WO2015021044A1 - Compositions et procédés pour la libération en réponse à des stimuli d'un agent thérapeutique - Google Patents

Compositions et procédés pour la libération en réponse à des stimuli d'un agent thérapeutique Download PDF

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WO2015021044A1
WO2015021044A1 PCT/US2014/049774 US2014049774W WO2015021044A1 WO 2015021044 A1 WO2015021044 A1 WO 2015021044A1 US 2014049774 W US2014049774 W US 2014049774W WO 2015021044 A1 WO2015021044 A1 WO 2015021044A1
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peptide
composition
hydrogel
peptides
therapeutic agent
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Amay Van HOVE
Danielle Benoit
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University Of Rochester
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L71/00—Compositions of polyethers obtained by reactions forming an ether link in the main chain; Compositions of derivatives of such polymers
    • C08L71/02—Polyalkylene oxides

Definitions

  • Peptide drugs have been identified for a variety of applications including to promote vascularization (Finetti et al., 2012, Biochem Pharm., 84(3):303-l 1; Santulli et al., 2009, J of Trans Med. 7:41), reduce inflammation (Akeson et al., 1996, J Biol Chem. 271(48):30517-23; Schultz et al, 2005, Biomaterials 26(15):2621-30), and as cancer therapeutics (Yang et al, 2003, Cancer Res. 63(4):831-7; Selivanova et al, Nat Med. 1997, 3(6):632-8).
  • Peptides typically mimic the bioactivity of larger proteins or growth factors, and offer many advantages over traditional protein delivery.
  • peptides Due to small sizes, peptides can be produced synthetically and delivered at concentrations higher than whole proteins while maintaining high specificity to targets, resulting in high potencies of action and relatively few off-target effects. Additionally, some peptides often do not require complex tertiary structures for bioactivity, resulting in lower susceptibility to denaturation and degradation in vivo (Finetti et al., 2012, Biochem Pharm., 84(3):303- 11). As peptides often do not fully recapitulate protein bioactivities, some reports indicate peptides may need to be delivered at higher doses to achieve therapeutic results (Ben- Sasson et al, 2003, Blood.
  • Diffusion-mediated peptide release can be achieved using polymers such as Hydron (Ben-Sasson et al, 2003, Blood. 102(6):2099-107; Failla et al, 2008, Blood. 111(7):3479-88) or hydrogels such as ReGel (PLGA-b-PEG-b-PLGA) (Choi et al, 2004, Pharm Res. 21(5):827-31), pluronics (Santulli et al, 2009, J of Trans Med. 7:41) or Matrigel (Van Slyke et al, 2009, Tissue Eng Pt A. 15(6): 1269-80).
  • polymers such as Hydron (Ben-Sasson et al, 2003, Blood. 102(6):2099-107; Failla et al, 2008, Blood. 111(7):3479-88) or hydrogels such as ReGel (PLGA-b-PEG-b-PLGA) (Choi
  • cardiac ischemia the leading cause of death worldwide in 2008, killing more than 7.2 million people (Mathers and Loncar, 2006, PLoS Medicine, 3(11)).
  • Cardiac ischemia often occurs as a result of myocardial infarction, and current treatments are unable to adequately repair ischemic cardiac tissue, with up to one-third of patients ultimately developing heart failure.
  • Other ischemic tissue disorders which could benefit from localized pro-angiogenic drug delivery, including peripheral vascular ischemia and diabetic ulcers, are characterized in part by a decrease in angiogenesis seen at the wound site.
  • pro-angiogenic proteins such as vascular endothelial growth factor (VEGF) or platelet derived growth factor (PDGF)
  • VEGF vascular endothelial growth factor
  • PDGF platelet derived growth factor
  • VEGF vascular endothelial growth factor
  • PDGF platelet derived growth factor
  • a composition comprising a plurality of synthetic monomers, and a plurality of prodrugs.
  • each prodrug comprises at least one therapeutic domain and one or more cleavable domains, wherein the cleavable domains couples the prodrug to one or more of the plurality of synthetic monomers.
  • the ratio of prodrug to synthetic monomer in the composition is greater than about 1 : 1. In one embodiment, the ratio of prodrug to synthetic monomer in the composition is greater than about 2: 1.
  • the prodrug comprises a therapeutic peptide coupled to a cleavable peptide sequence at its amino terminus and a cleavable peptide sequence at its carboxy terminus.
  • the synthetic monomer comprise poly(ethylene glycol) (PEG).
  • the PEG is functionalized with terminal norbornene groups.
  • the composition is a polymerized matrix. In one embodiment, polymerization of the matrix is induced by UV light. In one embodiment, the composition is a hydrogel. In one embodiment, the composition is a solution.
  • the at least one cleavage domain comprises a matrix metalloproteinase (MMP) -sensitive cleavage domain.
  • MMP matrix metalloproteinase
  • the MMP- sensitive cleavage domain comprises the amino acid sequence of SEQ ID NO: 1.
  • an increased expression, activity, or combination thereof, of a MMP induces cleavage of the MMP-sensitive cleavage domain.
  • the therapeutic domain comprises a pro-angiogenic amino acid sequence. In one embodiment, the therapeutic domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.
  • cleavage of the cleavage domains releases a released therapeutic agent.
  • the released therapeutic agent comprises a fragment of a first cleavage domain, wherein the fragment is positioned at a first terminal end of the released therapeutic agent.
  • the released therapeutic agent comprises a fragment of a second cleavage domain, wherein the fragment is positioned at a second terminal end of the released therapeutic agent.
  • the released therapeutic agent comprises a first fragment of a first cleavage domain, wherein the first fragment is positioned at a first terminal end of the released therapeutic agent, and a second fragment of a second cleavage domain, wherein the second fragment is positioned at a second terminal end of the released therapeutic agent.
  • the released therapeutic agent is a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12.
  • the composition provides localized and stimuli-induced release of the released therapeutic agent.
  • a method of delivering a therapeutic agent to a subject in need thereof comprising administering to a subject a composition described herein.
  • the method comprises administering a composition comprising a plurality of synthetic monomers, and a plurality of prodrugs.
  • each prodrug comprises at least one therapeutic domain and one or more cleavable domains, wherein the cleavable domains couples the prodrug to one or more of the plurality of synthetic monomers.
  • the ratio of prodrug to synthetic monomer in the composition is greater than about 1 : 1. In one embodiment, the ratio of prodrug to synthetic monomer in the composition is greater than about 2: 1.
  • the prodrug comprises a therapeutic peptide coupled to a cleavable peptide sequence at its amino terminus and a cleavable peptide sequence at its carboxy terminus.
  • the synthetic monomer comprise poly(ethylene glycol) (PEG).
  • PEG poly(ethylene glycol)
  • the PEG is functionalized with terminal norbornene groups.
  • the composition is a polymerized matrix. In one embodiment, polymerization of the matrix is induced by UV light. In one embodiment, the composition is a hydrogel. In one embodiment, the method comprises implanting the hydrogel into the subject at a treatment site.
  • the composition is a solution.
  • the method comprises polymerizing the composition ex vivo.
  • the method comprises the step of administering UV light to the subject in vivo, thereby polymerizing the composition to form a matrix.
  • the at least one cleavage domain comprises a matrix metalloproteinase (MMP) -sensitive cleavage domain.
  • MMP matrix metalloproteinase
  • the MMP- sensitive cleavage domain comprises the amino acid sequence of SEQ ID NO: 1.
  • an increased expression, activity, or combination thereof, of a MMP induces cleavage of the MMP-sensitive cleavage domain.
  • the therapeutic domain comprises a pro-angiogenic amino acid sequence.
  • the therapeutic domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.
  • cleavage of the cleavage domains releases a released therapeutic agent.
  • the released therapeutic agent comprises a fragment of a first cleavage domain, wherein the fragment is positioned at a first terminal end of the released therapeutic agent.
  • the released therapeutic agent comprises a fragment of a second cleavage domain, wherein the fragment is positioned at a second terminal end of the released therapeutic agent.
  • the released therapeutic agent comprises a first fragment of a first cleavage domain, wherein the first fragment is positioned at a first terminal end of the released therapeutic agent, and a second fragment of a second cleavage domain, wherein the second fragment is positioned at a second terminal end of the released therapeutic agent.
