Patent Application
20130115222
This invention relates to the identification of a ligand-receptor system involved in the microvascular functions of the heart, and to the manipulation of the system to prevent or limit microvascular dysfunction and damage following acute myocardial ischemia, thereby promoting myocardial recovery.
Treatments of acute myocardial infarction focus primarily on limiting the duration of ischemia, using angioplasty or thrombolysis to establish reperfusion. However, even with reestablishment of macrovascular patency, a significant proportion of patients exhibit microvascular damage in the infarcted tissue leading to tissue fibrosis, impaired cardiac contractility, and ultimately to a higher incidence of death (Eltzschig et al., Brit. Med. Bulletin, 70:71-86 (2004)). To date, the specific pro-inflammatory cytokines that mediate microvascular dysfunction or apoptosis following cardiac ischemia are unknown, and identification of such locally produced factor(s) would provide important potential therapeutic targets to limit microvascular dysfunction, and promote myocardial recovery following reperfusion.
Nerve growth factor (NGF) mRNA is rapidly induced following cardiac ischemia-reperfusion in rodents (Hiltunen et al., Journal of Pathology, 194(2):247-253 (2001); Hasan et al., Brain Research, 1124(1):142-154 (2006)). NGF, the prototypic member of the neurotrophin family which also includes brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), binds to the receptor tyrosine kinase TrkA to mediate cell survival and differentiation (Reichardt, Philos Trans R Soc Lond B Biol Science, 361(1473):1545-1564 (2006)). Neurotrophins are initially synthesized as precursors, or proneurotrophins, which can be proteolytically cleaved to release the C-terminal domain, or mature neurotrophin. Recent studies show that proNGF is not an inactive precursor, but acts as a signaling ligand, distinct from its mature counterpart, to mediate neuronal cell death (Lee, Science, 294: 1945-48 (2001), and reviewed in Hempstead, Neurotoxicity Research, 16: 255-60 (2009)). The pro-apoptotic action of proNGF requires the expression of a distinct receptor p75NTR, which encodes an intracellular death domain, and a co-receptor, sortilin (Nykjaer et al., Nature, 427(6977):843-848 (2004); Jansen et al., Nat Neuroscience, 10(11):1449-1457 (2007)).
In the cardiovascular system, the functions of the related neurotrophins BDNF and NT-3 have been established in promoting vascular integrity and regulating cardiac septation (Donovan et al., Nat Genet, 14(2):210-213 (1996); Donovan et al., American Journal of Pathology, 147(2): 09-324 (1995); and reviewed in Caporali et al., Physiol Review, 89(1):279-308 (2009)). However, the actions of cardiac NGF are less well characterized. NGF is synthesized by cardiomyocytes and cardiac vascular smooth muscle cells, and promotes sympathetic innervation (Glenbova and Ginty, J. Neurosci 24: 743 (2004)). In addition, myocyte-derived NGF can acutely modulate synaptic transmission between sympathetic neurons and cardiac myocytes (Lockhart et al., Journal of Neuroscience, 17(24):9573-9582 (1997)), in part by altering the firing properties of these neurons (Luther et al., Journal of Neurophysiology, 96(2):946-958 (2006)). Indeed, NGF-deficient (NGF−/−) mice die perinatally of presumed innervation defects without overt abnormalities of the cardiovascular system (reviewed in Caporali et al., Physiol Review, 89(1):279-308 (2009)).
ProNGF and p75NTR are expressed at low to undetectable levels in most adult tissues, but are rapidly induced following acute injury and promote apoptosis, best studied in the central and peripheral nervous systems (reviewed in Hempstead, Neurotox Research, 16(3):255-260 (2009)). Although no studies have evaluated proNGF levels following cardiac ischemia, the level of NGF mRNA increases within hours following cardiac ischemia/reperfusion, and elevated protein levels are maintained for several days before returning to baseline (Hiltunen et al., Journal of Pathology, 194(2):247-253 (2001)). In addition, local expression of p75NTR is increased in endothelial cells and vascular smooth muscle cells following acute aortic injury (Donovan et al., American Journal of Pathology, 147(2): 09-324 (1995)), and p75NTR activation promotes vascular smooth muscle cell apoptosis (Wang et al., Am J Pathology 157: 1247-58, 2000) and endothelial cell apoptosis (Kim et al., Journal of Biological Chemistry, 279(32):33538-33546 (2004)). p75NTR deficiency (p75NTR−/−) results in reduced apoptosis of vascular smooth muscle cells following carotid artery injury, suggesting a role for locally produced neurotrophins in regulating this vascular response (Kraemer, Circ Res, 91(6):494-500 (2002)). No studies to date have examined sortilin, or other members of the sortilin family members including SorCS1, SorCS2, SorCS3, in the vasculature.
Endothelial cells depend on complex and reciprocal interactions with pericytes during vascular development, and pericytes maintain microvascular structure and function in the adult vasculature (reviewed in Gaengel et al., Arterioscler Thromb Vasc Biol, 29:630-638 (2008)). Disruption of endothelial:pericyte communication leads to vascular hemorrhage and embryonic lethality, best exemplified by mice deficient in Pdgfb or Pdgfrb, which fail to recruit pericytes to specific vascular beds (Lindahl et al., Science, 277(5323):242-245 (1997); Hellstrom et al., Development 126:3047-55 (1999)). In adult mice, TGFβ and bone morphogenetic proteins play critical roles in maintaining pericyte survival and promoting microvascular integrity, as genetic deletion of Bmp1a in smooth muscle cells leads to cardiac dysfunction under hypoxic conditions, and blockade of TGFβ leads to abnormal vascular permeability (El-Bizri et al., Circ Res 102(3):380-388 (2008); Walshe et al., PLoS One 4(4):1-16 (2009)). In summary, dysregulation of endothelial cell:pericyte crosstalk during maturation of the cardiac microvasculature leads to cardiac dysfunction later on in life.
The inventors have identified a new ligand-receptor system, proNGF and p75NTR/SorCS2, to be involved in the microvascular functions of the heart. Accordingly, this disclosure provides methods for limiting microvascular damage following acute myocardial ischemia based on administration of an antagonist of this newly identified system, thereby promoting myocardial recovery.
