Peptides as active agents to stabilize biological barriers
Abstract
Peptide consisting of the amino acid sequence GX1RPX2X3X4 X5GGX6 (SEQ ID NO: 1) in which X1 is an amino acid selected from the group consisting of R and A; X2 is both omitted and is an amino acid selected from the group consisting of L and V; X3 is both omitted and is an amino acid sequence consisting of 1 to 5 amino acids; X4 is both omitted and is an amino acid sequence consisting of GG; X5 represents two amino acids selected from the group consisting of A, I and S; and X6 is both omitted and is an amino acid sequence consisting of 1 to 5 amino acids.
Term
4.7 yearsto projected expiry
Projected expiry 17 June 2031, counted from filing; an application has no term until it is granted.
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1 claim: 1 independent, 0 dependent
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138 paragraphs in 4 sections, as filed
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GEF activity was measured as fluorescence using Fluoroskan Ascent FL 2.6 from Thermo Electron Corporation. The wavelength of the excitation filter was set at 355 nm and the wavelength of the emission filter at 460 nm.
Table 1
Relative values compared to unstimulated control; (*) p <0.05 compared to tests without XIB1-a or XIB1-b
<dl><dt>ECV 304 cells </dt><dd>Half FROM </dd></dl>
<dl><dt>1 min control peptide </dt><dd> 1 0,5 </dd></dl>
<dl><dt>5 min control peptide </dt><dd> 1 0,5 </dd></dl>
<dl><dt>XIB1-a or XIB1-b 1 min </dt><dd> 1 0,4 </dd></dl>
<dl><dt>XIB1-a or XIB1-b 5 min </dt><dd> 1 0,3 </dd></dl>
<dl><dt>Thrombin 1 U / ml 1 min </dt><dd> 5 1,1 </dd></dl>
<dl><dt>Thrombin 1 U / ml 5 min </dt><dd> 3,4 0,9 </dd></dl>
<dl><dt>1 U / ml thrombin + XIB1-a or XIB1-b; 1 min</dt><dd> 2,5* 1 </dd></dl>
<dl><dt>1 U / ml thrombin + XIB1a or XIB1-b; 5 min</dt><dd> 1,5* 0,2 </dd></dl>
<dl><dt>LPS 100 µg / ml, 1 min </dt><dd> 3,2 1,1 </dd></dl>
<dl><dt>LPS 100 µg / ml, 5 min </dt><dd> 3,2 1,1 </dd></dl>
<dl><dt>LPS 100 µg / ml + XIB1-a or XIB1-b, 1 min </dt><dd> 1,7* 0,9 </dd></dl>
<dl><dt>LPS 100 µg / ml + XIB1a or XIB1-b, 5 min </dt><dd> 1,3* 0,8 </dd></dl>
<dl><dt>CaCo-2 </dt><dd>Half FROM </dd></dl>
<dl><dt>1 min control peptide </dt><dd> 1 0,2 </dd></dl>
<dl><dt>5 min control peptide </dt><dd> 1 0,3 </dd></dl>
<dl><dt>XIB1-a or XIB1-b 1 min </dt><dd> 1 0,5 </dd></dl>
<dl><dt>XIB1-a or XIB1.b 5 min </dt><dd> 1 0,5 </dd></dl>
<dl><dt>PMA 1 µg / ml 1 min </dt><dd> 2,5 0,5 </dd></dl>
<dl><dt>PMA 1 µg / ml 5 min </dt><dd> 1,8 0,8 </dd></dl>
<dl><dt>PMA 1 µg / ml + XIB1-a or XIB1-b; 1 min</dt><dd> 1,4* 0,2 </dd></dl>
<dl><dt>PMA 1 µg / ml + XIB1a or XIB1-b; 5 min</dt><dd> 1,5 0,3 </dd></dl>
14
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Table2
Relative values compared to unstimulated control; (*) p <0.05 compared to tests without XIB1-a or XIB1-b
<dl><dt>ECV 304 cells </dt><dd>Half FROM </dd></dl>
<dl><dt>1 min control peptide </dt><dd> 1 0,1 </dd></dl>
