TREATMENT OF MYOCARDIAL INFARCTION

Abstract

An injectable elastin-like recombinamer (ELR) hydrogel for use in a method of treating a mammal that has suffered a myocardial infarction to modulate the response of cardiac muscle damaged by the infarct is described. The hydrogel is injected into the cardiac muscle damaged by the infarct at least two days after the myocardial infarction, and results in clinically significant repair of cardiac muscle evidenced by at least one or more of reduced scarring, positive remodelling of the damaged muscle, restoring cardiac muscle function to a clinically significant extent after infarction an improvement in angiogenesis, or a decreased pro-inflammatory response in the infarct zone, and typically within 10, 20 or 30 days of administration.

Claims

1-13. (canceled)

14. A method of treating a mammal that has suffered a myocardial infarction to modulate the response of cardiac muscle damaged by the infarct, the method comprising administration of an injectable elastin-like recombinamer (ELR) hydrogel, in which the hydrogel is injected into the cardiac muscle damaged by the infarct at least two days after the myocardial infarction, and in an amount sufficient to modulate the response of cardiac muscle damaged by the infarct.

15. The method of claim 14, in which the hydrogel is injected into the cardiac muscle damaged by the infarct at least three days after the myocardial infarction.

16. The method of claim 14, in which the hydrogel is injected into the cardiac muscle damaged by the infarct about 2 to 7 days after the myocardial infarction.

17. The method of claim 14, in which the myocardial infarction is a partial myocardial infarction.

18. The method of claim 14, for limiting scarring, inducing positive remodelling of the damaged muscle, and/or restoring cardiac muscle function to a clinically significant extent after infarction.

19. The method of claim 14, in which the ELR hydrogel is injected into a peri-infarct zone of the damaged cardiac muscle.

20. The method of claim 14, in which the ELR hydrogel is injected into a plurality of spaced-apart sites of the damaged cardiac muscle.

21. The method of claim 14, in which the ELR hydrogel is administered to the damaged cardiac muscle at a dosage of 1-500 μL ELR hydrogel per Kg body weight.

22. The method of claim 14, in which the ELR hydrogel is administered to the damaged cardiac muscle at a dosage of 10-100 μL ELR hydrogel per Kg body weight.

23. The method of claim 14, in which the ELR hydrogel is chilled prior to administration.

24. The method of claim 14, in which the ELR hydrogel is chemically crosslinked.

25. The method of claim 14, in which the ELR is functionalised with azide and cyclooctyne groups.

26. The method of claim 14, in which the ELR is functionalised with glycan moieties.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0045] FIG. 1: Representative pictures of the epicardial injection mode of a trial quantity of an elastin-like recombinamer hydrogel in a lamb heart collected from an abattoir. The hydrogel was stained with Alcan Blue to improve its immediate visualisation after the injection.

[0046] FIG. 2: Representative pictures of the physical integration of the ELR hydrogel in the tissue immediately after epicardial injection in a lamb heart collected from the abbatoir. Tissue sections were stained with Verhoeff-van Gieson both in the area injected (right picture) with the hydrogel (right) and in the area far from the injection site as a control (left picture).

[0047] FIG. 3: TEM imaging of cardiomyocytes and ECM response in sheep 28 days after myocardial infarction.

[0048] (A, B): Muscle volume fraction in the ELR hydrogel treated (a) and MI-only group (B),

[0049] (C, D): Collagen volume fraction in ELR hydrogel-treated (C) and the MI-only group (D),

[0050] (E, F): Masson's Trichome staining of the infarcted area in ELR hydrogel-treated (E) and control MI group (F).

[0051] Scale bar 200 μm. n=4 t-students test *p<0.05.

[0052] FIG. 4: Confocal imaging of capillaries in the ischaemic zones after ELT hydrogel injection (A) and in the MI-only control group (B) 28 days after induction of myocardial infarction. Scale bars 100 μm. Positive staining for capillary structures was validated in 5 days old neonatal Sprague-Dawley rats (C). Scale bar 20 μm.

DETAILED DESCRIPTION OF THE INVENTION

[0053] All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

[0054] Definitions and General Preferences

[0055] Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

[0056] Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term “a” or “an” used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.

[0057] As used herein, the term “comprise,” or variations thereof such as “comprises” or “comprising,” are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term “comprising” is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps.

[0058] As used herein, the term “disease” is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses conditions arising from infection, trauma, injury, surgery, radiological ablation, poisoning or nutritional deficiencies.

[0059] As used herein, the term “treatment” or “treating” refers to an intervention (e.g. the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s) (for example, a decrease in fibrotic area, an increase muscle volume fraction in ischaemic zones, an improvement in preservation of small vessels, or a decreased pro-inflammatory response in the infarct zone). In this case, the term is used synonymously with the term “therapy”.

