WO2021201813A1 - The method of inhibiting of the fibrin polymerization - Google Patents
The method of inhibiting of the fibrin polymerization Download PDFInfo
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- WO2021201813A1 WO2021201813A1 PCT/UA2021/000030 UA2021000030W WO2021201813A1 WO 2021201813 A1 WO2021201813 A1 WO 2021201813A1 UA 2021000030 W UA2021000030 W UA 2021000030W WO 2021201813 A1 WO2021201813 A1 WO 2021201813A1
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- fibrin
- polymerization
- peptides
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- mixture
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/36—Blood coagulation or fibrinolysis factors
- A61K38/363—Fibrinogen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/36—Blood coagulation or fibrinolysis factors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
Definitions
- the invention relates to medicine and biotechnology and consists in suppression of intravascular thrombosis by inhibition of fibrin polymerization by mixture of synthetic peptides - structural analogues of the coiled-coil region fragments of fibrin(ogen); the invention can be used to create an effective antithrombotic drugs.
- Pathological activation of the blood coagulation system results in intravascular thrombosis caused by the appearance of fibrin in the bloodstream and its subsequent polymerization with the formation of a three-dimensional thrombus scaffold, is the leading cause of life-threatening diseases such as acute myocardial infarction, ischemic stroke, pulmonary embolism, etc. [1]. Therefore, the suppression of pathological thrombosis is the main goal of all existing antithrombotic agents. However, there are few agents that directly inhibit the fibrin polymerization and the formation of a three-dimensional thrombus scaffold.
- the closest analogue of the claimed method is a method of inhibiting fibrin polymerization with peptides obtained from D-fragment of human fibrin by hydrolysis and subsequent chromatographic purification, in particular peptides containing amino acid sequences:
- This method has a number of disadvantages, in particular it uses high molecular weight peptides that can be immunogenic. Also, there are no described conditions for standardization of the drug developed on the basis of a mixture of peptides. Since peptides are derived from the D- fragment of human fibrinogen, there is a risk of contamination of the drug with viruses and prions.
- an object of the invention is providing an effective and safe method of inhibiting fibrin polymerization using a mixture of low molecular weight non- immunogenic peptides of a known composition which effectively inhibits the formation of a three-dimensional fibrin - thrombus scaffold.
- the problem is solved by the use of compatible peptides - structural analogues of individual regions of the fibrinogen coiled-coil region, which have a synergistic inhibition effect on fibrin polymerization processes.
- a mixture of these impurity-free peptides was dissolved in a pH 7.4 buffer with 0.15 M NaCl.
- a 30 m ⁇ solution of peptides (0.33 mM final concentration), human blood donors’ plasma (70 m ⁇ ), APTT reagent (70 m ⁇ ) were added to the well of the well plate and incubated at 25 °C for 3 min.
- 130 m ⁇ of a 25 mM CaCl 2 solution in 0.02 M HEPES buffer pH 7.4 with 0.15 M NaCl is added to the well.
- the formation of a fibrin clot is recorded by changes in turbidity of the incubation media using automatic microplate reader at a wavelength of 405 nm. Measurements carried out for 10 minutes.
- Inhibition of fibrin polymerization is recorded by extending the lag-phase of fibrin clot formation - the time from the addition of the CaCl 2 solution into the incubation media to the beginning of the phase of exponential growth of media turbidity
- the grid was washed first with 100 mM ammonium acetate buffer (pH 7.9), then with 10 mM ammonium acetate buffer (pH 8.5).
- the grid is laid on a drop of contrast agent previously applied to the surface of the Teflon block and after 1 minute the contrast is removed using a water-flow pump.
- Electron microscope 1-600 at 75 kV, at 20000-50000 magnification is used to obtain electron microscopic images of the media in which fibrin polymerization occurs: 60 s after the addition of thrombin to the control sample, the formation of fibrin protofibrils and their lateral association is observed (Fig. 2), after 180 s mature fibrils were observed (Fig.
- the claimed method allows to effectively and specifically inhibit the fibrin polymerization by a mixture of synthetic peptides MEILRGDFSSANN, QKRQKQVKDN, NPDESSKPN - structural analogues of fragments of the fibrin(ogen) coiled-coil region, which is the basis for the development of a new effective antithrombotic drug.
