NOVEL DIAGNOSTIC MARKER FOR PANCREATIC CANCER
20220251625 · 2022-08-11
Inventors
Cpc classification
International classification
Abstract
The invention provides a compound characterized by formula (1): X1-Thr-Thr-Ala-Arg-X2, wherein cleavage of the compound into a fragment 1 comprising X1 and a fragment 2 comprising X2 generates a detectable signal. The invention further provides an in vitro method for detecting protease activity in a subject's body fluid, comprising contacting the body fluid with the compound of the invention and detecting a signal, wherein the body fluid may comprise a hydrolytic enzyme derived from pancreatic cancer cells. Furthermore, the invention provides a kit comprising the compound of the invention and a measurement buffer. In addition, the invention provides the use of the compound, the in vitro method or the kit of the invention for the detection of pancreatic cancer, or for monitoring a subject that is suspected of having pancreatic cancer, has an increased risk of developing pancreatic cancer, or has had pancreatic cancer. The invention also provides the use of the compound of the invention in a method of treating pancreatic cancer, the method comprising carrying out the in vitro method for detecting protease activity in a subject's body fluid, and treating pancreatic cancer in a subject for which protease activity, has been detected.
Claims
1. A compound characterized by formula 1:
X1-Thr-Thr-Ala-Arg-X2 (1), wherein cleavage of the compound into a fragment 1 comprising XI and a fragment 2 comprising X2 generates a detectable signal, wherein XI comprises or consist of a component C1 and X2 comprises or consists of a component C2, and the detectable signal itis generated upon spatial separation of C1 and C2, by hydrolytic cleaving 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, wherein the sequence Thr-Thr-Ala-Arg is accessible for a hydrolytic enzyme, in particular a hydrolytic enzyme cleaving the compound into XI-Thr-Thr-Ala-Arg-OH (fragment 1) and NH.sub.2-X2 (fragment 2).
3. The compound according to claims 1, wherein one of C1 and C2, in particular C2, is a chromophore having an absorption maximum 1 (AMI) at a wavelength 1, and the compound has an absorption maximum 2 (AM2) at a wavelength 2 that is different from wavelength 1.
4. The compound according to claim 1, wherein the pair of C1 and C2 is selected from the group consisting of 2-aminobenzoic acid (ABZ)/pNA, ABZ/ANB-NH.sub.2, ABR/DNP, ABZ/EDDNP, EDANS/DABCYL, TAM/D ANSYL, ABZ/Tyr(3-N0.sub.2), in particular the pair of C1 and C2 is selected from ABZ/pNA and. ABZ/ANB-NH.sub.2.
5. An in vitro method for detecting protease activity in a subject's body fluid, comprising contacting the body fluid with a compound and detecting a signal, wherein the body fluid may comprise a hydrolytic enzyme, in particular a protease, derived from pancreatic cancer cells, and wherein the compound is characterized by formula 1:
X1-Thr-Thr-Ala-Arg-X2 (1), wherein cleavage of the compound into a fragment 1 comprising XI and a fragment 2 comprising X2 generates a detectable signal.
6. The in vitro method according to claim 5 for detecting the presence or absence of pancreatic cancer in a subject, wherein the presence of protease activity in the body fluid indicates the presence of pancreatic cancer and the absence of protease activity in the body fluid indicates the absence of pancreatic cancer.
7. The in vitro method according to claim 5 for the diagnosis of pancreatic cancer.
8. The in vitro method according to claim 5, wherein the body fluid is urine.
9. The method according to claim 5, wherein the compound is provided. at a concentration. of 0.1-10 mg/ml, particularly 0.25-7.5 mg/ml, more particularly 0.5-5 mg/ml, more particularly 0.75-2 mg/ml, even more particularly about 1 mg/ml, in a measurement buffer having neutral or alkaline pH, preferably physiological pH, and the body fluid sample is added to the compound at a ratio of 1:2 to 1:10, particularly 1:3 to 1:8, more particularly 1:4 to 1:6, even more particularly about 1:5.
10. The method according to claim 5, wherein detecting the signal comprises measuring absorbance or fluorescence, particularly measuring absorbance intensity at 300-500 nm, more particularly 380-430 nm, preferably for 40-60 min at 25-40° C., in particular at 36-38° C.
11. A kit comprising a compound according to claim 1 and a measurement buffer.
12. Use of the compound characterized by formula 1:
X1-Thr-Thr-Ala-Arg-X2 (1), wherein cleavage of the compound into a fragment 1 comprising XI and a fragment 2 comprising X2 generates a detectable signal, the methods of claim 5 or the kit comprising said compound and a measurement buffer for the detection of pancreatic cancer, or for monitoring a subject that is suspected of having pancreatic cancer, has an increased risk of developing pancreatic cancer, or has had pancreatic cancer.