[0060] Additionally, the terms “treatment” or “treating” refers to an intervention (e.g. the administration of an agent to a subject) which prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence within a treated population. In this case, the term treatment is used synonymously with the term “prophylaxis”.

[0061] As used herein, an “effective amount” or a “therapeutically effective amount” of an agent defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, but one that is sufficient to provide the desired effect, e.g. a clinically significant repair of cardiac muscle evidenced by at least one or more of a clinically relevant decrease in fibrotic area, an increase muscle volume fraction in ischaemic zones, an improvement in preservation of small vessels, or a decreased pro-inflammatory response in the infarct zone, and typically within 10, 20 or 30 days of administration. The amount will vary from subject to subject, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate “effective” amount in any individual case using routine experimentation and background general knowledge. A therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement. A therapeutic result need not be a complete cure. In one embodiment, the ELR hydrogel is administered to the damaged cardiac muscle at a dosage of about 1- to about 500 μL ELR hydrogel per Kg body weight. In one embodiment, the ELR hydrogel is administered to the damaged cardiac muscle at a dosage of about 10- to about 100 μL ELR hydrogel per Kg body weight. In one embodiment, the ELR hydrogel is administered to the damaged cardiac muscle at a dosage of about 30- to about 100 μL ELR hydrogel per Kg body weight. In one embodiment, the ELR hydrogel is administered to the damaged cardiac muscle at a dosage of about 40- to about 60 μL ELR hydrogel per Kg body weight.

[0062] In the context of treatment and effective amounts as defined above, the term subject (which is to be read to include “individual”, “animal”, “patient” or “mammal” where context permits) defines any subject, particularly a mammalian subject, for whom treatment is indicated. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; and rodents such as mice, rats, hamsters and guinea pigs. In preferred embodiments, the subject is a human.

[0063] As used herein, the term “elastin-like recombinamer” or “ELR” means a biocompatible recombinant protein-based polymer comprising a repeat sequence found in the mammalian elastic protein elastin, or a modification thereof. The most well-known members within the ELR family are based on the pentapeptide VPGVG (or its permutations), and a wide variety of polymers with the general formula (VPGXG), where X represents any natural amino acid except proline. ELR's are described in the following references: [0064] Rodriguez-Cabello et al (Polymer, Volume 50, Issue 22, 20 October 2009, Pages 5159-5169; [0065] Girotti et al (Macromolecules, 37 (9) (2004) Page 3396-3400; [0066] Martina et al (macromolecular Bioscience, 2 (7) (2002, Pages 319-328; [0067] Miao et al (Journal of Biological Chemistry, 278 (49) (2003), Pages 48553-48562; [0068] Zio et al (Macromolecules, 36 (5) (2003) Pages 1553-1558; [0069] Urry et al (Journal of Bioactive and Compatible Polymers, 6 (3) (1991) Pages 263-282; [0070] Mayer et al (Biomacromolecules, 3 (2) (2002) Pages 357-367; [0071] Spanish Patent Application No: ES2012030474; and [0072] European Patent Application No: 2397150.

[0073] As used herein, the term “hydrogel” as applied to an ELR means a three-dimensional network of ELR polymers in a water dispersion medium. In one embodiment, the ELR polymers are crosslinked (ideally chemically cross-linked) to form the three-dimensional network. Typically, the hydrogel matrix is formed of ELR homopolymer. In one embodiment, the hydrogel is injectable (i.e. has a viscosity that allows the delivery of the hydrogel in-vivo by injection). In one embodiment, the hydrogel is suitable for implantation. Methods of generating ELR hydrogels are described below and in the references above.

[0074] As used herein, the term “cross-linked” as applied to an ELR polymer means that the ELR polymer chains are covalently cross-linked with a crosslinking agent to form a three-dimensional network. Cross-linked ELR hydrogels are described in the literature, for example in the references above. The hydrogel of the invention may be crosslinked, crosslinkable, or not crosslinked. The hydrogel may be crosslinked prior to administration, or it may be crosslinked during or after administration. For example, the hydrogel and crosslinking agent may be administered using a syringe that keeps the two components separate until delivery where the components are mixed to allow in-situ crosslinking of the hydrogel. This may be achieved using a Duploject injection system.

[0075] As used herein, the term “modulate the response” as applied to cardiac muscle damaged by an infarct means one or more of a limiting scarring, inducing positive remodelling of the damaged muscle, inducing proliferation of small vessels, inducing a decrease in the pro-inflammatory response, and restoring cardiac muscle function to a clinically significant extent after infarction.

