PL238575B1 - New compound, method for obtaining it, a kit containing the new compound and application of the new compound for detection of epithelial tumors - Google Patents

Description

Description of the invention

The present invention relates to a novel compound and a diagnostic use of the compound for the detection of epithelial neoplasms, in particular of the epithelial neoplasm of the urinary system.

Peptides for the detection of bladder cancer are known from patent publications such as WO 2011/038142 or WO 2014/042209. The disclosed peptides, however, have a different structure to the new peptide presented in this application, and their method of tumor detection is different, where by marking with markers they allow the detection of the disease state.

The subject of the research was the development of a compound (a substrate with chromogenic and fluorogenic properties) whose enzymatic hydrolysis is observed only in the presence of the urine of a patient diagnosed with epithelial cancer.

It is known that one of the effects of neoplastic disease is an increase in the proteolytic enzymes of the tumor and surrounding tissues resulting from many processes, which include increased angiogenesis, tissue remodeling, necrosis of healthy tissues due to tumor infiltration, and apoptosis of neoplastic cells.

An object of the present invention is to provide a compound and a method that could be used for the detection of epithelial neoplasms, preferably bladder cancer. Surprisingly, this problem has been substantially solved by the present invention.

Studies were carried out to identify a compound that would be susceptible to increased proteolytic activity in human urine diagnosed with cancer of the urinary tract epithelium. The results were carried out on the urine of sick people (combined samples of 60 sick people) and 25 healthy people.

As a result of these studies, a compound was developed, the enzymatic hydrolysis of which is observed by monitoring the released ANB-NH2 molecule (5-amino-2-nitrobenzoic acid amide) at a wavelength of 410 nm.

The subject of the invention is a compound of the general formula:

ABZ1-Met ² -Lys ³ -Val ⁴ -Trp ⁵ -ANB-NH2 ⁶ where:

ABZ means 2-aminobenzoic acid

ANB is 5-amino-2-nitrobenzoic acid

The subject of the invention is the diagnostic application of the above-mentioned the compound as a marker for the diagnosis of epithelial cancer, especially of the urinary tract, in the urine.

The diagnostic activity of the compound is based on the in vitro hydrolysis of the new compound at position 5 through proteolytic enzymes in the presence of an epithelial tumor. The hydrolysis is visualized after incubating this compound with a biological sample by measuring the absorbance resulting from enzymatic hydrolysis.

The method of obtaining the compound according to the invention consists in carrying out the following steps of the methodology:

a) TentaGel S RAM amide resin swelling process;

b) deprotection of the Fmoc envelope in a 20% solution of piperidine in NMP;

c) washing the resin (DMF, piperidine);

d) performing a chloranil test;

e) depositing 5-amino-2-nitrobenzoic acid;

f) washing the resin (DMF, DCM);

g) performing a chloranil test;

h) repeating steps b) to g) until the compound / peptide of claim 1 is obtained;

i) cleavage of the peptide from the resin while removing the covers with the mixture

TFA: phenol: water: TIPS (88: 5: 5: 2, v / v / v / v);

j) filtering, centrifuging, dissolving the precipitate in distilled water with the aid of ultrasound, then subjecting to lyophilization;

k) analysis and purification by HPLC, MS, NMR;

l) mass spectrometry to verify identity.

PL 238 575 B1

The terms used above and in the specification and claims have the following meanings:

Epithelial neoplasm - that is, the cells of which are derived from the epithelium of the glands of external and internal secretion and from the ducts of these glands, e.g. a tumor of the epithelium of the urinary system.

Chromogenic properties - means the formation of a colored product

Fluorogenic properties - means the formation of a fluorescent product

NMP - N-methylpyrrolidone

DMF - dimethylformamide

Boc - tert-butyloxycarbonyl group

Fmoc - 9-fluorenylmethoxycarbonyl group

DCM - dichloromethane

TFA - trifluoroacetic acid

TIPS - triisopropylsilane

TRIS-HCl - tris (hydroxymethyl) aminomethane hydrochloride

ABZ means 2-aminobenzoic acid

ANB is 5-amino-2-nitrobenzoic acid

The invention is illustrated by the example and the drawing.

Description of the figures:

Figures 1-4 - Graphs 1-4 - HPLC results of the samples.

Figure 5 - Shows the rate of ABZ-Met-Lys-Val-Trp-ANB-NH2 substrate hydrolysis in urine samples diagnosed with cancer. Arabic numerals indicate the number of a randomly selected urine sample.