  • the released therapeutic agent is a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 12.
  • the composition provides localized and stimuli-induced release of the released therapeutic agent.
  • the subject has a condition associated with reduced vascularization, wherein the therapeutic domain comprises a pro-angiogenic amino acid sequence.
  • the condition is selected from the group consisting of cardiac ischemia, coronary heart failure, peripheral vascular ischemia, peripheral arterial disease, ischemic bowel disease, bone fracture healing, and diabetic ulcers.
  • the subject comprises an implanted engineered tissue in need of
  • the subject is a human.
  • Figure 1 is a schematic detailing the development of pro-angiogenic biomaterials aimed at coordinating tissue vascularization.
  • ischemic cardiac tissue develops as a result of arterial disease or myocardial infarction.
  • the hydrogel precursors comprising of norbornene-functionalized poly(ethylene glycol) macromers and angiogenic peptides connected to MMP-degradable tethers, are injected using minimally-invasive laparoscopic techniques, and polymerized in situ via UV polymerization.
  • the MMP-degradable tether is cleaved, resulting in a prolonged release of the angiogenic peptide into the ischemic tissue.
  • this material could be used for the delivery of any number of therapeutic peptides, including anti-apoptotic, anti-inflammatory, or chemotherapeutic.
  • Figure 2 is a schematic depicting thiol-ene photopolymerization of the matrix.
  • A Upon exposure to the UV light, the photoinitiator is cleaved in two and abstracts a hydrogen atom from a thiol on a cysteine amino acid.
  • B The resulting thiyl radical propagates across the norbornene carbon-carbon double bond.
  • C This produces a norbornene radical,
  • D) which abstracts a hydrogen from another thiol,
  • E completing the thio-ether bond formation, and propagating the reaction.
  • Figure 3 depicts the results of experiments demonstrating the influence of PEG structure/molecular weight on hydrogel properties.
  • Figure 4 depicts representative MALDI-ToF results, which shows correct synthesis of Qk(2T). Correct synthesis of each peptide was confirmed by the observed and expected molecular weight peaks coinciding.
  • Figure 12 depicts a summary of the effects of the native and 2T forms of the tested peptides in the HUVEC proliferation and HUVEC tube formation assays described herein.
  • Figure 13 depicts the results of experiments demonstrating hydrogel degradation and peptide release.
  • Hydrogel degradation ( Figure 13 A) and peptide release ( Figure 13B) was quantified upon incubation in at 37 °C in PBS or PBS containing 250 ⁇ g/mL collagenase.
  • Figure 14 is a schematic demonstrating enzymatically responsive release of therapeutic peptides from poly(ethylene glycol) hydrogels.
  • PEG hydrogels are formed via thiol-ene photopolymerization between norbornene functionalized PEG macromers and thiol groups on cysteine amino acids on either end of the crosslinking peptide sequence.
  • Peptide drugs are flanked with the enzymatically degradable sequence
  • IPESjLRAG SEQ ID NO: 1
  • J indicates cleavage site.
  • Figure 15 depicts the results of experiments demonstrating the effects of residual "tails" on peptide bioactivity.
  • Figure 15 A Fold increase in HUVEC tube length over control media with peptide treatment and
  • Figure 15B percent increase in HUVEC proliferation, quantified by measuring DNA content, over control media after 3 days of peptide treatment.
  • White bars indicate "N” peptide; grey bars indicate “2T” peptide; black bars indicate control groups.
  • Figure 16 comprising Figure 16A through Figure 16G, is a set of illustrations depicting the predicted structures for peptides (Figure 16A) the degradable linker (DL) alone, (Figure 16B) SPARCn 8 (DL), ( Figure 16C) SmPho(DL), ( Figure 16D) SPARC 3X (DL), ( Figure 16E) SPARCn 3 (DL), ( Figure 16F) Scrambled (DL), and (Figure 16G) Qk(DL). Arrowheads on a-helix ribbons point to C-termini.
  • Figure 17, comprising Figure 17A through Figure 17D, depicts the results of experiments characterizing the physical properties of hydrogels and examining hydrogel degradation.
  • Figure 17B hydrogel swelling ratios,
  • Figure 17C % peptide incorporated into hydrgoels, and
  • Figure 17D % peptide not forming crosslinks, horizontal bars indicate groups that are statistically equivalent (p > 0.05), * p ⁇ 0.05, & p ⁇ 0.01, $ p ⁇ 0.001.
  • n 12; error bars represent SEM.
  • Figure 18, comprising Figure 18A and Figure 18B, depicts the results of experiments examining hydrogel degradation and peptide release.
  • Figure 18A Time to complete degradation (study ended at 10 days), and
  • Figure 18B amount of "2T" peptide released upon complete hydrogel degradation, a-f indicates groups that are statistically equivalent (p > 0.05), $ p ⁇ 0.001, # p ⁇ 0.0001.
  • n 6; error bars represent SEM (some obscured by symbol).
  • Figure 20 depicts the results of experiments investigating the in vitro efficacy of peptide-releasing hydrogels.
  • Figure 20A Representative images of and ( Figure 20B) fold increase in tube length over control media upon treatment with degraded peptide-releasing hydrogels.
  • HUVECs were treated with ⁇ 1/7,000 ⁇ of a degraded gel, corresponding to 100 nM of released SPARCn 8 (2T).
  • Figure 21 is a set of illustrations depicting the predicted structures for various "N” and “2T” peptides. Comparing peptides that lost (KRX-725 and SPARC 3X ) and retained (SPARCn 3 and Qk) bioactivity upon inclusion of the "2T", no clear change in structure between "N” and “2T” form was observed. Arrowheads on a-helix ribbons point to C-termini.
  • Figure 22 is a set of graphs demonstrating the stability of released peptides.
  • Figure 23, comprising Figure 23 A through Figure 23F, depicts the results of MALDI-ToF mass spectrometry analysis of degraded hydrogels.
  • Figure 23 A SPARCii 8 (DL),
  • Figure 23B SmPho(DL),
  • Figure 23C SPARC 3X (DL),
  • Figure 23D SPARCii 3 (DL),
  • Figure 23E Scrambled(DL) and
  • Figure 23F buffer alone.
  • SPARCiig(DL) gels were degraded in Brij-free buffer for mass spectrometry, as the Brij signal obscured the "2T" form of the peptide. All degraded gels have a clear single peak at the expected "2T" peptide molecular weight, indicating peptides are not being further cleaved by MMP2 after release from the hydrogel networks. An increase of 1 or 2 Da indicates the released peptide is protonated. No clear spectra could be obtained for degraded Qk(DL) gels.
  • Figure 24, is a set of graphs depicting the results of experiments examining the in vitro mass loss (left), swelling ratio (center), and "2T" peptide release from (right) enzymatically responsive hydrogels.
  • Gels were incubated in buffer alone (solid symbol and line) for 24 hours, at which point 10 nM MMP-2 was added (open symbol, dashed line).
  • Figure 24A SPARC n 3 (DL),
  • Figure 24B SPARCii 8 (DL), and
  • Figure 25 depicts the results of experiments examining the in vitro diffusive release of (Figure 25 A) Qk(2T), (Figure 25B) SPARCii 3 (2T), (Figure 25C) SPARCn 8 (2T), and (Figure 25D) Scrambled(2T) out of non-degradable PEG gels.
  • Figure 26 depicts the results of experiments examining the effect of peptide concentration on tube formation in cultures treated with native or 2T forms of Qk (Figure 26A), SPARCn 3 ( Figure 26B), or SPARCiis ( Figure 26C).
  • Figure 26A depicts the results of experiments examining the effect of peptide concentration on tube formation in cultures treated with native or 2T forms of Qk ( Figure 26A), SPARCn 3 ( Figure 26B), or SPARCiis ( Figure 26C).
  • n 6; error bars represent SEM.
  • Figure 27 depicts the results of experiments examining tube formation induced upon treatment with varying amounts of degraded hydrogel.
  • Figure 27A Tube formation images (at 1/7, 000 th of a gel per well).
  • Figure 27B - Figure 27D Dose response curves for degraded hydrogel products, in terms of fraction of a gel per well,
  • Figure 27B Qk, triangles,
  • Figure 27C SPARCn 3 , diamonds and
  • Figure 27D SPARCng, circles.
