# Patent

## Title
Controlled release of growth factors and signaling molecules for promoting angiogenesis

## Abstract

The present invention comprises compositions, methods, and devices for delivering angiogenic factors and signaling molecules to a target tissue, and controlling the release of these factors and signaling molecules to spatially and temporally restrict their release and dissemination, for the purpose of promoting angiogenesis in target tissues.

## Background
RELATED APPLICATIONS
This application is a national stage application, filed under 35 U.S.C. §371, of International Application No. PCT/US2009/045856, filed Jun. 1, 2009, which claims the benefit of provisional application U.S. Ser. No. 61/130,486, filed May 30, 2008, the contents of which are incorporated by reference in their entireties.


GOVERNMENT SUPPORT
This invention was made with Government support under RO1 HL069957 awarded by the National Institutes of Health. The Government has certain rights in the invention.


FIELD OF THE INVENTION
The present invention relates to the fields of cardiology, tissue repair, and preventative medicine.
BACKGROUND OF THE INVENTION
Angiogenesis refers to a process of new blood vessel formation. Subjects suffering from coronary arterial disease (CAD) and peripheral arterial disease (PAD) can be treated by promoting angiogenesis in the tissue lacking sufficient blood flow. However, current methods of administering angiogenic drugs are sub-optimal because they cannot control the presentation of multiple compounds separately. Moreover, systemic administration of drugs at concentrations that are therapeutically effective for the affected area cause surrounding, healthy, tissues to be exposed unnecessarily to pro-angiogenic growth factors and could lead to undesirable side effects.
SUMMARY OF THE INVENTION
The present invention provides compositions and methods for controlling the local presentations of pro-angiogenic growth factors and signaling molecules that are together used to achieve angiogenesis at the tissue or organ of interest. The compositions and methods of the present invention allow the concerted and joint presentations of delivered growth factors and signaling molecules to be controlled separately by changing physical and/or chemical properties of the polymer delivery material to achieve an appropriate local concentration of the factor/molecules at the target tissue site.
Subjects to be treated in the manner described herein have been diagnosed as suffering from or at risk of developing an ischemic condition. The methods are suitable to treatment of human patients, as well as being suitable for veterinary use (e.g., treatment of companion animal such as dogs and cats. In a preferred embodiment, the methods are used to develop treatments for chronic ischemia in coronary and peripheral artery disease for diabetic subjects. Alternatively, or in addition, the present invention is used to improve wound healing in ulcers for diabetic subjects. The impaired endothelial tissues of diabetic subjects often have a reduced response to regular pro-angiogenic factors, making the continuous activation of vascular growth induced by the sustained and distinct presentation of growth factors and signaling molecules provided by the present invention particularly valuable. This type of continuous, sustained, and distinct presentation of pro-angiogenic factors and signaling molecules is not currently possible. Current methods that attempt to achieve similar therapeutic outcomes also induce deleterious side-effects due to substantial systemic dissemination of these factors throughout the body.
A device, which overcomes the shortcomings of existing approaches, comprises a scaffold composition, a bioactive composition, and a regulatory agent. The bioactive composition and the regulatory agent are incorporated into or coated onto the scaffold, e.g., a polymeric gel, composition, and the regulatory agent controls the activity of the bioactive agent. The bioactive composition is released from the scaffold composition at a first rate and the regulatory agent is released from said scaffold composition at a second rate. For example, the bioactive composition exits from the scaffold composition slower or faster relative to the regulatory agent. For example, vascular endothelial growth factor (VEGF) as a bioactive composition exits from the scaffold composition for a time period of one or more, e.g., 4 weeks, while gamma secretase inhibitor as a regulatory agent exits from the same scaffold composition for a time period of one or more days, e.g., 3 days. The difference between the release rates of multiple factors is important in determining the final angiogenesis outcomes. For example, the desired time-frame for Notch inhibitor delivery will be 7-20 fold (e.g., 8, 10, 15, or 18-fold) shorter than that of VEGF or PDGF, i.e., Notch inhibitor is released within 1-3 days while VEGF or PDGF is released within 7-60 days (e.g., 7, 10, 15, 30, 45, or 60 days). An exemplary bioactive composition is a pro-angiogenic factor such as VEGF and/or PDGF and an exemplary regulatory agent is an inhibitor or enhancer of angiogenesis such as DAPT.
One or more bioactive compositions are incorporated into or coated onto the scaffold composition, and the scaffold composition temporally controls release of the bioactive composition. Alternatively, or in addition, the scaffold composition spatially controls release of a bioactive composition. In another embodiment, the bioactive composition incorporated into or coated onto the scaffold composition temporally or spatially regulates release of a second bioactive composition.
The present invention also comprises a method of inducing blood vessel growth in a target tissue of a mammal, comprising providing a device comprising a scaffold composition with a bioactive composition being incorporated therein or thereon and contacting a mammalian tissue with the device wherein said scaffold composition temporally controls release of the bioactive composition and wherein the bioactive composition induces angiogenesis within the target tissue. For example, a polymeric gel composition loaded with pro-angiogenic factors and a regulatory factor is injected directly into the target site, or into a site that is adjacent to or in close proximity to the target site in which angiogenesis is desired. For example, the site of administration is 10 mm, 25 mm, 50 mm, 1 cm, 5 cm, 10 cm, 50 cm from the target tissue site where angiogenesis is to occur. In addition, multiple simultaneous injections (in different spatial locations, e.g., encircling or surrounding an affected anatomical location/site), or repeating injections every 1-2 weeks, at the same site or every few mm or cm apart in an ischemic region may be desirable.
The present invention further comprises a method of augmenting blood vessel growth, comprising providing a device comprising a scaffold composition with a bioactive composition being incorporated therein or thereon and contacting a mammalian tissue with the device, wherein said scaffold composition temporally controls release of the bioactive composition and wherein the bioactive composition induces growth from existing blood vessels.
The bioactive composition of the devices of the present invention is non-covalently linked to said scaffold composition. Alternatively, the bioactive composition is covalently linked to said scaffold composition.
Bioactive compositions of the present invention consist of, consist essentially of, or comprise one or more factors, which are administered either by direct protein delivery or delivering gene sequences to have cells locally make proteins. Exemplary bioactive compositions can include receptor ligands, transcription factors, and/or regulatory molecules.
Receptor ligands include, but are not limited to, vascular endothelial growth factor (VEGF (A-F)), fibroblast growth factors (acidic and basic FGF 1-10), granulocyte-macrophage colony-stimulating factor (GM-CSF), insulin, insulin growth factor or insulin-like growth factor (IGF), insulin growth factor binding protein (IGFBP), placenta growth factor (PIGF), angiopoietin (Ang1 and Ang2), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), transforming growth factor (TGF-α, TGF-β, isoforms 1-3), platelet-endothelial cell adhesion molecule-1 (PECAM-1), vascular endothelial cadherin (VE-cadherin), nitric oxide (NO), chemokine (C-X-C motif) ligand 10 (CXCL10) or IP-10, interleukin-8 (IL-8), hypoxia inducible factor (HIF), monocyte chemotactic protein-1 (MCP-1), vascular cell adhesion molecule (VCAM), ephrin ligands (including Ephrin-B2 and -B4).
Transcription factors include, but are not limited to, HIF-1α, HIF-1β and HIF-2α, Ets-1, Hex, Vezf1, Hox, GATA, LKLF, COUP-TFII, Hox, MEF2, Braf, Prx-1, Prx-2, CRP2/SmLIM and GATA family members, basic helix-loop-helix factors and their inhibitors of differentiation.
Regulatory molecules include, but are not limited to, enzymes (matrix metalloproteinase (MMP), tissue plasminogen activator (PLAT or tPA), cyclooxygenase (COX), angiogenin), molecules regulating Notch signaling which consists of, consists essentially of, or comprises monoclonal antibodies to Notch ligands and receptors, RNA interference, antisense Notch, receptor and mastermind-like 1 (MAML1) decoys, beta and gamma-secretase inhibitors (GSI), or any other molecules that can activate or inhibit Notch signaling.
Devices of the present invention consist of, consist essentially of, or comprise one or more bioactive compositions. A second bioactive composition consists of, consists essentially of, or comprises a signaling molecule selected from the group consisting of monoclonal antibodies to Notch ligands and receptors, RNA interference, antisense Notch, receptor and mastermind-like 1 (MAML1) decoys, beta and gamma-secretase inhibitors (GSI), or any other molecules that can activate or inhibit Notch signaling.
Alternatively, or in addition, signaling molecules of the second bioactive composition are selected from the group of any other molecules that can inhibit or activate Notch signaling. In a preferred embodiment of the present invention, the signaling molecule is DAPT (N—[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester) (Sigma-Aldrich, St. Louis, Mo.). Signaling molecules of the second bioactive composition are released from scaffolds and devices simultaneously or sequentially with each other. Signaling molecules of the second bioactive composition are released from scaffolds and devices simultaneously or sequentially with bioactive compositions comprising angiogenic factors. Preferably, the regulatory molecule is a Notch inhibitor such as the gamma secretase inhibitor DAPT.
Scaffold compositions of the present invention degrade at a predetermined rate based on a physical parameter selected from the group consisting of temperature, pH, hydration status, and porosity. Alternatively, or in addition, scaffold compositions are enzymatically digested by a composition elicited by a contacting cell, said release of said bioactive composition being dependent upon the rate of enzymatic digestion. A contacting cell is defined as a cell belonging to a target cell wherein the scaffold composition or device resides that physically contacts or adheres to the scaffold composition or device.
Scaffold compositions of the present invention contain an external surface. Alternatively, or in addition, scaffold compositions contain an internal surface. External or internal surfaces of the scaffold composition are solid or porous. Pore size is less than about 10 nm, in the range of about 10 nm-20 μm in diameter, or greater than about 20 μm. A scaffold composition with multiple internal surfaces optionally comprises one or more compartments.
Devices of the present invention are administered by intramuscular injection. Alternatively, or in addition, scaffold compositions and devices are administered by intraperitoneal injection, or endoscopic delivery or other minimally invasive delivery approach, or surgically implanted. Preferably, the loaded gel composition is injected as a bolus using a standard syringe and injection needle at or near the target angiogenesis site, or it can be surgically implanted or delivered via a catheter.
The devices and methods of the invention provide a solution to several problems associated with previous angiogenesis-inducing protocols. The bioactive composition is incorporated into or coated onto the scaffold composition. The scaffold composition and/or bioactive composition temporally and spatially (directionally) controls release of one or more additional bioactive compositions.
This device includes a scaffold composition which incorporates into or is coated with a bioactive composition; the device releases one or more bioactive compositions comprised of pro-angiogenic factors and signaling molecules to stimulate local vascular growth. Release of the bioactive composition is regulated spatially and temporally. Depending on the application for which the device is designed, the device regulates release through the physical or chemical characteristics of the scaffold itself. For example, the scaffold composition is differentially permeable, allowing release only in certain physical areas of the scaffold. The permeability of the scaffold composition is regulated, for example, by selecting or engineering a material for greater or smaller pore size, density, polymer cross-linking, stiffness, toughness, ductility, or viscoelascticity.
The scaffold composition contains physical channels or paths through which bioactive compositions can move more easily towards a targeted area of release of the device or of a compartment within the device. The scaffold composition is optionally organized into compartments or layers, each with a different permeability, so that the time required for a bioactive composition to move through the device is precisely and predictably controlled. Release is also regulated by the degradation, de- or re-hydration, oxygenation, chemical or pH alteration, or ongoing self-assembly of the scaffold composition. These processes are driven by diffusion or catalyzed by enzymes or other reactive chemicals.
Alternatively, or in addition, release is regulated by a bioactive composition. By varying the concentration of extracellular matrix components, adhesion molecules and other bioactive compounds in different areas of the device, including agents with means to create pores and enzymatically digest the scaffold composition, the bioactive composition has means to control the rate at which other elements of the same bioactive composition or additional bioactive compositions escape, or are released from the scaffold composition or device. The device controls and directs the flow of bioactive compositions or elements through its structure.
Chemical affinities are used to channel bioactive compositions towards a specific area of release. By varying the density and mixture of those bioactive substances, the device controls the timing of the combination and release of elements of bioactive compositions or multiple bioactive compositions. In one embodiment, components of a bioactive composition or two compositions are separated initially, but combined when allowed to flow through channels in the scaffold composition towards an area of release. The density and mixture of these bioactive substances is controlled by initial doping levels or concentration gradient of the substance, by embedding the bioactive substances in scaffold material with a known leaching rate, by release as the scaffold material degrades, by diffusion from an area of concentration, by interaction of precursor chemicals diffusing into an area, or by production/excretion of compositions by neighboring cells.
Cells in close physical proximity to the scaffold compositions and devices of the present invention, or those in physical contact with the scaffold composition and devices, secrete enzymes that affect the one or more features of the scaffold composition. Neighboring or juxtaposed cells residing within target tissues increase or decrease the structural integrity of the scaffold composition through directly (e.g., release of enzymes) or indirectly (e.g. release of signals recruiting cells to the scaffold compositions that affect structural integrity). Neighboring or juxtaposed cells produce factors that increase or decrease the rigidity, increase or decrease the porosity, increase or decrease the potential for or rate of degradation, increase or decrease the adhesion or mobility, or increase or decrease the immunogenicity of the scaffold composition or device. Alternatively, the scaffold composition is comprised of materials that are unaffected by enzymatic activity and are unaffected by cellular secretions from local tissues.