One aspect of this disclosure is directed to a method of limiting microvascular damage following acute myocardial ischemia in a subject by administering a proNGF antagonist. A proNGF antagonist can include a neutralizing antibody that binds specifically to proNGF, an aptamer, an oligopeptide, a small molecule compound, or a nucleic acid molecule which reduces the level or activity of a proNGF mRNA, for example.
Another aspect of this disclosure is directed to a method of reducing microvascular dysfunction and the associated damage following acute myocardial ischemia by administering a SorCS2 antagonist. A SorCS2 antagonist can include an antibody, an aptamer, an oligopeptide, a small molecule compound, or a nucleic acid molecule which reduces the level or activity of the SorCS2 mRNA.
An antagonist or a cocktail of antagonists can be administered to a subject as soon as practical after acute myocardial ischemia (AMI) occurs. In some embodiments, an antagonist or a cocktail of antagonists is administered to the subject within 48 hours. In a specific embodiment, administration is achieved within 2-6 hours of the AMI.
An antagonist or a cocktail of antagonists can be combined with a pharmaceutically acceptable carrier for administration, and can be given to a subject via standard routes including ingestion, injections via an intravenous, intraperitoneal, subcutaneous, transdermal, intramuscular, intranasal, or sublingual route, or via catheter delivery at the time of percutaneous intervention or during an open heart surgery.
The inventors have identified a new ligand-receptor system involved in the microvascular functions of the heart, which can be exploited to prevent microvascular dysfunction in the ischemic heart or limit microvascular damage following acute myocardial ischemia, thereby promoting myocardial recovery.
More specifically, the inventors have determined that both the proNGF ligand and its receptors p75NTR and SorCS2, a sortilin family member, are induced in the infarcted murine heart. The inventors have generated a knock-in mouse in which one allele expresses a cleavage-resistant form of proNGF under the control of the endogenous Ngf promoter, and the other non-targeted allele expresses NGF (termed proNGF-HA/+ mice). These mice are viable, but develop a dilated cardiomyopathy in early adulthood, leading to mortality by 6 to 8 months of age. The inventors have shown that pericytes ensheathing cardiac capillaries express p75NTR and SorCS2, but not sortilin, during late embryogenesis. The inventors have also shown that proNGF-expressing animals deficient in p75NTR do not develop cardiomyopathy, whereas proNGF-expressing animals deficient in sortilin are not rescued from this phenotype. In addition, the inventors have demonstrated that in the proNGF-HA/+ mice, endothelial cells are activated, leading to increased vascular permeability.
Without intending to be bound by any particular theory, it is believed that induction of proNGF and p75NTR in the heart of the proNGF-HA/+ mice act upon pericytes to promote dysfunction of microvascular endothelial cells, leading to an activated endothelium and subsequent cardiomyopathy; and SorCS2, which binds to proNGF, functions as a co-receptor with p75NTR to alter pericyte function.
In accordance with the present disclosure, this ligand-receptor system (proNGF-p75NTR/SorCS2), newly identified by the inventors, can be manipulated to reduce, limit or prevent microvascular dysfunction and microvascular damage in the heart following acute myocardial ischemia.
Acute myocardial ischemia (or AMI) is commonly known as heart attack, which occurs when blood supply to a part of the heart is interrupted. The resulting oxygen shortage can cause damage or death (or “infarction”) of heart muscle tissue if left untreated for a period of time. Prior to this disclosure, treatments of acute myocardial infarction focus primarily on reestablishment of blood flow in coronary arteries. However, even with reestablishment of macrovascular patency, a significant proportion of patients exhibit microvascular damage in the infarcted tissue that leads to chronically impaired heart function and disability, and a higher incidence of death (Eltzschig et al., Brit. Med. Bulletin, 70:71-86 (2004)). In accordance with this disclosure, however, myocardial recovery following reperfusion can be improved by preventing or otherwise limiting the microvascular dysfunction or damage through the use of an antagonist of the proNGF-p75NTR/SorCS2 system.
The term “microvascular” as used herein refers to small blood vessels, such as arterioles, capillaries and venules. Such small blood vessels are generally composed of two cell types: endothelial cells, which form the inner vessel wall, and pericytes, which form a layer around the endothelial tube (Gaengel et al., Arterioscler Thromb Vasc Biol 29: 630-638, 2009).
The term “microvascular dysfunction” refers to abnormalities in the structure and/or function of small blood vessels. Microvascular dysfunction can be assessed and determined by analyzing cardiac contractility, vascular permeability, electromicroscopy of microvasculature, and analysis of the levels of endothelial and pericyte markers (such as CD-31, isolectin B4, PDGFRβ and NG2), among others.
The term “microvascular damage” refers to the loss or compromise of the structural integrity and/or function of small blood vessels as a result of microvascular dysfunction.
As disclosed herein, administration of an antagonist of the proNGF-p75NTR/SorCS2 ligand-receptor system can reduce or limit, or event completely prevent, microvascular dysfunction and the associated damage following acute myocardial ischemia.
The term “antagonist” as used herein, refers to a molecule that inhibits the expression level of a component of the proNGF-p75NTR/SorCS2 ligand-receptor system on pericytes (“expression antagonist”); or alternatively, inhibits the interaction or binding between the components of the proNGF-p75NTR/SorCS2 ligand-receptor system expressed on pericytes (“binding antagonist”), thereby reducing the amount, formation, function, and/or downstream signaling of this ligand-receptor system.
A molecule is considered to inhibit the expression level of a component of the proNGF-p75NTR/SorCS2 system if the molecule causes a significant reduction in the expression (either at the level of transcription or translation) of the component. Similarly, a molecule is considered to inhibit the binding between the components of the proNGF-p75NTR/SorCS2 ligand-receptor system if the molecule causes a significant reduction in the binding between the components and the ligand-receptor complex formed, which causes a significant reduction in downstream signaling and functions mediated by the ligand-receptor system, e.g., activated endothelial cells and increased vascular permeability. A reduction is considered significant, for example, if the reduction is at least about 25%, and in some embodiments at least about 50%, and in other embodiments at least about 90%.