13. A method of treating pancreatic cancer, the method comprising the steps of: a. carrying out the method according to claim 5, and b. treating pancreatic cancer in. a subject for which protease activity, in particular increased protease activity has been detected in step a.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0111]
[0112]
[0113]
[0114]
EXAMPLES
[0115] The invention is illustrated by the following, non-limiting examples of implementation.
Example 1
Synthesis of compound ABZ.SUP.1.-Thr.SUP.2.-Thr.SUP.3.-Ala.SUP.4.-Argy-ANB-NH.SUB.2..SUP.6
[0116] 1. Preparation of the Chromogenic Peptide [0117] a) The first stage of the synthesis was to obtain a chromogenic peptide by solid phase synthesis—on a solid support, using Fmoc/tBu chemistry, i.e. using the protection. A compound with the sequence ABZ.sup.1-Thr.sup.2-Thr.sup.3-Ala.sup.4-Arg.sup.5-ANB-NH.sub.2.sup.6, where ABZ is 2-aminobenzoic acid, ANB-NH.sub.2 is amide of 5-amino-2-benzoic acid, and ANB is 5-amino-2-benzoic acid, obtained in the process of chemical synthesis in the solid phase using the following amino acid derivatives. [0118] Boc-ABZ, Fmoc-Thr(tBu), Fmoc-Ala, Fmoc-Arg(Pbf), ANB [0119] The synthesis of the compound, i.e. a diagnostic marker for the detection of pancreatic cancer, whose diagnosis is associated with the hydrolysis of this compound under the influence of proteolytic enzymes, was carried out on a solid support enabling the conversion of 5-amino-2-benzoic acid into ANB-NH.sub.2 amide: [0120] —e.g. amide resin, for instance TentaGel S RAM from RAPP Polymere (Germany), e.g. with a deposit of 0.23 mmol/g. [0121] It is possible to use other commercially available amide resins, including Rink Amide (Germany). [0122] The compound was synthesized manually using a laboratory shaker. For most of the steps, a 25 mL sintered syringe for solid phase synthesis was used as a reactor. [0123] All the obtained final compounds contained ABZ 2-aminobenzoic acid in position 1 of their sequence (i.e. at the N-terminus), and ANB 5-amino-2-nitrobenzoic acid molecule in position 6 (at the C-terminus). ABZ acts as a fluorescence donor, while ANB (5-amino-2-benzoic acid) acts as a fluorescence quencher and chromophore. The peptides contained at least (and preferably) one reactive site in their sequence, located between the amino acid residues Arg-ANB-NH.sub.2: at the 5 position of the compound. The synthesis, involving the attachment of amino acid derivatives, is carried out from the residue 6 to 1, i.e. from the C- to N-terminus. [0124] b) Deposition of ANB on TentaGel S RAM resin: [0125] Peptide synthesis was performed on TentaGel S RAM resin (Rapp Polymere) with a deposition of 0.23 mmol/g. In the first stage, the resin was prepared, including its loosening by the wash cycle. Subsequently, the Fmoc amino group protection was removed from the solid support with a 20% solution of piperidine in NMP, and a solvent wash cycle was carried out. To confirm the presence of free amino groups, a chloranil test was performed. [0126] Solvent wash cycle: [0127] DMF 1×10 minutes [0128] IsOH 1×10 minutes [0129] DCM 1×10 minutes [0130] Removal of Fmoc protection: [0131] DMF 1×5 minutes [0132] 20% piperidine in NMP 1×13 minutes [0133] 20% piperidine in NMP 1×8 minutes [0134] Solvent wash cycle: [0135] DMF 3×2 minutes [0136] IsOH 3×2 minutes [0137] DCM 3×2 minutes [0138] c) Chloranil test: [0139] The chloranil test consisted of transferring (using a spatula) several grains of resin from the reactor—a syringe, into a glass ampoule, to which 100 μL saturated solution of p-chloranil in toluene and 50 μL fresh acetaldehyde were added. After 10 minutes, the control of grains colour was carried out. [0140] At this stage, after performing the test, a green grain colour was obtained, which indicated the presence of free amino groups. After confirming the removal of 9-fluorenylmethoxycarbonyl protection from the resin, it was possible to proceed to the next stage, the attachment of the ANB derivative (5-amino-2-nitrobenzoic acid) [0141] d) Deposition of 5-amino-2-nitrobenzoic acid on a solid support [0142] The first step in the synthesis of the peptide library—a mixture of peptides, 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 DMF, after which the Fmoc protection was removed from the solid support functional group. One cycle of removing Fmoc protection included the following steps: [0143] Removal of Fmoc protection: [0144] 20% piperidine in NMP 1×3 minutes [0145] 20% piperidine in NMP 1×8 minutes [0146] e) Washing [0147] DMF 3×2 minutes [0148] IsOH 3×2 minutes [0149] DCM 3×2 minutes [0150] f) Chloranil test for the presence of free amino groups. [0151] The resin with a free amino group was washed with a 5% solution of N-methylmorpholine (NMM) in DMF, followed by DMF. The procedure of removing Fmoc protection and the wash cycle were performed in a Merrifield vessel. In a separate flask, ANB was dissolved in DMF, and TBTU, DMAP, and finally diisopropylethylamine (DIPEA) were subsequently added in the following excess in relation to polymer deposition: 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 off 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-N,N,N′,N′-tetramethyluronium (HBTU) were used to carry out subsequent reactions of ANB attachment to the resin. In the last step, the resin was washed successively with DMF, DCM and isopropanol, and air dried. [0152] g) Attachment of the C-terminal amino acid residue to ANB [0153] The corresponding amino acid derivative (9-fold molar excess relative to resin deposition) was dissolved in pyridine and transferred to the flask containing the resin with ANB deposited. The whole was cooled to −15° C. (ice bath: 1 part by weight NH.sub.4Cl, 1 part by weight NaNO.sub.3, 1 part by weight ice). After reaching the desired temperature, POCl.sub.3 was added (in a 1:1 ratio to the amount of amino acid derivative used) and the whole 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 completing the reaction, the resin was filtered off under reduced pressure, washed with DMF and MeOH, and left to dry. [0154] In the next step, the residue (alanine) was attached in the P2 position. [0155] Every attachment of amino acid residues was preceded by washing the resin with DMF for 5 minutes. Diisopropylcarbodiimide was used as a coupling agent in subsequent attachments. The procedure was repeated twice. [0156] After each acylation, a resin wash cycle started, followed by the chloranil test to monitor the attachment of the amino acid derivative to the free amino groups of the resin. [0157] Solvent wash cycle: [0158] DMF 3×2 minutes [0159] IsOH 3×2 minutes [0160] DCM 3×2 minutes [0161] Chloranil test: [0162] As a result of the tests, after the first two couplings, the colour of the grains was first green and then grey, so it was necessary to carry out another acylation, as a result of which the grains of the resin tested with the chloranil test were colourless. This indicated the attachment of ANB to TentaGel S RAM resin, which enabled moving to the next stage of peptide synthesis. [0163] h) Attachment of further protected amino acid residues: [0164] The resin together with the attached ANB residue in the reactor was washed with DMF, followed by deprotection of the Fmoc from the amino group to attach the protected amino acid alanine derivative. [0165] Removal of Fmoc protection: [0166] iDMF 1×5 minutes [0167] 20% piperidine in NMP 1×3 minutes [0168] 20% piperidine in NMP 1×8 minutes [0169] Solvent wash cycle: [0170] DMF 3×2 minutes [0171] IsOH 3×2 minutes [0172] DCM 3×2 minutes [0173] Chloranil test: [0174] The chloranil test produced a positive result, as evidenced by the green colour of the resin grains. This enabled moving to the next stage—attachment of the Fmoc-Thr (tBu)—OH amino acid residue. [0175] Attachment of the amino acid derivative: [0176] The process of coupling was preceded by washing the resin in DMF. The composition of the coupling mixture remained unchanged when attaching the protected glutamic acid residue. [0177] At the end of each acylation, a solvent wash cycle was performed according to the given procedure, which was followed by a chloranil test for the presence of free amino groups in the solution. [0178] Solvent wash cycle: [0179] DMF 3×2 minutes [0180] IsOH 3×2 minutes [0181] DCM 3×2 minutes [0182] Chloranil test: [0183] The resin grains during the test carried out after the second acylation were colourless, which enabled moving to the next stage of the synthesis, i.e. the introduction of another protected amino acid derivative—threonine and 2-aminobenzoic acid molecule. The coupling processes followed the procedure discussed earlier. [0184] Tests carried out after attaching the above-mentioned residues showed positive results: the resin grains were colourless.
