Combination of bivalirudin and cardioprotective peptides

Abstract

The present invention relates to a combination of a direct thrombin inhibitor and an antiapoptotic peptide. The therapeutic uses of this combination, particularly in the treatment of acute myocardial infarction, are also described.

Description

[0001] CARDIOPROTECTIVE PEPTIDES AND THEIR USES

[0002] BACKGROUND

[0003] Myocardial infarction (Ml) is the leading cause of cardiovascular death worldwide. In France, Ml is now the leading cause of death in women, ahead of breast cancer, while the second leading cause of death in men (Kuhn et al. 2022).

[0004] Myocardial infarction (Ml) or acute myocardial infarction (AMI) - commonly known as a heart attack - is the interruption of blood supply to part of the heart, leading to cell death. This interruption is most often due to a coronary artery's occlusion (blockage).

[0005] Infarct size is a major determinant of myocardial functional recovery and mortality after AMI (Burns et al. 2002). It is also one of the best predictors of progression towards heart failure. Therefore, limiting infarct size is necessary to prevent post-ischaemic heart failure and improve patient survival.

[0006] Primary percutaneous coronary intervention (PPCI, also known as angioplasty) is currently the preferred treatment for ST-segment elevation Ml (STEMI). It is a minimally invasive procedure involving the use of a balloon catheter to reopen the obstructed or narrowed arteries or veins, and which may further involve placing a small permanent or bioresorbable stent in the newly opened artery (or vein) to help keep it open. In this way, normal circulation can be restored in the coronary arteries.

[0007] Several anticoagulation and antiplatelet protocols are recommended during and after angioplasty in order to reduce the incidence of thrombosis at the balloon dilatation site. In particular, PPCI includes periprocedural pharmacotherapy with an antithrombotic, notably unfractionated heparin such as danaparoid, a low molecular weight heparin such as enoxaparin, or a thrombin inhibitor such as bivalirudin or argatroban.

[0008] Heparins (unfractionated heparin (UFH) and the low molecular weight heparins (LMWH)) have remained as the anticoagulants of choice during the acute phase of ACS (Roffi 2015). UFH is recommended as Class I in ESC guidelines (Ibanez, 2017).

[0009] However, myocardial reperfusion can lead to side effects known as ischaemiareperfusion injury, for which there is currently no treatment (Naito et al. Therapeutic strategies for ischaemia reperfusion injury in emergency medicine. Acute Med Surg.

[0010] 7(1):e501, 2020). During the abrupt restoration of blood flow, cardiac cells (cardiomyocytes and vascular cells) fragilized by ischaemia die following the activation of intracellularcascades of complex events such as calcium overload, loss of ion homeostasis and production of oxygen free radicals leading to apoptotic processes. In particular, lethal ischaemiareperfusion injury occurs when apoptosis is triggered by reperfusion in cells that have survived ischaemia.

[0011] In this context, it has been shown that specific inhibition of the interaction of the FAS receptor with its adaptor proteins DAXX and FADD by pharmacological means via decoy peptides leads to powerful cardioprotection in a murine model (WO 2012 / 066376).

[0012] However, the efficacy of these decoy peptides has not yet been tested in patients. In particular, there is as yet no indication of the compatibility of these decoy peptides with existing treatments.

[0013] So, there is still a need for new treatments for myocardial infarction and reperfusion lesions.

[0014] SUMMARY OF THE DISCLOSURE

[0015] The present inventors have shown that, surprisingly, an anti -apoptotic peptide used in combination with a direct thrombin inhibitor leads to a marked reduction in apoptosis. In fact, the anti -apoptotic peptide in such a combination is still capable of entering cardiac cells and exerting its anti -apoptotic activity, thereby leading to a striking cardioprotective activity in conditions of myocardial reperfusion. In comparison, and surprisingly, another anticoagulant such as heparin inhibits the internalisation of this peptide and its anti-apoptotic activity. In particular, the peptide used in combination with heparin fails to show any cardioprotective effect in vivo. As a result, the anti -apoptotic peptide in such a combination leads to a reduction of the size of necrosed tissues, e.g., the size of an infarct, whereas no such reduction is observed when the anti -apoptotic peptide is combined with heparin.

[0016] The present disclosure therefore relates to a combination of a direct thrombin inhibitor and an anti -apoptotic peptide. The disclosure also relates to a kit-of-parts comprising a direct thrombin inhibitor and an anti-apoptotic peptide.

[0017] Preferably, the direct thrombin inhibitor present in the combination or kit-of-parts is bivalirudin (20 amino acid sequence set forth in SEQ ID NO:1).

[0018] The disclosure also relates to a combination or kit-of-parts comprising a peptide consisting of:a fragment of at most 740 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO: 2, wherein the fragment comprises the amino acid sequence set forth in SEQ ID NO: 3, or

[0019] a fragment of at most 208 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO: 4, wherein the fragment comprises the amino acid sequence set forth in SEQ ID NO: 5, or derivatives thereof,

[0020] wherein the peptide or its derivatives are capable of inhibiting cell apoptosis.

[0021] In a particular embodiment, the peptide or a derivative thereof of the combination or kit-of-parts disclosed herein is conjugated to a cell-penetrating peptide. Preferably, this peptide is the Tat peptide of sequence SEQ ID NO:6.

[0022] According to a particularly preferred embodiment, the peptide of the combination or kit-of-parts disclosed herein is a peptide Tat-DAXXp of sequence SEQ ID NO:7 or a peptide Tat-FADDp of sequence SEQ ID NO:8.

[0023] The disclosure also relates to the combination or kit-of-parts as described herein for use in a method of treating the human or animal body.

[0024] The disclosure also relates to the combination or kit-of-parts disclosed herein for use in a method of inhibiting cell apoptosis in the human or animal body.

[0025] The disclosure also relates to the combination or kit-of-parts disclosed herein for use in a method of treating acute myocardial infarction (AMI), cerebral infarction, organ transplants, cardiac interventions (extracorporeal circulation and temporary occlusion of a vessel), ischaemic lesions of the lower limbs, or acute disturbances of the circulation (shock), in the human or animal body.

[0026] The disclosure also relates to the combination or kit-of-parts disclosed herein for use in adjuvant treatment of primary percutaneous coronary intervention (PPCI) in the treatment of acute myocardial infarction (AMI), cerebral infarction, organ transplants, cardiac interventions (extracorporeal circulation and temporary occlusion of a vessel), ischaemic lesions of the lower limbs, or acute disturbances of the circulation (state of shock), in the human or animal body.

[0027] In a particular embodiment, the treatment is a treatment for acute myocardial infarction, preferably for ST-segment elevation myocardial infarction (STEMI).In a particular embodiment, (1) the direct thrombin inhibitor, e.g., bivalirudin, and (2) the anti -apopto tic peptide are administered parenterally. More particularly, (1) the direct thrombin inhibitor, for example, bivalirudin, is administered intravenously and (2) the anti-apoptotic peptide is administered intravenously or intra-arterially-r

[0028] In a particular embodiment, the methods of treatment disclosed herein comprise the simultaneous, separate, or sequential administration of (1) the direct thrombin inhibitor, for example, bivalirudin, and (2) the anti -apopto tic peptide.