A binding antagonist can act in two ways. A binding antagonist can compete with the ligand proNGF for the receptors thereby interfering with, blocking or otherwise preventing the binding of proNGF to p75NTR and/or SorCS2. This type of antagonist, which binds the receptor but does not trigger the expected signal transduction, is also known as a “competitive antagonist” and can include, for example, an oligopeptide designed based on a proNGF sequence, or an antibody directed to SorCS2. Alternatively, a binding antagonist can bind to and sequester the ligand, proNGF, with sufficient affinity and specificity to substantially interfere with, block or otherwise prevent binding of proNGF to p75NTR and/or SorCS2. This type of antagonist is also known as a “neutralizing antagonist”, and can include, for example, an antibody or aptamer directed to proNGF which binds specifically to proNGF.
An antagonist can also be characterized based on the target molecule which the antagonist is intended to antagonize. For example, a proNGF antagonist refers to a molecule which inhibits or reduces the expression of proNGF; or interferes with, blocks or otherwise prevents the interaction or binding of proNGF to p75NTR and/or SorCS2. On the other hand, a SorCS2 antagonist refers to a molecule which inhibits or reduces the expression of SorCS2; or interferes with, blocks or otherwise prevents the interaction between SorCS2 and proNGF and/or p75NTR.
In one embodiment, this disclosure provides a method of reducing microvascular dysfunction and the associated damage following acute myocardial ischemia by administration of a proNGF antagonist.
As disclosed herein, a proNGF antagonist can be a neutralizing antibody that is specific for proNGF, an aptamer, an oligopeptide, a small molecule compound, or a nucleic acid molecule which reduces the level or activity of a proNGF mRNA, for example.
In a specific embodiment, a proNGF antagonist is a neutralizing antibody that is specific for proNGF.
In this disclosure, a molecule (such as an antibody or aptamer) that is specific for proNGF is a molecule that binds with substantially greater affinity, and in some embodiments, binds nearly exclusively to proNGF, relative to a mature NGF and other proneurotrophins. By “substantially greater affinity” it is meant that the binding affinity (Kd) of a molecule for a proNGF is at least 5 fold, 10 fold, 50 fold, 100 fold, or 1000 fold or greater, of the binding affinity of the molecule for the mature NGF or other proneurotrophins.
In one particular embodiment, an antibody specific for proNGF is an antibody directed to the prodomain of proNGF.
The prodomain of proNGF is clearly distinct from those of other proneurotrophins (
The pro-domain of proNGF is highly conserved across species. As shown in
In certain embodiments, a proNGF-specific antibody is specifically directed to motifs or epitopes within the prodomain, including contiguous sequences of amino acids within the prodomain that are highly conserved across species as shown in
The term “antibody” as used herein includes intact immunoglobulin molecules, as well as molecules that include an antibody hypervariable region that binds specifically to an intended antigen, with or without an antibody constant region. The hypervariable region can include an entire antibody variable region. Thus, an antibody molecule that includes an antibody hypervariable region can be an intact antibody molecule, antibody fragments (including single chain antibodies) which retain the antigen binding specificity of intact antibodies, as well as chimeric and humanized antibodies. The antibody can be polyclonal or monoclonal, and can be of any class of immunoglobins, such as: IgG, IgM, IgA, IgD or IgE, and the subclass thereof.
Suitable antibodies can be produced in a non-human mammal, including for example, rabbits, rats, mice, horses, goats, camels, or primates. Monoclonal antibodies produced from a non-human mammal can be humanized to reduce the immunogenicity for use in humans following techniques documented in the art. For example, to humanize a monoclonal antibody raised in mice, one approach is to make mouse-human chimeric antibodies having the original variable region of the murine mAb, joined to constant regions of a human immunoglobulin. Chimeric antibodies and methods for their production are well known in the art. See, e.g., Cabilly et al., European Patent Application 125023 (published Nov. 14, 1984); Taniguchi et al., European patent Application 171496 (published Feb. 19, 1985); Morrison et al., European Patent Application 173494 (published Mar. 5, 1986); Neuberger et al., PCT Application WO 86/01533, (published Mar. 13, 1986); all of which are incorporated herein by reference. Alternatively, humanized antibodies can be made to by including constant regions of a human immunoglobulin, and additionally, substituting framework residues of the variable regions of a non-human antibody with the corresponding human framework residues, either leaving the non-human CDRs substantially intact, or even replacing the CDR with sequences derived from a human genome. See, e.g., Maeda et al., Hum. Antibod. Hybridomas 2: 124-134, 1991, and Padlan, Mol. Immunol. 28: 489-498, 1991. As an additional alternative, human antibodies can be produced from transgenic animals (e.g., transgenic mice) whose immune systems have been altered to correspond to human immune systems. An example of such a mouse is the so-called XenoMouse™ (Abgenix, Freemont, Calif.), described by Green, “Antibody Engineering via Genetic Engineering of the Mouse: XenoMouse Stains are a Vehicle for the Facile Generation of Therapeutic Human Monoclonal Antibodies,” J. Immunol. Methods 10; 231(1-2):11-23(1999).
In another specific embodiment, a proNGF antagonist is an aptamer that binds specifically to proNGF.
Aptamers are molecules, either nucleic acid or peptide, that bind to a specific target molecule. Nucleic acid aptamers are generally short strands of DNA or RNA that have been engineered through repeated rounds of in vitro selection known as SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets. Peptide aptamers can be selected using various systems, most frequently through the yeast two hybrid system. Peptide aptamers generally consist of a variable peptide loop (typically composed of ten to twenty amino acids), attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to an antibody.
In still another specific embodiment, a proNGF antagonist is an oligopeptide or a small molecule compound that binds to the receptors of proNGF (i.e., the p75 receptor and/or SorCS2) thereby blocking the binding of proNGF to its receptors, but does not lead to the downstream signaling or biological activity triggered by binding of proNGF to p75NTR/SorCS2.