- Fig. 1 Prolongation of the lag-period of fibrin polymerization in blood plasma activated by APTT reagent in the presence of peptides (0.33 mM), which mimic fragments of the fibrin(ogen) coiled-coil region, in a mixture and separately: Aa91-103 (MEILRGDFSSANN), Bb126-135 (QKRQKQVKDN), g69-77 (NPDESSKPN). K - control in the absence of inhibitory peptides Fig 2. Electron microscopy of the media in which fibrin polymerization occurs in the absence of inhibitor peptides 60 seconds after initiation of fibrin polymerization by thrombin.
- Fig. 3 Electron microscopy of the media in which fibrin polymerization occurs in the absence of inhibitor peptides 180 seconds after initiation of fibrin polymerization by thrombin.
- Fig. 4 Electron microscopy of the media in which fibrin polymerization occurs in the presence of an equimolar mixture of peptides that mimic fragments of the fibrin(ogen) coiled-coil region: Aa91-103 (MEILRGDFSSANN), Bb126-135 (QKRQKQVKDN), g69-77 (NPDESSKPN) 60 seconds after initiation of fibrin polymerization by thrombin.
- MEILRGDFSSANN MEILRGDFSSANN
- Bb126-135 QKRQKQVKDN
- NPDESSKPN g69-77
- Fig. 5 Electron microscopy of the media in which fibrin polymerization occurs in the presence of an equimolar mixture of peptides that mimic fragments of the fibrin(ogen) coiled-coil region: Aa91-103 (MEILRGDFSSANN), Bb 126- 135 (QKRQKQVKDN), g69-77 (NPDESSKPN) 180 seconds after initiation of fibrin polymerization by thrombin.
- MEILRGDFSSANN MEILRGDFSSANN
- Bb 126- 135 QKRQKQVKDN
- NPDESSKPN g69-77
Abstract
The invention relates to the method of the inhibition of the fibrin polymerization by synthetic peptides mimicking fragments of the coiled-coil region fibrin(ogen), in which is used a mixture of non-immunogenic synthetic structural peptide analogues of human fibrinogen molecule fragments such as MEILRGDFSSANN, QKRQKQVKDN, NPDESSKPN in equimolar ratio.
Description
THE METHOD OF INHIBITING OF THE FIBRIN POLYMERIZATION
The invention relates to medicine and biotechnology and consists in suppression of intravascular thrombosis by inhibition of fibrin polymerization by mixture of synthetic peptides - structural analogues of the coiled-coil region fragments of fibrin(ogen); the invention can be used to create an effective antithrombotic drugs.
Pathological activation of the blood coagulation system results in intravascular thrombosis caused by the appearance of fibrin in the bloodstream and its subsequent polymerization with the formation of a three-dimensional thrombus scaffold, is the leading cause of life-threatening diseases such as acute myocardial infarction, ischemic stroke, pulmonary embolism, etc. [1]. Therefore, the suppression of pathological thrombosis is the main goal of all existing antithrombotic agents. However, there are few agents that directly inhibit the fibrin polymerization and the formation of a three-dimensional thrombus scaffold.
There are methods to inhibit the polymerization of fibrin by low molecular weight compounds sUch as, for example [2, 3]. These methods have a number of disadvantages, namely, the manufacture of inhibitor compounds by chemical synthesis involves the use of starting compounds, as well as the emergence of intermediate compounds, which are often toxic and can be found in trace amounts in the final product. The compounds used in such methods have low specificity to the fibrin molecule, therefore can have cross-affinity towards other blood proteins, which would interfere with their normal functioning.
There are methods to inhibit the polymerization of fibrin by monoclonal antibodies or their Fab-fragments, with the epitopes overlapping the sites of fibrin polymerization [4-6]. The main disadvantages of such methods are the high
molecular weight of antibodies (150 kDa) and their Fab-fragments (40-45 kDa), which determines the potential immunogenicity. The possible induction of an immune response significantly complicates the use of antibodies and their Fab- fragments as antithrombotic agents.
