from the resin by washing with a 20% solvent solution.

Known processes for the preparation of chromogenic peptides involve the addition of individual components at appropriate times and under stoichiometric conditions. This coupling process consists of sequential steps during which individual elements (amino acid derivatives) are coupled, residues are removed by washing, the protecting groups are removed, and washing is repeated. This cycle is repeated for each amino acid residue. The resulting peptide is separated from the resin by reaction under acidic conditions. After this, the solution is separated from the resin by filtration, and then the peptide is precipitated from the resulting solution using a non-polar solvent. The peptide precipitate is then centrifuged.

Known methods for producing chromogenic peptides are based on a process performed on a solid support and partly in a buffer. An amide resin is used as a solid support, and a mixture of hydrophobic solvents is used as a solution.

The compounds of the present invention were prepared using the method described in Hojo et al. , Chem Pharm Bull (Tokyo), 2000. A detailed description of this synthesis is found below in the Examples section.

In addition, the peptide of the present invention can be coupled to a quantum dot. Quantum dots are characterized by strong luminescence, except for quenching in the presence of a metal nanoparticle. Viologens (eg methyl viologen or propyl viologen sulfonate (PVS)) can also be used as quenchers.

After cleavage of the consensus sequence by a protease, the quantum dots are released and emit light.

The first aspect of the present invention provides a compound characterized by Formula 1: X1-Thr-Thr-Ala-Arg -X2 (Formula 1), wherein cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 results in the formation of a detectable signal .

In preferred embodiments, the sequence Thr-Thr-Ala-Arg is accessible to a hydrolytic enzyme, in particular a hydrolytic enzyme that cleaves said compound into X1-Thr-Thr-Ala-Arg-OH (fragment 1) and NH ₂ -X2 (fragment 2 ).

The hydrolytic enzyme may be a protease or a combination of several proteases.

The detectable signal resulting from the cleavage of said compound can be selected from various suitable signals known to one skilled in the art. In preferred embodiments, the detected signal is an optical signal.

In preferred embodiments, X1 contains or consists of a component C1, and X2 contains or consists of a component C2, and a detectable signal occurs when C1 and C2 are spatially separated, i.e. during hydrolytic cleavage of the Thr-Thr-Ala-Arg peptide.

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. Thus, prior to cleavage, C1 and C2 may be separated by an amino acid sequence of 4-20 amino acids or 5-10 amino acids containing or consisting of Thr-Thr-Ala-Arg. In preferred embodiments, C1 and C2 are separated by no more than 10 amino acids.

In preferred embodiments, the detectable signal is a change in absorbance or fluorescence. Said change may be an increase or a decrease. In preferred embodiments, the detectable signal is an increase in absorption intensity at 300-500 nm, in particular 380-430 nm.

In preferred embodiments, 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 a wavelength 2 different from wavelength 1. Thus, when measuring absorption at wavelength 2 before and after cleavage of the compound, an increase in absorption will be detected.

Particularly preferred is the release of the chromophore in a free form upon cleavage. Upon cleavage, fragment 1 consists of X1-Thr-Thr-Ala-Arg, while fragment 2 consists of only NH ₂ -X2. Thus, in preferred embodiments, the chromophore is C2 rather than C1. If C2 is a chromophore, X2 preferably consists or essentially consists of C2. On the other hand, X1 may contain additional amino acids on either side of C1, so that prior to cleavage, C1 and C2 may be separated by a longer amino acid sequence than Thr-Thr-Ala-Arg.

In preferred embodiments, the chromophore is selected from para-nitroaniline (pNA), 5-amino-2-nitrobenzoic acid amide (ANB-NH ₂ ), 7-amido-4-trifluoromethylcoumarin (AFC), and 3-nitro-L-tyrosine (Tyr3 -NO ₂ ). In preferred embodiments, the chromophore is para-nitroaniline (pNA). In preferred embodiments, the chromophore is 5-amino-2-nitrobenzoic acid amide (ANB-NH ₂ ).

In preferred embodiments, C1 and C2 are a pair of a fluorescence donor and a fluorescence acceptor. If C1 and C2 are a fluorescence donor and acceptor pair and the compound is used in a method that measures the change in fluorescence upon cleavage of the compound, C1 and C2 are preferably separated by no more than 10 amino acids to ensure effective quenching of the fluorescence donor by the fluorescence acceptor. The skilled person knows that the distance between the fluorescence donor and the fluorescence acceptor is the most important parameter. Thus, if the amino acid sequence separating C1 and C2 is arranged in a condensed or coiled secondary structure, which brings C1 and C2 closer together than if a linear linker were used, it is permissible to use an even longer spacer between C1 and C2.

In preferred embodiments, the pair of C1 and C2 is selected from the group consisting of 2-aminobenzoic acid (ABZ)/pNA, ABZ/ANB-NH ₂ , ABZ/DNP, ABZ/EDDNP, EDANS/DABCYL, TAM/DANSYL, ABZ/ Tyr(3-NO ₂ ), in particular the pair of C1 and C2 is selected from ABZ/pNA and ABZ/ANB-NH ₂ . C1 and C2 preferably have a molecular weight of less than 500 g/mol, in particular less than 400 g/mol, more particularly less than 300 g/mol, more particularly 100 to 200 g/mol.

The pair of C1 and C2 may also be a fluorescence donor and acceptor protein pair 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. One skilled in the art knows methods for selecting suitable fluorescence donor and acceptor protein pairs based on their emission and absorption spectra.

If C1 and C2 are a fluorescence donor and acceptor protein pair, it is necessary to ensure that the relatively large size of the proteins does not prevent cleavage of the compound and that the Thr-Thr-Ala-Arg sequence is accessible for cleavage by the hydrolytic enzyme. This can be achieved, for example, by increasing the length of the amino acid sequence containing the peptide in accordance with SEQ ID NO: 1.

In preferred embodiments, the compound is characterized by Formula 2:

ABZ-Thr-Thr-Ala-Arg-ANB-NH ₂ (formula 2).

In preferred embodiments, the compound is characterized by Formula 3:

ABZ-Thr-Thr-Ala-Arg-pNA (formula 3).

After cleavage of compounds characterized by formula 2 or 3, free chromophore molecules (ANB-NH ₂ or pNA, respectively) are released. Thus, at a wavelength of 380-430 nm, an increase in absorption intensity can be detected. In addition, cleavage results in spatial separation of the fluorescence donor (ABZ) and the fluorescence acceptor (ANB-NH ₂ or pNA, respectively). Thus, the fluorescence emitted by ABZ is no longer quenched and an increase in fluorescence intensity can be detected with wavelength.

A second aspect of the present invention provides a method for detecting protease activity in a biological fluid of a subject in vitro , comprising contacting the biological fluid with a compound according to the first aspect of the present invention and detecting a signal, wherein the biological fluid may contain a hydrolytic enzyme, in particular a cell-derived protease. pancreatic cancer.

In preferred embodiments, the presence of protease activity in the biological fluid indicates the presence of pancreatic cancer, and the absence of protease activity in the biological fluid indicates the absence of pancreatic cancer.

In preferred embodiments, the present invention provides a method for diagnosing pancreatic cancer.

In preferred embodiments, the biological fluid is selected from blood or urine. In preferred embodiments, the biological fluid is urine. The inventors have surprisingly discovered that the compound according to the first aspect of the present invention is capable of detecting hydrolytic enzyme activity in a urine sample. Hydrolytic enzyme activity was significantly increased in subjects diagnosed with pancreatic cancer compared to healthy subjects (Figs. 1, 2).

In preferred embodiments, the subject has an increased risk of developing pancreatic cancer, is suspected of having pancreatic cancer, has a history of pancreatic cancer, or has a history of pancreatic cancer.

In preferred embodiments, the compound is provided at a concentration of 0.1-10 mg/ml, in particular 0.25-7.5 mg/ml, more specifically 0.5-5 mg/ml, more specifically 0.75-2 mg /ml, even more specifically about 1 mg/ml in a measurement buffer with a neutral or alkaline pH, preferably a pH of 6.8 to 8.5, more preferably a physiological pH, and a sample of the biological fluid is added to the compound in a ratio of 1: 2 to 1:10, in particular from 1:3 to 1:8, more specifically from 1:4 to 1:6, even more specifically about 1:5.

As used herein, the expression “neutral pH” refers to a pH of approximately 7.0. As used herein, the expression “physiological pH” refers to a pH of approximately 7.4.

In preferred embodiments, signal detection includes measuring absorbance or fluorescence.

In preferred embodiments, signal detection involves measuring absorption intensity at 300-500 nm, in particular 380-430 nm, preferably for 40-60 minutes at 25-40°C, in particular at 36-38°C.

In preferred embodiments, an increase in absorbance indicates the presence of hydrolytic enzyme activity.

In a third aspect of the present invention, there is provided a kit containing a compound according to the first aspect of the present invention and a measurement buffer.