Such small molecules and oligopeptides can be discovered by methods well known in the art. Typically, discovering such molecules involves providing a cell that expresses p75NTR and/or SorCS2, providing a small molecule or oligopeptide to be tested, and determining whether the small molecule or oligopeptide to be tested binds to p75NTR and/or SorCS2 and, optionally, results in the biological activity caused by binding of a proNGF to p75NTR/SorCS2. If the molecule binds with high affinity to p75NTR and/or SorCS2, it is a candidate for use in the present method to limit microvascular damage. If the molecule binds to p75NTR and/or SorCS2 with high affinity and blocks binding of proNGF to p75NTR/SorCS2, it is a stronger candidate. If, in addition to blocking binding, the molecule also fails to cause the biological activity expected from activating p75NTR/SorCS2, e.g. to activate endothelial cells, the molecule is a candidate for pre-clinical or clinical trials.
The oligopeptide has at least approximately four amino acid residues, and in some embodiments at least approximately five amino acid residues, and in other embodiments at least approximately six amino acid residues. The maximum number of amino acid residues is not important, as long as the oligopeptide has the desirable properties mentioned above. The oligopeptide may be linear or cyclic.
Some examples of oligopeptides include:
wherein Z represents any alpha amino acid and z represents any number from 0 to approximately 20, preferably from 0 to approximately 10, and more preferably from 0 to approximately 5. Any of these oligopeptides may be cyclic.
Small molecules include organic compounds, organometallic compounds, salts of organic and organometallic compounds, saccharides, amino acids, and nucleotides. Small molecules typically have molecular weights less than approximately 1000 Daltons, in some embodiments less than 800 Daltons. Small molecules include compounds that are found in nature as well as synthetic compounds.
In another specific embodiment, a proNGF antagonist administered is a nucleic acid molecule which reduces the level or activity of a proNGF mRNA. Such nucleic acid molecule includes an antisense RNA, a siRNA, a miRNA (or “microRNA”) or a transgene which codes for and is capable of expressing any such RNA molecule in the target tissue of a recipient. An antisense RNA is an RNA molecule that is complementary to endogenous mRNA and blocks translation from the endogenous mRNA by forming a duplex with the endogenous mRNA. siRNAs are small (typically 20-25 nucleotides in length) double-stranded RNAs which are known to be involved in the RNA interference pathway and interfere with the expression of a specific gene. Given the sequence of a target gene, siRNAs can be designed, and made either synthetically or in cells from an exogenously introduced vector (e.g., a plasmid) to achieve suppression of expression of a gene of interest. Similar to siRNAs, miRNAs are also small RNA molecules (generally about 21-22 nucleotides) that regulate gene expression. miRNAs are processed from long precursors transcribed from non-protein-encoding genes, and interrupt translation through imprecise base-pairing with target mRNAs. miRNA can be designed and introduced to cells or tissues to target and suppress the expression of a gene of interest (proNGF, SorCS2 or p75NTR) using techniques documented in the art. Modulation of miRNA can be accomplished by viral-mediated delivery of pro-miRNA or decoymiR or by delivery in plasma (as examples, Cordes K R, et al, Nature 460:705 (2009); Caporali A, et al., Circulation 123:282, (2011); Castoldi, M, J., Clin Invest. 121:1386 (2011); Vickers, K C et al., Nat Cell Biol 13: 423 (2011)).
In a further embodiment, this disclosure provides a method of reducing microvascular dysfunction and the associated damage following acute myocardial ischemia by administration of a SorCS2 antagonist.
As disclosed herein, a SorCS2 antagonist can be an antibody, an aptamer, an oligopeptide, a small molecule compound, or a nucleic acid molecule which reduces the level or activity of the SorCS2 mRNA.
In a specific embodiment, a SorCS2 antagonist is an antibody that binds specifically to SorCS2 and inhibits the interaction of SorCS2 with proNGF and/or p75NTR. An antibody that is specific for SorCS2 is an antibody that binds with substantially greater affinity, and in some embodiments, binds nearly exclusively to SorCS2, relative to other members of the sortlin family such as sortlin. By “substantially greater affinity” it is meant that the binding affinity of an antibody for SorCS2 is at least 5 fold, 10 fold, 50 fold, 100 fold, or 1000 fold or greater, of the binding affinity of the antibody for other members of the sortlin family.
In one particular embodiment, a SorCS2-specific antibody is directed to the ectodomain of SorCS2 (amino acids 20-1078 of human SorCS2). In certain embodiments, a SorCS2-specific antibody is specifically directed to specific motifs or epitopes within the ectodomain, such as the cystein-rich domain (amino acid residues 611-750 of human SorCS2), or the 10 bladed propeller domains (amino acids 45-610). The amino acid sequence of human SorCS2 is set forth in SEQ ID NO: 14 (Accession No. NP—065828).
Similar to proNGF antagonists as described above, SorCS2 antagonists are not limited to antibodies, but also include nucleic acid or peptide aptamers that bind specifically to SorCS2 and inhibit its interaction with proNGF and/or p75NTR, oligopeptides or small molecule compounds that block the interaction of SorCS2 with proNGF and/or p75NTR, as well as nucleic acid molecules (such as antisense, siRNA, or miRNAs) which reduce the level or activity of the SorCS2 mRNA.
Also provided herein is a cocktail of more than one antagonist molecule, which is also suitable for administration. The cocktail may, for example, include one or more antibody molecules, one or more aptamer molecules, one or more oligopeptides or small molecules, or various combinations thereof. The cocktail can also include one or more proNGF antagonists, in combination with one or more SorCS2 antagonists.
An antagonist or a cocktail of antagonists is administered to a subject as soon as practical after acute myocardial ischemia (AMI) occurs. However, antagonists administered as late as 48 h after the AMI occurs can still be effective. In certain embodiments, an antagonist is administered to the subject within 24 hours, 18 hours, 12 hours, 6 hours or even 2 hours of the AMI. In a specific embodiment, an antagonist is administered initially within 2-6 hours of the AMI. A repeat dose or doses can be administered as appropriate, which can be determined by a skilled physician.