There are methods to inhibit the polymerization of fibrin by polypeptides that have affinity for those regions of the fibrin molecule that contain polymerization sites and sites of intermolecular interactions that cause self- assembly of fibrin molecules. In particular, there are methods in which individual peptides or an unfractionated mixture of such peptides or low molecular weight fragments obtained by plasmin hydrolysis of the fibrin(ogen) molecule are used [7- 9]. The disadvantages of such methods consist in the use of human fibrinogen as a source of functionally active peptide inhibitors, which is dangerous due to the possible contamination of drugs with viruses and prions. Also, the disadvantages of using fragments of fibrin(ogen) to inhibit the polymerization of fibrin consist in their potential immunogenicity. Another important drawback is the difficulty of standardizing the process of obtaining tryptic fibrinogen fragments due to the large number of factors affecting the processes of proteolysis and chromatographic separation of mixtures.
There are methods that involve the use of synthetic antithrombotic peptides. In particular, the use of the TKPRPGP peptide can reduce the intensity of thrombosis [10]. The disadvantages of this method consist in the claimed effects of the peptide on the components of the fibrinolysis system and platelets, which makes it difficult to control the effectiveness of its use.
There is a method of using the GPRP peptide and its conjugates, which allows blocking the polymerization site "a" -hole, thus preventing fibrin formation [11]. The disadvantages of this method consist in the ability of the peptide to interact not only with fibrin but also with fibrinogen, the concentration of which in blood plasma is very high (normal range - 2-4 g/L), so that high concentrations of
the peptide or its conjugates are required to achieve an anti-polymerization activity.
The closest analogue of the claimed method is a method of inhibiting fibrin polymerization with peptides obtained from D-fragment of human fibrin by hydrolysis and subsequent chromatographic purification, in particular peptides containing amino acid sequences:
TRWY SMKTTMKIIPFNRLTIGQHHHLGAKQAGD V, QAGDV and X- QAGDV, where X is AK- or K [12]. This method has a number of disadvantages, in particular it uses high molecular weight peptides that can be immunogenic. Also, there are no described conditions for standardization of the drug developed on the basis of a mixture of peptides. Since peptides are derived from the D- fragment of human fibrinogen, there is a risk of contamination of the drug with viruses and prions.
Therefore an object of the invention is providing an effective and safe method of inhibiting fibrin polymerization using a mixture of low molecular weight non- immunogenic peptides of a known composition which effectively inhibits the formation of a three-dimensional fibrin - thrombus scaffold.
The problem is solved by the use of compatible peptides - structural analogues of individual regions of the fibrinogen coiled-coil region, which have a synergistic inhibition effect on fibrin polymerization processes.
Example 1.
To characterize the inhibition activity of the peptide mixture, their effect on fibrin polymerization in a fibrinogen-thrombin system was studied using a combination of turbidimetry and electron microscopy technique as described in [13]. Peptides - structural analogues of fibrin(ogen) fragments of the coiled-coil region, namely Aa91-103 MEILRGDF S S ANN (1453 Da), Bb126-135 QKRQKQVKDN (1270 Da), g69-77 NPDESSKPN (986 Da) are synthesized
according to standard procedure [14], characterized by MALDI-TOF analysis [15] and lyophilized.
A mixture of these impurity-free peptides was dissolved in a pH 7.4 buffer with 0.15 M NaCl. A 30 mΐ solution of peptides (0.33 mM final concentration), human blood donors’ plasma (70 mΐ), APTT reagent (70 mΐ) were added to the well of the well plate and incubated at 25 °C for 3 min. Then 130 mΐ of a 25 mM CaCl2 solution in 0.02 M HEPES buffer pH 7.4 with 0.15 M NaCl is added to the well. The formation of a fibrin clot is recorded by changes in turbidity of the incubation media using automatic microplate reader at a wavelength of 405 nm. Measurements carried out for 10 minutes. Inhibition of fibrin polymerization is recorded by extending the lag-phase of fibrin clot formation - the time from the addition of the CaCl2 solution into the incubation media to the beginning of the phase of exponential growth of media turbidity.
The addition of each of the studied peptides separately leads to a prolongation of the lag-period of polymerization by 1.3-1.4 times (Fig. 1). At the same time, the synergistic effect of three peptides - structural analogues of the coiled-coil region of the fibrin(ogen) (Aa91-103 MEILRGDF S S ANN, Bb126-135 QKRQKQVKDN, g69-77 NPDESSKPN) causes inhibition of fibrin self-assembly and prolongation of the lag-period of polymerization by 2-2.2 times (Fig. 1).