The physical or chemical structure of the scaffold also regulates the diffusion of bioactive agents through the device. The release profiles of multiple bioactive compositions are made distinct from each other by adjusting the properties and formulation of delivery vehicle. For example, the small-molecule-weight signaling molecule is first incorporated into microspheres followed by incorporation into alginate hydrogel, so that the signaling molecules have a more delayed release compared to the growth factors. The pore size, oxidization degree, molecular weight distribution of the alginate gel are varied to control the release rate of incorporated growth factors.
The bioactive composition includes one or more compounds that regulate cell function and/or behavior. The bioactive composition is covalently linked to the scaffold composition or non-covalently associated with the scaffold. For example, the bioactive composition is an extracellular matrix (ECM) component that is chemically crosslinked to the scaffold composition. Regardless of the tissue of origin, ECM components generally include three general classes of macromolecules: collagens, proteoglycans/glycosaminoglycans (PG/GAG), and glycoproteins, e.g., fibronectin (FN), laminin, and thrombospondin. ECM components associate with molecules on the cell surface and mediate adhesion and/or motility. Preferably, the ECM component associated with the scaffold is a proteoglycan attachment peptide or cyclic peptide containing the amino acid sequence arginine-glycine-aspartic acid (RGD). Proteoglycan attachment peptides are selected from the group consisting of G4RGDSP (SEQ ID NO: 1), XBBXBX (SEQ ID NO: 2), PRRARV (SEQ ID NO: 3), YEKPGSPPREVVPRPRPGV (SEQ ID NO: 4), RPSLAKKQRFRHRNRKGYRSQRGHSRGR (SEQ ID NO: 5), and RIQNLLKITNLRIKFVK (SEQ ID NO: 6), and cell attachment peptides are selected from the group consisting of RGD, RGDS, LDV, REDV, RGDV, LRGDN (SEQ ID NO: 7), IKVAV (SEQ ID NO: 8), YIGSR (SEQ ID NO: 9), PDSGR (SEQ ID NO: 10), RNIAEIIKDA (SEQ ID NO: 11), RGDT, DGEA, and VTXG.
Components of the ECM, e.g., FN, laminin, and collagen, interact with the cell surface via the integrin family of receptors, a group of divalent cation-dependent cell surface glycoproteins that mediate cellular recognition and adhesion to components of the ECM and to other cells. Ligands recognized by integrins typically contain an RGD amino acid sequence that is expressed in many ECM proteins. Exemplary molecules that mediate cell adhesion and/or movement include FN, laminin, collagen, thrombospondin 1, vitronectin, elastin, tenascin, aggrecan, agrin, bone sialoprotein, cartilage matrix protein, fibrinogen, fibrin, fibulin, mucins, entactin, osteopontin, plasminogen, restrictin, serglycin, SPARC/osteonectin, versican, von Willebrand Factor, polysaccharide heparin sulfate, cell adhesion molecules including connexins, selectins include collagen, RGD (Arg-Gly-Asp) and YIGSR (Tyr-Ile-Gly-Ser-Arg) peptides, glycosaminoglycans (GAGs), hyaluronic acid (HA), integrins, selectins, cadherins and members of the immunoglobulin superfamily. Carbohydrate ligands of the ECM include the polysaccharides hyaluronic acid, and chondroitin-6-sulfate.
The device optionally contains a second or third bioactive composition(s), e.g., a growth factor, differentiation factor, or signaling molecule. For example, the device includes vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), or fibroblast growth factor 2 (FGF2) or a combination thereof. Growth factors used to promote angiogenesis, wound healing, and other aspects of tissue regeneration are listed herein and are used alone or in combination to induce regeneration of bodily tissues by bioactive compositions released from an implanted scaffold device.
The bioactive composition(s) described above is non-covalently linked to the scaffold composition. Alternatively, or in addition, the bioactive composition is covalently associated with the scaffold. Noncovalent bonds are generally one to three orders of magnitude weaker than covalent bonds permitting diffusion of the factor out of the scaffold and into surrounding tissues. Noncovalent bonds include electrostatic, hydrogen, van der Waals, π aromatic, and hydrophobic. For example, a growth factor such as VEGF is associated with the device by noncovalent bonds and exits the device following administration of the device to a target site to promote angiogenesis within the target bodily tissue.
The polymer scaffold composition into or onto which the bioactive composition (growth factor, and/or regulatory molecule are loaded is biocompatible. The composition is bio-degradable/erodable or resistant to breakdown in the body. Relatively permanent (degradation resistant) scaffold compositions include metals and some polymers such as silk. Preferably, the scaffold composition degrades at a predetermined rate based on a physical parameter selected from the group consisting of temperature, pH, hydration status, and porosity, the cross-link density, type, and chemistry or the susceptibility of main chain linkages to degradation or it degrades at a predetermined rate based on a ratio of chemical polymers. For example, a high molecular weight polymer comprised of solely lactide degrades over a period of years, e.g., 1-2 years, while a low molecular weight polymer comprised of a 50:50 mixture of lactide and glycolide degrades in a matter of weeks, e.g., 1, 2, 3, 4, 6, 10 weeks. A calcium cross-linked gels composed of high molecular weight, high guluronic acid alginate degrade over several months (1, 2, 4, 6, 8, 10, 12 months) to years (1, 2, 5 years) in vivo, while a gel comprised of low molecular weight alginate, and/or alginate that has been partially oxidized, will degrade in a matter of weeks. A typical volume of alginate gel is 1 μL to 1 mL, with a degradation time ranging from 1 day to 6 weeks.
In one example, cells mediate degradation of the scaffold matrix, i.e., the scaffold composition is enzymatically digested by a composition elicited by a neighboring cell, and the release of the bioactive composition is dependent upon the rate of enzymatic digestion of the scaffold. In this case, polymer main chains or cross-links contain compositions, e.g., oligopeptides, which are substrates for collagenase or plasmin, or other enzymes produced by cells adjacent to the scaffold.
Exemplary scaffold compositions include polylactic acid, polyglycolic acid, PLGA polymers, alginates and alginate derivatives, gelatin, collagen, fibrin, hyaluronic acid, laminin rich gels, agarose, natural and synthetic polysaccharides, polyamino acids, polypeptides, polyesters, polyanhydrides, polyphosphazines, poly(vinyl alcohols), poly(alkylene oxides), poly(allylamines)(PAM), poly(acrylates), modified styrene polymers, pluronic polyols, polyoxamers, poly(uronic acids), poly(vinylpyrrolidone) and copolymers or graft copolymers of any of the above. One preferred scaffold composition includes alginate gels.
Porosity of the scaffold composition influences release of one or more bioactive compositions from the device. Pores are nanoporous, microporous, or macroporous. For example, the diameter of nanopores are less than about 10 nm; micropore are in the range of about 100 nm-20 μm in diameter; and, macropores are greater than about 20 μm (preferably greater than about 100 μm and even more preferably greater than about 400 μm). In one example, the scaffold is macroporous with aligned pores of about 400-500 μm in diameter.
In one preferred embodiment of the invention, one or more of the bioactive compositions contains an element with means to chemically induce pores in the scaffold composition that are nanoporous, microporous, or macroporous in size. The abundance of this element and the presence or absence of other elements that augment or inhibit the activity of this pore-making element determine pore size and density, and consequently, the rate at which one or more bioactive compositions passively escape or are actively released from the scaffold composition. Exemplary elements with means to chemically induce pores in the scaffold composition include, but are not limited to, potassium salts, calcium salts, magnesium salts, amino acids, week acids, carbohydrates, potassium bitartrate, creatine, aspargine, glutamine, aspartic acid, glutamic acid, leucin, neroleucine, inosine, isoleucine, magnesium citrate, magnesium phosphate, magnesium carbonate, magnesium hydroxide, and magnesium oxide. Pore-forming elements are also enzymes incorporated into or coated onto the scaffold composition. One or more of these elements are initially contained within separate compartments of the scaffold composition and later combined by having one or more of these elements flow through common channels or paths in the scaffold composition. These elements individually have means to induce pore formation in the scaffold composition. Alternatively, these elements are combined, and the resulting combination has means to induce pore formation in the scaffold composition.
The devices are manufactured in their entirety in the absence of cells or can be assembled around or in contact with cells (the material is gelled or assembled around cells in vitro or in vivo in the presence of cells and tissues). In one embodiment of the invention, the scaffold composition material with one or more bioactive compositions incorporated, is injected into a target tissue where local blood flow is restricted or would healing is impaired.
The device is manufactured in one stage comprising one layer or compartment. Alternatively, the device is manufactured in two or more (3, 4, 5, 6, . . . 10 or more) stages in which one layer or compartment is made and infused or coated with a bioactive composition followed by the construction of a second, third, fourth or more layers, which are in turn infused or coated with a bioactive composition in sequence. Each layer or compartment is identical to the others or distinguished from one another by the elements comprising the bioactive composition incorporated into or coated onto them as well as distinct chemical, physical and biological properties.
A method of making a scaffold is carried out by providing a scaffold composition and covalently linking or noncovalently associating the scaffold composition with a first bioactive composition. The scaffold composition is also contacted with a second bioactive composition. The second bioactive composition is non-covalently associated with the scaffold composition to yield a doped (loaded) scaffold, i.e., a scaffold composition that includes one or more bioactive substances. The contacting steps are optionally repeated to yield a plurality of doped scaffolds, e.g., each of the contacting steps is characterized by a different amount of the second bioactive composition to yield a gradient of the second bioactive composition in the scaffold device. Rather than altering the amount of composition, subsequent contacting steps involve a different bioactive composition, i.e., a third, fourth, fifth, sixth . . . , composition or mixture of compositions, that is distinguished from the prior compositions or mixtures of prior doping steps by the structure or chemical formula of the factor(s). The method optionally involves adhering individual niches, layers, or components to one another and/or insertion of semi-permeable, permeable, or nonpermeable membranes within or at one or more boundaries of the device to further control/regulate locomotion of cells or bioactive compositions.
Therapeutic applications of the device include vascular tissue generation, regeneration and repair. A mammalian tissue is contacted with the device. The scaffold composition and/or the bioactive composition spatially or directionally regulates release of a bioactive composition with means to promote angiogenesis in local tissues such as vascular, muscle, gastrointestinal, e.g., bowel, cardiac, brain, kidney, bone, nerve both central and peripheral nervous system (CNS and PNS) or any tissue characterized by a shortage of oxygen or nutrients or in which the vasculature is damaged, absent, or functionally impaired.
A method of inducing local angiogenesis in a target tissue is carried out by administering to a mammal a device containing a scaffold composition and a bioactive composition incorporated therein or thereon. The scaffold composition and/or bioactive composition induces release of pro-angiogenic factors and signaling molecules from the device into the local tissue environment. The release of these angiogenic factors is controlled spatially and temporally. Release can be sustained at a steady rate/dosage for a desired period of time, e.g., minutes; 0.2. 0.5, 1, 2, 4, 6, 12, 24 hours; 2, 4, 6, days; weeks (1-4), months (2, 4, 6, 8, 10, 12) or years, during which the cells are exposed to structural elements and bioactive compositions that lead to improved vascular growth.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. All references cited herein are incorporated by reference.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a bar graph showing the effect of VEGF on sprouting ratio. An in vitro model was used to test the significance of a controlled local concentration of VEGF.
FIG. 2 is a bar graph showing the effect of VEGF and GSI on sprouting ratio. In vitro model was used to test the significance of a combination of DAPT and VEGF.
FIG. 3 is a bar graph showing the effect of different amounts and ratios of amounts of VEGF and GSI on sprouting ratio. In vitro model establishing the significance of a distinct presentation of VEGF and DAPT.
FIGS. 4A-C are a series of line graphs showing the effect of VEGF and a GSI (DAPT) on angiogenesis. 4A, In vitro release profiles of VEGF and DAPT from injected alginate hydrogel system; 4B and 4C, In vivo model to test the effect of controlled presentation of VEGF and DAPT to recover blood flow in an ischemia situation. Blood flow recovery subject to hindlimb ligation. gel: represents the use of the alginate as the delivery vehicle. im: intramuscular injection. ip: intraperitoneal injection.
FIGS. 5A-B are bar graphs showing the effect of VEGF and DAPT on blood capillary density. An in vivo model was used to test the effect of controlled presentation of VEGF and DAPT on newly formed blood vessel density in an ischemia situation. +: use of the substance. −: lack of the substance. gel: alginate gel. im: intramuscular injection. ip: intraperitoneal injection.
FIG. 6 is a series of photomicrographs showing the effect of DAPT on gastrointestinal tissue. In vivo model was used to test the effect of DAPT delivered from alginate gel system and from intraperitoneal injection on the cells in small intestines. gel: alginate gel. ip: intraperitoneal injection. H&E, alcian blue, Ki67 and HES-1 are the four different staining methods to characterize the crypt cells in small intestines. Ki-67 was used to stain the proliferative cells. Loss of Notch signaling can alter the proliferation rate of crypt cells. HES-1 staining was to examine the expression of a known Notch target gene in crypts. Alcian blue staining was to examine the deposition of glycosaminoglycan molecules. Loss of Notch signaling can result in more deposition of glycosaminoglycan molecules. Expression of HES-1 (hairy and enhancer of split 1), a member of basic helix-loop-helix family of transcription factors and a known Notch target gene in crypts was examined. Loss of Notch signaling can alter the proliferation rate of crypt cells, as shown by Ki-67 staining the proliferative cells. In addition, Notch inhibition has been reported to alter the balance between proliferative crypt cells and goblet cells, resulting in more deposition of glycosaminoglycan molecules, as characterized by alcian blue staining Notch inhibition can also result in a significant alteration of the morphology of the small intestine as compared to controls, as demonstrated by hematoxylin and eosin (H&E) staining.
FIG. 7A is a bar graph showing the effect of VEGF, DAPT, and the combination on blood capillary density.
FIG. 7B is a line graph showing the effect of VEGF, DAPT, and the combination on blood flow.
FIG. 8A is a bar graph showing the effect of VEGF, PDGF, and the combination on blood capillary density.
FIG. 8B is a line graph showing the effect of VEGF, PDGF, and the combination on blood flow.
FIG. 9A is a bar graph showing the effect of VEGF, DAPT, PDGF, and combinations thereof on blood capillary density.
FIG. 9B is a line graph showing the effect of VEGF, DAPT, PDGF, and combinations thereof on blood flow.
FIG. 10A is series of photomicrographs showing the effect of VEGF, DAPT, PDGF, and combinations thereof on maturation of newly formed blood vessels in the local muscle tissues around the ischemic site.
FIGS. 10B-C are bar graphs showing the effect of VEGF, DAPT, PDGF, and combinations thereof on maturation of newly formed blood vessels as measured by density of smooth muscle actin (SMA) positive vessels per unit area (FIG. 10B) and the percentage of SMA positive vessels (FIG. 10C).