An antagonist can be combined with a pharmaceutically acceptable carrier in any convenient and practical manner, e.g., by admixture, solution, suspension, emulsification, encapsulation, absorption and the like, and can be made in formulations such as tablets, capsules, powder, syrup, suspensions that are suitable for injections, implantations, inhalations, ingestions or the like.
As used herein, a pharmaceutically acceptable carrier includes any and all solvents, dispersion media, isotonic agents and the like. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the effectiveness of the active ingredients contained therein, its use in practicing the methods disclosed herein is appropriate. The carrier can be liquid, semi-solid, e.g. pastes, or solid carriers. Examples of carriers include oils, water, saline solutions, alcohol, sugar, gel, lipids, liposomes, resins, porous matrices, binders, fillers, coatings, preservatives and the like, or combinations thereof.
The concentration of an antagonist in formulations may range from as low as about 0.1% to as much as 15 or 20% by weight and can be selected based on the nature of the particular antagonist used, the mode of administration selected, among other considerations. Thus, a typical formulation for injection could be made up to contain 1 mL sterile buffered water of phosphate buffered saline and 1-1000 mg, possibly 10-100 mg, of an antagonist such as an antibody-based antagonist, for example.
Depending on the nature of the antagonist or the circumstances of AMI, a pharmaceutical formulation containing an antagonist, can be given to the subject by standard routes, including ingestion, or injections via an intravenous, intraperitoneal, subcutaneous, transdermal, intramuscular, intranasal, or sublingual route, or via catheter delivery at the time of percutaneous intervention or during an open heart surgery. Generally speaking, an antibody or aptamer-based antagonist can be delivered intravenously. An RNA-based antagonist can be delivered directly into the heart, e.g., by catheter delivery at the time of percutaneous intervention, or with open heart surgery if bypass grafting is necessary.
The amount of antagonist administered to be effective may depend on the condition of the patient (e.g., age, body weight and health), the time interval since the occurrence of AMI, and the nature of the antagonist. The precise amount of an antagonist to be effective can be determined by a skilled physician.
The present description is further illustrated by the following examples, which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, and published patent applications as cited throughout this application) are hereby expressly incorporated by reference.
This example describes the materials and methods used in the experiments disclosed in Example 2.
Genomic PAC clone RPCI21-494-C12 was identified from a 129/SvevTACfBR female spleen library purchased from the UK HGMP MRC Geneservice (Cambridge, UK) using standard dot blotting techniques (Osoegawa et al., Genome Research, 10(1):116-128 (2000)). The hemagglutinin (HA) epitope tag was added by site-directed mutagenesis in-frame before the stop codon. The furin recognition site was mutated using site-directed mutagenesis (Stratagene) from KR to AA. A modified neomycin-resistance cassette, consisting of (from 5′ to 3′) frt-loxp-engrailed splice acceptor-pGK-neor-stop codon-frt-loxp sequences, was inserted 350 bp upstream from the start codon. A diphtheria toxin cassette was used for positive selection. Targeting efficiency was approximately 4%. Three positive independently-derived clones expressing Prongf-HA (called proNGF-HA/+ herein) were expanded and microinjected into C57B1/6 embryos. Three independent lines of proNGF-HA mice were obtained in this manner. Two lines of Ngf-HA knock-in mice (called wtNGF-HA/+) were generated in the same manner, except the furin cleavage site was not mutated in this construct. Southern blotting was performed using standard techniques. PCR was used to identify the knocked-in allele from the wild-type allele by amplifying the HA epitope tag: 5′-TGA AGC CCA CTG GAC TAA ACT T-3′ (SEQ ID NO: 15) and 5′-AAT CTG GAA CAT CGT ATG GG-3′ (SEQ ID NO: 16). The presence of the neomycin-resistance cassette was determined using a second pair of PCR primers: 5′-GAG ATC CAC TAG TTC TAG CCT CGA G-3′ (SEQ ID NO: 17) and 5′-CCC ACA CAC TGA CAC TGT CAC AC-3′ (SEQ ID NO: 18).
All procedures were approved by the Weill Cornell Medical College IACUC. The β-actin-cre delete strain was used to remove the neo-resistance cassette and enable expression of the knocked-in allele. The following primer pairs were used to identify the presence of the cre-recombinase allele: 5′-TTA TAA CAC CCT GTT ACG TAT AGC C-3′ (SEQ ID NO: 19) and 5′-TAT CTC TGA CCA GAG TCA TCC TTA G-3′ (SEQ ID NO: 20). p75NTR−/− mice were purchased from Jackson Laboratories, and backcrossed to >G8 on a C57B1/6 background, prior to using them in proNGF-HA/+ genetic rescue breeding. Other control strains included NGF+/− (Crowley et al., Cell, 76(6):1001-1011 (1994)) and sortilin−/− (Jansen et al., Nat Neuroscience, 10(11):1449-1457 (2007)) mice.
For immunohistochemical analysis, embryonic trunks and adult hearts were snap frozen in OCT:30% sucrose (1:1; Sakura). For protein and RNA analyses, hearts were stored at −80° C. until processing.
FITC-dextran (70 kDa; Sigma) was injected as a bolus through the tail vein of 3 mo mice (Camelleri 1983). After perfusion for 10 minutes, the animals were sacrificed and hearts harvested and processed for immunofluorescence.
Frozen cryostat sections were immediately fixed in acetone or in 4% PFA. For IHC, after 15 min in 0.1% H2O2/methanol at −20° C., the sections were rinsed in PBS, blocked (5% serum of secondary antibody/0.1% Triton x-100/PBS), and incubated with primary antibody overnight at 4° C. The primary antibodies used were: HA (Sigma, 1:400), p75NTR (ECD: R&D, 1:1000; ICD: Promega, 1:500), sortilin (R&D, 1:400), SorCS2 (ICD: generated by immunizing rabbits against huSorCS2 aa 1138-1159, coupled to KLH, 1:1000; or ECD: R&D, 1:100), activated caspase-3 (Cell Signaling, 1:200), tyrosine hydroxylase (Jackson Immunological, 1:100), phospho-cJun (Cell Signaling; 1:100), CD31 (BD Biosciences, 1:100), CD41 (BD Biosciences, 1:100), fibrin(ogen) (FITC-conjugated, Dako, 1:250), CD68 (TRITC-conjugated, Serotec, 1:100), Iba1 (Wako, 1:400), ICAM (eBioscience, 1:100). Biotinylated secondary antibodies were conjugated to ABC reagent and detected using the VIP kit (Vector Labs). IF was performed essentially as above. Alexa-conjugated secondary antibodies (Invitrogen) were used to detect primary antibodies. Confocal microscopy was performed using either a Zeiss LSM510 or LSM700 laser scanning microscope.