[0029] DESCRIPTION OF FIGURES

[0030] Fig. 1 shows that heparin inhibits the internalisation of Cy5-Tat-DAXXp and Cy5-Tat-FADDp in cardiomyocytes in vitro. (A) - Experimental and treatment protocol for the incubation of Cy5-Tat-FADDp (Cy5-TF) in the presence of heparin.

[0031] Fig.2 shows that bivalirudin has no effect on the internalisation of Cy5-Tat-FADDp in cardiomyocytes. (A) - Experimental and treatment protocol for the incubation of Cy5-Tat-DAXXp (Cy5-TD) and Cy5-Tat-FADDp (Cy5-TF) in the presence of bivalirudin.

[0032] Fig. 3 shows that heparin abolishes the cardioprotective effect of Tat-DAXXp in an ex vivo model of myocardial ischaemia-reperfusion injury.

[0033] (A) - Experimental ex vivo treatment protocol for the evaluation of the cardioprotective effect of Tat-DAXXp in the presence of heparin (0.036 mg / L) as an anticoagulant. Isolated hearts mounted on a Langendorff system were perfused through the aorta and subjected to 40 min of regional ischaemia (ligation of the left coronary artery) followed by 60 min of reperfusion. The Tat-DAXXp peptide (1 pM) was administered after the ischaemia period for 1 minute in a Krebs buffer solution containing 0.036 mg / L heparin. At the end of the protocol, the heart was removed from the Langendorff apparatus for histological analysis using the TTC method to assess infarct size.

[0034] (B) - Scatter diagram and mean ± SD of the infarct size (in % of the area at risk) of the hearts of mice treated with heparin. Under these conditions, the Tat-DAXXp peptide was unable to reduce infarct size (46.40 ± 3.46 for TD, n=7 compared with 52.47 ± 9.09 for IR, n=6; p ns>0.3129). The data were compared using the non -para metric Mann-Whitney test.

[0035] Fig. 4 shows that bivalirudin does not abolish the cardioprotective effect of Tat-DAXXp in an ex vivo model of myocardial ischaemia-reperfusion injury.(A) - Experimental ex vivo treatment protocol for the evaluation of the cardioprotective effect of Tat-DAXXp in the presence of bivalirudin (403 pg / L) as an anticoagulant. Isolated hearts mounted on a Langendorff system were perfused through the aorta and subjected to 40 min of regional ischaemia (ligation of the left coronary artery) followed by 60 min of reperfusion. The Tat-DAXXp peptide (1 pM) was administered after the ischaemia period for 1 minute in a Krebs buffer solution containing 403 pg / L of bivalirudin. At the end of the protocol, the heart was removed from the Langendorff apparatus for histological analysis using the TTC method to assess infarct size.

[0036] (B) - Scatter diagram and mean ± SD of the infarct size (in % of area at risk) of the hearts of mice treated with bivalirudin. Under these conditions, the Tat-DAXXp peptide reduced infarct size by -32.3% (36.74 ± 3.23 for TD, n=8 compared with 54.29 ± 4.66 for IR, n=7; p ***>0.003). The data were compared using the non -para metric Mann-Whitney test.

[0037] Figure 5 shows that heparin prevents Tat-FADDp cardioprotective activity in vivo.

[0038] (A): Experimental in vivo treatment protocol for the evaluation of the cardioprotective effect of TF in the presence of heparin anticoagulant. Mice were subjected to 60 minutes of ischaemia after the left coronary artery ligation and myocardial reperfusion during 60 minutes was achieved by releasing the knot. Heparin (66 U / kg) or physiological serum (Saline) was injected intravenously 30 minutes after the onset of the ischaemic period. Tat-FADDp (TF; 1 mg / kg) or Tat (1 mg / kg) were injected (in the left ventricle cavity, i.c.) 5 minutes before the onset of reperfusion. At the end of the protocol, the heart was removed for histological analysis using the TTC method to evaluate infarct size or for ELISA determination of apoptosis. (B, C) Scatter dot blots and mean ± SD for infarct size (% of area at risk) was measured after 1 h of reperfusion in mice subjected to 60 min-ischaemia, treated with Tat or TF injected intravenously 5 min before reperfusion at 1 mg / kg in mice in (B) physiological or (C) in the presence of heparin anticoagulant. (B): A drastic decrease in infarct size at 1 h of reperfusion was observed after TF treatment versus ischemiareperfusion (IR) (46.7%-decrease between 23.54 ± 3.45 for TF, n=7 versus 44.15 ± 7.93 for IR, n=6; p**=0.005) or versus Tat alone (42.66 ± 4.71 for TF, n=7 versus 42.66 ± 4.71 for Tat, n=6; p**=0.006). (C): Administration of heparin during ischaemia prevents TF cardioprotective effect since there was no difference (p=0.5135) between TF + Heparin (45.13 ± 7.47, n=7) and IR + Heparin (48.50 ± 3.84, n=6) or Tat +Heparin (47.78 ± 7.72, n=6) groups.

[0039] (D, E) Internucleosomal DNA fragmentation, a hallmark of apoptosis, was determined by ELISA in the hearts of IR mice not treated or treated with Tat or TF (1 mg / kg) after 1 h ofreperfusion in (D) absence or in (E) presence of heparin. Scatter dot blots and mean ± SD show the l / N I ratio corresponding to the ratio of soluble nucleosomes in the ischaemic versus the non-ischaemic portion of LV tissues. (D) A drastic decrease in soluble nucleosome ratio at 1 h of reperfusion was observed after TF treatment versus IR (2.52 ± 0.72 for TF, n=6 versus 5.07 ± 1.03 for IR, n=6; p*=0.01) or versus Tat (5.08 ± 0.95 for Tat, n=6; p*=0.01). (E) In presence of heparin, the antiapoptotic effect of TF was abolished and the was no difference (pns=0.26) for the DNA fragmentation (l / NI ratio) among IR+Heparin (4.27 ± 1.34, n=6), Tat+Heparin (5.49 ± 0.97, n=6) and TF+Heparin (5.03 ± 0.67, n=6). Data were compared using Kruskal-Wallis non -para metric test followed by the Dunn’s post-test for multiple comparisons.

[0040] Figure 6 shows that Tat-DAXXp used in combination with Bivalirudin allows to maintain it cardioprotective effects of in an in vivo mouse model of myocardial IR injury.