DETAILED DESCRIPTION OF THE INVENTION
The use of compounds that can modulate the signaling of growth factors, e.g., gamma-secretase inhibitors (GSI), to promote angiogenesis, has been proposed (US 2006/0264380 A1). However, these previous studies did not indicate a need to provide the compounds over particular time-frames, nor do they provide any method to achieve a sustained presence of the compounds. Previous studies mention the use of a combination of pro-angiogenic factors together with GSI. Functionally, a bolus injection of multiple pro-angiogenesis compounds simultaneously cannot control the presentation of each individual compound separately, as they may each need to have a distinct presentation. More importantly, single or multiple injections that introduce drugs into system circulation represent a sub-optimal method to reach the therapeutic level at the specific tissue of interest, and can result in high concentrations of drug accumulation at distant organs or tissues, which may lead to various side effects.
The current invention provides a method to control the local presentations of pro-angiogenic growth factors and signaling molecules that are together used to achieve angiogenesis at the tissue or organ of interest. The spatial and temporal presentation of delivered growth factors and signaling molecules can be controlled separately by fine-tuning the physical and chemical properties of the polymer delivery material.
Specifically, this invention will be especially useful for developing treatments for chronic ischemia in coronary or peripheral artery disease for diabetics, or wound healing in diabetes ulcers, as the impaired endothelium in these patients normally have a reduced response to regular pro-angiogenesis factors, thus a continuous activation by the sustained and distinct presentation of growth factors and signaling molecules may be critical. This is currently not achievable by other methods without inducing possible side-effects.
Angiogenesis refers to a process of new blood vessel formation. Patients suffering from coronary arterial disease (CAD) and peripheral arterial disease (PAD) can be treated by promoting angiogenesis in the tissue lacking sufficient blood blow. To deliver compounds to induce angiogenesis is therefore a promising therapeutic approach. Many growth factors, (e.g. vascular endothelial growth factor (VEGF), play a vital role in inducing angiogenesis by mediating the proliferation, migration and differentiation of endothelial cells. Molecules that can modulate the signaling pathways of growth factors, such as Notch inhibitors (e.g., gamma secretase inhibitors (GSI)), can also be used to augment the angiogenesis process. Therefore, delivering growth factors together with molecules mediating signaling pathways may have a beneficial effect.
Current delivery approaches mainly rely on injection of factors/molecules alone, i.e., in the absence of a scaffold composition. However, single injection is insufficient for circumstances where growth factors and signaling molecules need to be present over long time-frames, as they both have a short half-life. In addition, bolus injection of multiple compounds simultaneously cannot control the existence of each individual compound separately, if they each need to have a distinct presentation. More importantly, single or multiple injections that introduce drugs into system circulation are sub-optimal to reach the therapeutic level at the specific tissue of interest, and can result in high concentrations at distant organs or tissues, which may lead to various side effects.
The current invention utilizes an injectable biocompatible polymer material system incorporating pro-angiogenic growth factors, together with molecules that modulate the signaling pathway, to achieve angiogenesis at the tissue or organ of interest. The spatial and temporal presentation of delivered growth factor and signaling molecules can be controlled separately by fine-tuning the physical and chemical properties of the polymer material.
Angiogenesis
Angiogenesis is a physiological process wherein new blood vessels arise or extend from pre-existing vessels. The present invention encompasses methods of promoting blood vessel growth from existing vessels, as well as spontaneous blood vessel growth (also referred to as vasculogenesis) and arteriogenesis (collateral vessel formation). The term “angiogenesis” is meant to encompass all three methods of blood vessel formation named supra.
Angiogenesis is a normal process in growth and development, as well as in wound healing. Compositions and methods of the present invention induce or augment endogenous mechanisms for regulating angiogenesis and/or wound healing. Alternatively, or in addition, compositions and methods of the present invention introduce exogenous mechanisms for inducing or regulating angiogenesis and/or wound healing that do not normally occur in a target tissue. Furthermore, compositions and methods of the present invention induce, regulate, augment, or replace mechanisms for inducing or regulating angiogenesis and/or wound healing that are insufficient, aberrant, or incomplete compared to the endogenous mechanism due to genetic mutation, disease, infection, drug treatment, medical condition, or tissue transplant procedure.
Angiogenesis is also a fundamental step in the transition of neoplastic tumors from a dormant, or benign, state to a malignant, cancerous, or metastatic, state. Compositions and methods of the present invention disperse angiogenic factors and signaling molecules within a local tissue environment and do not allow for systemic administration or diffusion of bioactive compositions. This ability of the devices of the present invention to contain angiogenic factors to a confined target region demonstrates a significant clinical advantage over previous methods of delivery that are systemic in nature.
Controlled Release of Factors to Promote Angiogenesis
The release profiles of bioactive substances from scaffold devices is controlled by both factor diffusion and polymer degradation, the dose of the factor loaded in the system, and the composition of the polymer. Similarly, the range of action (tissue distribution) and duration of action, or spatiotemporal gradients of the released factors are regulated by these variables. The diffusion and degradation of the factors in the tissue of interest is optionally regulated by chemically modifying the factors (e.g., PEGylating growth factors). In both cases, the time frame of release determines the time over which effective angiogenesis by the device is desired.
In the current system, the degradation rate of alginate, the polymer carrier, is controlled by its composition (for example, content of guluronic acid of alginate molecules, differential molecular weight distribution of alginate), physical and chemical treatment (e.g., irradiation or oxidization), and the degree of crosslinking which is controlled by the choice and the amount of the crosslinking agents (for e.g., ionic crosslinker or covalent crosslinker). More specifically, increasing the content of guluronic acid of alginate molecules will increase cross-linking and slow degradation, decreasing molecular weight will speed up degradation, irradiation will decrease molecular weight and thus increase the degradation rate, oxidization will increase the degradation rate, increasing the amount of crosslinker will slow the degradation rate, and different crosslinker molecules may result in differential crosslinking degree and affect the degradation rate.
The doses of the factors loaded in the alginate carrier are altered to achieve effective doses at a desired tissue site, e.g., from 1 to 10 microgram for VEGF165, 1 to 10 microgram for PDGF-BB (dimeric glycoprotein composed of two B (-BB) chains), and 0.01 to 1 microgram for DAPT, for a mouse with an average weight of 10-50 g. An effective dosage amount or ratio of amounts is one that induces and promotes angiogenesis at the target tissue or organ site. The optimal dose is 3 to 10 microgram for VEGF165, 3 to 10 microgram for PDGF-BB, 0.05 to 1 microgram for DAPT in the presence of VEGF at a dose of 1 to 10 microgram, and 0.05 to 1 microgram for DAPT in the presence of PDGF-BB at a dose of 1 to 10 microgram, for a mouse with an average weight of 10-50 g. The amounts and ratio of amounts scale up proportionately for humans.
The relative ratio of VEGF, PDGF and the Notch signaling molecule (e.g., DAPT) is critical in determining the final outcomes of angiogenesis. There exists an effective range and optimal value of the relative ratio between the amount of VEGF and DAPT, the amount of PDGF and DAPT, and the amount of VEGF, PDGF and DAPT. The effective range of the relative ratio (by mole) is 1:1 to 1:200 for VEGF165 to DAPT, 1:1 to 1:200 for PDGF-BB to DAPT, and 1:0.1:1 to 1:10:200 for VEGF165, PDGF-BB and DAPT, in the alginate polymer system. The optimal relative ratio (by mole) is 1:31 for VEGF165 to DAPT, 1.8:31 for PDGF to DAPT, and 1:1.8:31 for VEGF165:PDGF-BB:DAPT. The optimal ratio may vary depending on the specific delivery systems to be used, the species model (e.g., rat, rabbit, pig, dog, human and etc), and any change of the composition and the release kinetics of each of these factors.
Arteriosclerosis
Arteriosclerosis, or arterial disease, is a general term used to describe the thickening and hardening of the arteries. One particular kind of arteriosclerosis that contributes to heart disease is atherosclerosis. Atherosclerosis is a progressive disease that is characterized by a buildup of plaque within the arteries that may partially, or totally, block blood flow through an artery. Plaque is formed from fatty substances, cholesterol, cellular waste, calcium, and fibrin. Atherosclerosis generally results in ischemia, or restriction of the blood supply, for the tissues supplied by the blood carried in the blocked artery.
Oxygen Supply and Deprivation
Because oxygen is mainly bound to hemoglobin in red blood cells, insufficient blood supply causes tissue to become hypoxic, or, in more severe situations, when no oxygen is supplied, anoxic. Oxygen deprivation can cause necrosis, or cell death. In very aerobic tissues such as heart and brain, at body temperature, necrosis due to ischemia becomes irreversible in 3-4 hours. Complete oxygen deprivation to organs such as the heart and brain for greater than 20 minutes causes irreversible damage.
Ischemia is a consequence of heart diseases, transient ischemic attacks, cerebrovascular accidents, ruptured arteriovenous malformations, and peripheral artery occlusive disease. The heart, kidneys, and brain are the most sensitive organs to inadequate blood supply. Stroke, aneurism, hemorrhage, and traumatic injury of the brain commonly result in ischemic conditions. Ischemia in brain tissue induces the ischemic cascade, in which proteolytic enzymes, reactive oxygen species, and other harmful chemicals damage and may ultimately kill brain tissue. Similarly, artery disease and blockages can occlude blood flow to the heart inducing ischemia and death of heart muscle tissue. A macroscopic region of necrotic cells is called an infarction. Heart attacks lead to significant cell death from prolonged oxygen deprivation, also referred to as myocardial infarction.
Restoration of blood flow after a period of ischemia may cause more damage than the ischemia. Reintroduction of oxygen causes a greater production of damaging free radicals, resulting in reperfusion injury and accelerated necrosis.