Frozen hearts and brains were homogenized in lysis buffer (0.1M Tris pH 7.4/1% Triton x-100/0.1% NP-40/0.05% SDS/10% glycerol/protease inhibitor cocktail [Sigma]). HA-tagged proteins were pulled down using anti-HA antibody (Sigma) at 4° C. Protein A-sepharose slurry (Sigma-Aldrich) were added to capture the immune-complex, washed extensively, and boiled in SDS-PAGE loading buffer. Blotted proteins were detected using HA.11 monoclonal antibody (Covance) and developed with ECL (Amersham). See
RT-PCR was performed following standard protocols and reagents (Invitrogen). PCR was performed using the HA primers described above, as well as β-actin: 5′-AAA GAG AAG CTG TGC TAT GTT GCT C-3′ (SEQ ID NO: 21) and 5′-GCA TAG AGG TCT TTA CGG ATG TCA A-3′(SEQ ID NO: 22). ELISA.
The NGF Emax kit (Promega) was used for all ELISA measurements, following manufacturer's instructions.
An Accuson Sequoia clinical ultrasound equipped with a 14 Hz probe (graciously provided by K. Hajjar, CUWMC) was used for this study. Each mouse was anesthesized using Avertin, allowed to recover baseline heart rate for at least 20 minutes on a warming pad set on low temperature, and depilated. The probe head, whose range was extended using a 1 cm gel offset, was applied to a layer of ultrasound gel on the mouse's chest, and the midline of the heart was detected by the localization of the papillary muscles in the left ventricle. Once the midline was identified, the probe was swept up and down the caudal-rostral axis of the heart to ensure observation at the widest part of the left ventricle, and then the probe was stabilized for 1 min of recording to VHS tape. A printout of the M mode was made, to facilitate calculation of the fractional shortening (% FS) using the following formula:
% FS=[(LVEDD−LVESD)/LVEDD]×100
where LVEDD=left ventricular end-diastolic diameter, and LVESD=left ventricular end-systolic diameter. All statistical analyses were performed using Student's t-test.
Anesthesia was induced with 4% inhaled isoflurane and maintained with 2% isoflurane. Mice were then intubated and mechanically ventilated. Core body temperature was monitored with a rectal probe and maintained at 37° C. and ECG was monitored throughout the surgery using a lead II configuration and PowerLab data acquisition system. A left thoractomy was performed in the 4th intercostal space and the pericardium was opened. The left anterior descending coronary artery (LAD) was reversibly ligated with an 8-0 suture for 40 minutes and then reperfused by release of the ligature. Occlusion was confirmed with ST segment elevation on the ECG, regional cyanosis, and wall motion abnormalities. Reperfusion was confirmed by return of color to the myocardium distal to the ligation and disappearance of ST elevation. The suture remained within the wound for identification of the ligature site, and the chest and skin were closed in layers. After surgery, animals were returned to individual cages and given regular food and water for 48 h before euthanasia and tissue harvest. Buprenex (0.1 mg/kg) was administered as needed to ensure that the animals were comfortable following surgery. All surgical procedures were performed under aseptic conditions.
All reagents were purchased from Electron Microscopy Sciences. Hearts were dissected from mice, rinsed well in cold PBS, and immersed in Karnovsky's fixative (2.5% glutaraldehyde, 4% paraformaldehyde, 0.02% picric acid in 0.1 M PBS) over night. One cm blocks were post-fixed in 1% osmium tetroxide/1.5% potassium-ferricyanide, stained with 1.5% uranyl acetate, and dehydrated through graded ethanol series. After embedding in Spurr's resin, sections were cut at 55-60 nm thickness using a Diatome diamond knife on an RMC MT-7000 ultramicrotome. Sections were contrasted with lead citrate and viewed on a JSM 100 CX-II electron microscope operated at 80 kV. Images were recorded on Kodak 4489 Electron Image film and then digitized at 900 dpi for publication.
This example describes experiments that demonstrate a rapid induction of proNGF and p75NTR following ischemia/reperfusion injury in the murine heart. This example also describes generation of a proNGF knock-in mouse and shows that proNGF acted upon p75NTR-expressing pericytes, leading to endothelial cell activation, enhanced vascular permeability and microvascular compromise, which culminated in a lethal dilated cardiomyopathy in adulthood in the proNGF knock-in mouse. Additionally, this example describes experiments which demonstrate that pericytes ensheathing cardiac capillaries express p75NTR and SorCS2, but not sortilin, during late embryogenesis, establishing that SorCS2, which binds to proNGF, functions as a co-receptor with p75NTR to alter pericyte function.