  • Figure 28 is a graph depicting tube formation dose response curves for degraded hydrogel products, in terms of "2T" peptide dose per well.
  • the scrambled peptide releasing gels did not affect tube formation at any concentration.
  • n 9; error bars represent SEM.
  • compositions and methods for stimuli-responsive release of a therapeutic agent are at least partially based upon the discovery that therapeutic agents can be incorporated into a hydrogel matrix, where the therapeutic agents can be induced to be released through the cleavage of a cleavage domain within the matrix.
  • a composition comprises a prodrug comprising at least one cleavage domain and at least one therapeutic domain.
  • cleavage of the cleavage domain is dependent upon the localized environment at a treatment site.
  • cleavage is dependent upon the expression and/or activity of a matrix metalloproteinase (MMP), whose expression and/or activity are known to be enhanced in tumors, ischemic tissue, and regions in need of vascularization.
  • MMP matrix metalloproteinase
  • a method to promote angiogenesis or vascularization comprises the stimuli-induced release of pro-angiogenic therapeutic agents from an implanted polymerized matrix.
  • the method may be used to promote vascularization of a tissue engineered construct in vivo.
  • the method may be used to promote vascularization of a tissue engineered construct ex vivo.
  • a method to treat a tumor comprises the stimuli- induced release of anti-tumor therapeutic agents, including for example a
  • a method to treat inflammation comprises the stimuli-induced release of an anti-inflammatory agent from an implanted matrix at a treatment site.
  • polymerization of a hydrogel matrix is initiated via administration of UV light, which, in some embodiments, results in the thiol-ene polymerization of the matrix.
  • the matrix comprises PEG functionalized with terminal norbornene groups, which upon administration of UV- light, binds to thiol-containing groups, which thereby allows for easy and effective incorporation of prodrugs into the matrix.
  • the composition comprises a matrix that is polymerized ex vivo, which is later implanted at a treatment site.
  • the composition comprises a solution of synthetic monomers and prodrugs, which can be injected at treatment site and polymerized in vivo through the application of UV light. Definitions
  • an element means one element or more than one element.
  • abnormal when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal” (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.
  • biocompatible refers to any material, which, when implanted in a mammal, does not provoke an adverse response in the mammal.
  • a biocompatible material when introduced into an individual, is not toxic or injurious to that individual, nor does it induce immunological rejection of the material in the mammal.
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
  • a disorder in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
  • a disease or disorder is "alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.
  • an “effective amount” or “therapeutically effective amount” of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered.
  • An “effective amount” of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound.
  • the term "gel” refers to a three-dimensional polymeric structure that itself is insoluble in a particular liquid but which is capable of absorbing and retaining large quantities of the liquid to form a stable, often soft and pliable, but always to one degree or another shape-retentive, structure.
  • the gel is referred to as a hydrogel.
  • the term “gel” will be used throughout this application to refer both to polymeric structures that have absorbed a liquid other than water and to polymeric structures that have absorbed water, it being readily apparent to those skilled in the art from the context whether the polymeric structure is simply a "gel” or a "hydrogel.”
  • “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous.
  • the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • an "instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of a composition, peptide, method, and the like in a kit for stimuli-induced release of a therapeutic agent, as recited herein.
  • the instructional material can describe one or more methods of providing stimuli-induced release.
  • the instructional material of the kit can, for example, be affixed to a container which contains the identified composition or precursors thereof or be shipped together with a container which contains the composition or precursors thereof. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the composition be used cooperatively by the recipient.
  • peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • polymer refers to a molecule composed of repeating structural units typically connected by covalent chemical bonds.
  • polymer is also meant to include the terms copolymer and oligomers.
  • polymerization refers to at least one reaction that consumes at least one functional group in a monomeric molecule (or monomer), oligomeric molecule (or oligomer) or polymeric molecule (or polymer), to create at least one chemical linkage between at least two distinct molecules (e.g., intermolecular bond), at least one chemical linkage within the same molecule (e.g., intramolecular bond), or any combination thereof.
  • a polymerization reaction may consume between about 0% and about 100% of the at least one functional group available in the system. In one embodiment, polymerization of at least one functional group results in about 100% consumption of the at least one functional group. In another embodiment, polymerization of at least one functional group results in less than about 100% consumption of the at least one functional group.
  • the term "patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof, whether in vitro or in situ, amenable to the methods described herein.
  • the patient, subject or individual is a mammal, non- limiting examples of which include a primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, and the like, that is in need of tissue vascularization.
  • the subject is a human being. In such
  • the subject is often referred to as an "individual” or a “patient.”
  • the terms “individual” and “patient” do not denote any particular age
  • a site in need of vascularization refers to any site or region within a subject which, for any reason, is in need vascularization or angiogenesis.
  • the site is a region of ischemic tissue.
  • a “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.
  • “treating a disease or disorder” means reducing the frequency with which a symptom of the disease or disorder is experienced by a patient. Disease and disorder are used interchangeably herein.
  • terapéuticaally effective amount refers to an amount that is sufficient or effective to prevent or treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a disease or condition, including alleviating symptoms of such diseases.
  • Ranges throughout this disclosure, various aspects can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • compositions and methods for stimuli-responsive treatment are compositions and methods for stimuli-responsive treatment.
  • the compositions and methods provide for the stimuli-responsive release of a therapeutic agent, wherein the release is dependent upon the local environment of a treatment site.
  • release of the therapeutic agent is dependent upon enzymatic activity present at the treatment site, where the enzymatic activity is associated with the need for treatment.
  • the compositions and methods provide for the controlled, local, and stimuli-responsive release of a therapeutic agent that can be used to treat any type of disease or disorder. That is, the particular therapeutic agent, and the stimuli-responsive release of the therapeutic agent, can be altered depending upon the type of disease being treated and the need at the treatment site.
  • compositions and method for stimuli-responsive release of pro-angiogenic therapeutic agents are provided.
  • the compositions and methods can be used for the controlled and local delivery of pro-angiogenic therapeutic agents for the treatment of diseases and disorders including, but not limited to cardiac ischemia, coronary heart failure, peripheral vascular ischemia, peripheral arterial disease, ischemic bowel disease, bone fracture healing, and diabetic ulcers.
  • the controlled and local delivery of pro- angiogenic therapeutic agents is used to promote the vascularization of engineered tissue.
  • the compositions and methods could also be used to enhance vascularization of transplanted cells and tissues, increasing viability of the tissue and the therapeutic efficacy of the treatment.
  • tissue engineering and regenerative medicine strategies of tissues could benefit from the angiogenic therapy provided by the compositions and methods described herein, to promote host vascularization, because simple diffusion is insufficient to deliver nutrients and remove waste from these constructs.
  • a composition for stimuli-responsive treatment comprises a plurality of synthetic monomers and a plurality of prodrugs.
  • the prodrugs are cleaved to release a therapeutic agent from a polymerized matrix when the local environment of the composition dictates that release is needed.
  • the therapeutic agent is a therapeutic peptide.
  • the prodrug comprises a stimuli-responsive releasing element.
  • the prodrug comprises a stimuli-responsive cleavable domain, which when cleaved releases the therapeutic agent.
  • the prodrug comprises a therapeutic peptide coupled to a cleavable peptide sequence at its amino terminus and a cleavable peptide sequence at its carboxy terminus.
  • the ratio of prodrug to synthetic monomer in the composition is greater than about 1 : 1. In one embodiment, the ratio of prodrug to synthetic monomer in the composition is greater than about 2: 1. In one embodiment, the ratio of prodrug to synthetic monomer in the composition is greater than about 4: 1. In one embodiment, the ratio of prodrug to synthetic monomer in the composition is greater than about 8: 1. In one embodiment, the ratio of prodrug to synthetic monomer in the composition is greater than about 16: 1.
  • the released therapeutic agent may comprise a peptide, protein, nucleic acid, small molecule, and combinations thereof.
  • the matrix comprises a prodrug comprising at least one therapeutic domain and at least one cleavable domain.
  • the peptide comprises a therapeutic domain positioned between two cleavable domains.