Coronary Arterial Disease
Patients diagnosed with Coronary Arterial Disease (CAD, also called coronary heart disease, coronary artery disease, ischaemic heart disease, and atherosclerotic heart disease) results from the accumulation of atheromatous plaques within the walls of the arteries that supply the myocardium (heart muscle) with oxygen and nutrients. CAD encompasses a wide spectrum of patients with varying disease severity and prognosis. Patients with mild CAD and the best prognoses are asymptomatic. Mild CAD individuals have atheromatous streaks within the walls of their coronary arteries that do not obstruct blood flow and the lumen of their coronary artery is normal in calibre (as assessed by coronary angiogram). As an individual progresses along this spectrum toward more severe phenotypes, the atheromatous streaks along the coronary walls increase in thickness. Atheromatous plaques begin to form initially and expand into the walls of the artery but, ultimately, expand into the luman of the vessel where they will begin to restrict blood flow.
Once the plaques obstruct more than 70% of the diameter of the vessel lumen, the individual develops symptoms of obstructive coronary artery disease and is diagnosed with ischemic heart disease. The first symptoms of ischemic heart disease are often exertional angina (chest pain) or decreased exercise tolerance. Angina that occurs regularly with activity, upon awakening, or at other predictable times is termed stable angina. Angina that changes in intensity, character or frequency is termed unstable. Unstable angina may precede myocardial infarction.
The degree of severity of CAD can progress to near-complete or complete blockage of the coronary artery. At this end of the spectrum, most individuals experience one or more heart attacks (myocardial infarctions) and all experience chronic ischemia. If the blood flow to the heart tissue is restored to any degree, ischemic tissue is capable of a partial or full recovery depending upon the degree of blood flow restoration. Tissue that has suffered from an infarction is dead, and the damage is irreversible.
Peripheral Arterial Disease
Peripheral Arterial Disease (PAD, also called peripheral artery occlusive disease (PAOD), peripheral vascular disease, and peripheral artery disease) is caused by the obstruction of large peripheral arteries, which can result from atherosclerosis or inflammatory processes and can lead to a narrowing of the artery (stenosis) or obstruction of the artery by thrombus (obstruction by blood clot) or embolism (obstruction by object carried in blood stream from alternate location). PAD/PAOD results in ischemia that is either acute (rapid onset, short duration) or chronic (long-term). Exemplary symptoms of PAD/PAOD include, but are not limited to, claudication (pain, weakness, or cramping in muscles due to decreased blood flow); sores, wounds, or ulcers that heal slowly or incompletely; change in color (blueness, paleness) or cooling compared to other limbs; diminished hair or nail growth on affected limbs compared to unaffected limbs.
PAD/PAOD occurrence is often associated with or caused by smoking, diabetes mellitus, dyslipidemia (e.g. elevated cholesterol, including total cholesterol, LDL cholesterol, and triglyceride levels), hypertension, increased or decreased levels of inflammatory mediators (for example, C-reactive protein, homocysteine, and fibrinogen), aging (especially individuals over 50), racial background (especially prevalent among African-American individuals), gender (more frequently seen in males), obesity, or individuals with personal histories of vascular disease, heart attack, or stroke. The present invention encompasses methods of administering compositions, scaffolds, and devices to all individuals listed supra, for the purposes of repairing or replenishing blood supply to blood- and oxygen-deprived tissues.
PAD/PAOD is diagnosed using a number of tests. The initial test is an ankle brachial pressure index (ABPI/ABI) which measures the fall in blood pressure in the arteries supplying the legs. A reduced ABPI, quantitatively, a score of less than 0.9, suggests a diagnosis of PAD/PAOD. Moderate PAD/PAOD is diagnosed with a reduced ABPI score of less than 0.8. Severe PAD/PAOD is diagnosed with a reduced ABPI score of less than 0.5. However, conditions other than PAD/PAOD can result in reduced ABPI scores of less than 0.9. Thus, additional tests are performed to confirm a diagnosis of PAD/PAOD. A secondary examination usually comprises a lower limb Doppler ultrasound examination of the femoral artery. Alternatively, or in addition, imaging examinations can be performed by angiography using art-recognized standard methods. Furthermore, a multi-slice computerized tomography (CT) scan is used to directly image the arterial system.
PAD/PAOD severity is divided in the Fontaine stages (Fontaine R, Kim M, Kieny R (1954). Helvetica Chirurgica Acta, Basel 21 (5/6):499-533): mild pain while walking (“claudication”)(stage I); severe pain on walking relatively shorter distances (intermittent claudication)(stage II); pain while resting (stage III); loss of sensation to the lower part of the extremity (stage IV); tissue loss (gangrene)(stage V).
Angiogenic Bioactive Compositions
Compositions and methods of the present invention comprise growth factors and signaling molecules that induce, regulate, or augment angiogenesis. Bioactive compositions of the present invention comprise one or more growth factors or signaling molecules incorporated into or coated onto the scaffold composition. Exemplary growth factors and signaling molecules encompassed by the present invention include, but are not limited to, vascular endothelial growth factor (VEGF (A-F)), fibroblast growth factors (acidic and basic FGF 1-10), granulocyte-macrophage colony-stimulating factor (GM-CSF), insulin, insulin growth factor or insulin-like growth factor (IGF), insulin growth factor binding protein (IGFBP), placenta growth factor (PIGF), angiopoietin (Ang1 and Ang2), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), transforming growth factor (TGF-α, TGF-β, isoforms 1-3), platelet-endothelial cell adhesion molecule-1 (PECAM-1), vascular endothelial cadherin (VE-cadherin), nitric oxide (NO), chemokine (C-X-C motif) ligand 10 (CXCL10) or IP-10, interleukin-8 (IL-8), hypoxia inducible factor (HIF), monocyte chemotactic protein (MCP), vascular cell adhesion molecule (VCAM), ephrin ligands (including Ephrin-B2 and -B4); Transcription factors such as HIF-1α, HIF-10 and HIF-2α, Ets-1, Hex, Vezf1, Hox, GATA, LKLF, COUP-TFII, Hox, MEF2, Braf, Prx-1, Prx-2, CRP2/SmLIM and GATA family members, basic helix-loop-helix factors and their inhibitors of differentiation; and regulatory molecules include enzymes (matrix metalloproteinase (MMP), tissue plasminogen activator (PLAT or tPA), cyclooxygenase (COX), angiogenin), molecules regulating Notch signaling which consists of monoclonal antibodies to Notch ligands and receptors, RNA interference, antisense Notch, receptor and mastermind-like 1 (MAML1) decoys, beta and gamma-secretase inhibitors (GSI), or any other molecules that can activate or inhibit Notch signaling.
Vascular Endothelial Growth Factor (VEGF, also known as VEGF-A)
The term “VEGF” broadly encompasses two families of proteins that result from the alternate splicing of a single gene, VEGF, composed of 8 exons. The alternate splice sites reside in the exons 6, 7, and 8. However, the alternate splice site in the terminal exon 8 is functionally important. One family of proteins arise from the proximal splice site and are denoted (VEGFxxx). Proteins produced by alternate splicing at this proximal location are PRO-angiogenic and are expressed conditionally (for instance, when tissues are hypoxic and secreted signals induce angiogenesis). The other family of proteins arise from the distal splice site and are denoted (VEGFxxxb). Proteins produced by alternate splicing at this distal location are ANTI-angiogenic and are expressed in healthy tissues under normal conditions.
VEGF exons 6 and 7 contain splice sites (result in the inclusion or exclusion of exons 6 and 7) that affect heparin binding affinity and amino acid number. Humans comprise VEGF121, VEGF121b, VEGF145, VEGF165, VEGF165b, VEGF189, and VEGF206. Heparin binding affinity, interactions with heparin surface proteoglycans (HSPGs) and neuropilin co-receptors on the cell surface mediated by amino acid sequences in exons 6 and 7 enhance the ability of VEGF variants to activate VEGF signaling receptors (VEGFRs).
Endogenous VEGF splice variants are released from cells as glycosylated disulfide-bonded dimers. Structurally VEGF belongs to the PDGF family of cysteine-knot growth factors comprising Placenta growth factor (PlGF), VEGF-B, VEGF-C and VEGF-D (the VEGF sub-family of growth factors). VEGF is sometimes referred to as VEGF-A to differentiate it from these related growth factors. The term “VEGF” used herein to describe the present invention is meant to refer to VEGF-A.
Members of the VEGF family stimulate cellular responses by binding to cell-surface tyrosine kinase receptors (the VEGFRs). VEGF-A binds to VEGFR-1 (also known as Flt-1) and VEGFR-2 (also known as KDR/Flk-1). VEGFR-2 is the predominant receptor for VEGF-A mediating almost all of the known cellular responses to this growth factor. The function of VEGFR-1 is unclear, although it is thought to modulate VEGFR-2 signaling. VEGFR-1 may also sequester VEGF from VEGFR-2 binding (which may be important during development).
Compositions, methods, and devices of the present invention comprise all VEGF polypeptides generated from alternative splicing including pro- and anti-angiogenic forms. Devices of the present invention administered to a subject contain only pro-angiogenic VEGF polypeptide splice forms. Alternatively, or in addition, devices of the present invention administered to a subject contain a mixture of pro- and anti-angiogenic VEGF polypeptide splice forms. Pro- and anti-angiogenic VEGF polypeptide splice forms are released by the scaffold composition of the device simultaneously or sequentially. For example, the opposing splice forms are released together in order to achieve a precise level of stimulation. Alternatively, the opposing splice forms are released sequentially to stimulate angiogenesis and subsequently attenuate the signal when the desired result has been achieved. In another embodiment, devices comprising pro-angiogenic VEGF polypeptide splice forms are placed at the target tissue site while devices comprising anti-angiogenic VEGF polypeptide splice forms are placed in surrounding tissues in order to prevent pro-angiogenic signals from disseminating into and stimulating non-target tissue.
Exemplary VEGF polypeptide splice forms comprised by the compositions, methods, and devices of the present invention include, but are not limited to, the polypeptides described by the following sequences and SED ID NOs. VEGF polypeptide splice forms are released from compositions, scaffolds, or devices of the present invention as naked, or glycosylated polypeptides. Alternatively, or in addition, VEGF polypeptide splice forms are monomers or disulfide-bonded dimers. In a preferred embodiment, VEGF polypeptide splice forms are released into target tissues from compositions, scaffolds, and/or devices of the present invention as glycosylated disulfide-bonded dimers.
Human VEGF148 is encoded by the following amino acid sequence (NCBI Accession No. NP—001020540 and SEQ ID NO: 12):