Example 2.
The effect of a mixture of peptides - structural analogues of the fibrin(ogen) fragments of the coiled-coil region, namely Aa91-103 MEILRGDFSSANN, Bb126-135 QKRQKQVKDN, g69-77 NPDESSKPN on the polymerization of fibrin is studied by transmission electron microscopy of negative stained samples [13]. A 1% aqueous solution of uranyl acetate is used as a negative contrast. The electron microscopy grids are covered with a layer of 0.3 % solution of the fOrmware in dichloroethane and fixed by spraying carbon with a vacuum evaporator. To prepare the samples, 0.3 mg/ml human fibrinogen, 0.025 M CaCl2
in 0.05 M ammonium formiate buffer (pH 7.9), - a total sample volume of 220 mΐ were added sequentially to a sterile glass tube. The polymerization of fibrin is initiated by the addition of thrombin to a final concentration of 0.25 NTH/ml. After 90 and 180 s, aliquots are taken from the polymerization medium and diluted to a final fibrinogen concentration of 7 pg/ml, the 10 mΐ solution of which is transferred on a carbon-coated lattice, which after 2 minutes is treated with 1% uranyl acetate solution. After 5 minutes incubation, the grid was washed first with 100 mM ammonium acetate buffer (pH 7.9), then with 10 mM ammonium acetate buffer (pH 8.5). Next, the grid is laid on a drop of contrast agent previously applied to the surface of the Teflon block and after 1 minute the contrast is removed using a water-flow pump. Electron microscope 1-600 at 75 kV, at 20000-50000 magnification is used to obtain electron microscopic images of the media in which fibrin polymerization occurs: 60 s after the addition of thrombin to the control sample, the formation of fibrin protofibrils and their lateral association is observed (Fig. 2), after 180 s mature fibrils were observed (Fig. 3), in the presence of three peptides - structural analogues of the fibrin(ogen) coiled-coil region, namely Aa91-103 MEILRGDF S S ANN, Bb126-135 QKRQKQVKDN, g69-77 NPDESSKPN, no protofibrils were detected after 60 s (Fig. 4), single protofibrils were detected after 180 s (Fig. 5), indicating effective inhibition of fibrin self- assembly by synergic action of the studied peptides.
Thus, the claimed method allows to effectively and specifically inhibit the fibrin polymerization by a mixture of synthetic peptides MEILRGDFSSANN, QKRQKQVKDN, NPDESSKPN - structural analogues of fragments of the fibrin(ogen) coiled-coil region, which is the basis for the development of a new effective antithrombotic drug.
The claimed method was developed and tested in the protein structure and function department of Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine.
Figure captions
Fig. 1. Prolongation of the lag-period of fibrin polymerization in blood plasma activated by APTT reagent in the presence of peptides (0.33 mM), which mimic fragments of the fibrin(ogen) coiled-coil region, in a mixture and separately: Aa91-103 (MEILRGDFSSANN), Bb126-135 (QKRQKQVKDN), g69-77 (NPDESSKPN). K - control in the absence of inhibitory peptides Fig 2. Electron microscopy of the media in which fibrin polymerization occurs in the absence of inhibitor peptides 60 seconds after initiation of fibrin polymerization by thrombin.
Fig. 3. Electron microscopy of the media in which fibrin polymerization occurs in the absence of inhibitor peptides 180 seconds after initiation of fibrin polymerization by thrombin.
Fig. 4. Electron microscopy of the media in which fibrin polymerization occurs in the presence of an equimolar mixture of peptides that mimic fragments of the fibrin(ogen) coiled-coil region: Aa91-103 (MEILRGDFSSANN), Bb126-135 (QKRQKQVKDN), g69-77 (NPDESSKPN) 60 seconds after initiation of fibrin polymerization by thrombin.
Fig. 5. Electron microscopy of the media in which fibrin polymerization occurs in the presence of an equimolar mixture of peptides that mimic fragments of the fibrin(ogen) coiled-coil region: Aa91-103 (MEILRGDFSSANN), Bb 126- 135 (QKRQKQVKDN), g69-77 (NPDESSKPN) 180 seconds after initiation of fibrin polymerization by thrombin.