[0076] As used herein, the term “administration” in the context of a hydrogel means administration into damaged cardiac muscle, and is usually performed by means of direct injection, typically direct epicardial injection, or a catheter technique. However, other routes of administering hydrogel into the cardiac muscle may also be employed, including intravenous delivery and intraperitoneal injection. In one embodiment, the injectable hydrogel is administered into the cardiac muscle by a method selected from epicardial injection at surgery, a minimally invasive procedure, catheter delivery percutaneously (typically as an adjunct to intervention procedures), and as a stand-alone procedure.

[0077] As used herein, the term “injectable” as applied to an ELR hydrogel means that the ELR hydrogel can be epicardially injected into heart muscle of a mammal. The term “epicardial injection” means injection into the cardiac muscle through the epicardium, the envelope of tissue that surrounds the heart of mammals. In one embodiment, the injection is carried out using a 25G syringe. Various systems and methods are available for direct injection into the heart, including the EpiAccess System (EpiEP, New Haven, Conn., USA).

[0078] As used herein, the term “peri-infarct zone” means the periphery of an area of infarct that separates the ischaemic cardiac muscle and surrounding healthy non-ischaemic cardiac muscle. In one embodiment, the treatment comprises administering ELR hydrogel at a plurality of locations around the peri-infarct zone, for example 2-10 injections, and preferably at least 3, 4 or 5.

[0079] As used herein, the term “anti-fibrosis effects” means a reduction in the area of fibrosis is the treated cardiac muscle.

[0080] As used herein, the term “anti-inflammatory effects” means a reduction in the level of pro-inflammatory mediators (e.g. proinflammatory cytokines) present in the treated cardiac muscle.

[0081] As used herein, the term “partial myocardial infarction” means a myocardial infarction characterised by a partial blockage of a coronary artery, otherwise referred to as subendocardial or non-ST-elevation MI (NSTEMI). As used herein, the term “full myocardial infarction” means a myocardial infarction characterised by a full blockage of a coronary artery otherwise referred to as ST-elevation MI (STEMI) or transmural infarction. Determining whether a myocardial infarction is partial or full can be determined by suitable imaging, for example a cardiac MRI scan, echocardiogram or interpretation of an ECG.

[0082] As used herein, the term “Type 1” myocardial infarction” means a spontaneous MI related to ischemia from a primary coronary event (e.g., plaque rupture, thrombotic occlusion). “Type 2” myocardial infarction is secondary to ischemia from a supply-and-demand mismatch. “Type 3” myocardial infarction is an MI resulting in sudden cardiac death. “Type 4a” myocardial infarction is an MI associated with percutaneous coronary intervention, and “Type 4b” is associated with in-stent thrombosis. “Type 5” is an MI associated with coronary artery bypass surgery.

[0083] As used herein, the term “transmural myocardial infarction” means a myocardial infarction characterised by ischaemic necrosis of the full thickness of the affected muscle segments, extending from the endocardium through the myocardium to the epicardium. A “non-transmural myocardial infarction” is defined as an area of ischemic necrosis that does not extend through the full thickness of myocardial wall segment(s). In a non-transmural MI (subendocardial or NSTEMI), the area of ischemic necrosis is limited to the endocardium or to the endocardium and myocardium. It is the endocardial and subendocardial zones of the myocardial wall segment that are the least perfused regions of the heart and the most vulnerable to conditions of ischemia.