A fourth aspect of the present invention provides the use of a compound according to the first aspect of the present invention, a method according to the second aspect of the present invention, or a kit according to the third aspect of the present invention for the detection of pancreatic cancer, or for monitoring a subject at increased risk of developing pancreatic cancer suspected of having pancreatic cancer. or a history of pancreatic cancer.

Depending on the purpose of the method according to the second aspect, the term “subject” may be characterized by various restrictions. For example, if the method is to be used to detect pancreatic cancer or screen subjects for pancreatic cancer, the presence of pancreatic cancer in the subject is unknown, i.e. he may or may not have pancreatic cancer. In this example, the subject preferably has an increased risk of developing pancreatic cancer, is suspected of having pancreatic cancer, or has a history of pancreatic cancer (ie, has recovered from detectable pancreatic cancer). “Increased risk” means that the subject may have one or more risk factors for developing general cancer or pancreatic cancer, preferably in accordance with the American Cancer Society definition for general cancer or pancreatic cancer. Examples of risk factors for pancreatic cancer include tobacco use (particularly smoking), heavy alcohol consumption, obesity, type 2 diabetes, occupational factors (workplace exposure to certain chemicals used in dry cleaning and the metalworking industry), chronic pancreatitis, age 50 years or more (particularly 65 years or more), male gender, ethnicity (particularly African American and Caribbean men of African descent), family history of pancreatic cancer (particularly first-degree relatives) degree of relationship), as well as the presence of a congenital predisposition syndrome (for example, hereditary breast and ovarian cancer syndrome caused by mutations in the BRCA1 or BRCA2 genes; hereditary breast cancer caused by mutations in the PALB2 gene; familial atypical melanoma with multiple nevi syndrome (FAMMM) , caused by mutations in the p16/CDKN2A gene and associated with melanoma of the skin and eyes; familial pancreatitis, usually caused by mutations in the PRSS1 gene; Lynch syndrome, also known as hereditary nonpolyposis colorectal cancer (HNPCC), most often caused by a defect in the MLH1 or MSH2 genes; or Peutz-Jeghers syndrome, caused by defects in the STK11 gene).

Pancreatic cancer can be of any subtype and stage as defined above, i.e. the presence or absence of cancer of any subtype and/or stage can be detected.

In preferred embodiments, the presence of a significant amount of protease activity or an amount of protease activity greater than that in the control sample indicates the presence of pancreatic cancer, and the absence of a significant amount of protease activity or an amount of protease activity equal to or less than that of the control sample indicates the absence of pancreatic cancer glands.

In a specific embodiment, the method of the second aspect further includes confirming detection of pancreatic cancer by using one or more additional means for detecting pancreatic cancer. Additional tools may be a cancer marker (or "biomarker") or traditional (non-marker) detection tools. The cancer marker may, for example, be a DNA methylation marker, a mutation marker (eg, SNP), an antigenic marker, a protein marker, a microRNA marker, a cancer-specific metabolite, or an expression marker. Conventional means may, for example, be a biopsy (eg, visual examination of a biopsy with or without staining techniques, such as protein or expression markers), an imaging technique, or a medical examination, such as tactile examination. In preferred embodiments, the additional means of detecting pancreatic cancer is selected from the group consisting of physical examination (enlarged liver or gallbladder, jaundice, i.e., yellowing of the skin or sclera), CT scan, MRI, cholangiopancreatography (particularly endoscopic retrograde cholangiopancreatography, magnetic resonance cholangiopancreatography or percutaneous transhepatic cholangiography), angiography (especially x-ray angiography, CT angiography or MR angiography), abdominal ultrasound, endoscopic ultrasound, PET scan, blood tests (especially for bilirubin , CA 19-9 or CEA) and biopsy.

The term “is an indicator” or “indicates” as used herein refers to the act of identifying or defining something to be indicated. As those skilled in the art will appreciate, such an estimate may not typically be correct for 100% of subjects, although it is preferably correct. At the same time, this term requires the correct indication to be performed in a statistically significant part of subjects. The statistical significance of a portion can be easily determined by one of ordinary skill in the art using various well-known statistical evaluation tools, for example, confidence intervals, p-value determination, Student's t test, Mann-Whitney test, etc. For details, see 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%, according to at least 90%, at least 95%. The p values are preferably 0.05, 0.01 or 0.005.

The phrase “method for detecting the presence or absence” as used herein, with respect to pancreatic cancer, means determining the presence or absence of cancer in a subject. As those skilled in the art will appreciate, such an estimate may not typically be correct for 100% of subjects, although it is preferably correct. At the same time, this term requires the correct indication to be performed in a statistically significant part of subjects. For a description of statistical significance and appropriate confidence intervals and p values, see above.

As used herein, the term “diagnosis” refers to determining the presence or absence of cancer in a subject. The protease activity assay diagnostics described herein can be supplemented with the tools described herein to confirm cancer detected by the protease activity assay. As those skilled in the art will appreciate, the diagnosis may not routinely be correct for 100% of subjects, although it is preferably correct. At the same time, this term requires correct diagnosis in a statistically significant part of subjects. For a description of statistical significance and appropriate confidence intervals and p values, see above.

As used herein, the phrase “screening a population of subjects” in relation to a cancer as described herein means applying the method of the first aspect using samples obtained from a population of subjects. These subjects are preferably characterized by an increased risk of, suspicion of, or history of cancer. In particular, subjects in the population may have one or more of the risk factors mentioned herein. In a particular embodiment, the same one or more of the risk factors may occur in all subjects of the population. For example, a population may consist of subjects characterized by heavy use of alcohol and/or tobacco. It should be understood that the term “screening” refers to the diagnosis described above in population subjects, and is preferably confirmed using additional tools described herein. As those skilled in the art will appreciate, a screening result may not typically be correct for 100% of subjects, although it is preferably correct. At the same time, this term requires obtaining the correct screening result in a statistically significant proportion of subjects. For a description of statistical significance and appropriate confidence intervals and p values, see above.

As used herein, the term "monitoring" refers to following a diagnosed cancer during a treatment procedure or for a specified period of time, typically for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 5 years, 10 years or any other period of time. The term “monitoring” means that states and, in particular, changes in these cancer states can be detected based on the amount of protease activity, in particular, on the basis of changes in this amount over periodic periods of time of any type, for example, daily or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 times a month (no more than one determination per day) during the course of treatment, which can be up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15 or 24 months. The amount or changes in amount can also be determined when treatment-specific events occur, for example, before and/or after each treatment cycle or drug administration. The cycle represents the time between one round of treatment and the start of the next round. Cancer treatment is usually not a one-time treatment, but a course of treatment. The course usually lasts from 4 to 6 months, but its duration may be more or less than indicated. During the course of treatment, 4 to 8 cycles of treatment usually take place. Typically, a treatment cycle includes a break from treatment to allow the body to recover. As those skilled in the art will appreciate, a monitoring result may not typically be correct for 100% of subjects, although it is preferably correct. At the same time, this term requires obtaining the correct monitoring result from a statistically significant part of the subjects. For a description of statistical significance and appropriate confidence intervals and p values, see above.

A fifth aspect of the present invention provides the use of a compound according to the first aspect of the present invention in a method for treating pancreatic cancer, said method comprising the steps of performing the method according to the second aspect of the present invention and treating pancreatic cancer in a subject who has protease activity, in particular increased protease activity.

In another aspect of the present invention, there is provided a method for treating pancreatic cancer, comprising performing a method according to the second aspect of the present invention and treating pancreatic cancer in a subject that has protease activity, in particular increased protease activity.

As used herein, the term “treatment” and its derivatives in relation to cancer means a therapeutic treatment aimed at reducing the progression of cancer. Beneficial or desirable clinical outcomes include alleviation of symptoms, decreased disease duration, stabilized (ie, not worsened) disease state, slowed disease progression, improvement of disease state, and remission (partial or complete), preferably detectable, but not limited to. Successful treatment does not necessarily mean a cure, and may also mean an increase in survival compared to the expected survival without treatment. In a preferred embodiment, the treatment is a first line treatment, i.e. the cancer has not been treated previously. Cancer treatment involves the use of a treatment regimen.

As used herein, the term “treatment regimen” refers to a method of treating a subject based on the disease, available procedures, and drug treatments. Non-limiting examples of cancer treatment regimens include chemotherapy, surgery and/or radiation, or combinations thereof. The early treatment of cancer provided by the present invention allows, in particular, surgical treatment, especially radical resection. In particular, the term "treatment regimen" refers to the administration of one or more anticancer agents or therapeutic agents, as defined below. As used herein, the term “anti-cancer agent or therapeutic agent” refers to chemical, physical or biological agents or therapeutic agents or surgery, including combinations thereof, having anti-proliferative, anti-tumorigenic and/or carcinostatic properties.

The anticancer chemical agent or therapeutic agent may be selected from the group consisting of alkylating agents, antimetabolites, plant alkaloids and terpenoids, and topoisomerase inhibitors. The alkylating agents are preferably platinum compounds. In one embodiment, the platinum compounds are selected from the group consisting of cisplatin, oxaliplatin, eptaplatin, lobaplatin, nedaplatin, carboplatin, iproplatin, tetraplatin, lobaplatin, DCP, PLD-147, JM118, JM216, JM335, and satraplatin.