References
1. Lugovskoy EV, Makogonenko EM, Komisarenko SV. Molecular mechanisms of formation and destruction of fibrin. Kiev: Naukova Dumka, 2013. 230 p.
2. Pat. US 90494A1. Apr. 11, 2013.
3. Pat. US 8906421 B2. Dec. 9, 2014
4. Pat. RU 2152801 C2. 20.07.2000
5. Pat. EA 023477 Bl. 2016.06.30.
6. Pat. US 005487892 A. Jan. 30, 1996.
7. Pat. US 006413931 BL Jul. 2, 2000.
8. Pat. US9777054B2. 03.10.2017.
9. Pat. US5376631 A. 27.12.1994.
10. Pat. RU 2290195 Cl. 27.12.2006.
11. Pat. WO 2011 /006069. 13.01.2011.
12. Pat. US4455290A. 19.06.1984.
13. Lugovskoy E. V., Gritsenko P. G., Kolesnikova I. N., Lugovskaya N. E., Komisarenko S. V. A neoantigenic determinant in coiled coil region of human fibrin beta-chain. Thromb Res. 2009; 123(5): 765-770.
14. Andreev S.M., Samoilova N.A., Davidovich Yu.A., Rogozhin S.V. Polymeric reagents in peptide synthesis. Usp. chem., 1987; 56: 629-655.
15. Chapman J. R. Mass Spectrometry of Proteins and Peptides. Humana Press, 2000. 538.
Claims
Claims
Method of inhibiting of the fibrin polymerization by synthetic peptides mimicking fragments of the coiled-coil region of fibrin(ogen), in which a mixture of structural peptide analogues of human fibrinogen molecule fragments is used; that is characterized by using of a mixture of non-immunogenic synthetic peptides MEILRGDF S S ANN, QKRQKQVKDN, NPDESSKPN in equimolar ratio.
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| UAA202002123A UA127258C2 (en) | 2020-03-30 | 2020-03-30 | METHOD OF INHIBITING FIBRIN POLYMERIZATION |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4455290A (en) * | 1981-04-02 | 1984-06-19 | Research Corporation | Inhibition of fibrin polymerization by a peptide isolated from fibrin Fragment D1 |
| RU2290195C1 (en) * | 2005-04-21 | 2006-12-27 | Институт Молекулярной Генетики Российской Академии Наук (Имг Ран) | Agent possessing anti-coagulating, anti-thrombocytic, anti-thrombosis, fibrin-depolymerizing and fibrinolytic activity |
| US9777054B2 (en) * | 2005-08-23 | 2017-10-03 | Ension Inc. | Soluble fibrin inhibitory peptides and uses thereof |
-
2021
- 2021-03-29 WO PCT/UA2021/000030 patent/WO2021201813A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4455290A (en) * | 1981-04-02 | 1984-06-19 | Research Corporation | Inhibition of fibrin polymerization by a peptide isolated from fibrin Fragment D1 |
| RU2290195C1 (en) * | 2005-04-21 | 2006-12-27 | Институт Молекулярной Генетики Российской Академии Наук (Имг Ран) | Agent possessing anti-coagulating, anti-thrombocytic, anti-thrombosis, fibrin-depolymerizing and fibrinolytic activity |
| US9777054B2 (en) * | 2005-08-23 | 2017-10-03 | Ension Inc. | Soluble fibrin inhibitory peptides and uses thereof |
Non-Patent Citations (2)
| Title |
|---|
| LUGOVSKOY E. V., GRITSENKO P. G., KAPUSTIANENKO L. G., KOLESNIKOVA I. N., CHERNISHOV V. I., KOMISARENKO S. V.: "Functional role of Bβ-chain N-terminal fragment in the fibrin polymerization process", THE FEBS JOURNAL, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 274, no. 17, 1 September 2007 (2007-09-01), GB , pages 4540 - 4549, XP055935614, ISSN: 1742-464X, DOI: 10.1111/j.1742-4658.2007.05983.x * |
| STABENFELDT SARAH E. ET AL.: "A new direction for anticoagulants: Inhibiting fibrin assembly with PEGylated fibrin knob mimics", BIOTECHNOLOGY AND BIOENGINEERING, vol. 108, no. 10, 2011, pages 2424 - 2433, XP071115303, DOI: 10.1002/bit.23184 * |
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