[0041] (A) - Experimental in vivo treatment protocol for the evaluation of the cardioprotective effect of Tat-DAXXp (TD) in presence of Heparin (H) or Bivalirudin (BV) anticoagulants compared to the control condition (saline). Mice were subjected to 60 min of ischaemia after the left coronary artery ligation and myocardial reperfusion during 60 min was achieved by releasing the knot. Heparin (66 U / kg), Bivalirudin (7.75 mg / kg) or physiological serum (Saline) were injected 30 min after the onset of the ischaemic period in the blood. TD (1 mg / kg) prepared in physiological solution or physiological serum (saline) alone were injected 5 min before the onset of reperfusion. At the end of the protocol, the heart was removed for histological analysis using the TTC method to evaluate infarct size (B,C,D) - Bar graphs and Scatter dot blots ± SD for infarct size (% of area at risk) in mice treated with saline solution (IR) or TD in control condition (saline; B) and in the presence of Heparin (C) or Bivalirudin (D). A drastic decrease in infarct size was observed after TD treatment versus IR in saline condition (52.6%-decrease between 44.15 ± 2.98 for IR, n=6 versus 20.94 ± 2.33 for TD, n=3; p*=0.023) and in presence of bivalirudin 52.70 ± 5.46 for IR+Bivalirudin, n=7 versus 30.34 ± 3.22 for TD+ Bivalirudin, n=6, p*=0.023. By contrast, TD in presence of heparin did not decrease infarct size compared to IR+Heparin: 48,50 ± 1.57 for IR+ Heparin, n=6 versus 39.41 ± 9.1 for TD+ Heparin, n=2; pns= 0,64. (E) - Scatter dot blots and mean ± SD for areas at risk (% of LV) measured in mice treated or not with TD (1 mg / kg) in saline, heparin or bivalirudin conditions. No difference between groups were determined (pns=0.4994)

[0042] DETAILED DESCRIPTION OF THE INVENTIONDefinitions

[0043] The term “about” or “approximately” refers to the normal range of error for a given value or range known to the person of skill in the art. It usually means within 20%, such as within 10%, or within 5% (or 1% or less) of a given value or range.

[0044] As used herein, “acute myocardial infarction” refers to myocardial necrosis caused by acute obstruction of a coronary artery. ST-segment elevation myocardial infarction (STEMI, transmural myocardial infarction)” as used here is myocardial necrosis with ST-segment elevation on the electrocardiogram that is not rapidly suppressed by nitroglycerin.

[0045] As used herein, “administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a S. repens extract) into a patient, such as by oral, mucosal (e.g., sublingual, buccal, rectal, vaginal, nasal, etc.), subcutaneous, cutaneous, intradermal, intravenous, intramuscular, intrathecal, ocular, otic, inhalation, nebulisation, cutaneous, or transdermal delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

[0046] A “direct thrombin inhibitor”, as used herein, refers to a compound that directly inhibits factor coagulation, independently of antithrombin, by binding to thrombin and blocking its catalytic site and / or the fibrinogen binding site. Direct thrombin inhibitors include argatroban, dabigatran etexilate, melagatran, ximelagatran, hirudin and derivatives thereof, such as desirudin, lepirudin, and bivalirudin. A preferred direct thrombin inhibitor is bivalirudin.

[0047] An “effective amount” or “therapeutically effective amount” of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to elicit the desired biological response in a subject. Such response includes alleviation of the symptoms of the disease or disorder being treated, prevention, inhibition or a delay in the recurrence of symptom of the disease or of the disease itself, an increase in the longevity of the subject compared with the absence of the treatment, or prevention, inhibition or delay in the progression of symptom of the disease or of the disease itself. An “effective amount” is, in particular, the amount of the agent effective to achieve the desired therapeutic or prophylactic result. More specifically, an “effective amount” as usedherein is an amount of the agent that confers a therapeutic benefit. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects.

[0048] The term “excipient” as used herein refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilising agent for drugs that imparts a beneficial physical property to a formulation, such as increased protein stability, increased protein solubility, and decreased viscosity. Examples of excipients include, but are not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, non-ionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See also, Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA, which is hereby incorporated by reference in its entirety.

[0049] As used herein, “percent sequence identity” refers to comparisons between amino acid sequences and is determined by comparing two optimally aligned sequences over a comparison window, wherein the part of the amino acid sequence in the comparison window may include additions or deletions (i.e. gaps) relative to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where the identical amino acid residue appears in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percentage of sequence identity. The percentage can also be calculated by determining the number of positions where the identical amino acid residue appears in both sequences or an amino acid residue is aligned with a gap to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percentage sequence identity. Those familiar with the subject will know that there are many established algorithms for aligning two sequences. Optimal alignment of the sequences to be compared can be achieved, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. ScL USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the GCG Wisconsin software package) or by visual inspection (see generally Current Protocols in Molecular Biology, F. M. Ausubel et al, eds, Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc. (supplement 1995) (Ausubel)). The BLAST and BLAST 2.0 algorithms, described in Altschul et al, 1990, J. Mol. Biol. 215: 403-410 and Altschul et al, 1977, Nucleic Acids Res. 3389-3402, respectively.

[0050] The term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia, or other generally recognised Pharmacopeia for use in animals, and more particularly in humans. More specifically, when referring to a carrier, the expression “pharmaceutically acceptable” means that the carrier(s) is compatible with the other ingredient(s) of the composition and is not deleterious to the recipient thereof. Accordingly, as used herein, the expression “pharmaceutically acceptable carrier” refers to a carrier or a diluent that does not inhibit the biological activity and characteristics of a compound for administration without stimulating a living organism. The type of carrier can be selected based upon the intended route of administration. The amount of each carrier used may vary within ranges conventional in the art. As a pharmaceutically acceptable carrier in the composition which is prepared as a liquid solution, physiological saline, sterilised water, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, and a mixture of one or more of them can be used as a sterilised carrier suitable for a living organism. If necessary, common additives like antioxidant, buffer solution, and bacteriostat may be added. Furthermore, by additionally adding a diluent, a dispersant, a surfactant, a binder, or a lubricant, the composition can be prepared as a formulation for injection like an aqueous solution, suspension, and emulsion, a pill, a capsule, a granule, or a tablet.

[0051] As used herein, the term "treat" or "treatment" means to reverse, alleviate, inhibit the progression of or prevent the disorder or condition to which that term applies.

[0052] As used herein, the terms “subject”, “individual”, or “patient”, are used interchangeably, refer to any animal, including mammals and primates (e.g., human), such as mice, rats, other rodents, rabbits, dogs, cats, swine, pig, cow, goat, cattle, sheep, horses, primates, and humans. In some embodiments, the subject is a human.

[0053] Combinations

[0054] The present inventors have shown that, surprisingly, an anti-apoptotic peptide in combination with a direct thrombin inhibitor leads to a marked reduction in apoptosis. The anti-apoptotic peptide in such a combination is still able to enter cardiac cells and exerts its anti-apoptotic activity. This results in a significant reduction of the size of the infarct, i.e.,the area of tissue that is undergoing or has undergone necrosis as a result of the Ml and additional reperfusion injury. This effect translates in vivo in a remarkable cardioprotective activity. In comparison, and surprisingly, another anticoagulant such as heparin inhibits the internalisation of this peptide. Indeed, no effect on the size of the infarct is observed further to the administration of the anti -apopto tic peptide in combination with heparin. In vivo, the combination of the peptide and heparin fails to show any cardioprotective activity.