<dl><dt>5 min control peptide </dt><dd> 1 0,3 </dd></dl>
<dl><dt>XIB1-a or XIB1-b 1 min </dt><dd> 1 0,5 </dd></dl>
<dl><dt>XIB1-a or XIB1-b 5 min </dt><dd> 1 0,4 </dd></dl>
<dl><dt>Thrombin 1 U / ml 1 min </dt><dd> 4,3 1,1 </dd></dl>
<dl><dt>Thrombin 1 U / ml 5 min </dt><dd> 3,2 1,3 </dd></dl>
<dl><dt>1 U / ml thrombin + XIB1-a or XIB1-b; 1 min</dt><dd> 2,1* 0,5 </dd></dl>
<dl><dt>1 U / ml thrombin + XIB1-a or XIB1-b; 5 min</dt><dd> 1,9* 0,4 </dd></dl>
<dl><dt>LPS 100 µg / ml; 1 min</dt><dd> 2,7 1,1 </dd></dl>
<dl><dt>LPS 100 µg / ml; 5 min</dt><dd> 2,9 1,1 </dd></dl>
<dl><dt>LPS 100 µg / ml + XIB1-a or XIB1-b, 1 min </dt><dd> 1, 6* 1,6 </dd></dl>
<dl><dt>LPS 100 µg / ml + XIB1-a or XIB1-b 5 min </dt><dd> 1,7* 1 </dd></dl>
<dl><dt>CaCo-2 </dt><dd>Half FROM </dd></dl>
<dl><dt>1 min control peptide </dt><dd> 1 0,3 </dd></dl>
<dl><dt>5 min control peptide </dt><dd> 1 0,2 </dd></dl>
<dl><dt>XIB1-a or XIB1-b 1 min </dt><dd> 1 0,4 </dd></dl>
<dl><dt>XIB1-a or XIB1-b 5 min </dt><dd> 1 0,2 </dd></dl>
<dl><dt>PMA 1 µg / ml 1 min </dt><dd> 2,3 1,2 </dd></dl>
<dl><dt>PMA 1 µg / ml 5 min </dt><dd> 2,0 0,6 </dd></dl>
<dl><dt>PMA 1 µg / ml + XIB1-a or XIB1-b 1 min </dt><dd> 1,4* 0,5 </dd></dl>
<dl><dt>PMA 1 µg / ml + XIB1-a or XIB1-b 5 min </dt><dd> 1,3* 0,1 </dd></dl>
<dl><dt>HpMec </dt><dd>Half FROM </dd></dl>
<dl><dt>1 min control peptide </dt><dd> 1 0,3 </dd></dl>
<dl><dt>5 min control peptide </dt><dd> 1 0,2 </dd></dl>
<dl><dt>XIB1-a or XIB1-b 1 min </dt><dd> 1 0,4 </dd></dl>
<dl><dt>XIB1-a or XIB1-b 5 min </dt><dd> 1 0,2 </dd></dl>
<dl><dt>Thrombin 1 U / ml 1 min </dt><dd> 3,8 1,1 </dd></dl>
<dl><dt>Thrombin 1 U / ml 5 min </dt><dd> 3,2 1,2 </dd></dl>
<dl><dt>Thrombin 1 U / ml + XIB1-a or XIB1-b 1 min </dt><dd> 2,1* 0,3 </dd></dl>
<dl><dt>Thrombin 1 U / ml + XIB1-a or XIB1-b 5 min </dt><dd> 1,3* 0,4 </dd></dl>
As can be derived from Table 2, in ECV 304 cells, thrombin and LPS stimulation produced an increase in RhoA activity compared to untreated control cells. ECV 304 cells stimulated with thrombin or LPS in the presence of XIB1-a or XIB1-b show a significant reduction in RhoA activity compared to
16
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(continuation)
<dl><dt>ECV 304 cells </dt><dd>Half FROM </dd></dl>
<dl><dt>Thrombin 1 U / ml 5 min </dt><dd> 4 1,4 </dd></dl>
<dl><dt>1 U / ml thrombin + XIB1-a or XIB1-b; 1 min</dt><dd> 1* 0,3 </dd></dl>
<dl><dt>1 U / ml thrombin + XIB1-a or XIB1-b; 5 min</dt><dd> 1* 1,1 </dd></dl>
<dl><dt>LPS 100 µg / ml 1 min </dt><dd> 3 1,2 </dd></dl>
<dl><dt>LPS 100 µg / ml 5 min </dt><dd> 3 1,3 </dd></dl>
<dl><dt>LPS 100 µg / ml + XIB1-a or XIB1-b, 1 min </dt><dd> 1* 1,1 </dd></dl>
<dl><dt>LPS 100 µg / ml + XIB1-a or XIB1-b, 5 min </dt><dd> 1* 1,2 </dd></dl>
<dl><dt>CaCo-2 </dt><dd>Half FROM </dd></dl>
<dl><dt>1 min control peptide </dt><dd> 0 0 </dd></dl>
<dl><dt>5 min control peptide </dt><dd> 0 0 </dd></dl>
<dl><dt>XIB1-a or XIB1-b 1 min </dt><dd> 0 0 </dd></dl>
