COMPOUNDS FOR THE PREVENTION, TREATMENT AND DIAGNOSIS OF THROMBI

Abstract

The present invention relates to compounds for medical use in the treatment or in the prevention or in the diagnosis of arterial or venous thromboembolism.

Claims

1. Ligands of the neutrophil extra-cellular traps (NETs) for medical use in the treatment or in the prevention or in the diagnosis of arterial or venous thromboembolism, wherein said ligands do not include digoxigenin and distamycin for interventional neuroradiology.

2. Ligands according to the preceding claim, wherein said neutrophil extra-cellular traps (NETs) are within a thrombus.

3. Ligands of the extra-cellular chromatin for medical use according to the previous claim 1 or 2, wherein said ligands are selected from: DNA ligands, and Topoisomerases, and Histone ligands.

4. Ligands of the extra-cellular chromatin for medical use according to the previous claim, wherein said DNA ligands comprise: Intercalating ligands, Groove-binding ligands, and DNA alkylating agents.

5. Ligands of the extra-cellular chromatin for medical use according to the previous claim 3, wherein said intercalating ligands are selected in the group comprising: ciprofloxacine, methylene blue, berberine, proflavin, daunomycin, doxorubicin and thalidomide.

6. Ligands of the extra-cellular chromatin for medical use according to the previous claim 3, wherein said groove-binding ligands may be minor groove-binding ligands or major groove-binding ligands, selected in the group comprising: sulindac, netropsin, tallimustine, hairpin polyamides, bis(benzimidazoles), aureolic acids and bisquaternary ammonium heterocycles.

7. Ligands of the extra-cellular chromatin for medical use according to the previous claim 3, wherein said DNA alkylating agents have one of the following general structure: I) ω-azido polyethylene glycol methanesolfonate derivatives ##STR00009## wherein n is from 1 to 6; II) a 4-(bis(2-haloethyl)amino)phenyl moiety joined to a ω-azido polyethylene glycol chain with a carboxamido spacer ##STR00010## wherein n is from 1 to 6 m is from 0 to 6 X is halogen selected from Cl, Br, I; III) a benzoheterocycle such as benzo[b]furan, indole, N-alkylindole, indazole, benzo[b]thiophene bearing at their 5-position a bis(2-haloethyl)amino) moiety and joined by carboxamide group to a ω-azido polyethylene glycol spacer ##STR00011## wherein m is from 1 to 6 X is halogen selected from: Cl, Br, I Z is —CH— or —N— Y is —O—, —S—, —NH or N—C.sub.1-6alkyl alkyl chain.

8. Ligands of the extra-cellular chromatin for medical use according to the previous claim 3, wherein said DNA alkylating agents are selected in the group comprising: all bis-chloroethylamine or bis-bromoethylamine derivatives such as chlorambucil, norchlorambucil, bendamustin, bromo benzoic mustard or melphalan, Busulfan, N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-5-(bis(2-chloroethyl)amino)benzofuran-2-carboxamide (MBF) and 4-[Bis-(2-bromoethyl)amino]benzoic acid.

9. Ligands of the extra-cellular chromatin for medical use according to any one of the previous claims, wherein said ligand is selected in the group comprising: Digoxigenin, Distamycin, Ciprofloxacin, Thioridazine, Netropsin, Trabectedin, Sulindac, Piperaquine, Atabrine (Mepacrine), Mitonafide, Chloroquine, Amsacrine, Indomethacin, Methylene blue, berberine, proflavin, daunomycin, doxorubicin, thalidomide, tallimustin, hairpin polyamides, bis(benzimidazole), aureolic acids and bisquaternary ammonium heterocycles) and derivatives of any one of the above.

10. Ligands of the neutrophil extra-cellular traps (NETs) for medical use according to any one of the preceding claims, wherein said treatment comprises the treatment of a condition selected in the group comprising: occlusive vascular conditions.

11. Ligands of the neutrophil extra-cellular traps (NETs) for medical use according to the preceding claim, wherein said occlusive vascular condition leads to acute organ ischemia.

12. Ligands of the neutrophil extra-cellular traps (NETs) for medical use according to any one of the preceding claims 1 to 9, wherein said prevention comprises the prevention of a condition selected in the group comprising: distal embolization and pulmonary embolism.

13. Ligands of the neutrophil extra-cellular traps (NETs), which are used for the adhesion to or the removal of a thrombus.

14. Ligands of the neutrophil extra-cellular traps (NETs) for medical use according to any one of the preceding claims, wherein said ligands are within a substrate.