[0055] The present description therefore relates to a combination of a direct thrombin inhibitor and an anti -apopto tic peptide.

[0056] Such a combination is particularly useful because it can both inhibit apoptosis of cardiac cells and prevent or treat thrombus formation, particularly during the reperfusion phase after an acute myocardial infarction. It is therefore particularly useful for preventing or treating reperfusion lesions.

[0057] Direct thrombin inhibitors

[0058] Direct thrombin inhibitors act directly by binding to thrombin, thereby blocking the conversion of fibrinogen into fibrin.

[0059] Any direct thrombin inhibitor can be used in the combinations described here. In an embodiment, the combinations disclosed herein comprise a direct thrombin inhibitor selected from the group consisting of: argatroban, dabigatran etexilate, melagatran, ximelagatran, and hirudin and derivatives thereof, such as desirudin, lepirudin, and bivalirudin.

[0060] According to a preferred embodiment, the combination disclosed herein comprises a hirudin derivative in particular a hirudin derivative selected from the group consisting of desirudin, lepirudin, and bivalirudin.

[0061] More preferably, the combination disclosed herein includes bivalirudin.

[0062] Bivalirudin, also known as Hirulog-8, is a synthetic derivative of hirudin, a thrombininhibiting peptide that occurs naturally in the saliva of the medicinal leech Hirudo medicinalis. However, unlike hirudin, bivalirudin is a reversible inhibitor, which is particularly advantageous for the temporary prevention of blood clotting during a PPCI procedure.

[0063] Bivalirudin is a peptide containing 20 residues with a pl of 3.91, a net charge = -4.0 at pH 7.0, and a molecular weight of 2,180 Da. Bivalirudin consists of the amino acid sequence:(D-Phe)-Pro-Arg-Pro-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-lle-Pro-Glu-Glu-Tyr-Leu (SEQ ID NO: 1). Methods for synthesising bivalirudin can include, but are not limited to, solid phase peptide synthesis, solution phase peptide synthesis, or a combination of solid phase and solution phase procedures (e.g., U.S. Pat. No. 5,196,404; U.S. Pat. No. 7582727; Okayama et al, Chem. Pharm. Bull. 44: 1344-1350, 1996; Steinmetzer et al. J. Biochem. 265: 598-605, 1999; WO 91 / 02750).

[0064] Peptides inhibiting cell apoptosis

[0065] The anti -apopto tic peptides used in the combinations described here are in particular peptides that inhibit the interaction between the FAS (First receptor Apoptosis Signal) (Roubille et al. 2007) and its adaptor proteins, FADD (Fas-associated protein with death domain) and DAXX (Death-associated protein).

[0066] Indeed, the inventors have previously shown that the peptide comprising the particular amino acid sequences SEQ ID NO:3 ("DAXXp"), which is a fragment of the DAXX protein (SEQ ID NO: 2); and the peptide comprising the particular amino acid sequence SEQ ID NO: 5 ("FADDp"), which is a fragment of the FADD protein (SEQ ID NO: 4), interact with the FAS receptor and are thus capable of decreasing cell apoptosis (see e.g., WO 2012 / 066103, WO 2012 / 066376).

[0067] The description relates to peptides consisting of:

[0068] a fragment of at most 740 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO:2, wherein the fragment comprises the amino acid sequence set out in SEQ ID NO:3, or

[0069] a fragment of at most 208 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO:4, wherein the fragment comprises the amino acid sequence set out in SEQ ID NO: 5,

[0070] or derivatives thereof,

[0071] wherein these peptides or their derivatives are capable of inhibiting cell apoptosis.

[0072] The peptides according to the disclosure are fragments of an amino acid sequence having at least 80% identity with SEQ ID NO: 3 or SEQ ID NO: 5. In particular embodiments, the anti-apoptotic peptides according to the disclosure are fragments of an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 2 or SEQ ID NO: 4.The peptides disclosed herein or their derivatives are particularly advantageous in that they are capable of inhibiting cellular apoptosis, in particular cardiomyocyte apoptosis, as demonstrated in applications WO 2012 / 066103 and WO 2012 / 066376. Inhibition of cell apoptosis by the peptides or their derivatives disclosed herein can be measured by any suitable method known to the person skilled in the art, generally using a cell apoptosis detection kit. An example of a kit suitable for measuring cell apoptosis is the kit ELISAPLUS® (Cat. No. 11 774425001, Roche Cell Death Detection Applied Science).

[0073] The anti -apopto tic peptides, according to the disclosure, consist of fragments of at most 130 amino acids. In particular embodiments, the anti-apoptotic peptides according to the disclosure consist of fragments of at most 120, 110, 100, 90, 80, 70, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or 9 amino acids.

[0074] When the anti-apoptotic peptides according to the disclosure comprise the amino acid sequence of SEQ ID NO:3, said peptides comprise at least 16 amino acids.

[0075] When the anti-apoptotic peptides according to the disclosure comprise the amino acid sequence of SEQ ID NO: 5, said peptides comprise at least 15 amino acids.

[0076] In a particular embodiment, the anti-apoptotic peptide according to the disclosure consists of SEQ ID NO:3.

[0077] In a particular embodiment, the anti-apoptotic peptide according to the disclosure consists of SEQ ID NO:5.

[0078] Anti-apoptotic peptide derivatives

[0079] The disclosure also relates to derivatives of the peptides described above, which retain the activity of inhibiting cell apoptosis. Indeed, it is possible to use such derivatives in the present combinations.

[0080] The derivatives generally consist of peptides as described, which have been chemically or biologically modified.

[0081] Examples of derivatives are peptides, according to the disclosure in which:

[0082] at least one amino acid of the peptide has been substituted, inserted, or deleted, and / or

[0083] at least one amino acid in the peptide has been chemically modified or derived.These “chemically modified or derived” amino acids include, for example, derivatives of natural amino acids, such as 4 -hydroxy pro line for proline, 5-hydroxylysine for lysine, homoserine for serine, ornithine for lysine, etc. Other “chemically modified or derived” amino acids include, for example, a label, such as fluorescein, tetramethylrhodamine or the cyanine dye Cy5.5; or one or more post-translational modifications such as acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, sulphation, glycosylation or lipidation. Indeed, certain chemical modifications, in particular N-terminal glycosylation, have been shown to increase the stability of peptides in human serum (Powell et al, Pharma Res 1993: 10: 1268-1273). Chemically modified or derived" amino acids also include those whose membrane permeability is increased by N-myristoylation (Brand, et al, Am J Cell Physiol 1996; 270:C1362-C1369). It is understood that a derivative of a peptide according to the disclosure may contain more than one of the amino acids “chemically modified or derived”.