Fig. 6 - shows the rates of ABZ-Met-Lys-Val-Trp-ANB-NH2 substrate hydrolysis depending on the presence of the inhibitor, in a human urine sample with established urinary epithelial carcinoma.

Fig. 7 - shows the dependence of the degree of hydrolysis of the ABZ-Met-Lys-Val-Trp-ANB-NH2 substrate on the pH environment.

Fig. 8 - shows the hydrolysis efficiency of ABZ-Met-Lys-Val-Trp-ANB-NH2 substrate depending on the concentration degree of human urine with diagnosed cancer of the urinary system epithelium. P r z k ł a d 1

I) Synthesis of a new compound:

a) The first stage of the synthesis was to obtain a chromogenic peptide, which was obtained by solid phase synthesis using Fmoc / tBu chemistry.

The compound with the sequence ABZ1-Met ² -Lys ³ -Val ⁴ -Trp ⁵ -ANB-NH2 ⁶ , where ABZ - is 2-aminobenzoic acid, was obtained by chemical solid-phase synthesis with the use of the following amino acid derivatives:

Boc-ABZ, Fmoc-Met, Fmoc-Lys (Boc), Fmoc-Val, Fmoc-Trp (tBu), ANB

The synthesis of peptide substrates was carried out on a solid support:

- TentaGel S RAM resin from RAPP Polymere (Germany) with a deposit of 0.23 mmol / g.

The synthesis of all peptides was carried out manually using a laboratory shaker. For most steps, the reactors were a syringe tipped with a 25 ml solid phase frit.

All synthesized substrates contained in their sequence at the N-terminus 2-aminobenzoic acid (ABZ) and a molecule of 5-amino-2-nitrobenzoic acid at the C-terminus (ANB). ABZ acted as a fluorescence donor, while ANB acted as a fluorescence quencher and a chromophore at the same time. The peptides in their sequence contained one reactive site located between the amino acid residues Trp-ANB-NH2.

Mounting of ANB on TentaGel S RAM resin:

The peptide synthesis was performed on Rapp Polymere's TentaGel S RAM resin with a deposit of 0.23 mmol / g. In the first stage, the resin was fluffed through a washing cycle. Subsequently, the Fmoc amino group protection was removed from the support - with the use of a 20% solution of piperidine in NMP. Then a cycle of washing with solvents was carried out. A chloranil test was performed to confirm the presence of free amino groups.

Solvent wash cycle: DMF 1x10 minutes

IsOH 1x10 minutes

DCM 1x10 minutes Removal of Fmoc sheath: DMF 1x5 minutes

PL 238 575 B1

20% piperidine in NMP 1x3 minutes

20% piperidine in NMP 1x8 minutes

Solvent wash cycle: DMF 3x2 minutes

IsOH 3x2 minutes

DCM 3x2 minutes Chloranil test:

The chloranil test consisted of transferring, using a spatula, a few grains of resin from a syringe-reactor to a glass ampoule, to which then 100 µl of a saturated p-chloranil solution in toluene and 50 µl of fresh acetaldehyde were added. After 10 minutes, the color of the grains was checked.

At this stage, after carrying out the test, a green color of the grains was obtained, which indicated the presence of free amino groups. After confirming the removal of the fluorenylmethoxycarbonyl shields from the resin, it was possible to proceed to the next step, adding the ANB derivative (5-amino-2-nitrobenosesic acid).

Supporting 5-amino-2-nitrobenzoic acid on a solid support

The first step in the synthesis of the peptide library was the deposition of the ANB on 1 g of resin. Prior to chromophore attachment, the resin used for the reaction was washed with the following solvents: DMF, DCM, and again DMF, and the Fmoc-shield was removed from the support functional group. One Fmoc- cover removal cycle included the following steps:

1. Removal of the Fmoc- cover:

20% piperidine solution in NMP 1x3 minutes 1x8 minutes

2. Washing

DMF 3 x 2 minutes isopropanol 3 x 2 minutes DCM 3 x 2 minutes

3. Test for the presence of free amino groups: Chloranilic [167].

The resin with the free amino group was washed with a 5% solution of N-methylmorpholine (NMM) in DMF followed by DMF. The Fmoc- sheath removal procedure and the wash cycle were performed in a Merrifield pan. In a separate flask, ANB was dissolved in DMF and I added successively TBTU, DMAP, and finally diisopropylethylamine (DIPEA) in the following excesses relative to polymer deposition: ANB / TBTU / DMAP / 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. O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium (HATU) hexafluorophosphate (HATU), followed by O- (benzotriazol-1-yl) hexafluorophosphate ) -N, N, N ', N'-tetramethyluronium (HBTU). In the last step, the resin was washed sequentially with DMF, DCM, and isopropanol and air dried.