[0185] 2. Removal of the Peptide From the Solid Support [0186] After synthesis, the amide of ABZ-Thr-Thr-Ala-Arg-ANB-NH.sub.2 peptide was removed from the solid support, along with the simultaneous removal of the side protection with the mixture: TFA: phenol: water: TIPS (88:5:5:2, v/v/v/v) in a round-bottomed flask on a magnetic stirrer. [0187] After 3 hours, the contents of the flask were filtered off under reduced pressure on a sintered (Schott) funnel and washed with diethyl ether. The resulting sediment was centrifuged on a SIGMA 2K30 centrifuge (Laboratory Centrifuges) for 20 minutes. The precipitate obtained after centrifugation is dissolved in water by means of ultrasound and lyophilised. [0188] Identity/characteristics of a new compound—HPLC analysis, MS [0189] HPLC conditions: RP Bio Wide Pore Supelco C8 250 mm 4 mm column, A phase system 0.1% TFA in water, B: 80% acetonitrile in A), flow rate 1 ml/min, UV detection at 226 nm. [0190] Obtaining the compound was confirmed.
Example 2
Preparation of the Compound With the formula: ABZ1-Thr2-Thr3-Ala4-Arg5-pNA6
[0191] The process is carried out in a similar manner to that described in Example 1, except that the corresponding amino acid derivatives and additional substituents are used, and the process is carried out partly in solution and partly on a solid support.
[0192] Preparation of p-nitroanilide Ala [0193] a) The first stage of the synthesis was to obtain a protected peptide by solid phase synthesis using Fmoc/tBu chemistry. [0194] ABZ.sup.1-Thr(tBu).sup.2-Thr(tBu).sup.3-Ala.sup.4-OH compound, where ABZ is 2-aminobenzoic acid, was obtained by solid phase chemical synthesis using the following amino acid derivatives: [0195] Boc-ABZ, Fmoc-Thr(tBu), Fmoc-Ala. [0196] The compound was synthesized on a solid support: [0197] 2-chloro-chlorotriyl resin, e.g. from Iris BIOTECH GMBH (Germany), with deposition of 1.6 mmol Cl/g groups. [0198] The compound was synthesized manually using a laboratory shaker. Throughout all stages, a 25 ml sintered syringe for solid phase synthesis was used as the reactor. Peptide synthesis was carried out on a solid support: 2-chloro-chlorotrityl resin, e.g. from Iris BIOTECH GMBH (Germany), with deposition of 1.6 mmol Cl/g groups. In the first stage, the resin was loosened in a wash cycle. Subsequently, the Fmoc amino group protection was removed from the solid support with a 20% solution of piperidine in NMP. Then a solvent wash cycle was carried out. To confirm the presence of free amino groups, a chloranil test was performed. [0199] Solvent wash cycle: [0200] DMF 1×10 minutes [0201] IsOH 1×10 minutes [0202] DCM 1×10 minutes [0203] Removal of Fmoc protection: [0204] DMF 1×5 minutes [0205] 20% piperidine in NMP 1×3 minutes [0206] 20% piperidine in NMP 1×8 minutes [0207] Solvent wash cycle: [0208] DMF 3×2 minutes [0209] IsOH 3×2 minutes [0210] DCM 3×2 minutes [0211] b) Chloranil test: [0212] The chloranil test consisted of transferring (with a spatula) several grains of resin from the reactor—a syringe, into a glass ampoule, and adding 100 μL saturated solution of p-chloranil in toluene and 50 μL fresh acetaldehyde. After 10 minutes, the control of grains colour was carried out. [0213] At this stage, after the test, a green colour of grains was obtained, which indicated the presence of free amino groups. After confirming the removal of the 9-fluorenylmethoxycarbonyl protection from the resin, the attachment of Fmoc-Ala derivative was initiated. [0214] c) Embedding Fmoc-Ala on a solid support [0215] The first step in the synthesis of the peptide library was the deposition of Fmoc-Ala on 1 g of resin. Before attachment of the amino acid derivative, the resin used for the reaction was washed with the following solvents: DMF (dimethylformamide), DCM (methylene chloride) and again with DMF, after which the Fmoc-protection was removed from the solid support functional group. One cycle of Fmoc-protection removal included the following steps: [0216] Removal of Fmoc protection: [0217] 20% piperidine in NMP 1×3 minutes [0218] 20% piperidine in NMP 1×8 minutes [0219] Washing [0220] DMF 3×2 minutes [0221] IsOH 3×2 minutes [0222] DCM 3×2 minutes [0223] Chloranil test for the presence of free amino groups. [0224] The resin with a free amino group was washed with DMF. In a separate flask, Fmoc-Ala was dissolved in DMF. Next, TBTU, DMAP, and finally diisopropylethylamine (DIPEA) were added in excess to the polymer deposition: Fmoc-Pro/TBTU/DMAP/DIPEA, 3:3:2:6. The obtained mixture was added to the resin and stirred for 3 hours. The resin was filtered off 