The physical anticancer agent or therapeutic agent may be selected from the group consisting of radiation therapy (eg, radical radiation therapy, adjuvant radiation therapy, palliative radiation therapy, external beam radiation therapy, brachytherapy, or metabolic radiation therapy), phototherapy (using, for example, hematoporphyrin or photofrin II) and hyperthermia.

The surgery may be radical resection, palliative surgery, prophylactic surgery, or cytoreductive surgery. It usually involves excision, such as intracapsular excision, marginal excision, extensive excision, or radical excision, as described in Baron and Valin (Rec. Med. Vet, Special Canc. 1990; 11(166):999-1007).

The biological anticancer agent or therapeutic agent may be selected from the group consisting of antibodies (e.g., antibodies that stimulate an immune response that destroys cancer cells, such as retuximab or alemtuzumab, antibodies that stimulate an immune response by binding to receptors on immune cells that generate inhibitory signals, not allowing the immune cell to attack its own cells, such as ipilimumab, antibodies that interfere with proteins essential for tumor growth, such as bevacizumab, cetuximab or panitumumab, or antibodies conjugated to a drug, preferably a cell-killing substance, such as a toxin , a chemotherapeutic or radioactive substance, for example, Y-ibritumomab-tiuxetan, I-tositumomab or ado-trastuzumab-emtansine), cytokines (for example, interferons or interleukins, for example, IFN-alpha and IL-2), vaccines (for example, vaccines containing antigens associated with cancer, e.g. sipuleucel-T), oncolytic viruses (e.g. naturally occurring oncolytic virus, e.g. reovirus, Newcastle disease virus or mumps virus, or genetically engineered viruses, e.g. measles virus, adenovirus, vaccinia virus or herpes virus, the primary targets of which are cells bearing antigens associated with cancer), gene therapy agents (for example, DNA or RNA that replace a modified tumor suppressor, block oncogene expression, improve the state of the subject's immune system, enhance the sensitivity of cancer cells to chemotherapy, radiation therapy, or other therapeutic agents that induce cell self-destruction or impart an antiangiogenic effect) and adoptive T cells (e.g., subject-derived tumor-infiltrating T cells selected for antitumor activity, or subject-derived T cells genetically modified to recognize an antigen associated with cancer).

In one embodiment, one or more of the anticancer drugs is selected from the group consisting of abiraterone acetate, ABVD, ABVE, ABVE-PC, AC, AC-T, ADE, ado-trastuzumab-emtansine, afatinib dimaleate, aldesleukin, alemtuzumab, aminolevulnic acid, anastrozole, aprepitant, arsenic trioxide, Erwinia chrysanthemi asparaginase, axitinib, azacitidine, BEACOPP, belinostat, bendamustine hydrochloride, BEP, bevacizumab, bexarotene, bicalutamide, bleomycin, bortezomib, bosutinib, brentuximab-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, liposomal cytarabine, dabrafenib, dacarbazine, dactinomycin, dasatinib, daunorubicin hydrochloride, decitabine, degarelix, denileukin-diftitox, denosumab, dexrazoxane hydrochloride, docetaxel, doxorubicin hydrochloride, liposomal hydrochloride doxorubicin chloride, eltrombopag-olamine, enzalutamide, hydrochloride epirubicin, EPOCH, eribulin mesylate, erlotinib hydrochloride, etoposide phosphate, everolimus, exemestane, FEC, filgrastim, fludarabine phosphate, fluorouracil, FU-LV, fulvestrant, gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtu zumab-ozogamicin, glucarpidase , goserelin acetate, bivalent HPV vaccine, recombinant quadrivalent HPV vaccine, hyper-CVAD, ibritumomab-tiuxetan, ibrutinib, ICE, idelalisib, ifosfamide, imatinib mesylate, imiquimod, iodo-131-tositumomab and tositumomab, ipilimumab hydrochloride irinotecan, ixabepilone , lapatinib ditosylate, lenalidomide, letrozole, leucovorin calcium, leuprolide acetate, liposomal cytarabine, lomustine, mechlorethamine hydrochloride, megestrol acetate, mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone hydrochloride, MOPP, nelarabine, nilotinib , obinutuzumab, ofatumumab, omacetaxine mepesuccinate, OEPA, OFF, OPPA, oxaliplatin, paclitaxel, albumin stabilized paclitaxel nanoparticle formulation, PAD, palifermin, palonosetron hydrochloride, pamidronate disodium, panitumumab, pazopanib hydrochloride, pegaspargase, peginterferon alfa-2b, pembrolizumab, pemetrexed disodium, pertuzumab, plerixafor, pomalidomide, ponatinib hydrochloride, pralatrexate, prednisone, procarbazine hydrochloride, radium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant bivalent HPV vaccine, recombinant quadrivalent HPV vaccine, recombinant interferon alfa-2b, regorafenib , rituximab, romidepsin, romiplostim, ruxolitinib phosphate, siltuximab, sipuleucel-T, sorafenib tosylate, STANFORD V, sunitinib malate, TAC, talc, tamoxifen citrate, temozolomide, temsirolimus, thalidomide, topotecan hydrochloride, toremifene, tositum omaba and iodine-131-tositumomab, TPF, trametinib, trastuzumab, vandetanib, VAMP, VeIP, vemurafenib, vinblastine sulfate, vincristine sulfate, liposomal vincristine sulfate, vinorelbine tartrate, vismodegib, vorinostat, XELOX, ziv-aflibercept and zoledronic acid or their salts.

In yet another aspect of the present invention, there is provided a method for preparing a compound in accordance with the first aspect of the present invention.

In preferred embodiments, a compound characterized by the formula ABZ ¹ -Thr ² -Thr ³ -Ala ⁴ -Arg ⁵ -ANB-NH ₂ ⁶ wherein ABZ is 2-aminobenzoic acid and ANB-NH ₂ is a 5-amino-amide 2-nitrobenzoic acid is prepared in accordance with a process carried out on a solid support, preferably containing an Fmoc group. Before initiating the process, the solid support is prepared, its volume is increased by repeated washing with hydrophobic solvents, preferably dimethylformamide, methylene chloride or N-methylpyrrolidone, and removal of the Fmoc protecting group, preferably by washing with a 10-30% solution of piperidine in solvents such as dimethylformamide, methylene chloride or N-methylpyrrolidone. A process is then carried out comprising the following steps:

a) Precipitate 5-amino-2-nitrobenzoic acid ANB onto the resin, pre-washing the solid support with a 3-6% solution of N -methylmorpholine (NMM) in DMF, followed by DMF. A solution of ANB in DMF is then prepared to which TBTU, DMAP and finally diisopropylethylamine (DIPEA) are added in the following excess relative to the precipitated polymer: ANB/TBTU/DMAP/DIPEA, 3:3:2:6. The resulting mixture is added to the resin and stirred until homogeneous, then the resin is filtered under reduced pressure and washed with solvents such as DMF, DCM and isopropanol. The binding of ANB to the resin is then continued using hexafluorophosphate- O- (7-azabenzotriazol-1-yl)-N ,N,N',N' -tetramethyluronium (HATU) and then hexafluorophosphate -O- (benzotriazol-1-yl) -N,N,N',N'-tetramethyluronium (HBTU) in excess, and when complete, the solid support is washed successively with DMF, DCM and isopropanol and gently dried.

b) The addition of an amino acid residue to the ANB is accomplished using a reaction with an Fmoc-Arg(Pbf)-OH amino acid derivative with at least a five-fold molar excess of the amino acid derivative relative to the resin dissolved in anhydrous pyridine and contacting the resin with the precipitated ANB. Then the entire mixture is cooled to a temperature of at least 20°C, POCl ₃ is added in a ratio of 1:1 relative to the amount of amino acid derivative used and mixed. The stirring process is carried out at room temperature and then at elevated temperature, after completion of the reaction, the resin is filtered under reduced pressure, washed with DMF and MeOH and dried carefully, the resulting intermediate is subjected to the acylation process.

c) Acylation of the resulting intermediate is performed using an amino acid derivative, preferably Fmoc-Ala-OH, then Fmoc-Thr(tBu)-OH, then Fmoc-Thr(tBu)-OH and finally Boz-Abz-OH. The acylation is performed in steps from 6 to 1 residue using excess diisopropylcarbodiimide as a coupling agent. At the end of each step, the resin is washed with DMF and (preferably) subjected to a chloranil test, which monitors the addition of the amino acid derivative.

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

e) Separation of the peptide from the resin is performed using a mixture of: TFA, phenol, water and TIPS, maintaining a ratio of 88:5:5:2 (v/v/v/v), respectively; the mixture is stirred for at least one hour, preferably three hours, and the resulting precipitate is filtered off under reduced pressure, washed with diethyl ether, and the resulting peptide is centrifuged.

f) Preparation of the final product is carried out by dissolving the peptide in water through sonication and lyophilization.