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Human VEGF165 is encoded by the following amino acid sequence (NCBI Accession No. NP—001020539 and SEQ ID NO: 13):









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Human VEGF165b is encoded by the following amino acid sequence (NCBI Accession No. NP—001028928 and SEQ ID NO: 14):









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Human VEGF183 is encoded by the following amino acid sequence (NCBI Accession No. NP—001020538 and SEQ ID NO: 15):









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Human VEGF189 is encoded by the following amino acid sequence (NCBI Accession No. NP—003367 and SEQ ID NO: 16):









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Human VEGF206 is encoded by the following amino acid sequence (NCBI Accession No. NP—001020537 and SEQ ID NO: 17):









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Gamma-Secretase Inhibitors (GSI)

Notch is a cell-surface receptor that regulates cell fate decisions throughout development and under selected conditions in adult tissues. Notch signaling results in widely variable outcomes depending on the cells and signaling molecules involved. However, it is generally known that binding of Notch ligands of the Delta and Jagged families results in the proteolytic cleavage of Notch. The Notch protein is first cleaved in the extracellular domain and then subsequently cleaved in the transmembrane domain. The second cleavage event is mediated by γ-secretase. Notch cleavage allows the intracellular domain of the receptor (the Notch IntraCellular Domain, NICD) to translocate to the nucleus where it regulates transcription. Thus, γ-secretase is a Notch activator.
Notch signaling is involved in angiogenesis and vascular remodeling. Moreover, Notch signaling regulates endothelial cell proliferation and migration events necessary to form new blood vessels during angiogenesis in normal tissues as well as malignant tumors. Methods of the present invention are drawn towards inducing angiogenesis in normal tissues, not malignant tissues. Furthermore, it is of great importance to avoid inducing a malignant state within a stable or benign tumor by introducing pro-angiogenic factors in the absence of factors to limit Notch activation. In one preferred embodiment of the present invention, pro-angiogenic factors are released from compositions, scaffolds, or devices, either simultaneously or sequentially, with notch-inhibitors, e.g. inhibitors of gamma-secretase (γ-secretase), to prevent stimulation of angiogenesis within neoplastic tissue.
Compositions, scaffolds, and devices of the present invention comprise all inhibitors of Notch activation to be released simultaneously or sequentially with pro-angiogenic factors. Inhibitors of Notch activity encompassed by the present invention block binding of one or more ligands to the Notch receptor. Alternatively, or in addition, inhibitors of Notch activity present intracellular signal transduction from the Notch receptor or cleavage of the Notch receptor polypeptide. Notch inhibitors of the present invention comprise endogenous or exogenous small molecules, compounds, single- or double-stranded RNA polynucleotides, single- or double-stranded DNA polynucleotides, polypeptides, antibodies, intrabodies, natural or synthetic ligands, genetically-engineered ligands, and genetically-manipulated γ-secretase proteins or fragments thereof. Exemplary inhibitors of Notch activation include, but are not limited to, monoclonal antibodies to Notch ligands and receptors, RNA interference, antisense Notch, receptor and mastermind-like 1 (MAML1) decoys, beta and gamma-secretase inhibitors (GSI).
Gamma-secretase is an integral membrane protein that is one part of a multi-subunit protease complex that cleaves single-pass transmembrane proteins at residues within the transmembrane domain. The gamma secretase complex comprises four individual proteins: presenilin, nicastrin, APH-1 (anterior pharynx-defective 1), and PEN-2 (presenilin enhancer 2). A fifth protein, known as CD147, is a non-essential regulator of the complex whose absence increases activity.
The proteins in the gamma secretase complex are heavily modified by proteolysis during assembly and maturation of the complex. Presenilin is an aspartyl protease comprising the catalytic subunit and is activated by autocatalytic cleavage of to N- and C-terminal fragments. Nicastrin maintains the stability of the assembled complex, regulates intracellular protein trafficking, and recognizes substrates via binding to the N-terminal ectodomain of the target protein. PEN-2 associates with the complex via binding of a transmembrane domain of presenilin and stabilizes the complex after presenilin proteolysis. APH-1, which is required for proteolytic activity, binds to the complex via a conserved alpha helix interaction motif and aids in initiating assembly of premature components.
The present invention comprises one or more inhibitors of γ-secretase which target one or more proteins of this complex and inhibit one or more functions of these proteins. Alternatively, or in addition, γ-secretase inhibitors of the present invention prevent assembly of the γ-secretase protease complex. Furthermore, contemplated γ-secretase inhibitors of the present invention inhibit intracellular signal transduction from an assembled γ-secretase protease complex. Contemplated γ-secretase inhibitors of the present invention bind one or more proteins of the g-secretase complex and partially or entirely block an activity or function. Exemplary γ-secretase inhibitors of the present invention decrease, prevent, or delay activation, as well as inactivate one or more protease components. Exemplary γ-secretase inhibitors of the present invention desensitize or down regulate the activity or expression of one or more proteins of the γ-secretase complex. Exemplary γ-secretase inhibitors of the present invention consist of, consist essentially of, or comprise endogenous or exogenous small molecules, compounds, single- or double-stranded RNA polynucleotides, single- or double-stranded DNA polynucleotides, polypeptides, antibodies, intrabodies, natural or synthetic ligands, genetically-engineered ligands, and genetically-manipulated γ-secretase proteins or fragments thereof.
Exemplary γ-secretase inhibitors of the present invention include, but are not limited to, DAFT and N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester.
Cell-Mediated Enzymatic Scaffold Degradation
Cells secrete enzymes that degrade the material of the scaffold, thereby controlling the rate at which cells exit the scaffold. Cells within close proximity to the implanted scaffold composition secrete enzymes, such as collagenases and plasmin, which degrade the polymer composition. This property is used in certain embodiments to control the release of bioactive compositions into the local cellular environment. The rate of release of bioactive composition may thus be regulated by controlling the density and susceptibility to these enzymes of oligopeptides used as either cross-links in the material or as components of the main chains. Certain materials are degraded in a preprogrammed manner independent of cell action (e.g. hydrolytic degradation of poly(lactide-co glyolide) as a degradable scaffold. The scaffolds may be prepared such that the degradation time may be controlled by using a mixture of degradable components in proportions to achieve a desired degradation rate. Scaffold compositions are sensitive to degradation by materials secreted by the cells located immediately adjacent to the scaffold. One example of this is the use of metalloproteinase (MMP)-sensitive substrate in the scaffold matrix; bioactive composition is released when the adjacent cells have secreted sufficient MMP to begin degradation of the matrix.
Scaffold Compositions and Architecture
Components of the scaffolds are organized in a variety of geometric shapes (e.g., beads, pellets), niches, planar layers (e.g., thin sheets). For example, multicomponent scaffolds are constructed in concentric layers each of which is characterized by different physical qualities (% polymer, % crosslinking of polymer, chemical composition of scaffold, pore size, porosity, and pore architecture, stiffness, toughness, ductility, viscoelasticity, and or composition of bioactive substances such as growth factors, homing/migration factors, differentiation factors. Each niche has a specific effect on a cell population, e.g., promoting or inhibiting a specific cellular function, proliferation, differentiation, elaboration of secreted factors or enzymes, or migration.
Cells incubated in the scaffold are educated and induced to migrate out of the scaffold to directly affect a target tissue, e.g., and injured tissue site. For example, stromal vascular cells and smooth muscle cells are useful in sheet-like structures are used for repair of vessel-like structures such as blood vessels or layers of the body cavity. For example, such structures are used to repair abdominal wall injuries or defects such as gastroschisis. Similarly, sheet-like scaffolds seeded with dermal stem cells and/or keratinocytes are used in bandages or wound dressings for regeneration of dermal tissue. The device is placed or transplanted on or next to a target tissue, in a protected location in the body, next to blood vessels, or outside the body as in the case of an external wound dressing. Devices are introduced into or onto a bodily tissue using a variety of known methods and tools, e.g., spoon, tweezers or graspers, hypodermic needle, endoscopic manipulator, endo- or trans-vascular-catheter, stereotaxic needle, snake device, organ-surface-crawling robot (United States Patent Application 20050154376; Ota et al., 2006, Innovations 1:227-231), minimally invasive surgical devices, surgical implantation tools, and transdermal patches. Devices can also be assembled in place, for example by sequentially injecting or inserting matrix materials. Scaffold devices are optionally recharged with cells or with bioactive compounds, e.g., by sequential injection or spraying of substances such as growth factors or differentiation factors.
A scaffold or scaffold device is the physical structure upon which or into which cells associate or attach, and a scaffold composition is the material from which the structure is made. For example, scaffold compositions include biodegradable or permanent materials such as those listed below. The mechanical characteristics of the scaffold vary according to the application or tissue type for which regeneration is sought. It is biodegradable (e.g., collagen, alginates, polysaccharides, polyethylene glycol (PEG), poly(glycolide) (PGA), poly(L-lactide) (PLA), or poly(lactide-co-glycolide) (PLGA) or permanent (e.g., silk) In the case of biodegradable structures, the composition is degraded by physical or chemical action, e.g., level of hydration, heat or ion exchange or by cellular action, e.g., elaboration of enzyme, peptides, or other compounds by nearby or resident cells. The consistency varies from a soft/pliable (e.g., a gel) to glassy, rubbery, brittle, tough, elastic, stiff. The structures contain pores, which are nanoporous, microporous, or macroporous, and the pattern of the pores is optionally homogeneous, heterogenous, aligned, repeating, or random.
Alginates are versatile polysaccharide based polymers that may be formulated for specific applications by controlling the molecular weight, rate of degradation and method of scaffold formation. Coupling reactions can be used to covalently attach bioactive epitopes, such as the cell adhesion sequence RGD to the polymer backbone. Alginate polymers are formed into a variety of scaffold types. Injectable hydrogels can be formed from low MW alginate solutions upon addition of a cross-linking agent, such as calcium ions, while macroporous scaffolds are formed by lyophilization of high MW alginate discs. Differences in scaffold formulation control the kinetics of scaffold degradation. Release rates of morphogens or other bioactive substances from alginate scaffolds are controlled by scaffold formulation to present morphogens in a spatially and temporally controlled manner. This controlled release not only eliminates systemic side effects and the need for multiple injections, but can be used to create a microenvironment that activates host cells at the implant site.




The scaffold comprises a biocompatible polymer matrix that is optionally biodegradable in whole or in part. A hydrogel is one example of a suitable polymer matrix material. Examples of materials which can form hydrogels include polylactic acid, polyglycolic acid, PLGA polymers, alginates and alginate derivatives, gelatin, collagen, agarose, natural and synthetic polysaccharides, polyamino acids such as polypeptides particularly poly(lysine), polyesters such as polyhydroxybutyrate and poly-epsilon-caprolactone, polyanhydrides; polyphosphazines, poly(vinyl alcohols), poly(alkylene oxides) particularly poly(ethylene oxides), poly(allylamines)(PAM), poly(acrylates), modified styrene polymers such as poly(4-aminomethylstyrene), pluronic polyols, polyoxamers, poly(uronic acids), poly(vinylpyrrolidone) and copolymers of the above, including graft copolymers.
The scaffolds are fabricated from a variety of synthetic polymers and naturally-occurring polymers such as, but not limited to, collagen, fibrin, hyaluronic acid, agarose, and laminin-rich gels. One preferred material for the hydrogel is alginate or modified alginate material. Alginate molecules are comprised of (1-4)-linked β-D-mannuronic acid (M units) and α L-guluronic acid (G units) monomers, which can vary in proportion and sequential distribution along the polymer chain. Alginate polysaccharides are polyelectrolyte systems which have a strong affinity for divalent cations (e.g. Ca+2, Mg+2, Ba+2) and form stable hydrogels when exposed to these molecules. See Martinsen A., et al., Biotech. & Bioeng., 33 (1989) 79-89.) For example, calcium cross-linked alginate hydrogels are useful for dental applications, wound dressings chondrocyte transplantation and as a matrix for other cell types.
An exemplary device utilizes an alginate or other polysaccharide of a relatively low molecular weight, preferably of size which, after dissolution, is at the renal threshold for clearance by humans, e.g., the alginate or polysaccharide is reduced to a molecular weight of 1000 to 80,000 daltons. Preferably, the molecular mass is 1000 to 60,000 daltons, particularly preferably 1000 to 50,000 daltons. It is also useful to use an alginate material of high guluronate content since the guluronate units, as opposed to the mannuronate units, provide sites for ionic crosslinking through divalent cations to gel the polymer. U.S. Pat. No. 6,642,363, incorporated herein by reference discloses methods for making and using polymers containing polysachharides such as alginates or modified alginates that are particularly useful for cell transplantation and tissue engineering applications.
Useful polysaccharides other than alginates include agarose and microbial polysaccharides such as those listed in the table below.