  • the therapeutic domain of the prodrug may comprise a peptide, protein, nucleic acid small molecule, and combinations thereof.
  • the cleavable domain of the prodrug may comprise a peptide, protein, nucleic acid small molecule, and combinations thereof.
  • the prodrug is a peptide.
  • the at least one cleavable domain of the prodrug is a peptide, while the at least one therapeutic domain is a small molecule drug.
  • the cleavable domain is cleaved by a particular enzyme, whose activity at a given treatment site regulates the release of the therapeutic agent.
  • the expression, activity, or both of the enzyme is increased at the time and location where the therapeutic agent is needed.
  • the composition is a hydrogel comprising polymerized matrix.
  • the composition is a solution comprising matrix materials or precursors, including for example non-polymerized synthetic monomers and prodrugs.
  • the composition comprises poly(ethylene glycol)
  • the composition comprises PEG monomers.
  • the composition comprises a polymerized matrix comprising PEG.
  • the PEG functionalized with terminal norbornene groups, which allows for incorporation of the prodrug into a matrix through the use of thiol-containing groups.
  • one or more arms of PEG is linked or conjugated to norbornene groups, thereby allowing for the linkage of the PEG- norbornene to a thiol-containing group of the prodrug.
  • the composition comprises a matrix comprising PEG bound or tethered to a prodrug, wherein the prodrug comprises at least one therapeutic domain and at least one cleavable domain.
  • the at least one cleavable domain is an amino acid sequence cleaved by a MMP, which is upregulated at a treatment site.
  • the at least one cleavable domain comprises the amino acid sequence IPESLRAG (SEQ ID NO: 1), which when cleaved forms the fragments IPES (SEQ ID NO: 2) and LRAG (SEQ ID NO: 3).
  • the therapeutic domain comprises a pro-angiogenic amino acid sequence.
  • the pro-angiogenic amino acid sequence may be selected from the group consisting of Qk (KLTWQELYQLKYKGI; SEQ ID NO : 4), SPARC m
  • TEGTK GHKLHLDY SEQ ID NO: 5
  • SPARCns K GHK; SEQ ID NO: 6
  • the prodrug comprises a therapeutic domain positioned between two cleavable domains. For example, upon cleavage of the cleavable domains, a therapeutic agent is released from the matrix. Therefore, in certain embodiments
  • the released therapeutic agent comprises the therapeutic domain, as well as a fragment of a first cleavable domain located at a first terminal end of the therapeutic agent and a fragment of the second cleavable domain located at a second terminal end of the therapeutic agent.
  • the released therapeutic agent is a released therapeutic peptide which comprises SEQ ID NO: 3 at the N-terminus.
  • the released therapeutic peptide comprises SEQ ID NO: 2 at the C- terminus.
  • the released therapeutic peptide is referred to herein as the "two-tailed" or "2T" version.
  • the compositions and methods described herein are partly based upon the discovery that the 2T version of pro- angiogenic peptides retain their therapeutic activity, and thus can be incorporated into the stimuli-responsive composition.
  • the therapeutic agent may be released from the matrix, but still bound to matrix materials (e.g. PEG), and still retain bioactivity.
  • Matrix e.g. PEG
  • compositions and methods of the application employ a matrix.
  • the matrix materials are formed into a 3 -dimensional scaffold.
  • the scaffold can contain one or more matrix layers.
  • the scaffold can contain at least two matrix layers, at least three matrix layers, at least four matrix layers, at least five matrix layers, or more.
  • Matrix materials of various embodiments are biocompatible materials.
  • the matrix is biodegradable.
  • the matrix is degraded through the cleavage of cleavage domains located throughout the matrix.
  • the matrix can be fabricated into structural supports, where the geometry of the structure (e.g., shape, size, porosity, micro- or macro-channels) is tailored to the application.
  • Suitable matrix materials are discussed in, for example, Ma and Elisseeff, ed. (2005) Scaffolding in Tissue Engineering, CRC, ISBN 1574445219; Saltzman (2004) Tissue Engineering: Engineering Principles for the Design of Replacement Organs and Tissues, Oxford ISBN 019514130X.
  • At least two matrix materials are used to fabricate a tissue module described herein.
  • the at least two matrix materials can be homogenously mixed throughout the scaffold, heterologously mixed throughout the scaffold, or separated into different matrix layers of the scaffold.
  • Matrices can be produced from proteins (e.g. extracellular matrix proteins such as fibrin, collagen, and fibronectin), polymers (e.g., polyvinylpyrrolidone), polysaccharides (e.g. alginate), hyaluronic acid, or analogs, mixtures, combinations, and derivatives of the above.
  • proteins e.g. extracellular matrix proteins such as fibrin, collagen, and fibronectin
  • polymers e.g., polyvinylpyrrolidone
  • polysaccharides e.g. alginate
  • hyaluronic acid e.g., hyaluronic acid, or analogs, mixtures, combinations, and derivatives of the above.
  • the matrix can be formed of synthetic polymers.
  • synthetic polymers include, but are not limited to, poly(ethylene) glycol, bioerodible polymers (e.g., poly(lactide), poly(glycolic acid), poly(lactide-co-glycolide), poly(caprolactone), polyester (e.g., poly-(L-lactic acid), polyanhydride, polyglactin, polyglycolic acid), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates), polyphosphazene, degradable polyurethanes, non- erodible polymers (e.g., polyacrylates, ethylene-vinyl acetate polymers and other acyl substituted cellulose acetates and derivatives thereof), non-erodible polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, polyvinyl pyrrolidone,
  • polyvinylimidazole chlorosulphonated polyolifms
  • polyethylene oxide polyvinyl alcohol (e.g., polyvinyl alcohol sponge), synthetic marine adhesive proteins, teflon®, nylon, or analogs, mixtures, combinations (e.g., polyethylene oxide-polypropylene glycol block copolymer; poly(D,L-lactide-co-glycolide) fiber matrix), and derivatives of the above.
  • the matrix can be formed of naturally occurring polymers or natively derived polymers.
  • polymers include, but are not limited to, agarose, alginate (e.g., calcium alginate gel), fibrin, fibrinogen, fibronectin, collagen (e.g., a collagen gel), gelatin, hyaluronic acid, chitin, and other suitable polymers and biopolymers, or analogs, mixtures, combinations, and derivatives of the above.
  • the matrix can be formed from a mixture of naturally occurring biopolymers and synthetic polymers.
  • the matrix can comprise a composite matrix material comprising at least two components described above.
  • a composite matrix material can comprise at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more, components.
  • the plurality of components can be homogenously mixed throughout the scaffold, heterologously mixed throughout the scaffold, or separated into different matrix layers of the scaffold, or a combination thereof.
  • one or more matrix materials are modified so as to increase biodegradability.
  • PCL is a biodegradable polyester by hydrolysis of its ester linkages in physiological conditions, and can be further modified with ring opening polymerization to increase its biodegradability.
  • the matrix comprises a plurality of PEG monomers, including for example a plurality of PEG macromers.
  • any suitable PEG macromer may be incorporated into the matrix.
  • the particular type of PEG macromer utilized may be dependent upon the particular application of the matrix/scaffold and the desired properties of the matrix/scaffold.
  • the matrix comprises an 8-arm lOkDa PEG macromer.
  • alternative forms of PEG including forms of differing size or structure, can be utilized in the matrix.
  • the PEG macromer is a multi-arm macromer comprising 4, 8, 16, or more arms.
  • the PEG macromer is functionalized with terminal norbornene groups, which in certain instances allow for linking of the PEG macromer to a thiol-containing prodrug.
  • one or more arms of PEG is linked or conjugated to norbornene groups, thereby allowing for the linkage of the PEG-norbornene to a thiol-containing group of the prodrug.
  • the norbornene functionalized PEG macromers can be linked to a peptide using thiol- norbornene chemistry, making use of thiol-containing amino acids. Therefore, the norbonene functionalized PEG macromers allows for easy incorporation of a peptide into the matrix.
  • the matrix is not limited to this particular method of matrix crosslinking. Rather, any type of crosslinking chemistry can be used in formation of the matrix.
  • the matrix comprises a prodrug linked to the PEG macromer.
  • the matrix comprises PEG macromers that are crosslinked together via the incorporation of a prodrug between two PEG macromers.