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), followed by hexafluorophosphate-O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium (HBTU) were used to conduct subsequent reactions of Fmoc-Pro attachment to the resin. In the last step, the resin was washed successively with DMF, DCM and isopropanol, and air dried. [0225] c. Attachment of further protected amino acid residues: [0226] The resin together with the attached Fmoc-Ala residue in the reactor was washed with DMF, followed by deprotection of Fmoc from the amino group to attach the protected threonine derivative. [0227] Removal of Fmoc protection: [0228] DMF 1×5 minutes [0229] 20% piperidine in NMP 1×3 minutes [0230] 20% piperidine in NMP 1×8 minutes [0231] Solvent wash cycle: [0232] DMF 3×2 minutes [0233] IsOH 3×2 minutes [0234] DCM 3×2 minutes [0235] Chloranil test: [0236] The chloranil test produced a positive result, as evidenced by the green colour of the resin grains. This enabled moving to the next stage—attachment of the Fmoc-Thr (tBu)-OH amino acid residue. [0237] Attachment of the amino acid derivative: [0238] The process of coupling was preceded by washing the resin in DMF. The composition of the coupling mixture remained unchanged when attaching the protected glutamic acid residue. [0239] At the end of each acylation, a solvent wash cycle was performed according to the given procedure, which was followed by a chloranil test for the presence of free amino groups in solution. [0240] Solvent wash cycle: [0241] DMF 3×2 minutes [0242] IsOH 3×2 minutes [0243] DCM 3×2 minutes [0244] Chloranil test: [0245] The resin grains during the test carried out after the second acylation were colourless, which enabled moving to the next stage of the synthesis, i.e. the introduction of another protected amino acid derivative—threonine and 2-aminobenzoic acid molecule. The coupling processes followed the procedure discussed earlier. [0246] Tests carried out after attaching the above-mentioned residues showed positive results: the resin grains were colourless. [0247] d) Removal of peptide from the solid support while maintaining side group protection [0248] After completing the synthesis, the protected ABZ-Thr(tBu)-Th (tBu)-Ala-OH peptide was removed from the solid support, retaining the side protection with the mixture: acetic acid: TFE (trifluoroethanol): DCM (2:2:6, v/v/v) in a round-bottomed flask on a magnetic stirrer. [0249] After 2 hours, the contents of the flask were filtered off under reduced pressure on sintered (Schott) funnels, washing with the astringent mixture. The solution was washed with hexane (1:10 v/v), evaporated under reduced pressure and then lyophilised. [0250] e) Chemical synthesis of paranitroanilide Arg derivatives [0251] The mixed anhydrides method was used to synthesize Fmoc-Arg(Pbf)-pNA. In the first step, 2 mmol of Fmoc-Arg (Pbf) were 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 were added. Reactions were carried out for 2 hours at −15° C., and then for 24 hours at room temperature. After completing the reaction, the solvent evaporated and the dry residue was dissolved in ethyl acetate. The resulting solution was washed successively with saturated aqueous NaCl solution, 10% citric acid, and 5% sodium bicarbonate. The resulting solution was dried over anhydrous sodium sulphate, ethyl acetate was distilled off under reduced pressure, and the dry residue was dried in a vacuum desiccator over P.sub.2O.sub.5 and KOH. [0252] f) Coupling of the protected peptide with paranitroanilide Arg (Pbf) [0253] The protected ABZ'-Thr(tBu).sup.2-Thr (tBu).sup.3-Ala.sup.4-OH peptide was dissolved in a small amount of DCM, and subsequently activated with TFFH (tetramethylfluoroformamide) for 30 minutes at 0° C. Then a catalytic amount of DMAP and Arg (Pbf)-pNA was added. The reaction was carried out for 24 hours at room temperature, after which the solvent evaporated. The resulting solution was poured with the mixture removing the side 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. [0254] After this time, cold diethyl ether was added to the flask, and the resulting precipitate was centrifuged in a high speed centrifuge at 5,000 rpm for 20 minutes. The precipitate obtained after centrifugation was dissolved in water by means of ultrasound and then lyophilised. [0255] Identity/characteristics of a new compound—HPLC analysis, MS [0256] HPLC conditions: RP Bio Wide Pore Supelco C8 250 mm 4 mm column, A phase system 0.1% TFA in water, B: 80% acetonitrile in A), flow rate 1 mL/min, UV detection at 226 nm. Obtaining of the compound was confirmed.