A knock-in mouse was generated in which the Ngf coding exon was replaced with an allele with a C-terminal hemagglutinin (HA) tag to facilitate detection of all forms of NGF (wtNGF-HA/+ mice). This strategy enhanced detection of NGF protein, which is expressed at subnanomolar concentration in the heart. The wtNGF-HA/+ mice were analyzed and were seen to express the knocked-in allele in a manner that was indistinguishable from the endogenous allele (see below and
To elucidate the pathophysiological effects of proNGF in the heart, knock-in mice were generated in which the Ngf coding exon was replaced with a mutant allele to impair furin cleavage (aa-K120R121 mutated to AA). In addition, a hemagglutinin (HA) epitope tag was added at the C-terminus to facilitate detection (Prongf-ha) (
Expression of Prongf-ha mRNA and Ngf-ha mRNA was confirmed using RT-PCR of heart tissue from embryonic or neonatal animals. As NGF is expressed at subnanomolar levels in target organs including the heart (Lommatzsch 2005), the inventors were unable to detect HA immunoreactivity (proNGF-HA or wtNGF-HA) in sections of the uninjured neonatal or adult heart (data not shown and
To analyze the effects of proNGF expression in the heart, adult proNGF-HA/+ mice were examined. Marked biventricular enlargement of the hearts was observed in 8 month old (mo) proNGF-HA/+ mice, as compared with NGF+/+ hearts in age-matched mice (
To corroborate the cardiac fibrosis in proNGF-HA/+ mice, cardiac ultrastructure was examined using transmission electron microscopy of mice at 4 months of age (
To determine the time course of functional impairment in the proNGF-HA/+mice, transthoracic echocardiography was performed on a cohort of mice beginning at 2 months of age (mo). Functional cardiac compromise was observed in the proNGF-HA/+ mice compared to NGF+/+ littermates and to wtNGF-HA/+ knock-in mice, beginning early in life (
To determine if the observed cardiomyopathy in proNGF-HA/+ mice was due to activation of established the proNGF receptors, p75NTR and sortilin, the inventors evaluated proNF-HA/+ mice that were deficient for either p75NTR (p75NTR−/−; proNGF-HA/+; n=14) or for sortilin (sort−/−; proNGF-HA/+; n=15). It was hypothesized that, if the observed dilated cardiomyopathy was due to the binding of proNGF to p75NTR and/or to sortilin, a deletion of either receptor for proNGF might prevent this phenotype. Indeed, p75NTR deletion rescued the cardiac hypocontractility as measured by echocardiography (
Surprisingly, sortilin deficiency did not rescue defects in fractional shortening, or histologic evidence of biventricular enlargement and cardiac fibrosis of proNGF-HA/+ mice (
To identify the mechanisms by which proNGF mediates cardiac dysfunction, hearts from young NGF+/+ and proNGF-HA/+ mice were analyzed (
To evaluate potential mechanisms that led to the histological and electron microscopic evidence of microvascular compromise in the young proNGF-HA/+ mice, three parameters were used to assess microvascular integrity. To evaluate whether endothelial cell activation in the proNGF-HA/+ animals led to platelet trapping and fibrin(ogen) deposition, CD41 (to detect platelets) and fibin(ogen) immunoreactivity were assessed. In the proNGF-HA/+ mice, increased CD41 immunoreactivity (
To gain a better understanding of how proNGF:p75NTR signaling led to microvascular damage in proNGF-HA expressing animals, proNGF receptors were localized in the developing and mature heart. The p75NTR receptor was readily detectable on tyrosine hydroxylase (TH)-expressing sympathetic processes extending into the adult NGF+/+ and the proNGF-HA/+ heart (
To uncover additional roles for proNGF we generated knock-in mice that misexpress the furin-resistant proNGF allele under the endogenous ngf promoter, to elucidate pathophysiological consequences in multiple organs.
A profound progressive deficit has been identified in cardiac contractility in the proNGF-HA/+ mice, which reflect a gain-of-function effect of proNGF, rather than a loss of function effect of reduced mature NGF, as documented by the absence of phenotype NGF haploinsufficient animals. Histological and ultrastructural analyses indicate that the cardiac hypocontractility and fibrosis was initiated by microvascular endothelial activation, leading to increased extracellular matrix deposition and subsequent scarring as well as pro-inflammatory cell infiltration. The pathologic role of proNGF:p75NTR activation in this cascade of tissue damage has been confirmed by genetic rescue of the cardiac phenotype in p75NTR−/− mice. Thus the heart, like the peripheral and central nervous system, utilizes p75NTR as the signaling component of the proNGF receptor complex. Interestingly, sortilin, a co-receptor for p75NTR and proNGF-mediated neuronal effects, did not play a role in this phenotype, since the sort−/−; proNGF-HA/+ genotype provided no rescue to the phenotype. SorCS2, another sortilin family member, has been identified herein as a p75NTR co-receptor in mediating the cardiac phenotype observed herein.
The identification of p75NTR+ pericytes, rather than endothelial cells, during embryonic cardiac development provides a surprising mechanism for proNGF-mediated cardiomyopathy in the young adult mouse. It is postulated herein that proNGF, secreted by cardiac myocytes, acts locally on p75NTR+ pericytes, to induce pericyte dysfunction, leading to a lack of trophic support of the microvascular endothelium.
This example illustrates how to develop and characterize a human antibody to the prodomain of proNGF, and demonstrate that the antibody is specific for proNGF but not related neurotrophins. This example also illustrates preclinical models to show that an anti-proNGF antibody blocks proNGF-induced cell death, and promotes cardioprotection in animal models of myocardial ischemia. This example further describes design of a small clinical study of patients with an acute myocardial infarction for establishing that delivery of anti-proNGF limits infarct size and promotes cardiac recovery.
ProNGF and the prodomain thereof can be produced recombinantly. Recombinant prodomain can be used as an immunogen to generate antibodies specific for the prodomain and hence specific for the proNGF. The antibodies can be analyzed for their efficacy of neutralization of activities of the recombinant proNGF.
High avidity monoclonal antibodies specific to different regions of the prodomain can be generated and identified. Both the pro-domain and mature domain of NGF are highly conserved across species. As shown in
Antibodies that are specific for the prodomain of proNGF, without recognizing the prodomains of other human proneurotrophins, or the mature domains, are documented. The prodomain of proNGF is clearly distinct from those of other proneurotrophins (
ELISA—To identify proNGF antibodies that interact only with proNGF but not other proneurotrophins or other growth factors that are known to play roles in cardiovascular cells, commercially available ELISA kits for NGF, BDNF, NT-3, NT-4, FGF, and VEGF will be modified by substituting the coating antibody with anti-proNGF in these sandwich ELISA assays. This approach is scalable and adaptable to positively identify a range of candidate proNGF antibodies and to rule out cross-reactivity with other growth factors.