15. A substrate comprising one or more of the ligands according to any one of claims 1 to 14.

16. A substrate comprising one or more of the ligands according to any one of claims 1 to 8, which is in the form of a coating.

17. The substrate according to the preceding claim 16, wherein said coating comprises one or more layer or a polymer or of a co-polymer.

18. The substrate according to the preceding claim 17, wherein said polymer is selected from the group comprising: polydopamine, PEG-bis-amine and copolymer polydopamine and PEG-bis-amine.

19. The substrate according to any one of the preceding claims 16 to 18, which comprises a secondary coating.

20. The substrate according to the preceding claim, wherein said secondary coating is a saccharidic or a polysaccharidic substrate or is a polyglycolic acid (PGA), polylactic acid (PLA) or polyvinyl alcohol (PVA) substrate.

21. The substrate according to the preceding claim, wherein said secondary coating comprises mannitol.

22. The ligand according to any one of the preceding claims 1 to 14, which is derivatized with a suitable group.

23. The ligand according to any one of the preceding claims 1 to 14, which is derivatized with a suitable derivatization group via a suitable linker.

24. The ligand according to the preceding claim, wherein said linker is selected from the group of the linkers having any one of the following structures:
—(O—CH.sub.2—CH.sub.2).sub.n— wherein n is from 3 to 5, or
—(CH.sub.2).sub.m—O—(CH.sub.2).sub.n— wherein m and n are independently 3 to 7, or
-[(—CH.sub.2).sub.n—O-].sub.z-(CH.sub.2).sub.m—N.sub.3, wherein n is from 1 to 10, preferably from 2 to 6 and more preferably from 2 to 4, z is from 1 to 6, preferably from 1 to 4 and more preferably from 1 to 3, m is from 1 to 10 and preferably from 2 to 5.

25. The ligand according to any one of the preceding claims 22 to 24, wherein said suitable derivatization group is represented by azide (—N.sub.3).

26. The ligand according to the preceding claim, wherein the linker has any one of the following structures: ##STR00012##

27. The ligand according to any one of the preceding claims 22 to 25, which is selected from the group comprising: N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-5-(bis(2-chloroethyl)amino)benzofuran-2-carboxamide (benzofuran azide mustard, MBF), 4-[Bis-(2-bromoethyl)amino]benzoic acid (BBM), N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-3-(4-(bis(2-chloroethyl)amino)phenyl)propanamide (PPM, norchlorambucil-azide), N-(2-(2-(2-azidoethoxy)ethoxy)ethyl)-4-(4-(bis(2-chloroethyl)amino)phenyl)butanamide (chlorambucil-azide), 4-(4-(4-((5-azidopentyl)oxy)butyl)piperazin-1-yl)-7-chloroquinone (Piperaquine-azide), 4-[Bis-(2-bromoethyl)amino]benzoic acid (azobenzene-azide, BBM), busulfan-azide (MsA).

28. A process for the preparation of a coating for a device comprising the one or more of the ligands according to any one of claims 15 to 21 comprising a first step for the preparation of a solution or of a suspension of the substrate.

29. A device comprising a portion coated with one or more of the ligands according to any one of claims 1 to 14 of or the substrate of any one of claims 15 to 21.

30. The device comprising a portion coated with the ligand according to any one of claims 1 to 14 or the substrate according to any one of claims 15 to 21, which is selected in the group comprising thrombectomy device, flowretriever, filters, embolic protection devices, distal protection devices, including balloon angioplasty, inferior vena cava filters.

31. The device according to claim 29 or 30 for use in the field of interventional neuroradiology.

32. The device according to any one of claims 29 to 31 for use in a field, which is other than the interventional neuroradiology.

33. A method for the treatment of venous or arterial thromboembolism in a subject comprising the step of contacting a thrombus comprising neutrophil extra-cellular traps (NETs) with a ligand according to any one of claims 1 to 14 or with a substrate according to claim any one of claims 15 to 21.

34. A method for the diagnosis venous or arterial thromboembolism in a subject comprising the step of contacting a thrombus comprising neutrophil extra-cellular traps (NETs) with a ligand according to any one of claims 1 to 14 or with a substrate according to any one of claims 15 to 21.

35. Use of a ligand according to any one of claims 1 to 14 or of a substrate according to any one of claims 15 to 21 for the adhesion to a thrombus comprising neutrophil extra-cellular traps (NETs).

36. Use of a ligand according to any one of claims 1 to 14 or of a substrate according to any one of claims 15 to 21 for the adhesion and the removal of thrombus comprising neutrophil extra-cellular traps (NETs).

