COMPOUNDS AND METHODS FOR THE IMMOBILIZATION OF MYOSTATIN-INHIBITORS ON THE EXTRACELLULAR MATRIX BY TRANSGLUTAMINASE

Abstract

The present invention relates to compounds and their use in the treatment of lesions, in tissue regeneration and/or tissue engineering. The compounds act as substrates for enzymes having transglutaminase activity and are suitable for their immobilization and/or attached therapeutic or diagnostic molecules on extracellular matrix (ECM) or synthetic ECM-derived materials, in particular for the immobilization of myostatin inhibitors.

Claims

1. A synthetic compound suitable for transglutaminase-mediated binding or attachment or incorporation of a therapeutic and/or diagnostic molecule into an extracellular matrix or a synthetic extracellular matrix component, wherein said compound comprises (a) at least one anchor domain and (b) at least one second domain, wherein said second domain possesses therapeutic and/or preventive activity, wherein said anchor domain is selected from the group consisting of: i) the D domain of Insulin Growth Factor-1 (IGF-I) as depicted in SEQ ID NO: 1, ii) a derivative of i) having at least 50% sequence identity with SEQ ID NO: 1, iii) a fragment of i) or ii), wherein said fragment comprises at least the four C-terminal amino acid residues depicted in SEQ ID NO: 1, or a fragment comprising the at least five, or a fragment comprising the at least six, or a fragment comprising the at least seven, or a derivative thereof having at least 75% identity to the amino acid sequences depicted in SEQ ID NO: 1, and wherein said second domain is directly or indirectly linked to any of the anchor domains referred to in i) to iii), and wherein said second domain comprises a molecule that inhibits and/or reduces and/or suppresses the activity of myostatin.

2. (canceled)

3. The synthetic compound according to claim 1, wherein the second domain is selected from the group consisting of a binder of myostatin, a small molecule, a protein, and a protein/peptide-polymer conjugate.

4. The synthetic compound according to claim 1, wherein the second domain comprises an amino acid sequence as defined in SEQ ID NO: 12 or a functional derivative thereof, wherein, said functional derivative hasan inhibitory activity on myostatin.

5. The synthetic compound according to claim 1, wherein said compound further comprises a cleavable linker between the anchor domain and the at least one second domain, wherein the linker has an amino acid sequence as depicted in SEQ ID NO: 13, or a functional derivative thereof.

6. A pharmaceutical composition or formulation comprising a synthetic compound as defined in claim 1.

7. The pharmaceutical composition or formulation according to claim 6, wherein said composition or formulation is suitable for localized administration.

8. A method for the treatment and/or prevention and/or diagnostic monitoring of a pathological condition selected from the group consisting of lesions. wounds, burns, excessive scar formation, post-operative scarring, fibrosis, lesioned sites or locations of injured or wounded and/or surgically treated tendons and/or ligaments, cosmetic surgically treated tissues, inflammation in lesions, impaired wound healing, complications of wound healing due to bacterial infections, due to age, due to diabetes, due to cardiovascular disorders, due to sensory neuropathies, and/or due to autoimmune diseases, wherein said method comprises administering, to a subject in need of such treatment and/or prevention and/or monitoring, a synthetic compound of claim 1.

9. The method according to claim 8, used for treatment and/or prevention, wherein the method further comprises administration of defined in any of the preceding claims or a pharmaceutical composition or formulation according to any of the preceding claims for use in the treatment and/or prevention of a an enzyme or fragment having transglutaminase activity.

10. The pharmaceutical composition according to claim 6, wherein said pharmaceutical composition is suitable for administration selected from transdermal, ophthalmic, nasal, otologic, enteral, pulmonal, urogenital, subcutaneous, intra-articular, intravenous, intracardiac, intramuscular, intraosseous and intraperitoneal administration.

11. A device comprising a synthetic compound as defined in claim 1.

12. The device according to claim 11, wherein the device is suitable as a delivery system for immediate and/or sustained release of said compound.

13. The device according to claim 12, wherein said device is selected from the group consisting of patches, implants, scaffolds, porous vascular grafts, stents, and/or wound dressings.