Polysaccharide Scaffold Compositions










Polymersa
Structure







Fungal




Pullulan (N)
1,4-;1,6-α-D-Glucan



Scleroglucan (N)
1,3;1,6-α-D-Glucan



Chitin (N)
1,4-β-D-Acetyl Glucosamine



Chitosan (C)
1,4-β.-D-N-Glucosamine



Elsinan (N)
1,4-;1,3-α-D-Glucan



Bacterial



Xanthan gum (A)
1,4-β.-D-Glucan with D-mannose;




D-glucuronic




Acid as side groups



Curdlan (N)
1,3-β.-D-Glucan (with branching)



Dextran (N)
1,6-α-D-Glucan with some 1,2;1,3-;




1,4-α-linkages



Gellan (A)
1,4-β.-D-Glucan with rhamose,




D-glucuronic acid



Levan (N)
2,6-β-D-Fructan with some




β-2,1-branching



Emulsan (A)
Lipoheteropolysaccharide



Cellulose (N)
1,4-β-D-Glucan







aN-neutral, A = anionic and C = cationic.






The scaffolds of the invention are porous or non-porous. For example, the scaffolds are nanoporous having a diameter of less than about 10 nm; microporous wherein the diameter of the pores are preferably in the range of about 100 nm-20 μm; or macroporous wherein the diameter of the pores are greater than about 20 μm, more preferably greater than about 100 μm and even more preferably greater than about 400 μm. In one example, the scaffold is macroporous with aligned pores of about 400-500 μm in diameter. Other methods of preparing porous hydrogel products are known in the art. (U.S. Pat. No. 6,511,650 incorporated herein by reference).
Bioactive Compositions
The device includes one or more bioactive compositions. Bioactive compositions are purified naturally-occurring, synthetically produced, or recombinant compounds, e.g., polypeptides, nucleic acids, small molecules, or other agents. The compositions described herein are purified. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity is measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
The bioactive composition comprises an element to improve a function of the scaffold composition or to promote angiogenesis. For example, at least one cell adhesion molecule is incorporated into or onto the polymer matrix to attach the scaffold composition to the local tissue site and prevent diffusion of the device. Such molecules are incorporated into the polymer matrix prior to polymerization of the matrix or after polymerization of the matrix. Examples of cell adhesion molecules include, but are not limited to, peptides, proteins and polysaccharides. More specifically, cell adhesion molecules include, but are not limited to, fibronectin, laminin, collagen, thrombospondin 1, vitronectin, elastin, tenascin, aggrecan, agrin, bone sialoprotein, cartilage matrix protein, fibrinogen, fibrin, fibulin, mucins, entactin, osteopontin, plasminogen, restrictin, serglycin, SPARC/osteonectin, versican, von Willebrand Factor, polysaccharide heparin sulfate, connexins, collagen, RGD (Arg-Gly-Asp) and YIGSR (Tyr-Ile-Gly-Ser-Arg) (SEQ ID NO: 9) peptides and cyclic peptides, glycosaminoglycans (GAGs), hyaluronic acid (HA), condroitin-6-sulfate, integrin ligands, selectins, cadherins and members of the immunoglobulin superfamily. Other examples include neural cell adhesion molecules (NCAMs), intercellular adhesion molecules (ICAMs), vascular cell adhesion molecule (VCAM-1), platelet-endothelial cell adhesion molecule (PECAM-1), L1, and CHL1.
Examples of some of these molecules and their function are shown in the following table.










ECM Proteins and peptides and


role in cell function












Seq.





ID



Protein
Sequence
No:
Role













Fibronectin
RGDS

Adhesion



LDV

Adhesion



REDV

Adhesion


Vitronectin
RGDV

Adhesion


Laminin A
LRGDN
7
Adhesion



IKVAV
8
Neurite extension


Laminin B1
YIGSR
9
Adhesion of many cells,





via





67 kD laminin receptor



PDSGR
10
Adhesion


Laminin B2
RNIAEIIKDA
11
Neurite extension


Collagen 1
RGDT

Adhesion of most cells



DGEA

Adhesion of platelets,





other





cells


Thrombo-
RGD

Adhesion of most cells


spondin
VTXG

Adhesion of platelets





Hubbell, J A (1995): Biomaterials in tissue engineering.


Bio/Technology 13: 565-576. One-letter abbreviations of amino acids are used, X stands


for any amino acid.






Additional examples of suitable cell adhesion molecules are shown below.










Amino acid sequences specific for proteoglycan


binding from extracellular matrix proteins










SEQ.



SEQUENCE
ID. NO.
PROTEIN





XBBXBX*
2
Consensus




sequence


 


PRRARV
3
Fibronectin


 


YEKPGSPPREVVPRPRPGV
4
Fibronectin


 


RPSLAKKQRFRHRNRKGYRSQRGHSRGR
5
Vitronectin


 


RIQNLLKITNLRIKFVK
6
Laminin









Particularly preferred cell adhesion molecules are peptides or cyclic peptides containing the amino acid sequence arginine-glycine-aspartic acid (RGD) which is known as a cell attachment ligand and found in various natural extracellular matrix molecules. A polymer matrix with such a modification provides cell adhesion properties to the scaffold, and sustains long-term survival of mammalian cell systems, as well as supporting cell growth.
Coupling of the cell adhesion molecules to the polymer matrix is accomplished using synthetic methods which are in general known to one of ordinary skill in the art and are described in the examples. Approaches to coupling of peptides to polymers are discussed in Hirano and Mooney, Advanced Materials, p. 17-25 (2004). Other useful bonding chemistries include those discussed in Hermanson, Bioconjugate Techniques, p. 152-185 (1996), particularly by use of carbodiimide couplers, DCC and DIC (Woodward's Reagent K). Since many of the cell adhesion molecules are peptides, they contain a terminal amine group for such bonding. The amide bond formation is preferably catalyzed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), which is a water soluble enzyme commonly used in peptide synthesis. The density of cell adhesion ligands, a critical regulator of cellular phenotype following adhesion to a biomaterial. (Massia and Hubbell, J. Cell Biol. 114:1089-1100, 1991; Mooney et al., J. Cell Phys. 151:497-505, 1992; and Hansen et al., Mol. Biol. Cell 5:967-975, 1994) can be readily varied over a 5-order of magnitude density range.
Device Construction
The scaffold structure is constructed out of a number of different rigid, semi-rigid, flexible, gel, self-assembling, liquid crystalline, or fluid compositions such as peptide polymers, polysaccharides, synthetic polymers, hydrogel materials, ceramics (e.g., calcium phosphate or hydroxyapatite), proteins, glycoproteins, proteoglycans, metals and metal alloys. The compositions are assembled into cell scaffold structures using methods known in the art, e.g., injection molding, lyophillization of preformed structures, printing, self-assembly, phase inversion, solvent casting, melt processing, gas foaming, fiber forming/processing, particulate leaching or a combination thereof. The assembled devices are then implanted or administered to the body of an individual to be treated.
The device is assembled in vivo in several ways. The scaffold is made from a gelling material, which is introduced into the body in its ungelled form where it gels in situ. Exemplary methods of delivering device components to a site at which assembly occurs include injection through a needle or other extrusion tool, spraying, painting, or methods of deposit at a tissue site, e.g., delivery using an application device inserted through a cannula. In one example, the ungelled or unformed scaffold material is mixed with bioactive substances and cells prior to introduction into the body or while it is introduced. The resultant in vivo/in situ assembled scaffold contains a mixture of these substances and cells.
In situ assembly of the scaffold occurs as a result of spontaneous association of polymers or from synergistically or chemically catalyzed polymerization. Synergistic or chemical catalysis is initiated by a number of endogenous factors or conditions at or near the assembly site, e.g., body temperature, ions or pH in the body, or by exogenous factors or conditions supplied by the operator to the assembly site, e.g., photons, heat, electrical, sound, or other radiation directed at the ungelled material after it has been introduced. The energy is directed at the scaffold material by a radiation beam or through a heat or light conductor, such as a wire or fiber optic cable or an ultrasonic transducer. Alternatively, a shear-thinning material, such as an ampliphile, is used which re-cross links after the shear force exerted upon it, for example by its passage through a needle, has been relieved.
Suitable hydrogels for both in vivo and ex vivo assembly of scaffold devices are well known in the art and described, e.g., in Lee et al., 2001, Chem. Rev. 7:1869-1879. The peptide amphiphile approach to self-assembly assembly is described, e.g., in Hartgerink et al., 2002, Proc. Natl. Acad. Sci. U.S.A. 99:5133-5138. A method for reversible gellation following shear thinning is exemplified in Lee et al., 2003, Adv. Mat. 15:1828-1832.
A multiple compartment device is assembled in vivo by applying sequential layers of similarly or differentially doped gel or other scaffold material to the target site. For example, the device is formed by sequentially injecting the next, inner layer into the center of the previously injected material using a needle, forming concentric spheroids. Non-concentric compartments are formed by injecting material into different locations in a previously injected layer. A multi-headed injection device extrudes compartments in parallel and simultaneously. The layers are made of similar or different scaffolding compositions differentially doped with bioactive substances. Alternatively, compartments self-organize based on their hydro-philic/phobic characteristics or on secondary interactions within each compartment.
Compartmentalized Device
A compartmentalized device is designed and fabricated using different compositions or concentrations of compositions for each compartment. For example, a first bioactive composition is encapsulated within hydrogels, using standard encapsulation techniques (e.g., alginate microbead formation). This compartment is then coated with a second layer of gel (e.g., double layered alginate microbeads). This second compartment is formed from the same material that contains bioactive composition elements, the same material in a distinct form (e.g., varying mechanical properties or porosity), or a completely different material that provides appropriate chemical/physical properties.
Alternatively, the compartments are fabricated individually, and then adhered to each other (e.g., a “sandwich” with an inner compartment surrounded on one or all sides with the second compartment). This latter construction approach is accomplished using the intrinsic adhesiveness of each layer for the other, diffusion and interpenetration of polymer chains in each layer, polymerization or cross-linking of the second layer to the first, use of an adhesive (e.g., fibrin glue), or physical entrapment of one compartment in the other. The compartments self-assemble and interface appropriately, either in vitro or in vivo, depending on the presence of appropriate precursors (e.g., temperature sensitive oligopeptides, ionic strength sensitive oligopeptides, block polymers, cross-linkers and polymer chains (or combinations thereof), and precursors containing cell adhesion molecules that allow cell-controlled assembly). An individual with ordinary skill in the art of stem cell biology and biomaterials can readily derive a number of potentially useful designs for combining or separating components of a bioactive composition.
Alternatively, the compartmentalized device is formed using a printing technology. Successive layers of a scaffold precursor doped with bioactive substances and/or cells is placed on a substrate then cross linked, for example by self-assembling chemistries. When the cross linking is controlled by chemical-, photo- or heat-catalyzed polymerization, the thickness and pattern of each layer is controlled by a masque, allowing complex three dimensional patterns to be built up when un-cross-linked precursor material is washed away after each catalyzation. (WT Brinkman et al., Photo-cross-linking of type 1 collagen gels in the presence of smooth muscle cells: mechanical properties, cell viability, and function. Biomacromolecules, 2003 July-August; 4(4): 890-895; W. Ryu et al., The construction of three-dimensional micro-fluidic scaffolds of biodegradable polymers by solvent vapor based bonding of micro-molded layers. Biomaterials, 2007 February; 28(6): 1174-1184; Wright, Paul K. (2001). 21st Century manufacturing. New Jersey: Prentice-Hall Inc.) Complex, multi-compartment layers are also built up using an inkjet device which “paints” different doped-scaffold precursors on different areas of the substrate. Julie Phillippi (Carnegie Mellon University) presentation at the annual meeting of the American Society for Cell Biology on Dec. 10, 2006; Print me a heart and a set of arteries, Aldhouse P., New Scientist 13 Apr. 2006 Issue 2547 p 19; Replacement organs, hot off the press, C. Choi, New Scientist, 25 Jan. 2003, v2379. These layers are built-up into complex, three dimensional compartments. The device is also built using any of the following methods: Jetted Photopolymer, Selective Laser Sintering, Laminated Object Manufacturing, Fused Deposition Modeling, Single Jet Inkjet, Three Dimensional Printing, or Laminated Object Manufacturing.
Growth Factors and Incorporation of Compositions into/onto a Scaffold Device
Bioactive substances that influence growth, development, movement, and other cellular functions are introduced into or onto the scaffold structures. Such substances include BMP, bone morphogenetic protein; ECM, extracellular matrix proteins or fragments thereof; EGF, epidermal growth factor; FGF-2, fibroblast growth factor 2; NGF, nerve growth factor; PDGF, platelet-derived growth factor; PIGF, placental growth factor; TGF, transforming growth factor, and VEGF, vascular endothelial growth factor. Cell-cell adhesion molecules (cadherins, integrins, ALCAM, NCAM, proteases) are optionally added to the scaffold composition.
Exemplary growth factors and ligands are provided in the tables below.