[0084] Other derivatives of the peptides according to the disclosure are peptidomimetics of said peptides. Peptidomimetics refer to a synthetic chemical compound that has essentially the same structural and / or functional characteristics as the anti-apoptotic peptides disclosed herein. The mimetic may be composed entirely of non-naturally occurring synthetic amino acid analogues or may be a chimeric molecule comprising one or more naturally occurring amino acids and one or more non-naturally occurring amino acid analogues. The mimetic may also incorporate any number of conservative substitutions of natural amino acids which do not destroy the activity of the mimetic. Routine tests can be used to determine whether a mimetic has the required activity, using Test A described in WO 2012 / 066103 and WO 2012 / 066376. The term “substantially identical”, when used in reference to a mimetic or peptidomimetic, means that the mimetic or peptidomimetic possesses one or more activities or functions of the reference molecule, for example inhibition of cell apoptosis. The techniques used to develop peptidomimetics are conventional. For example, peptide bonds can be replaced by non-peptide bonds or nonnatural amino acids that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original peptide. Other modifications can also be made by replacing the chemical groups of the amino acids with other chemical groups of similar structure. The development of peptidomimetics can be facilitated by determining the tertiary structure of the original fragment / peptide, free or bound to the intracellular region of the FAS receptor, using NMR spectroscopy, crystallography and / or computer-aided molecular modelling. Once a potential peptidomimetic compound is identified, it may be synthesised and its capability of inhibiting cell apoptosis can be assayed.Peptidomimetics can contain any combination of non-natural structural components, which generally fall into three structural groups: groups of residue linkages other than natural amine linkages ( peptide linkages'); non-natural residues in place of natural amino acid residues; residues that induce secondary structural mimicry (e.g. beta-turn, gammaturn, beta-sheet, alpha-helix conformation); or other changes that confer resistance to proteolysis.

[0085] For example, lysine mimetics can be generated by reacting lysinyl with succinic or other carboxylic acid anhydrides. Lysine and other alpha-amino-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transamidase-catalysed reactions with glyoxylate.

[0086] One or more residues can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality. Thus, any amino acid naturally occurring in the L- configuration (which can also be referred to as R or S, depending upon the structure of the chemical entity) can be replaced with the same amino acid or a mimetic, but of the opposite chirality, referred to as the D-amino acid, but which can additionally be referred to as the R- or S-form.

[0087] As will be appreciated by those skilled in the art, the peptidomimetics of the present invention may also comprise one or more of the modifications disclosed herein for “chemically modified or derived” amino acids, for example a label or one or more post-translational modifications.

[0088] The peptides, derivatives and peptidomimetics can be produced and isolated using any method known in the art. Peptides can be synthesised, whole or in part, using usual chemical methods. Techniques for generating peptide and peptidomimetic libraries are well known, and include, for example, multipin, tea bag, split-couple-mix techniques and SPOT synthesis.

[0089] Conjugates

[0090] The disclosure also relates to the combinations described above, in which the anti-apoptotic peptide or a derivative thereof is conjugated to a cell-penetrating peptide (CPP).

[0091] Indeed, it is particularly advantageous to conjugate these peptides or their derivatives with “cell penetrating peptides” (CPPs) to facilitate the uptake of the peptides or their derivatives disclosed herein across cell membranes, such as the plasma membrane and / or the nuclear membrane of a cell (see in particular WO 2012 / 066103 and WO 2012 / 066376).CPPs are well-known peptides that can be conjugated to cargoes to facilitate the transport of cargoes across membranes.

[0092] A CPP generally contains two or more cationic amino acids, with hydrophobic amino acids or spacer groups separating some of the cationic amino acids. For example, the cationic amino acid is arginine (R). As a general rule, a CPP contains at least 3 or 4 arginine residues. In some cases, the CPP contains 5, 6 or more arginine residues. It is also known that the presence of aminohexyl and beta-alanine in the CPP sequence is advantageous as it helps to minimise the immunogenicity of the peptide.

[0093] Any CPP can be used to improve cytoplasmic delivery of the anti-apoptotic peptides disclosed herein or their derivatives. The skilled person may for example refer to Sun et al. Biomedicines 11 (7):1971 , 2023 and Sadeghian et al. J Pharm Pharmacol. 74(8) : 1085-1116, 2022. The skilled person may also consult the database accessible via the website CPPsite 2.0 (http: / / crdd.osdd.net / raghava / cppsite / ). In particular, the CPPs described in WO 2012 / 066376 can be used, i.e. any one of Tat, RXR, Bpep and Pip2b.

[0094] Preferably, the CPP used is the peptide Tat: GRKKRRQRRRPPQ (SEQ ID NO:6).

[0095] More preferably, the anti-apoptotic peptide disclosed herein comprises:

[0096] the peptide Tat of sequence SEQ ID NO:6, conjugated to

[0097] the peptide DAXXp of SEQ ID NO:3 or the peptide FADDp of sequence SEQ ID NO: 5.

[0098] In a particularly preferred embodiment, the peptide of the combination disclosed herein is a peptide Tat-DAXXp of sequence SEQ ID NO:7 or a Tat-FADDp peptide of sequence SEQ ID NO:8.

[0099] The CPP is typically linked to the N-terminal or C-terminal end of the peptide or derivative thereof according to the invention, preferably to the C-terminal. Chemical linkage may be performed via a disulfide bond, thioether, or thiol-maleimide linkage.

[0100] In a particular embodiment, the peptide or derivative thereof according to the invention is linked to the CPP through a linker. Any type of linker can be used by the skilled person, provided that said linker allows chemical linkage of the peptide or derivative thereof to the CPP. A range of linkers is possible, including amino acid sequences having a C-terminal Cysteine residue that permits formation of a disulphide, thioether or thiol-maleimide linkage. Other ways of linking the peptide or derivative thereof according to the inventionto the CPP include use of a C-terminal aldehyde to form an oxime. Still another type of linkers use click chemistry.

[0101] Examples of linkers are sequences of amino acids or amino acids selected from the group comprising: C, BC, XC, GC, BBCC, BXCC, XBC, X, XX, B, BB, BX or XB, wherein:

[0102] X = aminohexyl, B-alanyl, p-aminobenzoyl, isonipecotyl or 4-ami nobutyryl.

[0103] B = beta-alanine.

[0104] Pharmaceutical compositions

[0105] In another aspect, the description relates to a pharmaceutical composition comprising the combination of direct thrombin inhibitor and anti -apopto tic peptide disclosed herein. Preferably, the pharmaceutical composition contains, in addition to the combination, various diluents, fillers, salts, buffers, stabilisers, solubilisers, and other materials well known in the art.