14. An in vitro method of tissue engineering comprising the steps: providing an extracellular matrix substrate comprising a specific amino acid sequence serving as a target for transglutaminase, providing a synthetic compound as defined in claim 1, exposing the extracellular matrix substrate and the compound as defined in steps (i) and (ii) to an enzyme having transglutaminase activity under conditions and in a medium suitable for transamidation.

15. An artificial tissue obtainable by a method according to claim 14.

16. A method for the treatment and/or prevention of a pathological condition selected from the group consisting of lesions, wounds, burns, excessive scar formation, post-operative scarring, fibrosis, lesioned sites or locations of injured or wounded and/or surgically treated tendons and/or ligaments, cosmetic surgically treated tissues, inflammation in lesions, impaired wound healing, complications of wound healing due to bacterial infections, due to age, due to diabetes, due to cardiovascular disorders, due to sensory neuropathies, and due to autoimmune diseases wherein said method comprises the use of an artificial tissue according to claim 15.

17. The synthetic compound according to claim 1, wherein said second domain comprises a molecule that inhibits and/or reduces and/or suppresses the activity of human myostatin.

18. The synthetic compound according to claim 3, wherein the second domain is an antibody, an antibody fragment, an antibody derivative, an aptamer, a non-Ig-scaffold, an antibody-drug conjugate, an antagonist, a myostatin inhibitor or a myostatin suppressor.

19. The method according to claim 9, wherein said enzyme is selected from the group consisting of FXIII, TG1, TG2, TG3, TG4, TG5, TG6 and TG7.

20. The method according to claim 9, wherein said enzyme is human Factor XIIIa.

21. The device according to claim 13, wherein said device is made of biocompatible fleece, hydrocolloid, polyacrylate, alginate, hydrogel, or foam, and/or an artificially produced tissue, bone-replacement, polymer network, fibrin, collagen, elastin, hyaluronic acid or silk protein.

Description

DESCRIPTION OF FIGURES

[0190] FIG. 1: By topical application of the myostatin inhibitor together with the crosslinking enzyme in one hydrogel/TTS onto the wound, the organism is supported in the recovery process.

[0191] FIG. 2: ECM compositions in healthy skin and during wound healing.

[0192] FIG. 3: Schematic illustration of in situ ECM immobilization of the myostatin inhibitor (MI) mediated by human fXIIIa. (A) In situ MI immobilization on the ECM protein fibronectin through integration of IGF-I's D domain. (B) Integration of a protease cleavable linker (e.g. derived from collagen type I) to enable a faster release as response to matrix metalloproteinase upregulation in inflamed environment.

[0193] FIG. 4: Immobilization of the MI-D chain on decellularized ECM. (A) Coupling efficacy in presence and absence of TG. (B) Confocal Laser Scanning Microscopy images of MI labeled with Atto594-maleimide on ECM in presence (panels A) and absence (panels B) of FXIIIa. The red fluorescence (1) shows immobilized MI and the green fluorescence (2) an antibody-staining of fibronectin (3 represents the overlay). (C) Amount of free myostatin after exposure to ECM with immobilized MI under different conditions measured by ELISA. (D) Bioactivity of MI immobilized on ECM determined by differentiation of myoblasts in presence of myostatin-containing medium and MI bound on ECM in presence or absence of TG.

[0194] FIG. 5: (A) Release kinetics of MI modified with a PCL—responding to elevated MMP levels—and the D chain for immobilization on ECM (“MI1”) in comparison to MI modified with the D chain at the C-terminus for direct immobilization on ECM (“MI2”). The MI was labelled with a fluorescent dye before immobilization on the ECM and the release of MI exposed to MMP-1, -8, -9 and -13 for different periods of time was analyzed by HPLC with fluorescence detection. (B) Bioactivity of released MI determined by myoblast proliferation in presence of myostatin and MI after release from ECM triggered by MMPs.

[0195] FIG. 6: Illustration of the in situ delivery of MI-D chain to injured tissue. In the injured tissue, transglutaminase catalyzes the crosslinking of the MI with endogenous fibronectin on the ECM, resulting in local accumulation of the MI, thereby neutralizing upregulated myostatin at the wound site.

REFERENCES

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