Growth factors used for angiogenesis









Growth factor
Abbreviation
Relevant activities





Vascular
VEGF
Migration, proliferation and


endothelial

survival of ECs


growth factor


Basic fibroblast
bFGF-2
Migration, proliferation and survival of


growth factor

ECs and many other cell types


Platelet-derived
PDGF
Promotes the maturation of blood


growth factor

vessels by the recruitment of smooth




muscle cells


Angiopoietin-1
Ang-1
Strengthens EC-smooth muscle




cell interaction


Angiopoietin-2
Ang-2
Weakens EC-smooth muscle cell




interaction


Placental
PIGF
Stimulates angiogenesis


growth factor


Transforming
TGF
Stabilizes new blood vessels by


growth factor

promoting matrix deposition



















Growth factors used for wound healing









Growth Factor
Abbreviation
Relevant activities





Platelet-derived
PDGF
Active in all stages of healing process


growth factor


Epidermal growth
EGF
Mitogenic for keratinocytes


factor


Transforming
TGF-β
Promotes keratinocyte migration,


growth factor-β

ECM synthesis and remodeling, and




differentiation of epithelial cells


Fibroblast growth
FGF
General stimulant for wound healing


factor



















Growth Factors Used for Tissue-Engineering













Moleular

Representative




weight

supplier of rH growth


Growth factor
Abbreviation
(kDa)
Relevant activities
factor














Epidermal growth factor
EGF
6.2
Proliferation of epithelial, mesenchymal,
PeproTech Inc.





and fibroblast cells
(Rocky Hill, NJ, USA)


Platelet-derived
PDGF-AA
28.5
Proliferation and chemoattractant agent for
PeproTech Inc.


growth factor
PDGF-AB
25.5
smooth muscle cells; extracellular matrix



PDGF-BB
24.3
synthesis and deposition


Transforming
TFG-α
5.5
Migration and proliferation of
PeproTech Inc.


growth factor-α


keratinocytes; extracellular matrix





synthesis and deposition


Transforming
TGF-β
25.0
Proliferation and differentiation of bone
PeproTech Inc.


growth factor-β


forming cells; chemoattractant for





fibroblasts


Bone
BMP-2
26.0
Differentiation and migration of bone
Cell Sciences Inc.


morphogenetic
BMP-7
31.5
forming cells
(Norwood, MA, USA)


protein


Basic fibroblast
bFGF/FGF-2
17.2
Proliferation of fibroblasts and initiation of
PeproTech Inc.


growth factor


angiogenesis


Vascular endothelial
VEGF165
38.2
Migration, proliferation, and survival of
PeproTech Inc.


growth factor


endothelial cells





rH, recombinant human
















Immobilized ligands used in tissue engineering









Immobilized ligand*
ECM molecule source
Application





RGD
Multiple ECM molecules,
Enhance bone and cartilage tissue formation in



including fibronectin,
vitro and in vivo



vitronectin, laminin, collagen
Regulate neurite outgrowth in vitro and in vivo



and thrombospondin
Promote myoblast adhesion, proliferation and




differentiation




Enhance endothelial cell adhesion and




proliferation


IKVAV (SEQ ID NO: 8), YIGSR
Laminin
Regulate neurite outgrowth in vitro and in vivo


(SEQ ID NO: 9), RNIAEIIKDI (SEQ


ID NO: 11)


Recombinant fibronectin fragment
Fibronectin
Promote formulation of focal contacts in pre-


(FNIII7-10)

osteoblasts


Ac-GCRDGPQ-GIWGQDRCG
Common MMP substrates,
Encourage cell-mediated proteolytic degradation,


(SEQ ID NO: 18)
(e.g. collagen, fibronectin,
remodeling and bone regeneration (with RGD and



laminin)
BMP-2 presentation) in vivo





*Sequences are given in single-letter amino acid code. MMP, matrix metalloproteinase.






The release profiles of bioactive substances from scaffold devices is controlled by both factor diffusion and polymer degradation, the dose of the factor loaded in the system, and the composition of the polymer. Similarly, the range of action (tissue distribution) and duration of action, or spatiotemporal gradients of the released factors are regulated by these variables. The diffusion and degradation of the factors in the tissue of interest is optionally regulated by chemically modifying the factors (e.g., PEGylating growth factors). In both cases, the time frame of release determines the time over which effective cell delivery by the device is desired.
Carrier systems for tissue regeneration are described in the table below.










Polymeric carriers used to deliver various growth factors


and the type of tissues regenerated









Growth factor
Carrier
Tissue regenerated





EGF
Gelatin
Dermis



PET suture
Tendon



PVA sponge
Dermis


PDGF
Chitosan-PLLA scaffold
Craniofacial bone



CMC gel
Dermis



Fibrin
Ligament



Porous HA
Long Bone


TGF-β
Alginate
Cartilage



PLA
Long Bone



CaP-titanium mesh
Craniofacial bone



Polyoxamer; PEO gel
Dermis


rhBMP-2
Collagen sponge
Long bone




Craniofacial bone



HA-TCP granules
Spinal bone



HA-collagen
Long bone



PLA-DX-PEG
Ectopic and hip bone


rHBMP-7
HA
Spinal bone



Collagen-CMC
Spinal bone



Porous HA
Craniofacial bone


bFGF
Chitosan
Dermis



Heparin-alginate
Blood vessels



EVAc microspheres
Blood vessels



Fibrin matrices
Blood vessels


VEGF
PLG scaffold
Blood vessels



PLG scaffold
Blood vessels



PLG microspheres
Blood vessels



Fibrin mesh
Blood vessels





Abbreviations:


PET, poly (ethylene terepthalate);


PVA, polyvinyl alcohol;


PLLA, poly(L-lactic acid);


CMC, carboxymethylcellulose;


HA, hydroxyapatite;


PLA, poly(D,L-lactic acid);


CaP, calcium phosphate;


PEO, poly (ethylene oxide);


TCP, tricalcium phosphate;


PEG, poly(ethylene glycol);


-DX-, -p-dioxanone-;


EVAc, ethylene vinyl acetate;


PLG, poly (lactide-co-glycolide).






The bioactive substances are added to the scaffold compositions using known methods including surface absorption, physical immobilization, e.g., using a phase change to entrap the substance in the scaffold material. For example, a growth factor is mixed with the scaffold composition while it is in an aqueous or liquid phase, and after a change in environmental conditions (e.g., pH, temperature, ion concentration), the liquid gels or solidifies thereby entrapping the bioactive substance. Alternatively, covalent coupling, e.g., using alkylating or acylating agents, is used to provide a stable, long-term presentation of a bioactive substance on the scaffold in a defined conformation. Exemplary reagents for covalent coupling of such substances are provided in the table below.










Methods to covalently couple peptides/proteins to polymers









Functional

Reacting groups


Group of

on proteins/


Polymer
Coupling reagents and cross-linker
peptides





—OH
Cyanogen bromide (CNBr)
—NH2



Cyanuric chloride



4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-



methyl-morpholinium chloride



(DMT-MM)


—NH2
Diisocyanate compounds
—NH2



Diisothoncyanate compounds
—OH



Glutaraldehyde



Succinic anhydride


—NH2
Nitrous Acid
—NH2



Hydrazine + nitrous acid
—SH




—Ph—OH


—NH2
Carbodiimide compounds (e.g., EDC,
—COOH



DCC)[a] DMT-MM


—COOH
Thionyl chloride
—NH2



N-hydroxysuccinimide



N-hydroxysulfosuccinimide + EDC


—SH
Disulfide compound
—SH





[a]EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride;