  • the prodrug of the matrix is degradable or cleavable, which promotes the degradation of the matrix.
  • the cleavage of the prodrug to release a therapeutic agent is dependent upon a given stimulus, for example, the activity of a specific enzyme.
  • the prodrug comprises at least one therapeutic domain and at least one cleavable domain. In certain embodiments, cleavage of the at least one cleavable domain, releases a therapeutic agent from the matrix.
  • the at least one cleavable domain is an amino acid sequence which is cleaved by a MMP.
  • the cleavage domain is an MMP-sensitive cleavage domain.
  • the MMP-sensitive cleavage domain may be cleaved by any known MMP, including but not limited to MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP 8, MMP9, MMP10, MMP11, MMP 12, MMP13, MMP 14, MMP15, MMP 16, MMP 17, MMP 18, MMP 19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, MMP27, MMP28, and the like.
  • the at least one cleavable domain comprises the amino acid sequence IPESLRAG (SEQ ID NO: 1), which when cleaved forms the fragments IPES (SEQ ID NO: 2) and LRAG (SEQ ID NO: 3).
  • the cleavage domain comprising SEQ ID NO: 1 is optimized for cleavage by MMP 14, but is also susceptible to cleavage by MMP1, MMP2, MMP3, MMP7, and MMP9.
  • the composition of encompasses a cleavable domain that is cleaved by a specific stimulus.
  • the composition is not limited to MMP-sensitive cleavage domains. Rather, in certain embodiments, the cleavage domain is a substrate of a known enzyme with lytic activity.
  • the cleavage domain is cleaved via the activity of cathepsins.
  • the at least one therapeutic domain of the prodrug may comprise any bioactive molecule which demonstrates therapeutic activity, including but not limited to, a protein, isolated nucleic acid, antibody, small molecule, isolated peptide, and conjugates thereof.
  • the therapeutic domain comprises a pro- angiogenic peptide.
  • the amino acid sequence of the pro-angiogenic peptide may be selected from the group consisting of Qk (KLTWQELYQLKYKGI; SEQ ID NO: 4), SPARCns (TLEGTKKGHKLHLDY; SEQ ID NO: 5), and SPARCns (KKGHK; SEQ ID NO: 6).
  • the prodrug comprises a therapeutic domain positioned between two cleavable domains. In one embodiment, the prodrug comprises one or more therapeutic domains positioned between two cleavable domains. That is, in certain embodiments, the prodrug comprises one or more repeats of the therapeutic domain, where all of the repeats are positioned between two cleavable domains. In another embodiment, the prodrug comprises alternating repeats of the cleavable domain and therapeutic domain.
  • a therapeutic agent upon cleavage of the cleavable domains, a therapeutic agent is released from the matrix. Therefore, in certain embodiments, the released therapeutic agent comprises the at least one therapeutic domain, as well as a fragment of a first cleavable domain located at the first terminal end of the therapeutic agent and a fragment of the second cleavable domain located at the second end of the therapeutic agent.
  • the released therapeutic agent is a released therapeutic peptide, where the released therapeutic peptide comprises SEQ ID NO: 3 at the N-terminus. In one embodiment, the released therapeutic peptide comprises SEQ ID NO: 2 at the C-terminus.
  • the prodrug of the matrix is a peptide comprising the amino acid sequence of IPESLRAGKLTWQELYQLKYKGIPESLRAG (SEQ ID NO: 7), such that the released therapeutic peptide comprises the amino acid sequence of LRAGKLT WQEL YQLKYKGIPE S (Qk(2T); SEQ ID NO: 8).
  • the prodrug of the matrix is a peptide comprising the amino acid sequence of IPESLRAGTLEGTK GHKLHLDYIPESLRAG (SEQ ID NO: 9), such that the released therapeutic peptide comprises the amino acid sequence of LRAGTLEGTK GHKLHLDYIPES (SPARC n 3 (2T); SEQ ID NO: 10).
  • the prodrug of the matrix is a peptide comprising the amino acid sequence of IPESLRAGKKGHKIPESLRAG (SEQ ID NO: 1 1), such that the released therapeutic peptide comprises the amino acid sequence of LRAGKKGHKIPES (SPARC i i 8 (2T); SEQ ID NO: 12).
  • the prodrug of the composition comprises terminal cysteine residues, which in certain instances, allow for linkage to the norbornene functionalized PEG.
  • the prodrug comprises the amino acid sequence of CIPESLRAGKLTWQELYQLKYKGIPESLRAGC (SEQ ID NO: 13), CIPESLRAGTLEGTKKGHKLHLDYIPESLRAGC (SEQ ID NO: 14), or CIPESLRAGKKGHKIPESLRAGC (SEQ ID NO: 15).
  • the matrix can be formed by any method known in the art.
  • the matrix is constructed from the polymerization of monomer, polymer, or macromer matrix materials.
  • the matrix is formed through the polymerization of PEG macromers.
  • the peptides described herein crosslink PEG macromers.
  • polymerization of the matrix is achieved using "click" chemistry.
  • polymerization of the matrix is performed using thiol-norbornene chemistry.
  • the PEG macromer is functionalized with terminal norbornene groups.
  • the thiol-norbornene chemistry used to construct the matrix allows for rapid formation under cytocompatible conditions.
  • initiation of the polymerization of the matrix occurs via the administration of UV light.
  • the polymerization of the matrix makes use of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator.
  • LAP lithium phenyl-2,4,6-trimethylbenzoylphosphinate
  • the matrix is polymerized ex vivo, and is later implanted into a target treatment site.
  • a solution comprising matrix materials is administered to a target treatment site
  • the composition is a hydrogel comprising the matrix, as described herein.
  • Hydrogels can generally absorb a great deal of fluid and, at equilibrium, typically are composed of 60-90% fluid and only 10-30% polymer. In a preferred embodiment, the water content of hydrogel is about 70-80%). Hydrogels are particularly useful due to the inherent biocompatibility of the polymeric network (Hill- West, et al.,1994, Proc. Natl. Acad. Sci. USA 91 :5967-5971). Hydrogel biocompatibility can be attributed to hydrophilicity and ability to imbibe large amounts of biological fluids (Brannon-Peppas. Preparation and Characterization of Cross-linked Hydrophilic
  • hydrogels can be prepared by crosslinking hydrophilic biopolymers or synthetic polymers.
  • construction of hydrogels comprises the polymerization and/or copolymerization of monomers, macromers, polymers and the like.
  • hydrogel formation comprises copolymerization of two or more types of biopolymers and/or synthetic polymers.
  • hydrogels formed from physical or chemical crosslinking of hydrophilic biopolymers include but are not limited to, hyaluronans, chitosans, alginates, collagen, dextran, pectin, carrageenan, polylysine, gelatin or agarose, (see.: W. E. Hennink and C. F. van Nostrum, 2002, Adv. Drug Del. Rev. 54, 13-36 and A. S.
  • hydrogels based on chemical or physical crosslinking synthetic polymers include but are not limited to (meth)acrylate-oligolactide-PEO- oligolactide-(meth)acrylate, poly(ethylene glycol) (PEO; PEG), poly(propylene glycol) (PPO), PEO-PPO-PEO copolymers (Pluronics), poly(phosphazene), poly(methacrylates), poly(N-vinylpyrrolidone), PL(G)A-PEO-PL(G)A copolymers, poly(ethylene imine), PEG monoethylether methacrylate, etc.
  • Hydrogels closely resemble the natural living extracellular matrix (Ratner and Hoffman. Synthetic Hydrogels for Biomedical Applications in Hydrogels for Medical and Related Applications, Andrade, Ed. 1976, American Chemical Society: Washington, D.C., pp 1-36). Hydrogels can also be made degradable in vivo by incorporating PLA,
  • hydrogels can be modified with fibronectin, laminin, vitronectin, or, for example, RGD for surface modification, which can promote cell adhesion and proliferation (Heungsoo Shin, 2003, Biomaterials 24:4353-4364; Hwang et al, 2006 Tissue Eng. 12:2695-706).
  • RGD for surface modification
  • altering molecular weights, block structures, degradable linkages, and cross-linking modes can influence strength, elasticity, and degradation properties of the instant hydrogels (Nguyen and West, 2002, Biomaterials 23(22):4307-14; Ifkovits and Burkick, 2007, Tissue Eng. 13(10):2369-85).