<dl><dt>XIB1-a or XIB1-b 5 min </dt><dd> 0 0 </dd></dl>
<dl><dt>PMA 1 µg / ml 1 min </dt><dd> 3 0,3 </dd></dl>
<dl><dt>PMA 1 µg / ml 5 min </dt><dd> 3 1,2 </dd></dl>
<dl><dt>PMA 1 µg / ml + XIB1-a or XIB1-b; 1 min</dt><dd> 1* 1,1 </dd></dl>
<dl><dt>PMA 1 µg / ml + XIB1-a or XIB1-b; 5 min</dt><dd> 2* 1,3 </dd></dl>
<dl><dt>HpMec </dt><dd>Half FROM </dd></dl>
<dl><dt>Control peptide; 1 min</dt><dd> 0 0 </dd></dl>
<dl><dt>Control peptide; 5 min</dt><dd> 0 0 </dd></dl>
<dl><dt>XIB1-a or XIB1-b 1 min </dt><dd> 0 0 </dd></dl>
<dl><dt>XIB1-a or XIB1-b 5 min </dt><dd> 0 0 </dd></dl>
<dl><dt>Thrombin 1 U / ml 1 min </dt><dd> 4 2 </dd></dl>
<dl><dt>Thrombin 1 U / ml 5 min </dt><dd> 4 1,8 </dd></dl>
<dl><dt>1 U / ml thrombin + XIB1-a or XIB1-b; 1 min</dt><dd> 1* 0,5 </dd></dl>
<dl><dt>1 U / ml thrombin + XIB1-a or XIB1-b; 5 min</dt><dd> 0,3* 0,3 </dd></dl>
As can be derived from Table 3, in ECV 304 cells, thrombin and LPS stimulation induced MLC phosphorylation and actin tension fiber formation. ECV 304 cells stimulated with thrombin or LPS in the presence of XIB1-a or XIB1-b show a significant reduction in MLC phosphorylation and actin stress fiber formation compared to treatment with thrombin or LPS alone.
In CaCo-2 cells, a stimulus with PMA induced an increase in MLC phosphorylation and actin stress fiber formation after 1 min and after 5 min of stimulation. The magnitude of cytoskeletal activation after 1 and after 5 min was significantly reduced when CaCo-2 cells were co-treated with PMA and XIB1-a or XIB1-b.
In HepMec cells, thrombin induced an increase in MLC phosphorylation and actin stress fiber formation
18
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after 1 min and after 5 min of stimulation. Co-treatment of cells with XIB1-a or XIB1-b significantly reduced the magnitude of cytoskeletal activation after 1 min and after 5 min. Treatment with XIB1-a or XIB1-b alone did not alter the basic cytoskeletal activity.
The results demonstrate that the peptides of the present invention such as XIB1-a or XIB1-b reduce phosphorylation
5 of MLC and actin stress fiber formation induced by different stimulating agents in epithelial and endothelial cells. The phosphorylation of MLC and the formation of actin stress fibers are controlled by RhoA activity as described above. The results demonstrate that the peptides of the present invention such as XIB1-a and / or XIB1-b are decreasing the phosphorylation of MLC and actin tension fiber by inhibiting GEF activity and subsequent RhoA activity and thus are useful in the treatment and / or prevention of diseases or disorders
10 associated with a localized or systemic rupture of epithelial or endothelial barrier functions. Particular diseases and disorders include burns, acute lung injury (APL), acute respiratory distress syndrome (ARDS), respirator-induced lung injury (LPIR), systemic inflammatory response syndrome (SRIS), acute renal injury (ARF), septicemia , multi-organ dysfunction syndrome (SDMO) or edema.