Attachment of the C-terminal amino acid residue to the ANB

The appropriate amino acid derivative (9 fold molar excess to the resin deposition) was dissolved in pyridine and transferred to the flask containing the ANB-loaded resin. It was cooled down to -15 ° C (ice bath: 1 part by weight of NH4Cl, 1 part by weight of NaNO3, 1 part by weight of ice). After the desired temperature was reached, POCl3 (1: 1 ratio to the amount of amino acid derivative used) was added 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 completion of the reaction, the resin was filtered off under reduced pressure, washed with DMF and MeOH and allowed to dry.

In the next step, the residue at position P2 (serine) was attached.

All amino acid additions were preceded by washing the resin with DMF for 5 minutes. Subsequent attachments used diisopropylcarbodiimide as a coupling agent. The procedure was repeated twice.

After each acylation, the resin wash cycle was initiated, followed by a chloranil assay to monitor the attachment of the amino acid derivative to the free amino groups of the resin.

Solvent wash cycle: DMF 3x2 minutes

IsOH 3x2 minutes

DCM 3x2 minutes Chloranil test:

PL 238 575 B1

As a result of the tests, after the first two couplings, the color of the grains was first green and then gray, so it was necessary to perform another acylation, as a result of which the resin grains tested with the chloranil test were colorless. This indicated the attachment of ANB to the TentaGel S RAM resin, which allowed to proceed to the next stage of peptide synthesis.

Attaching further protected amino acid residues:

The resin along with the attached ANB residue in the reactor was washed with DMF, and then the Fmoc shell of the amino group was deprotected to attach the protected amino acid derivative of glycine.

Removal of the Fmoc cover: DMF 1x5 minutes

20% piperidine in NMP 1x3 minutes

20% piperidine in NMP 1x8 minutes

Solvent wash cycle: DMF 3x2 minutes

IsOH 3x2 minutes

DCM 3x2 minutes Chloranil test:

As a result of the chloranil test, a positive result was obtained, as evidenced by the green color of the resin grains. This allowed for the commencement of the next stage - the attachment of the Fmoc-Trp-OH amino acid residue.

Attaching an amino acid derivative:

Performed couplings were preceded by washing the resin in DMF. During the attachment of the protected glycine residue, the composition of the coupling mixture remained unchanged.

After completion of each acylation, the solvent washes were carried out according to the procedure given, followed by a chloranil test for the presence of free amino groups in the solution.

Solvent wash cycle: DMF 3x2 minutes

IsOH 3x2 minutes

DCM 3x2 minutes Chloranil test:

The resin grains of the test carried out after the second acylation were colorless, which allowed for the next stage of synthesis, which was the introduction of further protected amino acid derivatives - valine, lysine, methionine and 2-aminobenzoic acid molecule. Coupling processes took place according to the procedure discussed earlier.

Tests carried out after the attachment of the abovementioned residues showed positive results, the resin grains were colorless.

b) Removing the peptide from the support

After the synthesis was completed, the peptide amide was removed from the support along with the removal of the side covers with the mixture: TFA: phenol: water: TIPS (88: 5: 5: 2, v / v / v / v) in a round bottom flask on a magnetic stirrer.

After 3 hours, the contents of the flask were vacuum filtered on a Schott funnel, washing with diethyl ether. The resulting pellet was centrifuged on a SIGMA 2K30 centrifuge (Laboratory Centrifuges) for 20 minutes. The pellet obtained after centrifugation was dissolved in water with the use of ultrasound, and then it was lyophilized.

c) New compound identity / characterization - HPLC, MS and NMR analysis

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

tR 20.1

MS MALDI TOF matrix alpha cinnamic acid MW 845.4 Da calculated 844.4.

Incubation of this compound under the following conditions:

- 3 μl of urine from patients diagnosed with cancer of the urinary tract epithelium or urine from a healthy patient

- 177 μl buffer (50 mM Tris-HCl, pH 8.3)

- 20 μl of ABZ-Met-Lys-Val-Trp-ANB-NH2 substrate (0.5 mg / ml)

- time 60 minutes

- the temperature of 37 ° C gives the results which are shown below.