  • Hydrogels can also be modified with functional groups for covalently attaching a variety of proteins (e.g., collagen) or compounds such as therapeutic agents.
  • Therapeutic agents which can be linked to the matrix include, but are not limited to, analgesics, anesthetics, antifungals, antibiotics, anti-inflammatories, anthelmintics, antidotes, antiemetics, antihistamines, antihypertensives, antimalarials, antimicrobials, antipsychotics, antipyretics, antiseptics, antiarthritics, antituberculotics, antitussives, antivirals, cardioactive drugs, cathartics, chemotherapeutic agents, a colored or fluorescent imaging agent, corticoids (such as steroids), antidepressants, depressants, diagnostic aids, diuretics, enzymes, expectorants, hormones, hypnotics, minerals, nutritional supplements, parasympathomimetics, potassium supplements, radiation sensitizers, a radioisotope,
  • the therapeutic agent can also be other small organic molecules, naturally isolated entities or their analogs, organometallic agents, chelated metals or metal salts, peptide -based drugs, or peptidic or non-peptidic receptor targeting or binding agents. It is contemplated that linkage of the therapeutic agent to the matrix can be via a protease sensitive linker or other biodegradable linkage.
  • Molecules which can be incorporated into the hydrogel matrix include, but are not limited to, vitamins and other nutritional supplements; glycoproteins (e.g., collagen); fibronectin; peptides and proteins; carbohydrates (both simple and/or complex); proteoglycans; antigens; oligonucleotides (sense and/or antisense DNA and/or R A); antibodies (for example, to infectious agents, tumors, drugs or hormones); and gene therapy reagents.
  • the hydrogel comprises the prodrug described elsewhere herein.
  • the hydrogel comprises crosslinked PEG macromer, wherein the PEG macromers are linked to prodrug comprising at least one cleavage domain and at least one therapeutic domain.
  • PEG hydrogels have been used to deliver therapeutic molecules such as peptides and proteins, and offer numerous advantages as controlled release systems (Slaughter et al, 2009, Adv Mater. 21(32-33):3307-29).
  • PEG hydrogels are highly hydrophilic, inert, and biocompatible. Additionally, due to their synthetic nature, PEG hydrogels have highly tunable degradation profiles and mechanical properties (Lin et al., 2009, Pharm Res. 26(3):631-43). Moreover, PEG hydrogels can be formed using a number of synthetic schemes that are compatible with peptide incorporation.
  • norbornene-functionalized PEG PEGN
  • PEGN norbornene-functionalized PEG
  • thiol-containing crosslinkers to from hydrogel networks via step-growth photopolymerizations
  • This polymerization strategy is cytocompatible, produces homogeneous PEG-peptide networks, and allows for facile incorporation of peptides that include cysteine (thiol R-group) amino acids (Fairbanks et al, 2009, Adv Mater. 21(48):5005-10).
  • Thiol-ene based PEG hydrogels can also be rendered
  • Enzymatically-responsive peptide sequences have been used to control the temporal availability of the cell adhesion peptide RGD within non-degradable PEG hydrogels (Salinas et al, 2008, Biomaterials. 29(15):2370-7), and to provide responsive release of tethered vascular endothelial growth factor (VEGF) (Phelps et al, 2010, P Natl Acad Sci USA 107(8):3323-8).
  • VEGF vascular endothelial growth factor
  • enzymatically-responsive VEGF delivery results in greater vascular reperfusion compared to bolus injection of VEGF, presumably due to extended therapeutic protein delivery (Phelps et al, 2010, P Natl Acad Sci USA 107(8):3323-8).
  • one or more multifunctional cross-linking agents may be utilized as reactive moieties that covalently link biopolymers or synthetic polymers.
  • Such bifunctional cross-linking agents may include glutaraldehyde, epoxides (e.g., bis-oxiranes), oxidized dextran, p-azidobenzoyl hydrazide, N-[a.- maleimidoacetoxy]succinimide ester, p-azidophenyl glyoxal monohydrate, bis-[P-(4- azidosalicylamido)ethyl]disulfide, bis[sulfosuccinimidyl]suberate, dithiobis[succinimidyl proprionate, disuccinimidyl suberate, l-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), N-hydroxysuccinimi
  • polyacrylated materials such as ethoxylated (20) trimethylpropane triacrylate
  • ethoxylated (20) trimethylpropane triacrylate may be used as a nonspecific photo-activated cross-linking agent.
  • Components of an exemplary reaction mixture would include a thermoreversible hydrogel held at 39°C, polyacrylate monomers, such as ethoxylated (20) trimethylpropane triacrylate, a photo-initiator, such as eosin Y, catalytic agents, such as l-vinyl-2-pyrrolidinone, and triethanolamine.
  • the hydrogel comprises a UV sensitive curing agent which initiates hydrogel polymerization.
  • a hydrogel comprises the photoinitiator 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone.
  • polymerization is induced by 4-(2-hydroxyethoxy)phenyl-(2-hydroxy- 2-propyl)ketone upon application of UV light.
  • UV sensitive curing agents include 2-hydroxy-2-methyl-l-phenylpropan-2-one, 4-(2-hydroxyethoxy)phenyl (2-hydroxy-2-phenyl-2-hydroxy-2-propyl)ketone, 2,2-dimethoxy-2-phenyl-acetophenone 1 -[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2 -methyl- 1 -propane- 1 -one, 1 - hydroxycyclohexylphenyl ketone, trimethyl benzoyl diphenyl phosphine oxide and mixtures thereof.
  • the polymerization of the hydrogel is induced by the lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator.
  • LAP lithium phenyl-2,4,6-trimethylbenzoylphosphinate
  • the stabilized cross-linked hydrogel matrix may be further stabilized and enhanced through the addition of one or more enhancing agents.
  • enhancing agent or “stabilizing agent” is intended any compound added to the hydrogel matrix, in addition to the high molecular weight components, that enhances the hydrogel matrix by providing further stability or functional advantages.
  • Suitable enhancing agents which are admixed with the high molecular weight components and dispersed within the hydrogel matrix, include many of the additives described earlier in connection with the thermoreversible matrix discussed above.
  • the enhancing agent can include any compound, especially polar compounds, that, when incorporated into the cross-linked hydrogel matrix, enhance the hydrogel matrix by providing further stability or functional advantages.
  • Preferred enhancing agents for use with the stabilized cross-linked hydrogel matrix include polar amino acids, amino acid analogues, amino acid derivatives, intact collagen, and divalent cation chelators, such as ethylenediaminetetraacetic acid (EDTA) or salts thereof.
  • Polar amino acids are intended to include tyrosine, cysteine, serine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, lysine, and histidine.
  • the preferred polar amino acids are L-cysteine, L-glutamic acid, L-lysine, and L-arginine. Suitable concentrations of each particular preferred enhancing agent are the same as noted above in connection with the thermoreversible hydrogel matrix.
  • Polar amino acids, EDTA, and mixtures thereof, are preferred enhancing agents.
  • the enhancing agents can be added to the matrix composition before or during the crosslinking of the high molecular weight components.
  • enhancing agents are particularly important in the stabilized cross- linked bioactive hydrogel matrix because of the inherent properties they promote within the matrix.
  • the hydrogel matrix exhibits an intrinsic bioactivity that will become more evident through the additional embodiments described hereinafter. It is believed the intrinsic bioactivity is a function of the unique stereochemistry of the cross-linked macromolecules in the presence of the enhancing and strengthening polar amino acids, as well as other enhancing agents.
  • a method comprising manufacture of a hydrogel comprising the prodrug described herein is provided.
  • Manufacture of a hydrogel may comprise any known methods or techniques known in the art.
  • the method comprises forming a solution comprising the prodrug and optionally, one or more suitable biopolymers or synthetic polymers.
  • the manufacture of the hydrogel comprises administering of a crosslinker to a hydrogel solution.
  • the hydrogel is fabricated by
  • the hydrogel can have a structure, e.g., including one or more of a film, pad, cylinder, tube, micro thin film, a micro pad, a micro thin fiber, a nanosphere or a microsphere.
  • the hydrogel may be formed to be of any size or geometry as needed by its application.