[0106] As used herein, a “pharmaceutically acceptable carrier” encompasses all physiologically compatible solvents, buffers, saline solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, etc. The type of carrier may be selected according to the intended route of administration. The type of vehicle may be chosen according to the intended route of administration. In various embodiments, the vehicle is suitable for parenteral administration, for example, intravenous or intra-arterial, , intraperitoneal, subcutaneous, intramuscular, topical, transdermal, or oral. Pharmaceutically acceptable vehicles include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of vehicles and agents for substances pharmaceutically active is well known in the art. A typical pharmaceutical composition for intravenous infusion may contain 250 ml of sterile solution and 100 mg of the combination. The present methods of preparing parenterally administrable compounds are known or apparent to those skilled in the art and are described in more detail, for example, in Ringer's Remington's Pharmaceutical Science, 17th edition, Mack Publishing Company, Easton, Pa. (1985), and its 18thand 19theditions.

[0107] Each of the components of the combination contained in the composition, namely the direct thrombin inhibitor and the anti-apoptotic peptide, is preferably formulated in an effective amount. An “effective amount” refers to an amount that is effective, at the doses and for the periods required, to achieve the desired result, such as prevention or inhibition of coagulation or inhibition of cell apoptosis. A “therapeutically effective amount” is an amount sufficient to influence the therapeutic course of a particular disease state. Forexample, an effective amount of the anti-apoptotic peptide is an amount that is effective for reducing the size of the infarct resulting from an Ml and reperfusion injury. A therapeutically effective amount is also a amount in which the toxic or harmful effects of the agent are offset by beneficial therapeutic effects.

[0108] Kit of parts

[0109] In another aspect, the present description also provides a kit-of-parts comprising a direct thrombin inhibitor and an anti-apoptotic peptide.

[0110] More specifically, the present disclosure also relates to a kit-of-parts comprising:

[0111] (i) a first component comprising at least one direct thrombin inhibitor, and

[0112] (ii) a second component comprising an anti-apoptotic peptide or derivative thereof as disclosed herein.

[0113] The above components may each be provided as a pharmaceutical composition in the kit-of-parts. For example, the first component comprises at least one direct thrombin inhibitor and a pharmaceutically acceptable excipient. In another example, the second component comprises an anti-apoptotic peptide described below or a derivative thereof and a pharmaceutically acceptable excipient. Preferably, both the first and second components comprise a pharmaceutically acceptable excipient.

[0114] The kit-of-parts generally comprises the components in appropriate containers. For example, each container may be in the form of vials, bottles, squeeze bottles, jars, sealed sleeves, envelopes or pouches, tubes, or blister packs, or any other suitable form, provided that the container is configured to prevent premature mixing of the components. Each of the different components may be supplied separately, or some of the different components may be supplied together (i.e., in the same container).

[0115] The components of the kit-of-parts described here can be administered simultaneously (i.e., at the same time via the same or different administration routes) or separately (i.e., sequentially at different time points and / or via different administration routes). A sequential administration scheme is also referred to as “time-staggered” administration. The time-staggered administration of the different components of the kit-of-parts can ensure that the distinct mechanisms triggered by said components do not adversely affect each other.According to certain preferred embodiments, the components of the kit-of-parts disclosed herein are administered simultaneously (i.e. , at the same time by the same route of administration or by different routes of administration). According to certain preferred embodiments, the components of the kit-of-parts disclosed herein are administered separately (i.e., sequentially at different times and / or by different administration routes).

[0116] In a preferred embodiment, there is disclosed herein a kit-of-parts comprising:

[0117] (i) a first component comprising at least one direct thrombin inhibitor, and

[0118] (ii) a second component comprising an anti-apoptotic peptide or derivative thereof as disclosed herein;

[0119] for the therapeutic use disclosed herein, wherein the first and second components are administered together or independently at the same time or separately in time intervals. Therapeutic applications

[0120] The combinations, pharmaceutical compositions, and kit-of-parts disclosed herein are particularly useful, since they possess both cellular apoptosis inhibition activity, particularly in cardiac cells (cardiomyocytes and vascular cells), and anticoagulant activity via thrombin inhibition. This is strikingly illustrated by their ability to reduce the size of the necrosed myocardial and vascular tissues. They can therefore be advantageously used in all pathological situations where there is a need to inhibit cell apoptosis and prevent or inhibit coagulation.

[0121] In particular, the description relates to the combinations, pharmaceutical compositions and kit-of-parts disclosed herein for use in the treatment of acute myocardial infarction (AMI), cerebral infarction, organ transplants, cardiac interventions (extracorporeal circulation and temporary occlusion of a vessel), ischaemic lesions of the lower limbs, or acute disturbances of the circulation (shock), in humans or animals.

[0122] In another embodiment, a method is described for treating acute myocardial infarction (AMI), cerebral infarction, organ transplants, cardiac interventions (extracorporeal circulation and temporary occlusion of a vessel), lower limb ischaemic lesions, or acute circulatory disturbances (shock), said method comprising administering a combination, pharmaceutical composition or kit-of-parts disclosed herein to a human or animal in need thereof.The disclosure also relates to the use of the combinations, pharmaceutical compositions and kit-of-parts disclosed herein in the manufacture of a medicament for treating acute myocardial infarction (AMI), cerebral infarction, organ transplants, cardiac interventions (extracorporeal circulation and temporary occlusion of a vessel), ischaemic lesions of the lower limbs, or acute disturbances of the circulation (shock), in humans or animals.

[0123] In particular, the combinations, pharmaceutical compositions and kit-of-parts disclosed herein can be used as adjuvant therapy of PPCI in the treatment of acute myocardial infarction (AMI), cerebral infarction, organ transplants, cardiac interventions (extracorporeal circulation and temporary occlusion of a vessel), ischaemic lesions of the lower limbs, or acute disturbances of the circulation (shock), in humans or animals.

[0124] In a preferred embodiment, the combinations, pharmaceutical compositions, and kit-of-parts disclosed herein are used for the treatment of acute myocardial infarction. More preferably, the myocardial infarction is an ST-segment elevation myocardial infarction (STEMI).

[0125] In another preferred embodiment, the combinations, pharmaceutical compositions, and kit-of-parts disclosed herein are used for the treatment of reperfusion injury.

[0126] Preferably, the treatment comprises joint or independent administration of the direct thrombin inhibitor, for example, bivalirudin, and an anti -apopto tic peptide.

[0127] Preferably, (1) the direct thrombin inhibitor, for example, bivalirudin, and (2) the anti-apoptotic peptide are administered together or independently at the same time or separately in time intervals.

[0128] Even more preferably, (1) the direct thrombin inhibitor, for example, bivalirudin, and (2) the anti -apopto tic peptide are administered simultaneously, separately, or sequentially.

[0129] The invention in all its aspects is illustrated further in the following examples. The examples do not, however, limit the scope of the invention, which is defined by the appended claims.