37. Use of a ligand according to any one of claims 1 to 14 or of a substrate according to any one of claims 15 to 21 in the field of interventional neuroradiology comprising neutrophil extra-cellular traps (NETs).

38. Use of a ligand according to any one of claims 1 to 14 or of a substrate according to any one of claims 15 to 21 in the field other than interventional neuroradiology.

Description

FIGURES

[0164] FIGS. 1 to 13 and 22 to 28 show the structure of some of the ligands of the invention.

[0165] FIG. 14 shows the results obtained with the procedure of Example 1 obtained with fluorescence microscopy.

[0166] FIG. 15 reports the graph with data on the Integrated Density (Average Signal Density multiplied by the area).

[0167] FIGS. 16 and 17 show the results of the comparative assay on the platelet and chromatin binding properties of some of the ligands of the invention as compared to neutrophil extra-cellular traps (NETs) as in Example 2.

[0168] FIGS. 18 and 19 report the structure of some of the linkers of the invention.

[0169] FIG. 20 shows the results of the comparative assay on the platelet and chromatin binding properties of some of the linkers used for piperaquine as the ligand.

[0170] FIG. 21 shows the steps of the experiment according to Example 3.

[0171] FIG. 29 reports the structure of polydopamine.

EXAMPLE 1

Analysis of Thrombus Material Captured by the Surfaces

[0172] Nitinol disks were dip-coated in three serial baths composed of

[0173] 1. Polydopamine (1 mg/ml in tris buffer, pH 8.5, 22±2 hours)

[0174] 2. DBCO-PEG 4-Amine

[0175] 3. Azide-derivatized DNA-binding compounds (ciprofloxacin-N.sub.3 or thioridazine-N.sub.3)

[0176] A fresh thrombus was prepared with human whole blood additioned with PMA 100 nM and ionomycin 1 μM to promote strong leukocyte activation leading to the release of extracellular chromatin within the thrombus. Slice of the same thrombus were layered onto coated and bare metal disks in the wells of a 48-well plate, covered with human plasma and incubated at 37° C. on an orbital shaker for 5 minutes. The thrombus slices were mechanically removed using tweezers, the disk washed in PBS and stained with a solution made of Hoechst 33342 (1 μg/ml, to stain DNA) and Evans Blue (3%, to stain proteins) in PBS for 5 minutes. After careful washing, the presence of thrombus material-DNA (DAPI channel) and proteins (Rhodamine channel)—captured by the experimental surfaces was revealed by fluorescence microscopy. A representative image of the disk observed in the two channels is shown in FIG. 12. The image analysis was performed using the Image J open software. Integrated Density (Average Signal Density multiplied by the area) analysis showed that ciprofloxacin and thioridazine-coated surfaces consistently adhere to and capture more DNA and proteins from the thrombus.

EXAMPLE 2

Evaluation of the Chromatin and Platelet Binding

[0177] FIG. 16 reports the results of an experiment carried out in order to assay the selective binding properties exerted by different coating on blood platelets as compared to extracellular chromatin (NETs).

[0178] Experimental Setting

[0179] Nitinol disks are left untouched (bare metal) or coated with the tested chromatin ligands following a polydopamine, DBCO-PEG4-amine and azide derivative ligand sequential dip-coating layering procedure. Platelet adhesion (left panel) was evaluated by image analysis of the surface after staining with phalloidin coupled to AlexaFluor®488 (green fluorescence) after immersion of the disks for 10 min in blood withdrawn in PPAK (thrombin inhibitor providing a transient anticoagulation).

[0180] An example of disk displaying a low platelet binding (MBF coating, top) as compared to high binding to bare metal (bottom) is shown.

[0181] The extracellular chromatin binding was evaluated by applying for 3 minutes the disks on the bottom of wells in which fresh human neutrophils were stimulated with nigericin for 4 hours (PMID: 28574339; doi: 10.7554/eLife.24437), to promote the formation of neutrophil extracellular traps (NETs). Extracellular chromatin adhering to the disk surface was stained with the cell impermeant nuclear dye Sytox Green. An example of disk displaying a high (MBF coating, top) as compared to the low binding to bare metal (bottom) chromatin binding is shown.

[0182] FIG. 17 reports the results of an experiment carried out in order to assay the binding properties exerted by different coating on blood platelets as compared to extracellular chromatin (NETs).

[0183] Example of the binding properties exerted by different coatings (N.sup.o=6/coating) on blood platelets as compared to extracellular chromatin (NETs).

Two-Way Dot Plot:

[0184] Each dot corresponds to the mean binding (% of max, mean±SEM) of chromatin (X-axis) and platelet (Y-axix) with the standard error bars parallel to the respective axis.