HPLC analysis. The results are shown in Figures 1-4.

PL 238 575 Β1

The proteolytic activity of enzymes present in the tested biological material (urine samples with diagnosed cancer) is proportional to the intensity of the observed absorbance signal, released as a result of the hydrolysis of the ANB peptide bond. The hydrolysis of the substrate ABZ ¹ -Met ² -Lys ³ -Val ⁴ -Trp ⁵ -ANB-NH2 ⁶ takes place in position 5, where we obtain ABZ ¹ -Met ² -Lys ³ -Val ⁴ -Trp ⁵ and ANB-NH2.

The peptide (substrate) with the sequence ABZ ¹ -Met ² -Lys ³ -Val ⁴ -Trp ⁵ -ANB-NH2 ⁶ shows low absorption at 405/410 nm. During the hydrolysis, free ANB-NH2 molecule is released, which shows a maximum absorbance at 405/410. In other words, peptide hydrolysis causes an increase in ANB-NH2 concentration and thus an increase in absorbance at 405/410 nm.

From the graph (Fig. 5) we can read that in the case of samples 5, 6, 4 and 60, the decomposition of the ABZ-Met-Lys-Val-Trp-ANB-NH2 substrate occurs more efficiently than in the case of materials 40, 3, 9 or 7. Such a result may be due to the difference in activity as well as the amount of enzymes responsible for proteolysis.

Inhibitor Type A class of inhibited enzymes Final concentration in the measuring well [M] Cartilzomib Irreversible trconin proteinases (chymotrypsin activity of the human 20S protcasome) 1,000 X 10 ^s Bestatin irreversible aminopeptidases 3.625 x 10 ° C AEBSF irreversible serine proteinases 5.215 χ JO ⁴ E-64 irreversible cysteine proteinases 3.495 x 10 ° C Leupeptin reversible serine and cysteine proteinases 2.630 χ W ⁴

Proteolytic activity is dependent on the presence of specific compounds called inhibitors, so we added effective concentrations to inhibit specific classes of proteolytic enzymes to pooled urine samples of sick people (urinary tract cancer). The result of this experiment is shown in Fig. 6.

As a result of this experiment, it was found that in the tested material there are at least two enzymes showing maximum activity at acidic and alkaline pH.

The use of inhibitors of different classes of enzymes did not decrease the proteolytic activity. Only incubation of urine with leupeptin resulted in a reduction in the hydrolysis efficiency. Such a result might suggest the presence of serine or cysteine proteinases. The other inhibitors used also inhibit these enzyme classes, hence the conclusion that the enzymatic activity in the urine of people diagnosed with bladder cancer is due to enzymes belonging to a different type than those listed in the table above (Fig. 6). .

The enzymatic activity is influenced by many factors, one of which is the pH of the reaction medium. The conducted experiment may indicate that there are enzymes in the tested urine that prefer different conditions. We observe two maxima of proteolytic activity, suggesting the presence of at least two proteinases - at least one of which hydrolyzes under acidic conditions, the other basic (Fig. 7).

To increase the sensitivity of the detection method, the obtained biological material (human urine with diagnosed cancer of the urinary system epithelium) was concentrated. For this purpose, Amicon filter columns were used. As a result of this process, a four times densified material was obtained with an activity approximately four times higher than that of the starting material (Fig. 8). The fact of a linear response to increasing enzyme concentration confirms the selectivity of the compound obtained.

Claims (4)

Patent claims 1. A compound of the general formula: ABZ ¹ -Met ² -Lys ³ -Val ⁴ -Trp ⁵ -ANB-NH ₂ ⁶ where: ABZ means 2-aminobenzoic acid ANB is 5-amino-2-nitrobenzoic acid. PL 238 575 B1 2. Diagnostic marker with the general formula ABZ ¹ -Met ² -Lys ³ -Val ⁴ -Trp ⁵ -ANB-NH2 ⁶ , where: ABZ means 2-aminobenzoic acid, ANB means 5-amino-2-nitrobenzoic acid for use in diagnostics epithelial tumors. 3. The marker for use according to claim 1 The method of claim 2, wherein the detected neoplasm is an epithelial neoplasm of the urinary system. 4. The marker for use according to claim 1 The method of claim 2, wherein the use relates to biological material in the form of urine.