[0130] EXAMPLES

[0131] 1. Chemical characterisation of Tat-DAXXp and Tat-FADDp

[0132] Both peptides have virtually the same cardioprotective effect in a murine myocardial ischaemia-reperfusion model (see patents WO 2012 / 066103 and WO 2012 / 066376). Inaddition, the two peptides have virtually the same physical properties: similar length, similar pl values, similar hydrophobicity and high net positive charge (see Table 1).

[0133] [Table 1]

[0134] Sequence: GRKKRRQRRRPPQKKSRKEKKQTGSGPLG (SEQ ID NO:7)

[0135] Length: 29

[0136] Masse: 3428,0358

[0137] Isoelectric point (pl): 12.89

[0138] Net charge: +13

[0139] Hydrophobicity: +51.05 Kcal * mol1

[0140] Sequence: GRKKRRQRRRPPQVGKRKLERVQSGLDL (SEQ ID NO:8)

[0141] Length: 28

[0142] Masse 3397,0301

[0143] Isoelectric point (pl): 12.71

[0144] Net charge: +10

[0145] Hydrophobicity: +42.68 Kcal * mol1

[0146]

[0147] As a result, the two peptides can be interchanged when assessing side effects in the presence of anticoagulants.

[0148] 2. In vitro study showing that heparin inhibits internalisation of Tat-FADDp and Tat-DAXXp in cardiomyocytes

[0149] To study internalisation of Tat-DAXXp (TD) or Tat-FADDp (TF), in vitro experiments on cultured cardiomyocytes (rat H9c2 cells) were performed with the fluorescent peptides (Cy5-TD, 1 pM or Cy5-TF, 1 pM) in the absence or presence of heparin. The dose of heparin administered per patient (5,000 U) was extrapolated to 1 mg / mL for cell incubation. In addition, cells were also incubated with a 5-fold higher dose of heparin (5 mg / mL) to obtaina dose-dependent effect (Figure 1A). In the absence of heparin, the Cy5 fluorophore signal was observed with high intensity when the cells were incubated with Cy5-TD (Figure 1 B) and with Cy5-TF (Figure 1C). These results thus clearly demonstrate that heparin inhibits the internalisation of Cy5-TD and Cy5-TF in a dose-dependent manner.

[0150] Representative images of the internalisation of Cy5-TD (Figure 1B) and Cy5-TF (Figure 1C) revealed a dose-dependent reduction in red fluorescence as a function of co-incubation with heparin. H9c2 cells (non-fixed) were imaged by confocal microscopy (CLSM) after 4 hours incubation with Cy5 peptide (1 pM) in DMEM FluoroBrite in the absence or presence of heparin (1 mg / mL or 5 mg / mL). 10 minutes before the end of incubation, Hoechst dye (blue = nuclei) and WGA-Alexa488 (green = cell membrane) were added. Cells were washed twice with D-PBS and overlaid with DMEM FluoroBrite before imaging. White bars represent 20 pm.

[0151] 3. In vitro study showing that bivalirudin does not interfere with the internalisation of Tat- FADDp and Tat-DAXXp in cardiomyocytes

[0152] To study the influence of the anticoagulant bivalirudin on therapeutic peptide internalisation, cardiomyocytes (rat H9c2 cells) were incubated with were performed with the fluorescent Tat-DAXXp (Cy5-TD) or fluorescent Tat-FADDp (Cy5-TF) in the absence or presence of bivalirudin (Figure 2A).

[0153] For this purpose, the dose of bivalirudin administered per patient (bolus i.v. of 0.75 mg / kg followed by an infusion i.v. of 1.75 mg / kg / h up to 5 h post-procedure) was extrapolated to 403 pg / mL for cell incubation. In addition, cells were also incubated with a dose 5-fold higher (2.015 bivalirudin pg / mL) to obtain a dose-dependent effect (Figure 2B, C). These results show that the internalisation of the two peptides Cy5-TD and Cy5-TF was not abolished by bivalirudin, independently of the doses applied. Even at a dose of 5 mg / mL (used for heparin), bivalirudin did not affect the internalisation of either Cy5-TD or Cy5-TF (data not shown).

[0154] Representative images of the internalisation of Cy5-TD (Figure 2B) and Cy5-TF (Figure 2C) revealed that in the presence of bivalirudin, there was no dose-dependent reduction in red fluorescence. H9c2 cells (unattached) were imaged by confocal microscopy (CLSM) after 4 h incubation with Cy5-TF (1 pM) in DMEM FluoroBrite in the absence or presence of bivalirudin (403 pg / mL or 2.015 pg / mL). 10 min before the end of incubation, Hoechst dye (blue = nuclei) and WGA-Alexa488 (green = cell membrane) were added. Cells were washed twice with D-PBS and overlaid with DMEM FluoroBrite before imaging. White bars represent4. Ex vivo study showing that heparin inhibits the cardioprotective activity of Tat-DAXXp in mouse hearts

[0155] To comply with the conditions imposed by the recommendations of cardiology societies, the Tat-DAXXp (TD) peptide was administered in the presence of heparin as an anticoagulant. For this, we used a dose of heparin equivalent to what patients receive at the start of endocoronary angioplasty (5,000 III). The cardioprotective effect of Tat-DAXXp was evaluated ex vivo in a Langendorff-type perfusion system on mouse hearts exposed to 40 min of regional ischaemia (ligation of the left coronary artery) followed by 60 min of reperfusion. The peptide, prepared in the perfusion solution containing heparin, was administered as a bolus (1 pM) for 1 min during the start of reperfusion (see protocol Figure 3A). Analysis of infarct size was performed using the TTC method as previously described in Boisguerin et al 2020 (Boisguerin et al. A novel therapeutic peptide targeting myocardial reperfusion injury. Cardiovasc Res. ; 116:633-644, 2020).

[0156] The results presented in Figure 3B show that Tat-DAXXp, in the presence of heparin in the perfusion fluid, has no cardioprotective effect (p = ns for Tat-DAXXp versus IR) (Figure 3B).

[0157] 5. Ex vivo study showing that bivalirudin does not inhibit the cardioprotective activity of Tat-DAXXp in mouse hearts

[0158] To comply with the conditions imposed by the recommendations of cardiology societies, we administered the Tat-DAXXp (TD) peptide in the presence of bivalirudin, another anticoagulant molecule recommended in clinical guidelines. For this, we used a dose of bivalirudin equivalent to what patients receive at the start of endo-coronary angioplasty (403 pg / L). The cardioprotective effect of TD was evaluated ex vivo in a Langendorff-type perfusion system on mouse hearts exposed to 40 min of regional ischaemia (ligation of the left coronary artery) followed by 60 min of reperfusion. The peptide, prepared in the perfusion solution containing bivalirudin, was administered as a bolus (1 pM) for 1 min during the start of reperfusion (see protocol Figure 4A). Infarct size analysis was performed using the TTC method as previously described in Boisguerin et al 2020 (Boisguerin et al. A novel therapeutic peptide targeting myocardial reperfusion injury. Cardiovasc Res.; 116:633-644, 2020).