[0084] As used herein, the term “cardioprotective agents” refers to agents that exert certain effects, such as anti-apoptotic, anti-necrotic/cell death, anti-inflammatory, anti-fibrotic, and/or regenerative effects, that are beneficial to the patient or subject suffering from an acute myocardial infarction. Cardioprotective agents of the invention may exert their beneficial effects on cells such as for example myocytes in tissues such as heart tissues, but may also exert their effects on cells such as epithelial or endothelial cells present for example in arteries supplying the heart. When supplied sufficiently early after the occurrence of myocardial infarction the beneficial effects may include a reduction or prevention of cell death, such as through necrosis or apoptosis, of myocytes at the periphery of developing myocardial infarcts and/or an induction of growth of surviving myocytes. The cardioprotective agents may also stimulate growth or prevent cell death of epithelial or endothelial cells present in arteries supplying the heart. These effects may lead to a reduction in size and/or partial or complete restoration of the moderately injured and/or necrotic tissue of the infarcted area. Additional beneficial effects may include dissolving blood clots or other organic material leading to arterial constrictions or occlusions, such as vulnerable atherosclerotic plaques. Cardioprotective agents disclosed herein that exert anti-apoptotic effects include TGFβ1, IGF1, eNOS and bFGF and its subtypes. Other cellular factors that are involved in tissue repair or maintenance, stem cell factors, anti-apoptotic factors and/or growth factors may also be useful as cardioprotective agents. Such agents include granulocyte colony stimulating factor (GCSF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), fibroblast growth factors (FGFs), Angiotensin II, and stromal cell-derived factor 1 (SDF-1). TGF-β is a secreted protein that exists in three isoforms TGF-β1, TGF-β2 and TGF-β3. The TGF-β family is part of a superfamily of proteins known as the transforming growth factor beta superfamily, which includes inhibins, activin, anti-müllerian hormone, bone morphogenetic protein, decapentaplegic and Vg-1. The insulin-like growth factors (IGFs) are polypeptides with high sequence similarity to insulin and comprise of cell-surface receptors (e.g. IGF1R and IGF2R), ligands (e.g. IGF-1 and IGF-2), IGF binding proteins (e.g. IGFBP 1-6), as well as associated IGFBP proteases. Insulin-like growth factor 1 (IGF-1) is mainly secreted by the liver as a result of stimulation by growth hormone (GH) and plays a role in the promotion of cell proliferation and the inhibition of cell death (apoptosis). Insulin-like growth factor 2 (IGF-2) is thought to be a primary growth factor required for early development while IGF-1 expression is required for achieving maximal growth. Many cardioprotective agents disclosed herein, such as TGFβ1 and IGF1, are commercially available in purified or recombinant form and have been suggested for many therapeutic uses, such as for example wound healing (U.S. Pat. Nos. 4,861,757; 4,983,581 and 5,256,644), or induction of bone growth (U.S. Pat. No. 5,409,896 and 5,604,204).

Exemplification

[0085] The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

[0086] Hydrogel Preparation and Injection Conditions

[0087] Elastin-like recombinamers (ELRs) functionalised with azide (HRGD6) and cyclooctyne (HE5) groups for crosslinking were weighed under sterile conditions. ELRs were resuspended to maintain the HE5-cycloctyne to HRGD6-azide ratio at 1.79:1. ELRs were stored at 4° C. until the day before they were required for injection. Therefore, each ELR was dissolved in 750 μl of sterile molecular grade water overnight at 4° C. Once the solutions were totally clear, 250 μl of HRGD6-azide was placed in a sterile eppendorf tube, where 250 μl of HE5-cycloctyne was added to initiate crosslinking of the recombinamers and hydrogel formation. Cold sterile tips were used to carry out this procedure and the eppendorf tube containing the mixed ELRs was kept on ice for exactly 10 minutes before injection. The feasibility of the ELRs hydrogel epicardial injection and its physical integration in the myocardial tissue was tested ex vivo in a lamb heart collected from the abattoir (FIG. 1 and FIG. 2).

[0088] Large Animal (Sheep) In Vivo Study

[0089] Myocardial infarction was induced in five months old male sheep (weight 30 kg) by transmural suture ligation at intervals parallel to and along the left anterior descending artery. This novel concept was developed to mimic as closely as possible, the real-life type of myocardial infarction seen in clinical practice, unlike previous models of simple LAD ligation. Cardiothoracic surgeon (Mark Da Costa) and the veterinary team ensured that the surgical procedure was performed according to current European animal laboratory guidelines and practice. One week after MI induction the animal was re-operated and the hydrogel solution was injected into the pen-infarct zone by the repeated administration of 500 μl of ELR solution by epicardial injection. A sterile syringe having a maximum capacity of 1 ml and a 25G needle was used to perform each epicardial injection through the wall of the heart. Three hydrogels of 500 μl volume each were injected per sheep through the peri-infarct zone, which is the edge of the region between the site where ischemia was induced by coronary ligation and the healthy zone of the myocardial walls. The needle was carefully retracted after each injection to avoid backflow of the hydrogel solution. Echocardiography measurements were performed before surgery and at day 28 days post MI induction and 21 days post injection of the hydrogel.

[0090] Histological Analysis

[0091] According to our first preliminary analysis on a first batch of animals, the hydrogel-treated group showed a decreased fibrotic area and an increased muscle volume fraction in the ischemic zones by TEM imaging (FIG. 3(c) FIG. 3(d)). Masson's Trichrome staining confirmed the deposition of collagen in the fibrotic areas which was quantified to validate the first set of analysis once the harvesting of all the tissue was completed (FIG. 3(e); FIG. 3(f)). The preservation of small vessels was observed in both the elastin treated group and the control group by immunofluorescence experiments for CD31 and GS-1 lectin markers. Specifically GS-1 staining for capillaries and endothelial structures—which had been validated as a control in neonatal rat myocardial tissue—showed a higher expression in the ELR hydrogel treated animals (FIG. 4).

Equivalents

[0092] The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.