In preferred embodiments, a compound characterized by the formula ABZ ¹ -Thr ² -Thr ³ -Ala ⁴ -Arg ⁵ -pNA ⁶ where ABZ is 2-aminobenzoic acid and pNA is para-nitroaniline is prepared in accordance with the process performed by on a solid support, preferably containing an Fmoc group. Before initiating the process, the solid support is prepared, its volume is increased by repeated washing with hydrophobic solvents, preferably dimethylformamide, methylene chloride or N-methylpyrrolidone, and removal of the Fmoc protecting group, preferably by washing with a 10-30% solution of piperidine in solvents such as dimethylformamide, methylene chloride or N-methylpyrrolidone; then perform a process including the following steps:

a) The addition of a third amino acid residue (Fmoc-Ala) to the resin is accomplished using a derivative of the corresponding amino acid in a 9-fold molar excess relative to the precipitated resin, dissolved in anhydrous methylene chloride. The mixture is then stirred for at least two hours at room temperature; after completion of the reaction, the resin was filtered under reduced pressure, washed with DMF and MeOH, and then dried.

b) In subsequent steps, the addition of an amino acid residue is performed, hereinafter referred to as acylation, and the derivative Fmoc-Thr(tBu)-OH is used, and then Fmoc-Thr(tBu)-OH and Boc-ABZ-OH; Before each step, wash the DMF resin, preferably for 5 minutes; subsequent additions use a coupling agent, preferably diisopropylcarbodiimide, which is used in excess. This procedure is repeated twice and after each step the resin is washed with DMF and preferably subjected to the chloranil test, which monitors the addition of the amino acid derivative.

c) Removal of the Fmoc protecting group is carried out by washing with a 10-30% solution of piperidine in DMF and subsequent washing with each of the solvents: DMF, isopropanol and methylene chloride.

d) Repeat steps b) to c) to obtain the compound (ABZ ¹ -Thr(tBu) ² -Thr (tBu) ³ -Ala ⁴ -OH); the synthesis is carried out from residue 6 to 3, and then the resulting compound is detached from the solid support using a mixture of: TFA: phenol: water: TIPS in the proportions 88: 5: 5: 2 (v/v/v/v), respectively, with stirring . After at least two hours, the contents of the flask are filtered under reduced pressure and the precipitate is washed with diethyl ether. The precipitate is then centrifuged for preferably 20 minutes, dissolved in water by sonication and then lyophilized.

e) The synthesis of Fmoc-Arg (Pbf)-pNA is carried out in stages; In the first step, 2 mmol Fmoc-Arg (Pbf) is dissolved in anhydrous tetrahydrofuran (THF) in the presence of 2 mmol N-methylmorpholine (NMM), and the carboxyl group of the amino acid derivative is activated with 2 mmol isobutyl chloride. After 10 minutes of activation, 3 mmol of p-nitroaniline is added and the reaction is carried out for (preferably) 2 hours at (preferably) -15°C and then for a day at room temperature. After completion of the reaction, the solvent is evaporated and the dry residue is dissolved in ethyl acetate, then the resulting solution is washed successively with an aqueous solution of NaCl, 10% citric acid, 5% sodium bicarbonate and dried over anhydrous sodium sulfate; ethyl acetate is distilled under reduced pressure, and the dry residue is dried.

f) The combination of protected peptide ABZ ¹ -Thr(tBu) ² -Thr(tBu) ³ -Ala ⁴ -OH with para-nitroanilide Arg (Pbf) is based on the following process: protected peptide ABZ ¹ -Thr(tBu) ² -Thr( tBu) ³ -Ala ⁴ -OH is dissolved in a small amount of DCM and then activated with TFFH (tetramethylfluoroformamide) preferably for 30 minutes and preferably at 0°C, after which a catalytic amount of DMAP and Fmoc-Arg(Pbf) ⁵ -pNA is added ⁶ . The reaction is preferably carried out for 24 hours at room temperature, after which the solvent is evaporated and the resulting solution is poured into a mixture to deprotect the side group: TFA: phenol: water: TIPS (88:5:5:2, (v/v/v /vol)), and stir preferably for 3 hours.

g) The finished product is prepared by dissolving the peptide in water by sonication and then lyophilizing.

Brief description of drawings

In fig. Figure 1 shows the rate of hydrolysis of substrate (1) ABZ-Thr-Thr-Ala-Arg-ANB-NH ₂ in urine samples from patients diagnosed with cancer (samples 1-10) and in the urine of healthy individuals (11-25). The number of the selected urine sample is indicated in Arabic numerals. Figure 1 shows that all samples 1-10 degraded, however, in samples 3 and 10, the destruction of the ABZ-Thr-Thr-Ala-Arg-ANB-NH ₂ substrate occurred with greater efficiency than for materials 2 or 9. This result may be due to differences in the activity and quantity of enzymes responsible for proteolysis. In addition, Figure 1 shows that incubating a solution of compound of formula 2 with urine samples from healthy individuals (without diagnosed cancer) does not lead to an increase in absorption and, therefore, hydrolysis of the test compound does not occur. This result indicates the absence of proteolytic enzymes characteristic of pancreatic cancer.

In fig. Figure 2 shows the rate of hydrolysis of substrate (2) ABZ-Thr-Thr-Ala-Arg-pNA in urine samples from patients diagnosed with cancer (samples 1-10) and in the urine of healthy individuals (11-25). The number of the selected urine sample is indicated in Arabic numerals. A result similar to that described above for FIG. 1.

In fig. Figure 3 shows the relationship between the degree of hydrolysis of the substrate (1) ABZ-Thr-Thr-Ala-Arg-ANB NH ₂ and the pH of the medium. Tested pH values are indicated on the x-axis. The relationship between proteolytic activity and pH of the reaction medium was assessed. As a result of this experiment, it was found that the material tested contained at least one enzyme that exhibited maximum activity at alkaline pH values.

In fig. Figure 4 shows RP-HPLC analysis of a randomly selected system containing urine from a person diagnosed with pancreatic cancer. Both compounds (1) (ABZ-Thr-Thr-Ala-Arg-ANB-NH ₂ , shown) and (2) (ABZ-Thr-Thr-Ala-Arg-pNA, not shown) were degraded into the ABZ-Thr peptide fragment -Thr-Ala-Arg and chromophore (ANB-NH ₂ or pNA, respectively). A: substrate ABZ-Thr-Thr-Ala-Arg-ANB NH2 in 200 mM Tris-HCl buffer, pH 8.0. B: Hydrolysis of ABZ-Thr-Thr-Ala-Arg-ANB NH2 substrate in the urine of patients diagnosed with pancreatic cancer.

Examples

The present invention is illustrated by the following non-limiting examples of implementation.

Example 1: Synthesis of the compound ABZ ¹ -Thr ² -Thr ³ -Ala ⁴ -Arg ⁵ -ANB-NH ₂ ⁶

1. Preparation of chromogenic peptide

a) The first stage of the synthesis was the production of a chromogenic peptide through solid-phase synthesis - on a solid support, using Fmoc/tBu chemistry, i.e. using protection.

A compound with the sequence ABZ ¹ -Thr ² -Thr ³ -Ala ⁴ -Arg ⁵ -ANB-NH ₂ ⁶ , where ABZ is 2-aminobenzoic acid and ANB-NH ₂ is 5-amino-2 benzoic acid amide, was prepared according to a solid phase chemical synthesis process using amino acid derivatives.

Boc-ABZ, Fmoc-Thr(tBu), Fmoc-Ala, Fmoc-Arg(Pbf), ANB

Synthesis of the compound, i.e. diagnostic marker for the detection of pancreatic cancer, the diagnosis of which is associated with the hydrolysis of this compound under the action of proteolytic enzymes, was performed on a solid support that allowed the conversion of 5-amino-2-benzoic acid into the amide ANB-NH ₂ :

- for example, amide resin, for example TentaGel S RAM manufactured by RAPP Polymere (Germany), for example using a precipitate of 0.23 mmol/g.

Other commercially available amide resins can be used, including Rink Amide (Germany).

The compound was synthesized manually using a laboratory shaker. For most steps, a 25 mL ceramic solid-phase syringe was used as the reactor.

All resulting compounds contained ABZ 2-aminobenzoic acid at position 1 of their sequence (ie at the N-terminus), and an ANB 5-amino-2-nitrobenzoic acid molecule at position 6 (at the C-terminus). ABZ acted as a fluorescence donor, while ANB (5-amino-2-benzoic acid) acted as a fluorescence quencher and chromophore. The peptide sequence contained at least (and preferably) one reactive site located between the amino acid residues Arg-ANB-NH ₂ : at position 5 of the compound. Synthesis, including addition of amino acid derivatives, was performed from residue 6 to residue 1, i.e. from C-terminus to N-terminus.

b) Deposition of ANB on TentaGel S RAM resin:

Peptide synthesis was performed on TentaGel S RAM resin (Rapp Polymere) using a 0.23 mmol/g precipitate. The first stage was the preparation of the resin, including its loosening through a washing cycle. Subsequently, the Fmoc-protection of the amino group from the solid support was removed with a 20% solution of piperidine in NMP, and a solvent washing cycle was performed. The chloranil test was performed to confirm the presence of free amino groups.