[0185] The best compounds are those in the right bottom quadrant, where the coatings display a high binding towards chromatin and poor binding towards blood platelets. At the opposite, the worst situation e.g. high platelet and low chromatin binding properties, is displayed in the left upper quadrant (bare metal surface).

[0186] The results obtained are shown in FIGS. 17 and 20 and are reported in the following table:

TABLE-US-00002 Chomatin Platelet binding binding Chromatin/Platelet Ligand (% of max) (% of max) binding ratio Bare metal  4.03 ± 1.15  74.03 ± 11.02 0.05 PDA 36.39 ± 6.16 48.32 ± 2.53 0.75 Ciprofloxacin 23.63 ± 9.59 27.57 ± 3.22 0.86 Distamycin 12.26 ± 4.14 12.42 ± 1.72 0.99 Anti-H1-antibody  5.7 ± 1.71  8.89 ± 2.42 0.64 PPM 36.75 ± 5.78  48.8 ± 5.59 0.75 MBF  66.07 ± 13.21 23.18 ± 4.05 2.85 Chlorambucil 39.38 ± 9.9  37.32 ± 2.76 1.06 Pipe-2 57.5 7.05   16 ± 5.78 3.59

EXAMPLE 3

Evaluation of the Effect of the Linker on the Binding Properties of a Given Ligand on Piperaquine

[0187] FIG. 19 shows the result of the experiment carried out to evaluate the effect of the linker on the binding properties of a given ligand on piperaquine.

[0188] Experimental Setting

[0189] Different linkers were combined with piperaquine and the resulting azide derivatives were used for coating nitinol disks (N=6 for each compound), following the described 3-step dip-coating procedure. Platelet adhesion (left panel) was evaluated by analyzing the surface and intensity of the phalloidin staining (green fluorescence) after immersion of the disks (N.sup.o=6/coating) for 10 min in blood withdrawn in PPAK (thrombin inhibitor providing a transient anticoagulation). The binding of extracellular chromatin was evaluated by applying for 3 minutes the disks on the bottom of wells in which fresh human neutrophils were stimulated with nigericin for 4 hours, to promote the formation of neutrophil extracellular traps (NETs). Extracellular chromatin was stained with the cell impermeant nuclear dye Sytox Green.

[0190] The “mirror” bars in this figure indicate the platelet (left) as compared to the chromatin (right) binding (% of max, mean±SEM) binding for each of the displayed coatings, sorted (from the top to the bottom) according to the ability of each compound to clutch chromatin (best binding profile on the top).

EXAMPLE 4

Evaluation of the Clot-Device Detaching Time

[0191] FIG. 21 shows the steps of the experiment carried out to evaluate the time required for the detaching of the captured clot from a stent-retriever.

[0192] For the purposes of this assay, several identical artificial clots are prepared using fresh human blood as previously described (PMID: 29695602; doi: 10.1136/neurintsurg-2017-013675) with the addition of nigericin to allow enrichment of NETs within the clot (PMID: 28574339; doi: 10.7554/eLife.24437). The clots are positioned at the bottom of individual small polypropylene tubes.

[0193] The experiment comprises the following phases:

[0194] 1) Sheathed clinical grade thrombectomy devices (bare metal or coated with indicated ligands, following a sequential dip-coating layering procedure) are passed between the clot and the wall of the tube and deployed once they reach the bottom of the tube. Their ability to capture the clot is monitored by direct video recording (A).

[0195] 2) The device is extracted from the tube and the occurrence of its tight adhesion to the clot is video-recorded (B).

[0196] 3) The device with the adhering clot is transferred into a 50-ml tube containing saline (PBS) (C).

[0197] 4) The device+adhering clot are vigorously agitated within the fluid (D).

[0198] 5) The detachment of the clot is directly monitored by continuous video-recording (E).

[0199] The time required for the clot to detach from the device is quantified by blinded operators on the recorded videos (experiments in duplicate) and the data are as follows:

TABLE-US-00003 Device type Time delay to clot detachment (seconds) Bare metal  0 ± 0 Piperaquine  5 ± 2 Chlorambucil 15 ± 3 Polydopamine 23 ± 2

[0200] From the above description, the advantages of the present invention will be clear to the person skilled in the art.

[0201] State of the art strategies aim at dissolving the extracellular chromatin to allow effective pharmacologic thrombolysis.

[0202] Accordingly, the preset invention proposes, instead, to exploit the presence of the chromatin fibers to anchor the thrombus matter to ease its removal from the adhering vascular wall and stably retain its fragments to prevent secondary embolization.