  • the hydrogel may be formed with a pre-determined size and geometry, or alternatively may be cut into a desired size and geometry after formation.
  • the hydrogel may be formed to comprise any suitable amount of the prodrug.
  • the concentration of the prodrug within the hydrogel may be altered by increasing or decreasing the amount of prodrug added to a hydrogel solution.
  • the desired amount of the prodrug comprised in the hydrogel depends on the activity of the released therapeutic agent, the disease state of the patient, and the drug delivery characteristics of the released therapeutic agent.
  • the hydrogel is formulated to provide sustained release of the therapeutic agent.
  • the hydrogel provides sustained release of the therapeutic agent for at least 1 hour, 1 day, 3 days, 1 week, 2 weeks, 1 month, 3 months, 6 months, 1 year, 5 years, 10 years, or more.
  • the release rate of the therapeutic agent is dependent on the type of cleavage domain and the stimuli which cleave the cleavage domain, as described elsewhere herein.
  • the release rate of the therapeutic agent is dependent on the type of cleavage domain and the stimuli which cleave the cleavage domain, as described elsewhere herein.
  • the release of the therapeutic agent is dependent on the type of cleavage domain and the stimuli which cle
  • characteristics of the therapeutic agent are dependent on the expression, activity, or both of MMPs within the target tissue.
  • the hydrogel is modified to improve its functionality.
  • the hydrogel may be coated with any number of compounds in order enhance its biocompatibility, reduce its immunogenicity, enhance stability, enhance degradation, and/or enhance drug delivery.
  • the hydrogel may be shaped into any number of desirable configurations to satisfy any number of overall system, geometry or space restrictions.
  • the matrix or hydrogel may be shaped to conform to the dimensions and shapes of the whole or a part of the tissue.
  • the hydrogel may be shaped in different sizes and shapes to conform to the organs of differently sized patients.
  • the matrix or hydrogel may also be shaped in other fashions to accommodate the special needs of the patient.
  • the hydrogel comprises one or more cells.
  • the hydrogel comprises one or more cells embedded within the hydrogel.
  • the hydrogel comprises one or more cells on the hydrogel surface.
  • the hydrogel is seeded with one or more populations of cells.
  • the cells may be autologous, where the cell populations are derived from the subject's own tissue, or allogenic, where the cell populations are derived from another subject within the same species as the patient.
  • the cells may also be xenogenic, where the different cell populations are derived form a mammalian species that is different from the subject.
  • the cells may be derived from organs of mammals such as humans, monkeys, dogs, cats, mice, rats, cows, horses, pigs, goats and sheep.
  • Cells may be isolated from a number of sources, including, for example, biopsies from living subjects and whole-organ recover from cadavers.
  • the isolated cells are preferably autologous cells, obtained by biopsy from the subject intended to be the recipient. For example, a biopsy of skeletal muscle from the arm, forearm, or lower extremities, or smooth muscle from the area treated with local anesthetic with a small amount of lidocaine injected subcutaneously, and expanded in culture.
  • the biopsy may be obtained using a biopsy needle, a rapid action needle which makes the procedure quick and simple.
  • Cells may be isolated using techniques known to those skilled in the art.
  • the tissue or organ may be disaggregated mechanically and/or treated with digestive enzymes and/or chelating agents that weaken the connections between neighboring cells making it possible to disperse the tissue into a suspension of individual cells without appreciable cell breakage.
  • Preferred cell types include, but are not limited to, mesenchymal cells, especially smooth or skeletal muscle cells, endothelial cells, endothelial progenitor cells, myocytes (muscle stem cells), fibroblasts, chondrocytes, adipocytes, fibromyoblasts, and ectodermal cells, including ductile and skin cells, hepotocytes, Islet cells, cells present in the intestine, and other parenchymal cells, osteoblasts and other cells forming bone or cartilage. In some cases, it may also be desirable to include nerve cells.
  • mesenchymal cells especially smooth or skeletal muscle cells, endothelial cells, endothelial progenitor cells, myocytes (muscle stem cells), fibroblasts, chondrocytes, adipocytes, fibromyoblasts, and ectodermal cells, including ductile and skin cells, hepotocytes, Islet cells, cells present in the intestine, and other
  • stem cells including for example, embryonic stem cells, adult stem cells, mesenchymal stem cells, hematopoietic stem cells, induced pluripotent stem cells, cord blood derived stem cells, and the like.
  • Isolated cells may be cultured in vitro to increase the number of cells available for coating the biocompatible scaffold.
  • the use of allogenic cells, and more preferably autologous cells, is preferred to prevent tissue rejection.
  • the subject may be treated with immunosuppressive agents such as, cyclosporin or FK506, to reduce the likelihood of rejection.
  • immunosuppressive agents such as, cyclosporin or FK506, to reduce the likelihood of rejection.
  • chimeric cells, or cells from a transgenic animal may be coated onto the biocompatible scaffold.
  • Isolated cells may be normal or genetically engineered to provide additional or normal function.
  • Methods for genetically engineering cells with retroviral vectors, polyethylene glycol, or other methods known to those skilled in the art may be used. These include using expression vectors which transport and express nucleic acid molecules in the cells. (See Goeddel; Gene Expression Technology: Methods in
  • DNA or R A may be introduced into cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 3nd Edition, Cold Spring Harbor Laboratory press (2012)), and other laboratory textbooks.
  • Seeding of cells onto the matrix or hydrogel may be performed according to standard methods. For example, the seeding of cells onto polymeric substrates for use in tissue repair has been reported (see, e.g., Atala, A. et al, J. Urol. 148(2 Pt 2): 658-62 (1992); Atala, A., et al. J. Urol. 150 (2 Pt 2): 608-12 (1993)).
  • Cells grown in culture may be trypsinized to separate the cells, and the separated cells may be seeded on the hydrogel.
  • cells obtained from cell culture may be lifted from a culture plate as a cell layer, and the cell layer may be directly seeded onto the hydrogel without prior separation of the cells.
  • a seeded hydrogel is formed by adding one or more isolated cells to a hydrogel precursor solution, such that polymerization of the precursor solution results in a polymerized hydrogel with the isolated cell or cells encapsulated or embedded within the hydrogel.
  • the hydrogel may be coated with one or more cell adhesion-enhancing agents. These agents include but are not limited collagen, laminin, and fibronectin.
  • the hydrogel may also contain cells cultured on the hydrogel to form a target tissue substitute.
  • the target tissue that may be formed using the scaffold may be an arterial blood vessel, wherein an array of microfibers is arranged to mimic the configuration of elastin in the medial layer of an arterial blood vessel.
  • the composition includes an isolated peptide.
  • the composition comprises a prodrug, comprising at least one cleavage domain and at least one therapeutic domain.
  • the at least one cleavage domain, the at least one therapeutic domain, or both comprise a peptide.
  • the prodrug is an isolated peptide comprising at least one cleavage domain and at least one therapeutic domain.
  • cleavage of the peptide at the one or more cleavage domains releases a therapeutic peptide comprising the therapeutic domain from the composition.
  • the at least one cleavable domain comprises the amino acid sequence IPESLRAG (SEQ ID NO: 1), which when cleaved forms the fragments IPES (SEQ ID NO: 2) and LRAG (SEQ ID NO: 3).
  • the therapeutic domain comprises a pro-angiogenic amino acid sequence.
  • the pro-angiogenic amino acid sequence may be selected from the group consisting of Qk (KLT WQEL YQLKYKGI ; SEQ ID NO: 4), SPARCm
  • the released therapeutic peptide upon cleavage of the cleavable domains, a therapeutic peptide is released from the matrix. Therefore, in certain embodiments, the released therapeutic peptide comprises at least therapeutic domain, as well as a fragment of a first cleavable domain located at the N-terminus of the therapeutic peptide and a fragment of the second cleavable domain located at the C- terminus of the therapeutic peptide.
  • the released therapeutic peptide comprises SEQ ID NO: 3 at the N-terminus.
  • the released therapeutic peptide comprises SEQ ID NO: 2 at the C-terminus.