Solvent Wash Cycle:

DMF 1 x 10 minutes

IsOH 1 x 10 minutes

DXM 1 x 10 minutes

Removing Fmoc protection:

DMF 1 x 5 minutes

20% piperidine in NMP 1 x 3 minutes

20% piperidine in NMP 1 x 8 minutes

Solvent Wash Cycle:

DMF 3 x 2 minutes

IsOH 3 x 2 minutes

DXM 3 x 2 minutes

c) Chloranyl test:

The chloranil test consisted of transferring (using a spatula) several resin beads from a syringe reactor into a glass ampoule into which 100 μl of a saturated solution of p-chloranil in toluene and 50 μl of fresh acetaldehyde were added. After 10 minutes, the color of the granules was checked.

At this stage, after performing the test, a green color of the granules was obtained, indicating the presence of free amino groups. Once the removal of the 9-fluorenylmethoxycarbonyl protection from the resin was confirmed, it was possible to proceed to the next step, the addition of the ANB (5-amino-2-nitrobenzoic acid) derivative.

d) Precipitation of 5-amino-2-nitrobenzoic acid on a solid support

The first step in the synthesis of a peptide library - a mixture of peptides - was the deposition of ANB onto 1 g of resin. Before chromophore attachment, the resin used for the reaction was washed with the following solvents: DMF, DCM and DMF again, after which the Fmoc-protection was removed from the functional group of the solid support. One Fmoc protection removal cycle included the following steps:

Removing Fmoc protection:

20% piperidine in NMP 1 x 3 minutes

20% piperidine in NMP 1 x 8 minutes

e) Flushing

DMF 3 x 2 minutes

IsOH 3 x 2 minutes

DXM 3 x 2 minutes

f) Chloranyl test for the presence of free amino groups.

The resin with free amino groups was washed with a 5% solution of N -methylmorpholine (NMM) in DMF, and then with DMF. The Fmoc deprotection procedure and wash cycle were performed in a Merrifield vessel. ANB was dissolved in DMF in a separate flask and TBTU, DMAP and finally diisopropylethylamine (DIPEA) were added sequentially in the following excess relative to the precipitated polymer: ANB/TBTU/DMA/DIPEA, 3:3:2:6. The mixture thus obtained was added to the resin and stirred for 3 hours. The resin was filtered under reduced pressure, washed with DMF, DCM and isopropanol, and the entire acylation procedure was repeated twice. Hexafluorophosphate- O- (7-azabenzotriazol-1-yl)-N ,N,N ',N' -tetramethyluronium (HATU) and then hexafluorophosphate- O- (benzotriazol-1-yl) were used to perform sequential addition reactions of ANB to the resin )- N,N,N ' N '-tetramethyluronium (HBTU). At the last stage, the resin was washed successively with DMF, DCM and isopropanol and dried in air.

g) Attachment of C-terminal amino acid residue to ANB

The corresponding amino acid derivative (in a 9-fold molar excess relative to the precipitated resin) was dissolved in pyridine and transferred to a flask containing the ANB-precipitated resin. The mixture was cooled to -15°C (ice bath: 1 part by weight NH ₄ Cl, 1 part by weight NaNO ₃ , 1 part by weight ice). After reaching room temperature, POCl ₃ was added (in a 1:1 ratio to the amount of amino acid derivative used), and the mixture was stirred on a magnetic stirrer: 20 minutes at -15°C, 30 minutes at room temperature, and 6 hours at 40°C (oil bath). After completion of the reaction, the resin was filtered under reduced pressure, washed with DMF and MeOH, and left to dry.

In the next step, the residue (alanine) was added at position P2.

Before each addition of amino acid residues, the resin was washed with DMF for 5 minutes. In subsequent addition reactions, diisopropylcarbodiimide was used as a coupling agent. This procedure was repeated twice.

After each acylation, a resin wash cycle was initiated and then a chloranil test was performed to monitor the addition of the amino acid derivative to the free amino groups of the resin.

Solvent Wash Cycle:

DMF 3 x 2 minutes

IsOH 3 x 2 minutes

DXM 3 x 2 minutes

Chloranyl test:

Tests after the first two attachments resulted in the beads being initially green and then grey, so it was necessary to perform another acylation, which resulted in the resin beads tested using the chloranil test being colorless. This indicated the attachment of ANB to the TentaGel S RAM resin, allowing the next step of peptide synthesis to proceed.

h) Addition of further protected amino acid residues:

The resin, along with the attached ANB residue, was washed with DMF in the reactor, and then Fmoc deprotection of the amino groups was performed to attach the protected alanine amino acid derivative.

Removing Fmoc protection:

DMF 1 x 5 minutes

20% piperidine in NMP 1 x 3 minutes

20% piperidine in NMP 1 x 8 minutes

Solvent Wash Cycle:

DMF 3 x 2 minutes

IsOH 3 x 2 minutes

DXM 3 x 2 minutes

Chloranyl test:

The chloranil test result was positive, as indicated by the green coloration of the resin granules. This made it possible to proceed to the next stage - the addition of the amino acid residue Fmoc-Thr (tBu)-OH.

Addition of an amino acid derivative:

Before the joining process, the resin was washed with DMF. The composition of the addition mixture remained unchanged upon addition of the protected glutamic acid residue.

At the end of each acylation, a solvent wash cycle was performed according to a specified procedure, followed by a chloranil test to determine the presence of free amino groups in solution.

Solvent Wash Cycle:

DMF 3 x 2 minutes

IsOH 3 x 2 minutes

DXM 3 x 2 minutes

Chloranyl test:

The resin granules were colorless during the test performed after the second acylation, which allowed us to proceed to the next stage of the synthesis, i.e. the introduction of another protected amino acid derivative - threonine and a 2-aminobenzoic acid molecule. The joining process was performed according to the previously discussed procedure.

Tests performed after the attachment of the above-mentioned residues gave positive results: the resin granules were colorless.

2. Release of peptide from solid support

After synthesis, the peptide amide ABZ-Thr-Thr-Ala-Arg-ANB-NH ₂ was removed from the solid support while simultaneously deprotecting the side chain with a mixture of: TFA : phenol : water : TIPS (88:5:5:2, (v/v /v/v)) in a round-bottomed flask on a magnetic stirrer.

After 3 hours, the contents of the flask were filtered under reduced pressure using a ceramic funnel (Schott) and washed with diethyl ether. The resulting sediment was centrifuged in a SIGMA 2K30 centrifuge (Laboratory Centrifuges) for 20 minutes. The precipitate obtained after centrifugation was dissolved in water by sonication and lyophilized.

Identity/characteristics of the new compound - HPLC analysis, MS

HPLC conditions: Column RP Bio Wide Pore Supelco C8 250 mm 4 mm, phase system A 0.1% TFA in water, B: 80% acetonitrile in phase A), flow rate 1 ml/min, UV detection at wavelength 226 nm.

The connection was confirmed.

Example 2: Preparation of a compound of the formula: ABZ1-Thr2-Thr3-Ala4-Arg5-pNA6

The process was carried out similarly to that described in Example 1, except that derivatives of the corresponding amino acids and additional substituents were used, and the process was carried out partly in solution and partly on a solid support.

Preparation of p-nitroanilide Ala

a) The first stage of synthesis was the protected peptide through solid phase synthesis using Fmoc/tBu chemistry.

The compound ABZ ¹ -Thr(tBu) ² -Thr(tBu) ³ -Ala ⁴ -OH, where ABZ was 2-aminobenzoic acid, was prepared by solid phase chemical synthesis using the following amino acid derivatives:

Boc-ABZ, Fmoc-Thr(tBu), Fmoc-Ala.

The compound was synthesized on a solid support:

- 2-chloro-chlorotrityl resin, for example, produced by Iris BIOTECH GMBH (Germany), with precipitation of 1.6 mmol Cl/g groups.

The compound was synthesized manually using a laboratory shaker. In all stages, a 25-mL ceramic syringe for solid-phase synthesis was used as a reactor.

Peptide synthesis was performed on a solid support: 2-chloro-chlorotrityl resin, for example, produced by Iris BIOTECH GMBH (Germany), with the precipitation of 1.6 mmol Cl/g groups. In the first stage, the resin was loosened by a washing cycle. The Fmoc-protection of the amino group was then removed from the solid support with a 20% solution of piperidine in NMP. A solvent wash cycle was then performed. The chloranil test was performed to confirm the presence of free amino groups.