Example 3
[0257] The study on the application of new compounds was performed on a group of 10 patients diagnosed with pancreatic cancer. For this purpose, the compound with formula 2: ABZ-Thr-Thr-Ala-Arg-ANB-NH.sub.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 buffer (200 mM Tris-HCl, pH 8.0) and 80 μL of urine of a person with pancreatic cancer. The measurement was made in a 96-well plate designed for measuring absorbance, and each sample was analysed in triplicate at 37° C. The duration of the measurement was 60 minutes. During the measurement, the wavelength characteristic of the released chromophore (ANB-NH.sub.2 or pNA) was monitored at 405 nm (range 380-430 nm).
[0258] As a result of the measurement, the colour of the solution increased over time in all urine samples from patients diagnosed with pancreatic cancer. The observed absorbance increase over time was different for each of the tested samples. A different effect was obtained for 15 samples of healthy people, as none of the 15 urine samples tested had an increase in absorbance in the diagnostic range.
[0259] 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 compound is based on its enzymatic hydrolysis in such a place, which leads to the release of free chromophore molecules, respectively, ANB-NH.sub.2-amide of 5-amino-2-nitrobenzoic acid or pNA-para-nitroaniline, which shows absorbance at 320-480 nm, especially 380-430 nm.
TABLE-US-00001 TABLE 1 Values depicted 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 equal 0
TABLE-US-00002 TABLE 2 Values depicted 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 equal 0
TABLE-US-00003 TABLE 3 Values depicted 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
[0260] This invention further pertains to
1. Chemical compound—diagnostic marker of the general formula:
ABZ.sup.1-Thr.sup.2-Thr.sup.3-Ala.sup.4-Arg.sup.5-X.sup.6 (1) [0261] where: [0262] ABZ is 2-aminobenzoic acid, [0263] X is ANB-NH.sub.2 or pNA, [0264] wherein ANB-NH.sub.2 is amide of 5-amino-2-benzoic acid, [0265] ANB is 5-amino-2-nitrobenzoic acid, [0266] and pNA is para-nitroaniline.
2. Method of obtaining a chemical compound—a diagnostic marker
ABZ.sup.1-Thr.sup.2-Thr.sup.3-Ala.sup.4-Arg.sup.5-ANB-NH.sub.2.sup.6 , (2)
where:
ABZ is 2-aminobenzoic acid,
ANB-NH.sub.2 is amide of 5-amino-2-nitrobenzoic acid, is based on the fact that the process is performed on a solid support, preferably having the 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 removing the Fmoc protecting group, preferably by washing with a 10-30% solution of piperidine in solvents such as dimethylformamide, methylene chloride or N-methylpyrrolidone. Next, the process is carried out in subsequent stages: [0267] a) Deposition of 5-amino-2-nitrobenzoic acid ANB on the resin is preceded by washing the solid support 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,
[0268] DMAP and finally diisopropylethylamine (DIPEA) are added in the following excess in relation to the polymer deposition: ANB/TBTU/DMAP/DIPEA, 3:3:2:6. The obtained mixture is added to the resin and mixed until homogeneous, then the resin is filtered off under reduced pressure and washed with solvents such as DMF, DCM and isopropanol. Next, the binding of ANB to the resin is continued by 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 excess, and after finishing, the solid support is washed successively with DMF, DCM and isopropanol, and gently dried, [0269] b) Attachment of the amino acid residue to ANB is carried out using the reaction with the amino acid derivative of Fmoc-Arg (Pbf)-OH, at least a five-fold molar excess of the amino acid derivative in relation to the resin is dissolved in anhydrous pyridine and contacted with the resin with deposited ANB. Next, the whole is cooled to a temperature not lower than 20° C. and POCl.sub.3 is added in a ratio of 1:1 to the amount of amino acid derivative used, and mixed. The mixing process is carried out at room temperature and then at an elevated temperature; after the reaction is completed, the resin is filtered off under reduced pressure, washed with DMF and MeOH and gently dried; the obtained intermediate compound is subjected to the process of acylation. [0270] c) Acylation of the resulting intermediate compound is carried out using an amino acid derivative, preferably Fmoc-Ala-OH, and then Fmoc-Thr (tBu)-OH followed by Fmoc-Thr(tBu)-OH and finally by Boz-Abz-OH. Acylation is carried out in stages from residue 6 to 1, using diisopropylcarbodiimide as a coupling agent, which is used in excess. At the end of each step, the resin is washed with DMF and (preferably) subjected to the chloranil test, in which the attachment of the amino acid derivative is monitored. [0271] d) Removing the Fmoc protecting group is carried out by washing with a 10-30% piperidine solution in DMF, and subsequent washing with each of the solvents: DMF, isopropanol and methylene chloride. [0272] e) Separation of the peptide from the resin is carried out using the mixture: TFA, phenol, water, and TIPS, while maintaining a ratio of 88:5:5:2 v/v/v/v, respectively; the mixture is stirred for at least one hour, preferably for three hours, and the resulting precipitate is filtered off under reduced pressure, washed with diethyl ether, and the resulting peptide is centrifuged. [0273] f) Preparation of the finished product is carried out by dissolving the peptide in water by means of ultrasound, and lyophilisation.