Co-immunoprecipitation—The ability of the anti-proNGF monoclonal antibodies to recognize human, murine, potentially porcine or primate proNGF can be assessed to confirm utility in preclinical models. This can be performed using the media of cells expressing proNGF from other species, and co-immunoprecipitation analysis with the candidate monocloncal antibodies.
Uptake Assay—After identifying a panel of anti-proNGF antibodies, the antibodies are assessed in their abilities in blocking proNGF binding and internalization using HT1080 cells that co-express p75NTR and sortilin or SorCS2. Recombinant proNGF can be labeled with Alexa-594 and uptake assay can be carried out as recently described (Feng et al., J Mol Biol. 396:967, 2010). Anti-proNGF antibodies can be added to the culture medium 30 min before the proNGF addition. The amount of internalized Alexa-conjugated proNGF can be quantified 24 hrs later by semi-automated fluorescent microscopy as documented (Feng et al., J Mol Biol. 396:967, 2010). This assay has been developed for high through-put screening. As a control, comparable studies can be performed with Alexa-conjugated mature NGF. The anti-prodomain antibody should immunodeplete proNGF, but not mature NGF.
Cell Death Assay—Once anti-proNGF antibodies have been identified that block proNGF uptake, their utility in blocking proNGF-induced cell death can be verified using HT-1080 cells that co-express p75 with sortilin or SorCS2, and with primary superior cervical ganglion (SCG) neurons. This latter model has been extensively utilized to document the pro-apoptotic properties of the proneurotrophins (see Lee, Science, 294: 1945-48 (2001); Teng et al., J Neurosci, 25:5455 (2005); Yano et al., J Neurosci., 29: 14790 (2009), for example).
Antibody optimization would be carried out including affinity maturation and half-life extension.
Preclinical analysis of anti-proNGF efficacy. Studies of the biotherapeutic potential of anti-proNGF are performed in cardiac ischemia/reperfusion models in rodents, and subsequently extended to porcine/dog or primate models. Current polyclonal anti-proNGF antibodies could serve as a control.
In brief, animals are intubated, and subjected to 40 minutes of coronary ischemia by occlusion of the left anterior descending artery, followed by reperfusion. Anti-proNGF antibodies are delivered beginning at 2 or 6 hours following re-perfusion (to coincide with delivery times achievable in humans). Animals are dosed daily (or as optimal based on pharmacological studies). Ongoing studies that quantitate proNGF levels at 1, 3, 7, 10 days post ischemia/reperfusion injury in rodents can guide the duration of treatment. Three methodologies are used to determine efficacy. First, a cohort of animals is sacrificed at 3 or 10 days, and blood and cardiac tissue are analyzed for levels of proNGF (by ELISA and Western blot), and the shed ectodomains of p75 and sortilin/SorCS2 (a marker of ligand binding and receptor activation), as well as microvascular markers (by Western blot). Second, a cohort of rodents is sacrificed at 3 or 10 days, and hearts are examined histologically. This enables an examination of infarct size, and protection of the microvasculature (morphometric evaluation). Lastly, a cohort of rodents is imaged using Doppler-flow echocardiography at 3 and 10 days post ischemia reperfusion to assess the functional effects of anti-proNGF delivery on regional wall motion abnormalities (as compared to equivalent concentrations of isotype matched non-immune IgG). Comparable studies are undertaken on a larger mammal (pig) which is typically used for cardioprotection studies.
As an additional analysis, studies are performed on the proNGF “knock-in” mouse model. Immunoneutralization of proNGF should prevent the progressive cardiomyopathy observed in this animal. For these studies, a cohort of proNGF expressing mice is treated with anti-proNGF antibodies for two months (age 2-4 months) and Doppler imaging is performed to document improved cardiac function, and histological analysis is undertaken to document microvessel preservation and reduced expression of proNGF.
Assessment of plasma levels of proNGF in humans post myocardial ischemia. Total NGF levels in human plasma have been detected, consistent with prior reports. A quantitative ELISA can be developed using anti-prodomain antibodies to determine the magnitude of proNGF induction in human post-MI, using banked specimens or in a prospective study conducted at Weill Medical Center. Blood can be collected at presentation to the emergency room, immediately post-stenting/dilation, and then at 24 hour intervals for 4 days, and then at follow-up at 1 and 2 weeks. This analysis can be extended to a “biomarker panel” assessing the levels of shed ectodomain of p75, as an immediate endpoint for the clinical trials, as well as conventional assessment of cardiac injury (troponin). It should be noted that p75 ectodomain are present at low levels in human plasma. In preclinical models, the shed ectodomains increase following ligand binding.
Patient selection. Only patients with no prior history of myocardial infarction are considered (no prior medical history, or prior normal stress test or cardiac imaging), and no other significant co-morbidities, and have onset/duration of positive symptoms of less than 6 hours (window of 2-6 hours permitted). Patients must be evaluable by cardiac catheterization, and amenable to stent placement or dilation (no contraindications).
Patient evaluation and drug delivery. Utilizing cardiac catheterization, patients must demonstrate a reduction in ejection fraction of at least 15%, with an identifiable coronary lesion, regional wall motion abnormalities, no sustained atrial or ventricular tachyarrythmias, and restoration of flow with stent placement/dilation. Ideally, drug (anti-proNGF) or control can be delivered within 4-8 hours following reperfusion.
PK/PD studies. Biomarkers are evaluated every 6 hours for 24 hours, then daily for 4 days, then at 1 and 2 weeks following delivery. This includes plasma for proNGF (ELISA), shed ectodomain of p75.
Analysis of efficacy. Patients are evaluated by cardiac MRI, echocardiography to assess functional wall motion abnormalities, and recovery of function at 2 and 4 weeks post-delivery.
This application claims the benefit of priority of U.S. Provisional Application No. 61/397,663, filed Jun. 15, 2010, the entire content of which is incorporated herein by reference.
This invention was made with Government Support under Contract Nos. PO1HL04603 and R01 NS030687, awarded by the National Institutes of Health. The Government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US11/40447 | 6/15/2011 | WO | 00 | 1/25/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61397663 | Jun 2010 | US |