Solvent Wash Cycle:

DMF 1 x 10 minutes

IsOH 1 x 10 minutes

DXM 1 x 10 minutes

Removing Fmoc protection:

DMF 1 x 5 minutes

20% piperidine in NMP 1 x 3 minutes

20% piperidine in NMP 1 x 8 minutes

Solvent Wash Cycle:

DMF 3 x 2 minutes

IsOH 3 x 2 minutes

DXM 3 x 2 minutes

b) Chloranyl test:

The chloranil test consisted of transferring (using a spatula) a few resin beads from a syringe reactor into a glass ampoule and adding 100 μl of a saturated solution of p-chloranil in toluene and 50 μl of fresh acetaldehyde. After 10 minutes, the color of the granules was checked.

At this stage, after the test, a green color of the granules was obtained, indicating the presence of free amino groups. Once the removal of the 9-fluorenylmethoxycarbonyl protection from the resin was confirmed, the addition of the Fmoc-Ala derivative was initiated.

c) Embedding Fmoc-Ala on a solid support

The first step in the synthesis of the peptide library was the precipitation of Fmoc-Ala onto 1 g of resin. Before attaching the amino acid derivative, the resin used for the reaction was washed with the following solvents: DMF (dimethylformamide), DCM (methylene chloride) and DMF again, after which the Fmoc-protection was removed from the functional group of the solid support. One Fmoc protection removal cycle included the following steps:

Removing Fmoc protection:

20% piperidine in NMP 1 x 3 minutes

20% piperidine in NMP 1 x 8 minutes

Flushing

DMF 3 x 2 minutes

IsOH 3 x 2 minutes

DXM 3 x 2 minutes

Chloranyl test for the presence of free amino groups.

The resin with free amino groups was washed with DMF. In a separate flask, Fmoc-Ala was dissolved in DMF. TBTU, DMAP and finally diisopropylethylamine (DIPEA) were then added in excess to the precipitated polymer: Fmoc-Pro/TBTU/DMAP/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, washed with DMF, DCM and isopropanol, and the entire acylation procedure was repeated twice. Hexafluorophosphate- O- (7-azabenzotriazol-1-yl) -N,N,N',N' -tetramethyluronium (HATU) was used to perform sequential addition reactions of Fmoc-Pro to the resin, followed by hexafluorophosphate- O- (benzotriazol-1 -yl) -N,N,N'N' -tetramethyluronium (HBTU). At the last stage, the resin was washed successively with DMF, DCM and isopropanol and dried in air.

c. Addition of further protected amino acid residues:

The resin along with the attached Fmoc-Ala residue in the reactor was washed with DMF, and then the Fmoc-amino group was deprotected to attach the protected threonine derivative.

Removing Fmoc protection:

DMF 1 x 5 minutes

20% piperidine in NMP 1 x 3 minutes

20% piperidine in NMP 1 x 8 minutes

Solvent Wash Cycle:

DMF 3 x 2 minutes

IsOH 3 x 2 minutes

DXM 3 x 2 minutes

Chloranyl test:

The chloranil test result was positive, as indicated by the green coloration of the resin granules. This made it possible to proceed to the next stage - the addition of the amino acid residue Fmoc-Thr (tBu)-OH.

Addition of an amino acid derivative:

Before the joining process, the resin was washed with DMF. The composition of the addition mixture remained unchanged upon addition of the protected glutamic acid residue.

At the end of each acylation, a solvent wash cycle was performed according to a specified procedure, followed by a chloranil test to determine the presence of free amino groups in solution.

Solvent Wash Cycle:

DMF 3 x 2 minutes

IsOH 3 x 2 minutes

DXM 3 x 2 minutes

Chloranyl test:

The resin granules were colorless during the test performed after the second acylation, which allowed us to proceed to the next stage of the synthesis, i.e. the introduction of another protected amino acid derivative - threonine and a 2-aminobenzoic acid molecule. The joining process was performed according to the previously discussed procedure.

Tests performed after the attachment of the above-mentioned residues gave positive results: the resin granules were colorless.

a) Removal of peptide from solid support while maintaining side group protection

After completion of the synthesis, the protected peptide ABZ-Thr(tBu)-Th(tBu)-Ala-OH was removed from the solid support while maintaining the side group protection using the following mixture: acetic acid: TFE (trifluoroethanol): DCM (2:2:6 ( v/v/v)) in a round-bottomed flask on a magnetic stirrer.

After 2 hours, the contents of the flask were filtered under reduced pressure on a ceramic funnel (Schott) and washed with a viscous mixture. The solution was washed with hexane (1:10 (v/v)), evaporated under reduced pressure and then lyophilized.

b) Chemical synthesis of para-nitroanilide and Arg derivatives

The mixed anhydride method was used to synthesize Fmoc-Arg(Pbf)-pNA. In the first step, 2 mmol Fmoc-Arg (Pbf) was dissolved in anhydrous tetrahydrofuran (THF) in the presence of 2 mmol N-methylmorpholine (NMM). The carboxyl group of the amino acid derivative was activated by 2 mmol of isobutyl chloride. After 10 minutes of activation, 3 mmol of p-nitroaniline was added. Reactions were performed for 2 hours at -15°C and then for 24 hours at room temperature. After completion of the reaction, the solvent was evaporated, and the dry residue was dissolved in ethyl acetate. The resulting solution was washed successively with saturated aqueous NaCl, 10% citric acid and 5% sodium bicarbonate. The resulting solution was dried over anhydrous sodium sulfate, ethyl acetate was distilled under reduced pressure, and the dry residue was dried in a vacuum desiccator over P ₂ O ₅ and KOH.

c) Attachment of protected peptide to Arg para-nitroanilide (Pbf)

The protected peptide ABZ ¹ -Thr(tBu) ² -Thr (tBu) ³ -Ala ⁴ -OH was dissolved in a small amount of DCM and subsequently activated with TFFH (tetramethylfluoroformamide) for 30 minutes at 0°C. A catalytic amount of DMAP and Arg(Pbf)-pNA were then added. The reaction was carried out for 24 hours at room temperature, after which the solvent was evaporated. The resulting solution was poured into the mixture, deprotecting the side group: TFA : phenol : water : TIPS (88:5:5:2, (v/v/v/v) and stirred in a round-bottom flask on a magnetic stirrer for 3 hours.

After this, cold diethyl ether was added to the flask, and the resulting precipitate was centrifuged in a high-speed centrifuge at 5000 rpm for 20 minutes. The precipitate obtained after centrifugation was dissolved in water by sonication and then lyophilized.

Identity/characteristics of the new compound - HPLC analysis, MS

HPLC conditions: Column RP Bio Wide Pore Supelco C8 250 mm 4 mm, phase system A 0.1% TFA in water, B: 80% acetonitrile in phase A), flow rate 1 ml/min, UV detection at wavelength 226 nm. The connection was confirmed.

Example 3

A study of the new compound was carried out in a group of 10 patients diagnosed with pancreatic cancer. For this purpose, the compound of formula 2? ABZ-Thr-Thr-Ala-Arg-ANB-NH ₂ or formula 3: ABZ-Thr-Thr-Ala-Arg-pNA was dissolved in dimethyl sulfoxide (at a concentration of 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 a person with pancreatic cancer. The measurement was performed in a 96-well absorbance plate and each sample was analyzed in triplicate at 37°C. The measurement duration was 60 minutes. During the measurement, the spectral characteristics of the released chromophore (ANB-NH ₂ or pNA) were monitored at 405 nm (range 380-430 nm).

As a result of the measurement, the color of the solution increased over time in all urine samples from patients diagnosed with pancreatic cancer. The observed increase in absorption over time differed for each sample tested. A different effect was obtained for 15 samples from healthy individuals, as none of the 15 urine samples tested showed an increase in uptake within the diagnostic range.

The analysis confirmed the use of the compounds according to the examples in the diagnosis of pancreatic cancer. The mechanism of action of the new compounds is based on their enzymatic hydrolysis under such conditions, which leads to the release of free chromophore molecules, ANB-NH ₂ - 5-amino-2-nitrobenzoic acid amide or pNA - para-nitroaniline, respectively, which are characterized by absorption at 320- 480 nM, especially at 380-430 nm.

Table 1 - values shown in Fig. 1 1 0.128 0.152 0.2109 2 0.00532 0.00469 0.04912 3 0.07457 0.07826 0.07178 4 0.03727 0.03636 0.03044 5 0.02734 0.01944 0.02312 6 0.04416 0.04476 0.04487 7 0.02666 0.02332 0.025 8 0.02045 0.02486 0.02345 9 0.00647 0.00797 0.00596 10 0.08668 0.11456 0.08522 All other values 11-25 were equal to 0.

Table 2 - values shown in Fig. 2 1 0.22 0.189 0.2109 2 0.07457 0.07826 0.07178 3 0.01632 0.01669 0.01723 4 0.01727 0.01636 0.01044 5 0.05734 0.05944 0.06152 6 0.05416 0.05476 0.05487 7 0.02666 0.02332 0.025 8 0.02045 0.02486 0.02345 9 0.01947 0.01797 0.01896 10 0.11668 0.11456 0.10522 All other values 11-25 were equal to 0.