[0159] The results presented in Figure 4B show that TD, in the presence of bivalirudin in the perfusion fluid, has a cardioprotective effect, showing a reduction in infarct size of -32.3% (p***= 0.0003 for TD versus IR) (Figure 3B).6. Heparin prevents Tat-FADDp cardioprotective activity in vivo (murine model of myocardial ischaemia-reperfusion injury)

[0160] To assess in vivo the cardioprotective effect of Tat-FADDp in the presence of heparin (66 U / kg, equivalent to the human dose) in the blood, thus mimicking the clinical situation during PCI (percutaneous coronary intervention), mice were subjected to 60 min of ischaemia followed by 60 min of reperfusion (Figure 5A). Tat-FADDp (TF) was administered 5 min before the onset of reperfusion. Analysis of infarct size was performed using the TTC method as previously described (Boisguerin et al. A novel therapeutic peptide targeting myocardial reperfusion injury. Cardiovasc Res.; 116:633-644, 2020). The results presented in Figure 5B show that TF significantly decreased infarct size versus ischemia-reperfusion (IR) in saline condition. The cardioprotective effect of TF corresponding to a 46.7% decrease (p* for TF versus IR or versus Tat) in infarct size. This effect was lost when the mice were previously treated by one dose of heparin (66 U / kg) during the ischaemic phase (7%-decrease, p=ns for TF+Heparin versus IR+Heparin). Additional controls were added in this in vivo study, both Tat or Heparin alone had not effect on infarct size compared to IR control (p=ns). Please note that there was no difference among groups for areas at risk (% of LV), which confirmed that coronary ligation was performed at the same position in all hearts, inducing the same IR injury. Regarding the mechanisms underlying cardioprotection, we evidenced that the antiapoptotic effect of TF peptide providing a 50.3 %-decrease (p* for TF versus IR or versus Tat) in specific DNA fragmentation (amount of soluble oligonucleosomes of 180 base pairs) was completely lost when heparin (66 U / kg) was administered in the blood of the mice during the ischaemic period of the protocol (p=ns for TF+Heparin versus IR+Heparin or versus Tat +Heparin) (See Figure 5D).

[0161] 7. In vivo study showing that Tat-DAXXp used in combination with Bivalirudin allows to maintain its cardioprotective effects in an in vivo mouse model of myocardial IR injury.

[0162] To assess the cardioprotective effect of Tat-DAXXp (TD) in the presence of Heparin or Bivalirudin anti -coagulants, mice were subjected to 60 min of ischaemia followed by 60 min of reperfusion (Figure 6A). Tat-DAXXp (TD) or physiological serum (non-treated IR) was injected 5 minutes before the onset of reperfusion. Analysis of infarct size was performed using the phthalocyanine blue dye and 2, 3, 5-triphenyltetrazolium chloride (TTC) method as previously described (Boisguerin et al. A novel therapeutic peptide targeting myocardial reperfusion injury. Cardiovasc Res.; 116:633-644, 2020).

[0163] The results presented in Figure 6B shows that TD significantly decreased infarct size versus the non-treated IR condition by 52,6% (p* for TD versus IR). The cardioprotectiveeffect of TD was lost when the mice were previously treated by heparin (66 U / kg, equivalent to the human dose) during the ischaemic phase (30 min after the onset of ischaemia): pnsfor TD+H versus IR+H (Figure 6C). In the presence of Bivalirudin (7.75 mg / kg, equivalent to the human dose) injected 30 min after the onset of ischaemia, TD peptide provided a strong cardioprotective effect allowing to decrease infarct size by 42,4 % (p** TD+Bivalirudin versus I R+Bivalirudin; Figure 6D). There was no difference among groups for areas at risk (% of LV), which confirmed that coronary ligation was performed in the same position in all hearts, inducing the same IR injury (Figure 6E).

[0164] 8. Conclusion

[0165] Despite promising results in animal models, there is currently no adjuvant pharmacological treatment available to eliminate the adverse effects of reperfusion and retain only the beneficial effects on post-ischaemic cardiac recovery.

[0166] The data therefore show the absolute superiority of the activity of peptides Tat-DAXXp or Tat-FADDp when co-administered with bivalirudin during PPCI in AMI patients compared with the total absence of the peptides therapeutic effect in the presence of heparin.

Claims

25CLAIMS1. A combination comprising:bivalirudin anda peptide capable of inhibiting cell apoptosis, said peptide consisting of:a fragment of at most 130 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO: 2, wherein the fragment comprises the amino acid sequence set out in SEQ ID NO: 3, ora fragment of at most 130 amino acids of an amino acid sequence having at least 80% identity with SEQ ID NO:4, wherein the fragment comprises the amino acid sequence set out in SEQ ID NO: 5.

2. The combination according to claim 1, wherein the peptide is conjugated to a cellpenetrating peptide (CPP), optionally via a linker.

3. The combination according to claim 2, wherein the CPP is Tat SEQ ID NO:6.

4. The combination according to any one of claims 1 to 3, wherein the peptide is a peptide Tat-DAXXp of sequence SEQ ID NO:7 or a peptide Tat-FADDp of sequence SEQ ID NO:8.

5. A kit-of-parts including:bivalirudin anda peptide capable of inhibiting cell apoptosis as defined in any one of claims 1 to 4.

6. The combination according to any one of claims 1 to 4, or the kit-of-parts of claim 5, for use in the treatment of acute myocardial infarction (AMI), preferably ST-segment elevation myocardial infarction (STEMI), cerebral infarction, organ transplantation, cardiac interventions (extracorporeal circulation and temporary occlusion of a vessel), ischaemic lesions of the lower limbs, or acute circulatory disturbances (shock) in humans or animals.

7. The combination according to any one of claims 1 to 4, or the kit-of-parts of claim 5, for use in adjuvant treatment of primary percutaneous coronary intervention (PPCI) in the treatment of acute myocardial infarction (AMI), preferably ST-segment elevation myocardial infarction (STEMI), cerebral infarction, organ transplantation, cardiacinterventions (extracorporeal circulation and temporary occlusion of a vessel), ischaemic lesions of the lower limbs, or acute circulatory disturbances (state of shock), in humans or animals.

8. The combination according to any one of claims 1 to 4, or the kit-of-parts of claim 5, for use in the treatment of reperfusion injury in the human or animal body.

9. The combination or the kit-of-parts for the use of any of claims 6 to 8, in which (1) bivalirudin and (2) the anti -apopto tic peptide are administered parenterally, in particular bivalirudin intravenously and the anti -apopto tic peptide intravenously or intra-arterially.

10. The combination or kit-of-parts for the use of any of claims 6 to 9, wherein (1) bivalirudin and (2) the anti -apopto tic peptide are administered simultaneously, separately or sequentially.

Images