[0274] 3. Method for obtaining a chemical compound—a diagnostic marker:
ABZ.sup.1-Thr.sup.2-Thr.sup.3-Ala.sup.4-Arg.sup.5-pNA.sup.6 (3)
where:
ABZ is 2-aminobenzoic acid,
pNA is paranitroanilide,
[0275] is based on the fact that the process is carried out on a solid support, preferably having the 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 removing 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 the process is carried out in subsequent stages: [0276] a) Attachment of the third amino acid residue (Fmoc-Ala) to the resin is carried out using the appropriate amino acid derivative at a 9-fold molar excess in relation to the resin deposition, which is dissolved in anhydrous methylene chloride. The mixture is then stirred for at least two hours at room temperature; after completing the reaction, the resin is filtered off under reduced pressure and washed with DMF and MeOH, and then dried. [0277] b) In the subsequent steps, attachment of the amino acid residue, hereinafter referred to as acylation, is performed, and the derivative of Fmoc-Thr(tBu)-OH is used, followed by Fmoc-Thr(tBu)-OH and Boc-ABZ-OH; each step is preceded by washing the resin with DMF, for preferably 5 minutes; in subsequent attachments, a coupling agent is used, 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, in which the attachment of the amino acid derivative is monitored. [0278] c) Removal of the Fmoc protecting group is carried out by washing with a 10-30% piperidine solution in DMF, and subsequent washing with each of the solvents: DMF, isopropanol and methylene chloride. [0279] d) Repeating steps b) to c) until the compound (ABZ'-Thr(tBu).sup.2-Thr (tBu).sup.3-Ala.sup.4-OH) is obtained; synthesis is carried out from residue 6 to 3, and then the resulting compound is detached from the solid support using the mixture: TFA: phenol: water: TIPS in the proportions 88:5:5:2, v/v/v/v, respectively, while stirring. After at least two hours, the contents of the flask are filtered off under reduced pressure, and the precipitate is washed with diethyl ether. The precipitate is then centrifuged for preferably 20 minutes, dissolved in water by means of ultrasound, and then lyophilised. [0280] e) Fmoc-Arg (Pbf)-pNA synthesis is carried out in stages; in the first stage, 2 mmol of Fmoc-Arg (Pbf) 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 10 minutes of activation, 3 mmol of p-nitroaniline are added, and the reaction is carried out for (preferably) 2 hours at a temperature (preferably) of —15° C., followed by a day at room temperature. When the reaction is completed, the solvent is evaporated and the dry residue is dissolved in ethyl acetate; then the resulting solution is washed successively with saturated aqueous NaCl solution, 10% citric acid, 5% sodium bicarbonate, and dried over anhydrous sodium sulphate; ethyl acetate is distilled under reduced pressure, and the dry residue is dried. [0281] f) Combining the protected peptide ABZ.sup.1-Thr(tBu).sup.2-Thr(tBu).sup.3-Ala.sup.4-OH with paranitroanilide Arg (Pbf) is based on the following process: the protected peptide ABZ.sup.1-Thr(tBu).sup.2-Thr(tBu).sup.3-Ala.sup.4-OH is dissolved in a small amount of DCM, and then activated with TFFH (tetramethylfluoroformamide) for preferably 30 minutes at a preferable temperature of 0° C., after which a catalytic amount of DMAP and Fmoc-Arg(Pbf).sup.5-pNA.sup.6 is added. The reaction is preferably carried out for 24 hours at room temperature, after which the solvent is evaporated, and the resulting solution is poured with the mixture removing the side protection: TFA: phenol: water: TIPS (88:5:5:2, v/v/v/v), and mixed for preferably 3 hours. [0282] g) The finished product is prepared by dissolving the peptide in water using ultrasound, and then subjecting to lyophilisation.