Table 3 - values shown in Fig. 3 3 0 0 0 4 0 0 0 5 0 0 0 6 0 0 0 7 0 0 0 8 0.07661 0.07534 0.09735 9 0 0 0

The present invention further relates to

1. Chemical compound - diagnostic marker of the general formula:

ABZ ¹ -Thr ² -Thr ³ -Ala ⁴ -Arg ⁵ -X ⁶ (formula 1)

Where:

ABZ - 2-aminobenzoic acid,

X - ANB-NH ₂ or pNA,

where ANB-NH ₂ represents 5-amino-2-benzoic acid amide,

ANB - 5-amino-2-nitrobenzoic acid,

and pNA is para-nitroaniline.

2. Method for obtaining a chemical compound - a diagnostic marker

ABZ ¹ -Thr ² -Thr ³ -Ala ⁴ -Arg ⁵ -ANB-NH ₂ ⁶ , (formula 2)

Where:

ABZ - 2-aminobenzoic acid,

where ANB-NH ₂ represents 5-amino-2-nitrobenzoic acid amide,

based on the fact that said process is performed on a solid support, preferably containing an Fmoc group. Before initiating the process, the solid support is prepared, its volume is increased by repeated washing with hydrophobic solvents, preferably dimethylformamide, methylene chloride or N-methylpyrrolidone, and removal of the Fmoc protecting group, preferably by washing with a 10-30% solution of piperidine in solvents such as dimethylformamide, methylene chloride or N-methylpyrrolidone. A process is then carried out comprising the following steps:

a) Precipitate 5-amino-2-nitrobenzoic acid ANB onto the resin, pre-washing the solid support with a 3-6% solution of N -methylmorpholine (NMM) in DMF, followed by DMF. A solution of ANB in DMF is then prepared to which TBTU, DMAP and finally diisopropylethylamine (DIPEA) are added in the following excess relative to the precipitated polymer: ANB/TBTU/DMAP/DIPEA, 3:3:2:6. The resulting mixture is added to the resin and stirred until homogeneous, then the resin is filtered under reduced pressure and washed with solvents such as DMF, DCM and isopropanol. The binding of ANB to the resin is then continued using hexafluorophosphate- O- (7-azabenzotriazol-1-yl)-N ,N,N ',N' -tetramethyluronium (HATU) and then hexafluorophosphate -O- (benzotriazol-1-yl) -N,N,N',N'-tetramethyluronium (HBTU) in excess, and when complete, the solid support is washed successively with DMF, DCM and isopropanol and gently dried.

b) The addition of an amino acid residue to the ANB is accomplished using a reaction with an Fmoc-Arg(Pbf)-OH amino acid derivative with at least a five-fold molar excess of the amino acid derivative relative to the resin dissolved in anhydrous pyridine and contacting the resin with the precipitated ANB. Then the entire mixture is cooled to a temperature of at least 20°C, POCl ₃ is added in a ratio of 1:1 relative to the amount of amino acid derivative used and mixed. The stirring process is carried out at room temperature and then at elevated temperature, after completion of the reaction, the resin is filtered under reduced pressure, washed with DMF and MeOH and dried carefully, the resulting intermediate is subjected to the acylation process.

c) Acylation of the resulting intermediate is performed using an amino acid derivative, preferably Fmoc-Ala-OH, then Fmoc-Thr(tBu)-OH, then Fmoc-Thr(tBu)-OH and finally Boz-Abz-OH. The acylation is performed in steps from 6 to 1 residue using excess diisopropylcarbodiimide as a coupling agent. At the end of each step, the resin is washed with DMF and (preferably) subjected to a chloranil test, which monitors the addition of the amino acid derivative.

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

e) Separation of the peptide from the resin is performed using a mixture of: TFA, phenol, water and TIPS, maintaining a ratio of 88:5:5:2 (v/v/v/v), respectively; the mixture is stirred for at least one hour, preferably three hours, and the resulting precipitate is filtered off under reduced pressure, washed with diethyl ether, and the resulting peptide is centrifuged.

f) Preparation of the final product is carried out by dissolving the peptide in water through sonication and lyophilization.

3. Method for obtaining a chemical compound - a diagnostic marker:

ABZ ¹ -Thr ² -Thr ³ -Ala ⁴ -Arg ⁵ -pNA ⁶ , (formula 3)

Where:

ABZ - 2-aminobenzoic acid,

pNA - para-nitroanilide,

based on the fact that said process is performed on a solid support, preferably containing an Fmoc group. Before initiating the process, the solid support is prepared, its volume is increased by repeated washing with hydrophobic solvents, preferably dimethylformamide, methylene chloride or N-methylpyrrolidone, and removal of the Fmoc protecting group, preferably by washing with a 10-30% solution of piperidine in solvents such as dimethylformamide, methylene chloride or N-methylpyrrolidone; then perform a process including the following steps:

a) The addition of a third amino acid residue (Fmoc-Ala) to the resin is accomplished using a derivative of the corresponding amino acid in a 9-fold molar excess relative to the precipitated resin, dissolved in anhydrous methylene chloride. The mixture is then stirred for at least two hours at room temperature; after completion of the reaction, the resin is filtered under reduced pressure, washed with DMF and MeOH, and then dried.

b) In subsequent steps, the addition of an amino acid residue is performed, hereinafter referred to as acylation, and the derivative Fmoc-Thr(tBu)-OH is used, and then Fmoc-Thr(tBu)-OH and Boc-ABZ-OH; Before each step, wash the DMF resin, preferably for 5 minutes; subsequent additions use a coupling agent, preferably diisopropylcarbodiimide, which is used in excess. This procedure is repeated twice and after each step the resin is washed with DMF and preferably subjected to the chloranil test, which monitors the addition of the amino acid derivative.

c) Removal of the Fmoc protecting group is carried out by washing with a 10-30% solution of piperidine in DMF and subsequent washing with each of the solvents: DMF, isopropanol and methylene chloride.

d) Repeat steps b) to c) to obtain the compound (ABZ ¹ -Thr(tBu) ² -Thr (tBu) ³ -Ala ⁴ -OH); the synthesis is carried out from residue 6 to 3, and then the resulting compound is detached from the solid support using a mixture of: TFA: phenol: water: TIPS in the proportions 88: 5: 5: 2 (v/v/v/v), respectively, with stirring . After at least two hours, the contents of the flask are filtered under reduced pressure and the precipitate is washed with diethyl ether. The precipitate is then centrifuged for preferably 20 minutes, dissolved in water by sonication and then lyophilized.

e) The synthesis of Fmoc-Arg (Pbf)-pNA is carried out in stages; In the first step, 2 mmol Fmoc-Arg (Pbf) is dissolved in anhydrous tetrahydrofuran (THF) in the presence of 2 mmol N-methylmorpholine (NMM), and the carboxyl group of the amino acid derivative is activated with 2 mmol isobutyl chloride. After 10 minutes of activation, 3 mmol of p-nitroaniline is added and the reaction is carried out for (preferably) 2 hours at (preferably) -15°C and then for a day at room temperature. After completion of the reaction, the solvent is evaporated and the dry residue is dissolved in ethyl acetate, then the resulting solution is washed successively with an aqueous solution of NaCl, 10% citric acid, 5% sodium bicarbonate and dried over anhydrous sodium sulfate; ethyl acetate is distilled under reduced pressure, and the dry residue is dried.

f) The combination of protected peptide ABZ ¹ -Thr(tBu) ² -Thr(tBu) ³ -Ala ⁴ -OH with para-nitroanilide Arg (Pbf) is based on the following process: protected peptide ABZ ¹ -Thr(tBu) ² -Thr( tBu) ³ -Ala ⁴ -OH is dissolved in a small amount of DCM and then activated with TFFH (tetramethylfluoroformamide) preferably for 30 minutes and preferably at 0°C, after which a catalytic amount of DMAP and Fmoc-Arg(Pbf) ⁵ -pNA is added ⁶ . The reaction is preferably carried out for 24 hours at room temperature, after which the solvent is evaporated and the resulting solution is poured into a mixture to deprotect the side group: TFA: phenol: water: TIPS (88:5:5:2, (v/v/v /vol)), and stir preferably for 3 hours.

g) The finished product is prepared by dissolving the peptide in water by sonication and then lyophilizing.

4. The method for diagnosing pancreatic cancer is based on the following process: a chemical compound of general formula 1 in the concentration range of 0.1-10 mg/ml (preferably 1 mg/ml) is incubated in a measurement buffer at neutral or alkaline pH values, preferably physiological, with a slight amount of human urine in a ratio range of 1:2 to 1:10 (preferably 1:5 urine sample to measurement buffer), and measure the absorption intensity in the range of 300-500 nm (preferably 380-430 nm) for 40-60 minutes at a temperature in the range of 25-40°C (preferably 36-38°C).

In addition, the present invention relates, inter alia, to the following elements:

1. A compound characterized by formula 1:

X1-Thr-Thr-Ala-Arg-X2 (formula 1),

wherein cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 leads to the formation of a detectable signal.