Example 5: Endothelial and epithelial permeability
fifteen The following cell lines were used to measure permeability through endothelial and epithelial barriers: Caco-2, ECV 304 and HpMec. All cells were grown at confluence at standard conditions on a Transwell system (Costar) with a pore size of 4 µm. At the beginning of the experiment, the growth media were removed and replaced with Hank buffered saline. The upper chamber was supplemented with 2 mg / ml of FITC-labeled dextran (Sigma Aldrich). Cells were stimulated as indicated in Table 4. 30 min samples of the
twenty Lower chambers were collected and determined for fluorescence (Fluoroskan Ascent FL, Thermo Electron). 50 µg / ml of XIB1-a and XIB1-b were added when indicated.
Table4
Relative values compared to unstimulated control; (*) p <0.05 compared to tests without XIB1-a or XIB1-b; (*) p <0.05 compared to tests without XIB1-a or XIB1-b
<dl><dt>ECV 304 cells </dt><dd>Half FROM </dd></dl>
<dl><dt>Control peptide </dt><dd> 1 0,1 </dd></dl>
<dl><dt>XIB-1b </dt><dd> 1 0,3 </dd></dl>
<dl><dt>1 U / ml thrombin </dt><dd> 3 1,3 </dd></dl>
<dl><dt>Thrombin 1 U / ml + XIB1-a or XIB1-b </dt><dd> 1,5* 1 </dd></dl>
<dl><dt>LPS 100 µg / ml </dt><dd> 2,3 1 </dd></dl>
<dl><dt>LPS 100 µg / ml + XIB1 </dt><dd> 1,1* 0,6 </dd></dl>
<dl><dt>CaCo-2 </dt><dd>Half FROM </dd></dl>
<dl><dt>Control peptide </dt><dd> 1 0,3 </dd></dl>
<dl><dt>XIB1-a or XIB1-b </dt><dd> 1 0,3 </dd></dl>
<dl><dt>PMA 1 µg / ml 1 min </dt><dd> 2,5 1 </dd></dl>
<dl><dt>PMA 1 µg / ml + XIB1-a or XIB1-b </dt><dd> 1,4* 0,6 </dd></dl>
<dl><dt>HpMec </dt><dd>Half FROM </dd></dl>
<dl><dt>Control peptide </dt><dd> 1 0 </dd></dl>
<dl><dt>XIB1-a or XIB1-b </dt><dd> 1 0,5 </dd></dl>
19
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Contents4
1 sheet
Sheet 1
12 members in 8 offices
Priority claims9
| Document | Office | Kind | Date |
|---|---|---|---|
| 10102010 | Austria | A | |
| 10102010 | Austria | A | |
| 10102010 | Austria | – | |
| 2011060105 | European Patent Office (EPO) | W | |
| 2011060105 | European Patent Office (EPO) | W | |
| 10102010 | – | – | – |
| AT20100001010 | – | – | – |
| PCTEP2011060105 | – | – | – |
| WO2011EP60105 | – | – | – |
Members12
| Document | Office | Kind | |
|---|---|---|---|
| CA2802635A1 | Canada | A1 | |
| WO2011157819A2 | World Intellectual Property Organization (WIPO) | A2 | |
| WO2011157819A3 | World Intellectual Property Organization (WIPO) | A3 | |
| CN103038253A | China | A | |
| EP2582720A2 | European Patent Office (EPO) | A2 | |
| US2013143791A1 | United States of America | A1 | |
| JP2013529916A | Japan | A | |
| EP2582720B1 | European Patent Office (EPO) | B1 | |
| RU2012157399A | Russian Federation | A | |
| ES2486321T3This record | Spain | T3 | |
| US9012403B2 | United States of America | B2 | |
| JP5709985B2 | Japan | B2 |
Numbers
- Publication
- 2486321
- Publication, DOCDB
- 2486321
- Publication, EPODOC
- ES2486321T
- Application
- 11732400
- Application, DOCDB
- 11732400
- Application, EPODOC
- ES20110732400T
Titles2
- Spanish
- Péptidos como agentes activos para estabilizar barreras biológicas
- English
- Peptides as active agents to stabilize biological barriers
Classification
- CPC, 9
- C07K14/705
- C07K7/06
- A61K38/00
- A61P11/00
- A61P17/02
- A61P29/00
- A61P31/04
- A61P43/00
- C07K7/08
- IPC, 1
- C07K14 75