[0283] 4. The method for diagnosing pancreatic cancer is based on the following process: the chemical compound of general formula 1 in a concentration range of 0.1-10 mg/mL (preferably 1 mg/mL) is incubated in a measurement buffer at neutral or alkaline pH, preferably physiological, with a small amount of human urine in the proportion range 1:2 to 1:10 (preferably 1:5 urine sample to the measurement buffer), and absorbance intensity in the range 300-500 nm, (preferably 380-430 nm) is measured over 40-60 minutes at a temperature in the range of 25-40° C. (preferably 36-38° C.).
[0284] Additionally, the invention relates inter alia to the following items: [0285] 1. A compound characterized by formula 1:
X1-Thr-Thr-Ala-Arg-X2 (1),
wherein cleavage of the compound into a fragment 1 comprising X1 and a fragment 2 comprising X2 generates a detectable signal. [0286] 2. The compound according to item 1, wherein the sequence Thr-Thr-Ala-Arg is accessible for a hydrolytic enzyme, in particular a hydrolytic enzyme cleaving the compound into X1-Thr-Thr-Ala-Arg-OH (fragment 1) and NH.sub.2-X2 (fragment 2). [0287] 3. The compound according to item 1 or 2, wherein X1 comprises or consists of a component C1 and X2 comprises or consists of a component C2, and the detectable signal is generated upon spatial separation of C1 and C2. [0288] 4. The compound according to any of items 1 to 3, wherein one of C1 and C2, in particular C2, is a chromophore having an absorption maximum 1 (AM1) at a wavelength 1, and the compound has an absorption maximum 2 (AM2) at a wavelength 2 that is different from wavelength 1. [0289] 5. The compound according to any of items 1 to 4, wherein C1 and C2 are a pair of a fluorescence donor and a fluorescence acceptor. [0290] 6. The compound according to any of items 1 to 5, wherein the pair of C1 and C2 is selected from the group consisting of 2-aminobenzoic acid (ABZ)/pNA, ABZ/ANB-NH.sub.2, ABZ/DNP, ABZ/EDDNP, EDANS/DABCYL, TAM/DANSYL, ABZ/Tyr(3-NO.sub.2), in particular the pair of C1 and C2 is selected from ABZ/pNA and ABZ/ANB-NH.sub.2. [0291] 7. An in vitro method for detecting protease activity in a subject's body fluid, comprising contacting the body fluid with the compound according to any of items 1 to 6 and detecting a signal, wherein the body fluid may comprise a hydrolytic enzyme, in particular a protease, derived from pancreatic cancer cells. [0292] 8. The in vitro method according to item 7 for detecting the presence or absence of pancreatic cancer in a subject, wherein the presence of protease activity in the body fluid indicates the presence of pancreatic cancer and the absence of protease activity in the body fluid indicates the absence of pancreatic cancer. [0293] 9. The in vitro method according to item 7 or 8 for the diagnosis of pancreatic cancer. [0294] 10. The in vitro method according to any of items 7 to 9, wherein the body fluid is urine. [0295] 11. The method according to any of items 7 to 10, wherein the compound is provided at a concentration of 0.1-10 mg/ml, particularly 0.25-7.5 mg/ml, more particularly 0.5-5 mg/ml, more particularly 0.75-2 mg/ml, even more particularly about 1 mg/ml, in a measurement buffer having neutral or alkaline pH, preferably physiological pH, and the body fluid sample is added to the compound at a ratio of 1:2 to 1:10, particularly 1:3 to 1:8, more particularly 1:4 to 1:6, even more particularly about 1:5. [0296] 12. The method according to any of items 7 to 11, wherein detecting the signal comprises measuring absorbance or fluorescence, particularly measuring absorbance intensity at 300-500 nm, more particularly 380-430 nm, preferably for 40-60 min at 25-40° C., in particular at 36-38° C. [0297] 13. A kit comprising a compound according to any of items 1 to 6 and a measurement buffer. [0298] 14. Use of the compound according to any of items 1 to 6, the methods of any of items 7 to 12 or the kit according to item 13 for the detection of pancreatic cancer, or for monitoring a subject that is suspected of having pancreatic cancer, has an increased risk of developing pancreatic cancer, or has had pancreatic cancer. [0299] 15. The compound according to any of items 1 to 6 for use in a method of treating pancreatic cancer, the method comprising the steps of: [0300] a. carrying out the method according to any of items 7 to 12, and [0301] b. treating pancreatic cancer in a subject for which protease activity, in particular increased protease activity has been detected in step a.