2. A compound according to claim 1, characterized in that the sequence Thr-Thr-Ala-Arg is accessible to a hydrolytic enzyme, in particular a hydrolytic enzyme that cleaves said compound into X1-Thr-Thr-Ala-Arg-OH (fragment 1) and NH ₂ -X2 (fragment 2).

3. The connection according to claim 1 or 2, characterized in that X1 contains or consists of a component C1, and X2 contains or consists of a component C2, and a detectable signal occurs when C1 and C2 are spatially separated.

4. Connection according to any one of paragraphs. 1-3, characterized in that one of C1 and C2, in particular C2, is a chromophore characterized by absorption maximum 1 (AM1) at wavelength 1, and the compound is characterized by absorption maximum 2 (AM2) at wavelength 2, different from wavelength 1.

5. Connection according to any one of paragraphs. 1-4, characterized in that C1 and C2 are a pair of a fluorescence donor and a fluorescence acceptor.

6. Connection according to any one of paragraphs. 1-5, characterized in that the pair of C1 and C2 is selected from the group consisting of 2-aminobenzoic acid (ABZ)/pNA, ABZ/ANB-NH ₂ , ABZ/DNP, ABZ/EDDNP, EDANS/DABCYL, TAM/ DANSYL, ABZ/Tyr(3-NO ₂ ), in particular, the pair of C1 and C2 is selected from ABZ/pNA and ABZ/ANB-NH ₂ .

7. A method for detecting protease activity in a biological fluid of a subject in vitro , comprising bringing the biological fluid into contact with a compound according to claims. 1-6 and signal detection, wherein said biological fluid may contain a hydrolytic enzyme, in particular a protease, derived from pancreatic cancer cells.

8. The method of claim 7 for detecting the presence or absence of pancreatic cancer in a subject in vitro , wherein the presence of protease activity in the biological fluid indicates the presence of pancreatic cancer, and the absence of protease activity in the biological fluid indicates the absence of pancreatic cancer.

9. In vitro method according to claim 7 or 8 for diagnosing pancreatic cancer.

10. In vitro method according to any one of paragraphs. 7-9, characterized in that the biological fluid is urine.

11. Method according to any one of paragraphs. 7-10, characterized in that the compound is presented in a concentration of 0.1-10 mg/ml, in particular 0.25-7.5 mg/ml, more specifically 0.5-5 mg/ml, more specifically 0 .75-2 mg/ml, even more specifically approximately 1 mg/ml in a measurement buffer with a neutral or alkaline pH, preferably physiological pH, and a sample of biological fluid is added to the compound in a ratio of 1:2 to 1:10, in in particular, from 1:3 to 1:8, more specifically, from 1:4 to 1:6, even more specifically, approximately 1:5.

12. Method according to any one of paragraphs. 7-11, characterized in that signal detection involves measuring absorption or fluorescence, in particular measuring absorption intensity at 300-500 nm, more specifically 380-430 nm, preferably for 40-60 min at 25-40°C, in particular at 36-38°C.

13. A kit containing a compound according to any one of paragraphs. 1-6 and buffer for measurements.

14. Use of the compound according to any one of paragraphs. 1-6, method according to any one of paragraphs. 7-12 or the kit of claim 13 for detecting pancreatic cancer or for monitoring a subject suspected of having pancreatic cancer, at increased risk of developing pancreatic cancer, or having previously suffered from pancreatic cancer.

15. Connection according to any one of paragraphs. 1-6 for use in a method of treating pancreatic cancer, said method comprising the steps of:

a. performing the method according to any one of paragraphs. 7-12, and

b. treating pancreatic cancer in a subject in which protease activity, in particular increased protease activity, is detected in step a.

--->

LIST OF SEQUENCES

<110>URTESTE Sp. z o.o.

<120> New marker for diagnosing pancreatic cancer

<130> 1091-2 PCT

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 4

<212> PROTEIN

<213> Homo sapiens

<400> 1

Thr Thr Ala Arg

1

<---

Claims (37)

1. A compound for diagnosing pancreatic cancer, characterized by formula 1: X1-Thr-Thr-Ala-Arg-X2 (formula 1), wherein the cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 leads to the formation of a detectable signal, wherein X1 contains or consists of a C1 component and X2 contains or consists of a C2 component, and the detectable signal arises from the spatial separation of C1 and C2 by proteolytic cleavage of the compound, and wherein C1 and C2 are a pair of a fluorescence donor and a fluorescence acceptor. 2. The compound according to claim 1, characterized in that the sequence Thr-Thr-Ala-Arg is accessible to a proteolytic enzyme that cleaves said compound into X1-Thr-Thr-Ala-Arg-OH (fragment 1) and NH ₂ -X2 ( fragment 2). 3. Connection according to any one of paragraphs. 1 or 2, characterized in that one of C1 and C2, in particular C2, is a chromophore characterized by absorption maximum 1 (AM1) at wavelength 1, and the compound is characterized by absorption maximum 2 (AM2) at wavelength 2, different from wavelength 1. 4. Connection according to any one of paragraphs. 1-3, characterized in that the pair of C1 and C2 is selected from the group consisting of 2-aminobenzoic acid (ABZ)/pNA, ABZ/ANB-NH ₂ , ABZ/DNP, ABZ/EDDNP, EDANS/DABCYL, TAM/DANSYL, ABZ/Tyr(3- NO ₂ ), in particular the pair of C1 and C2 is selected from ABZ/pNA and ABZ/ANB-NH ₂ . 5. A method of detecting protease activity in a biological fluid of a subject in vitro , comprising contacting the biological fluid with a compound and detecting a signal, wherein said biological fluid may contain a proteolytic enzyme derived from pancreatic cancer cells, and wherein the compound is characterized by formula 1: X1-Thr-Thr-Ala-Arg-X2 (formula 1), wherein the cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 leads to the formation of a detectable signal, wherein X1 contains or consists of a C1 component and X2 contains or consists of a C2 component, and the detectable signal arises when C1 and C2 are separated spatially by proteolytic cleavage of the compound, and wherein C1 and C2 are a pair of a fluorescence donor and a fluorescence acceptor, wherein the subject is a subject suspected of having pancreatic cancer, at increased risk of developing pancreatic cancer, or having previously suffered from pancreatic cancer. 6. The in vitro method of claim 5 for detecting the presence or absence of pancreatic cancer in a subject, wherein the presence of protease activity in the biological fluid indicates the presence of pancreatic cancer, and the absence of protease activity in the biological fluid indicates the absence of pancreatic cancer. 7. In vitro method according to claim 5 or 6 for diagnosing pancreatic cancer. 8. In vitro method according to any one of paragraphs. 5-7, characterized in that the biological fluid is urine. 9. Method according to any one of paragraphs. 5-8, characterized in that the specified compound is presented in a concentration of 0.1-10 mg/ml, in particular 0.25-7.5 mg/ml, more specifically 0.5-5 mg/ml, more specifically 0.75 -2 mg/ml, even more specifically 1 mg/ml in a measurement buffer with neutral or alkaline pH, preferably physiological pH, and a sample of biological fluid is added to the compound in a ratio of 1:2 to 1:10, in particular from 1:3 to 1:8, more specifically, from 1:4 to 1:6, even more specifically, 1:5. 10. Method according to any one of paragraphs. 5-9, characterized in that signal detection involves measuring absorption or fluorescence, in particular measuring absorption intensity at 300-500 nm, more specifically 380-430 nm, preferably for 40-60 min at 25-40°C, in particular at 36-38°C. 11. Pancreatic cancer diagnostic kit containing: - connection according to any one of paragraphs. 1-4, characterized by formula 1: X1-Thr-Thr-Ala-Arg-X2 (formula 1), wherein the cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 leads to the formation of a detectable signal, wherein X1 contains or consists of a C1 component and X2 contains or consists of a C2 component, and the detectable signal arises when C1 and C2 are separated spatially by proteolytic cleavage of the compound, and wherein C1 and C2 are a pair of a fluorescence donor and a fluorescence acceptor; And - buffer for measurements. 12. Use of the compound according to any one of paragraphs. 1-4, characterized by formula 1: X1-Thr-Thr-Ala-Arg-X2 (formula 1), wherein the cleavage of the compound into fragment 1 containing X1 and fragment 2 containing X2 leads to the formation of a detectable signal, wherein X1 contains or consists of a C1 component and X2 contains or consists of a C2 component, and the detectable signal arises when C1 and C2 are separated spatially by proteolytic cleavage of the compound, and wherein C1 and C2 represent a pair of a fluorescence donor and a fluorescence acceptor, the method according to any one of claims. 5-10 or the kit of claim 11 containing said compound and a measurement buffer for the detection of pancreatic cancer or for monitoring a subject suspected of having pancreatic cancer, at increased risk of developing pancreatic cancer, or having previously suffered from pancreatic cancer. 13. A method for treating pancreatic cancer, including the steps: a) performing the method according to any one of paragraphs. 5-10, and b) treating pancreatic cancer in a subject in which protease activity is detected in step a). 14. The method according to claim 13, characterized in that the protease activity is increased protease activity.