DCC: dicyclohexylcarbodiimide






The following materials and methods were used to generate the data described herein.
Preparation and Loading of Polymer/Gel Compositions
Ultrapure alginates were purchased from ProNova Biomedical (Norway). MVG alginate, a high-G-containing alginate (M/G ratio of 40/60 as specified by the manufacturer), was used as the high molecular weight (molecular mass=250,000 Da) component to prepare gels. Low molecular weight alginate (molecular mass=50,000 Da) was obtained by gamma-irradiating high molecular weight alginate with a cobalt-60 source for 4 h at a gamma-dose of 3.0 Mrad (Radiation Lab, Massachusetts Institute of Technology). The alginate used to form gels was a combination of the two different molecular weight polymers at a ratio of 3:1. Both alginate polymers were diluted to 1% w/v in double-distilled H20, and 1% of the sugar residues in the polymer chains were oxidized with sodium periodate (Aldrich, St Louis, Mo.) by maintaining solutions in the dark for 17 h at room temperature. An equimolar amount of ethylene glycol (Fisher, Pittsburgh, Pa.) was added to stop the reaction, and the solution was subsequently dialyzed (MWCO 1000, Spectra/Por, Rancho Dominguez, Calif.) over 3 days. The solution was sterile filtered, frozen (−20 degree C. overnight), lyophilized and stored at −20 degree C. To prepare gels, modified alginates were reconstituted in EBM-2 (Lonza, Walkersville, Md.) to obtain a 2% w/v solution(75% low molecular weight, 25% high molecular weight MVG used in all experiments) prior to gelation. The 2% w/v alginate solutions were cross-linked with aqueous slurries of a calcium sulfate solution (0.21 g CaS04 mL/L distilled H20) at a ratio of 25:1 (40 microliter of CaS04 per 1 mL of 2% w/v alginate solution) using a 1 mL syringe. Reconstituted alginate was stored at 4 degree C. For incorporation of VEGF, PDGF and DAPT, alginate solutions were mixed with recombinant human VEGF 165, PDGF-BB (R&D systems, MN) or DAPT (N—[N-(3,5-Difluorophenacetyl-L-alanyl)]-Sphenylglycine t-Butyl Ester) (EMD Chemicals, NJ) by using two syringes coupled by a syringe connector. Calcium slurry (Sigma, St Louis, Mo.) was then mixed with the resulting alginate solution using two syringes coupled by a syringe connector to facilitate the mixing process and prevent entrapment of air bubbles during mixing. The mixture was allowed to gel for 30 min, and then was maintained at 4 degrees C. prior to animal injections.
Murine Hindlimb Model of Ischemia
The animals used were 6-week old severe combined immunodeficiency (SCID) mice on a C57BL/6J background (Jackson Laboratory, ME). Unilateral hindlimb ischemia was created as follows. The animals were anesthetized by intraperitoneal injections of ketamine (80 mg/kg) and xylazine (5 mg/kg). The external iliac and femoral artery and vein were ligated, and 50 μL alginate hydrogel incorporating 3 μg VEGF and/or 86-8600 ng DAPT was injected near the distal end of the ligation site. As controls, VEGF and DAPT in PBS were also injected intramuscularly or intraperitoneally (bolus delivery). Incisions were closed by 5-0 Ethicon sutures (Johnson & Johnson, N.J.). Blood flow in the hindlimb was monitored by a laser Doppler perfusion imaging (LDP/) system (Perimed AB, Sweden), and the results were normalized to the control unligated limb of the same animal.
Histology and Immunohistochemistry
Hindlimb muscle tissues between the two suture knots defining the ligation site were dissected and fixed by Z-fix (Anatech, M/) overnight and changed into 70% EtOH for storage prior to histology processing. Samples were embedded in paraffin and sectioned (5 μm thick) onto slides by Paragon (Paragon Bioservices, MD). Sections were incubated with primary anti-mouse CD31 antibody (1:250) (Pharmingen, Calif.) or α-smooth muscle actin antibody, followed by incubation with an anti-rat mouse biotinylated secondary (1:200) (Vector Laboratories, CA), and amplified by a Tyramide Signal Amplification (TSA) Biotin System (Perkin Elmer Life Sciences, MA). Staining was developed using DAB+ substrate chromogen (DAKO, CA) and counterstained with Mayer's Hematoxylin. Capillary densities were quantified by counting the CD31 positive capillary numbers, normalized to the tissue area, in 30 randomly chosen high-power (200×, 400×) fields. Images were captured with an Olympus-IX81 light microscope connected to an Olympus DP70 digital image capture system.
EXAMPLE 1
In vitro Model to Test the Significance of a Controlled Local Concentration of VEGF
Endothelial cells isolated from diabetics are shown to have a reduced response to VEGF as compared to age-matched non-diabetics (FIG. 1, *P<0.05), as reflected in their capability of forming sprouts, the first step in angiogenesis. In addition, there is an optimal VEGF concentration to induce most sprouts. This suggests the need of a controlled concentration of growth factors in the local area to reach the best therapeutic effect.
EXAMPLE 2
In vitro Model to Test the Significance of a Combination of DAPT and VEGF
Endothelial cells isolated from diabetics produce more sprouts in the presence of a combination of both gamma-secretase inhibitors with VEGF. (FIG. 2, *P<0.05) than with either of them alone, which implies the need of a combination of both growth factor and DAPT.
EXAMPLE 3
In vitro Model Establishing the Significance of a Distinct Presentation of VEGF and DAPT
This examples illustrates the concept that while the combination of VEGF and DAPT is superior to either single factor alone, the optimal concentration for each individual compound does not coincide. As shown in FIG. 3 (*P<0.05), the concentration of VEGF that gives the most sprouts if used alone (50 ng/ml) is less superior to a lower concentration (10 ng/ml) when in combined use with DAPT (2.5 μM). Moreover, increasing both the concentration of VEGF (50 ng/ml) and DAPT (10 μM) actually reduces the number of sprouts. This indicates that the presentation of VEGF and DAPT may need to be separately controlled to achieve an optimal effect.
EXAMPLE 4
In vivo Model to Test the Effect of Controlled Presentation of VEGF and DAPT to Recover Blood Flow in an Ischemia Situation
Alginate is used as the delivery vehicle. Injectable alginate hydrogels incorporating VEGF and GSI is injected into the hindlimb ischemia site created by femoral artery and vein ligation in a murine model. Release profiles of VEGF and DAPT from alginate hydrogels are distinct, as shown in FIG. 4A. Blood flow before and after ligation surgery is measured by laser doppler perfusion imaging (LDPI) to indicate the extent of angiogenesis in the ischemia area. As shown in FIGS. 4B and 4C, (*P<0.05), when DAPT and VEGF are both incorporated in the alginate hydrogel the blood flow recovery is superior to simple bolus injection of them together, either drug alone from alginate gel or by bolus injection (including direct intramuscular and intraperitoneal injection), and blank control. This points to the significance of a controlled presentation of multiple pro-angiogenic compounds as compared to bolus injection.
EXAMPLE 5
In vivo Model to Test the Effect of Controlled Presentation of VEGF and DAPT on Newly-Formed Blood Vessel Density in an Ischemia Situation
A combination of VEGF and DAPT delivered by alginate gel systems gave rise to the highest blood vessel density as compared to blank control, either alone released from gels (FIG. 5A), or administered via intramuscular or intraperitoneal injection (FIG. 5B). These data indicate the significance of controlled delivery of both VEGF and DAPT by the alginate gel system, i.e. controlled and coordinated release of growth factors and signaling molecules leads to an improved clinical result compared to conventional delivery methods.
EXAMPLE 6
In vivo Evaluation of the Effect of DAPT Delivered From Alginate Gel System and From Intraperitoneal Injection on the Cells in Small Intestines
As compared to normal controls, DAPT delivered from alginate gel system does not alter cell differentiation at distant sites as much as DAPT delivered via intraperitoneal injection (DAPT delivered via injection disrupts normal intestinal structure, as indicated by H&E staining, presence of extra glycosaminoglycans by alcian blue staining, loss of proliferating cells by Ki67 staining, and downregulation of Notch target gene expression by HES-1 staining) DAPT delivered from the gel system only has effects in the local region while DAPT delivered via intraperitoneal injection goes into the systemic circulation and led to adverse effects at distant organs (e.g., small intestines as an example). These data indicate that a controlled local but not systemic presentation of delivered DAPT is a preferred delivery method.
EXAMPLE 7
Blood Vessel Density and Blood Flow Recovery
In vivo evaluation of the effect of controlled presentation of VEGF and DAPT on newly formed blood vessel density and blood flow recovery was carried out in an ischemic type I diabetic mouse model. The results (FIGS. 7A-B) indicate that a combination of controlled delivery of a prescribed previously determined ratio, e.g., an optimal level, of VEGF and DAPT increased the blood vessel density and recovered blood flow.
EXAMPLE 8
Comparison of Controlled Presentation of Factors Compared to Delivery of Factors Alone
The effect of controlled presentation of VEGF and PDGF on newly formed blood vessel density and blood flow recovery was evaluated in vivo using the ischemic type I diabetic mouse model. The results (FIGS. 8A-B) indicate that a combination of optimal level of VEGF and PDGF is superior to VEGF or PDGF alone in recovering blood flow.
EXAMPLE 9
Controlled Presentation of a Combination of Factors VEGF, PDGF and DAPT on Angiogenesis
In vivo evaluation of the effect of controlled presentation of VEGF, PDGF and DAPT on newly formed blood vessel density and blood flow recovery was carried out in an ischemic type I diabetic mouse model. These results (FIGS. 9A-B) indicate that a combination of optimal level of VEGF and DAPT is superior to a combination of VEGF and PDGF in recovering blood flow.
EXAMPLE 10
Maturation of Newly Formed Blood Vessels
The effect of controlled presentation of VEGF, PDGF and DAPT on the maturation of newly formed blood vessels was evaluated using the same in vivo ischemic type I diabetic mouse model. The result (FIGS. 10A-C) indicates that a combination of optimal level of VEGF PDGF and DAPT is superior to a combination of VEGF and PDGF in generating more matured blood vessels.
The data generated described herein indicate that compositions and methods not only reliably induce and promote angiogenesis in bodily tissues and organs but also promote and support maturation of those vessels into functional vasculature to improve blood flow to ischemic, damaged, injured, or otherwise compromised tissues and organs.
Other Embodiments
The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

## Claims

1. A method of inducing growth of new blood vessels either from existing blood vessels, or creation of de novo vessels, or a combination thereof, in a target tissue of a mammal, comprising contacting a mammalian cell by injecting into a tissue a device comprising an alginate hydrogel scaffold composition comprising N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) and vascular endothelial growth factor (VEGF), wherein said scaffold composition temporally controls release of the DAPT and the VEGF, wherein the VEGF said bioactive composition induces angiogenesis, arteriogenesis, or vasculogenesis, and wherein the VEGF comprises VEGF121, VEGF145, VEGF165, VEGF189, or VEGF206.

2. The method of claim 1, wherein the DAPT is released before the VEGF.

3. The method of claim 1, wherein said VEGF is released over a period of weeks and said DAPT is released over a period of less than one week.

4. The method of claim 1, wherein said mammal is a human.

5. The method of claim 1, wherein said mammal has diabetes.

6. The method of claim 1, wherein said hydrogel scaffold comprises nanopores.

7. The method of claim 1, wherein the DAPT is released from the scaffold composition at a first rate and the VEGF is released from the scaffold composition at a second rate, and wherein the second rate is slower than the first rate.

8. The method of claim 7, wherein the VEGF exits from the scaffold composition for a time period of one or more weeks.

9. The method of claim 7, wherein the DAPT exits from the scaffold composition for a period of one or more days.

10. The method of claim 1, wherein the device comprises a molar ratio of 1:1 to 1:200 for VEGF165 to DAPT.

11. The method of claim 10, wherein the device comprises a molar ratio of 1:31 for VEGF165 to DAPT.

12. The method of claim 1, wherein the mammal has coronary arterial disease (CAD) or peripheral arterial disease (PAD).

13. The method of claim 1, wherein the mammal has acute or chronic ischemia due to peripheral arterial disease (PAD).

14. The method of claim 1, wherein the mammal has a diabetic ulcer or a wound due to peripheral arterial disease (PAD).

15. A method of inducing growth of new blood vessels either from existing blood vessels, or creation of de novo vessels, or a combination thereof, in a target tissue of a mammal, comprising contacting a mammalian cell by injecting into a tissue a device comprising an alginate hydrogel scaffold composition comprising N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) and a bioactive composition selected from the group consisting of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), wherein said scaffold composition temporally controls release of the DAPT and the bioactive composition, wherein the bioactive composition induces angiogenesis, arteriogenesis, or vasculogenesis, and wherein the VEGF comprises VEGF121, VEGF145, VEGF165, VEGF189, or VEGF206.

16. The method of claim 15, wherein said DAPT is released within 1-3 days and said VEGF or PDGF is released within 7-60 days.

17. The method of claim 15, wherein the DAPT is released before the bioactive composition.

18. The method of claim 15, wherein the PDGF comprises PDGF-BB.

19. The method of claim 15, wherein the device comprises a molar ratio of 1:1 to 1:200 for VEGF165 to DAPT, or 1:1 to 1:200 for PDGF-BB to DAPT.

20. The method of claim 19, wherein the device comprises a molar ratio of 1.8:31 for PDGF-BB to DAPT.

21. The method of claim 15, wherein the mammal has coronary arterial disease (CAD) or peripheral arterial disease (PAD).

22. The method of claim 15, wherein the mammal has acute or chronic ischemia due to peripheral arterial disease (PAD).

23. The method of claim 15, wherein the mammal has a diabetic ulcer or a wound due to peripheral arterial disease (PAD).

24. A method of inducing growth of new blood vessels either from existing blood vessels, or creation of de novo vessels, or a combination thereof, in a target tissue of a mammal, comprising contacting a mammalian cell by injecting into a tissue a device comprising a hydrogel scaffold composition comprising N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF), wherein said scaffold composition temporally controls release of the DAPT, VEGF, and PDGF, and wherein the VEGF and PDGF induce angiogenesis, arteriogenesis, or vasculogenesis.

25. The method of claim 24, wherein the device comprises a molar ratio of 1:0.1:1 to 1:10:200 for VEGF165, PDGF-BB and DAPT.

26. The method of claim 25, wherein the device comprises a molar ratio of 1:1.8:31 for VEGF165, PDGF-BB and DAPT.

27. The method of claim 24, wherein the mammal has coronary arterial disease (CAD) or peripheral arterial disease (PAD).

28. The method of claim 24, wherein the mammal has acute or chronic ischemia due to peripheral arterial disease (PAD).

29. The method of claim 24, wherein the mammal has a diabetic ulcer or a wound due to peripheral arterial disease (PAD).