  • the peptide of the matrix comprises the amino acid sequence of IPESLRAGKLTWQELYQLKYKGIPESLRAG (SEQ ID NO: 7), such that the released therapeutic peptide comprises the amino acid sequence of
  • the peptide of the matrix comprises the amino acid sequence of IPESLRAGTLEGTK GHKLHLDYIPESLRAG (SEQ ID NO: 9), such that the released therapeutic peptide comprises the amino acid sequence of
  • the peptide of the matrix comprises the amino acid sequence of IPESLRAGK GHKIPESLRAG (SEQ ID NO: 11), such that the released therapeutic peptide comprises the amino acid sequence of LRAGK GHKIPES
  • the peptide of the matrix comprises terminal cysteine residues, which in certain instances, allow for linkage to the norbornene functionalized PEG.
  • the peptide of the matrix comprises the amino acid sequence of CIPESLRAGKLTWQELYQLKYKGIPESLRAGC (SEQ ID NO: 13), CIPESLRAGTLEGTK GHKLHLDYIPESLRAGC (SEQ ID NO: 14), or CIPESLRAGKKGHKIPESLRAGC (SEQ ID NO: 15).
  • the peptide also encompasses peptide variants.
  • the variants of the peptides may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the peptide is an alternative splice variant of the peptide, (iv) fragments of the peptides and/or (v) one in which the peptide is fused with another peptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His- tag) or for detection (for example, Sv5 epitope tag).
  • the fragments include peptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.
  • the "similarity" between two peptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide to a sequence of a second peptide.
  • Variants are defined to include peptide sequences different from the original sequence, preferably different from the original sequence in less than 40% of residues per segment of interest, more preferably different from the original sequence in less than 25% of residues per segment of interest, more preferably different by less than 10%> of residues per segment of interest, most preferably different from the original protein sequence in just a few residues per segment of interest and at the same time sufficiently homologous to the original sequence to preserve the functionality of the original sequence and/or the ability to bind to ubiquitin or to a ubiquitylated protein.
  • compositions described herein includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence.
  • the degree of identity between two peptides is determined using computer algorithms and methods that are widely known for the persons skilled in the art.
  • the identity between two amino acid sequences is preferably determined by using the BLASTP algorithm [BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J. Mol. Biol. 215: 403-410 (1990)].
  • the peptides can be post-translationally modified.
  • post- translational modifications include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc.
  • Some modifications or processing events require introduction of additional biological machinery.
  • processing events such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489) to a standard translation reaction.
  • the peptides may include unnatural amino acids formed by post- translational modification or by introducing unnatural amino acids during translation.
  • a variety of approaches are available for introducing unnatural amino acids during protein translation.
  • special tRNAs such as tRNAs which have suppressor properties, suppressor tRNAs, have been used in the process of site-directed non-native amino acid replacement (SNAAR).
  • SNAAR site-directed non-native amino acid replacement
  • a unique codon is required on the mRNA and the suppressor tRNA, acting to target a non-native amino acid to a unique site during the protein synthesis (described in WO90/05785).
  • the suppressor tRNA must not be recognizable by the aminoacyl tRNA synthetases present in the protein translation system.
  • a non-native amino acid can be formed after the tRNA molecule is aminoacylated using chemical reactions which specifically modify the native amino acid and do not significantly alter the functional activity of the aminoacylated tRNA. These reactions are referred to as post-aminoacylation modifications.
  • post-aminoacylation modifications For example, the epsilon-amino group of the lysine linked to its cognate tRNA (tRNA L Ys), could be modified with an amine specific photoaffinity label.
  • a peptide may be conjugated with other molecules, such as proteins, to prepare fusion proteins. This may be accomplished, for example, by the synthesis of N- terminal or C-terminal fusion proteins provided that the resulting fusion protein retains the functionality of the peptide.
  • the composition comprises cyclic derivatives of the peptides or chimeric proteins.
  • Cyclization may allow the peptide or chimeric protein to assume a more favorable conformation for association with other molecules. Cyclization may be achieved using techniques known in the art. For example, disulfide bonds may be formed between two appropriately spaced components having free sulfhydryl groups, or an amide bond may be formed between an amino group of one component and a carboxyl group of another component. Cyclization may also be achieved using an azobenzene- containing amino acid as described by Ulysse, L., et al, J. Am. Chem. Soc. 1995, 117, 8466-8467.
  • the components that form the bonds may be side chains of amino acids, non- amino acid components or a combination of the two.
  • cyclic peptides may comprise a beta-turn in the right position. Beta-turns may be introduced into the peptides by adding the amino acids Pro-Gly at the right position.
  • a more flexible peptide may be prepared by introducing cysteines at the right and left position of the peptide and forming a disulphide bridge between the two cysteines.
  • the two cysteines are arranged so as not to deform the beta-sheet and turn.
  • the peptide is more flexible as a result of the length of the disulfide linkage and the smaller number of hydrogen bonds in the beta-sheet portion.
  • the relative flexibility of a cyclic peptide can be determined by molecular dynamics simulations.
  • the subject peptide therapeutic agents are peptidomimetics of the peptide.
  • Peptidomimetics are compounds based on, or derived from, peptides and proteins.
  • the peptidomimetics typically can be obtained by structural modification of a known peptide inhibitor sequence using unnatural amino acids, conformational restraints, isosteric replacement, and the like.
  • peptidomimetics constitute the continuum of structural space between peptides and non- peptide synthetic structures; peptidomimetics may be useful, therefore, in delineating pharmacophores and in helping to translate peptides into nonpeptide compounds with the activity of the parent peptides.
  • mimetopes of the subject peptide can be provided.
  • Such peptidomimetics can have such attributes as being non-hydrolyzable (e.g., increased stability against proteases or other physiological conditions which degrade the corresponding peptide), increased specificity and/or potency, and increased cell permeability for intracellular localization of the
  • peptide analogs can be generated using, for example, benzodiazepines (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, pl23), C-7 mimics (Huffman et al. in Peptides: Chemistry and Biologyy, G. R. Marshall ed., ESCOM Publisher: Leiden,
  • compositions specifically contemplate the use of conformationally restrained mimics of peptide secondary structure.
  • Numerous surrogates have been developed for the amide bond of peptides. Frequently exploited surrogates for the amide bond include the following groups (i) trans-olefms, (ii) fluoroalkene, (iii) methyleneamino, (iv) phosphonamides, and (v) sulfonamides.
  • mimetopes include, but are not limited to, protein-based compounds, carbohydrate-based compounds, lipid-based compounds, nucleic acid-based compounds, natural organic compounds, synthetically derived organic compounds, anti-idiotypic antibodies and/or catalytic antibodies, or fragments thereof.
  • a mimetope can be obtained by, for example, screening libraries of natural and synthetic compounds for compounds capable of binding to the peptide inhibitor.
  • a mimetope can also be obtained, for example, from libraries of natural and synthetic compounds, in particular, chemical or combinatorial libraries (i.e., libraries of compounds that differ in sequence or size but that have the same building blocks).
  • a mimetope can also be obtained by, for example, rational drug design.
  • the three-dimensional structure of a compound can be analyzed by, for example, nuclear magnetic resonance (NMR) or x-ray crystallography.
  • NMR nuclear magnetic resonance
  • the three-dimensional structure can then be used to predict structures of potential mimetopes by, for example, computer modelling, the predicted mimetope structures can then be produced by, for example, chemical synthesis, recombinant DNA technology, or by isolating a mimetope from a natural source (e.g., plants, animals, bacteria and fungi).
  • a natural source e.g., plants, animals, bacteria and fungi
  • a peptide, or chimeric protein may be synthesized by conventional techniques.
  • the peptide inhibitors or chimeric proteins may be synthesized by chemical synthesis using solid phase peptide synthesis. These methods employ either solid or solution phase synthesis methods (see for example, J. M. Stewart, and J. D.
  • Peptides may be developed using a biological expression system. The use of these systems allows the production of large libraries of random peptide sequences and the screening of these libraries for peptide sequences that bind to particular proteins.
  • Libraries may be produced by cloning synthetic DNA that encodes random peptide sequences into appropriate expression vectors, (see Christian et al 1992, J. Mol. Biol.
  • Libraries may also be constructed by concurrent synthesis of overlapping peptides (see U.S. Pat. No. 4,708,871).
  • the peptides and chimeric proteins may be converted into pharmaceutical salts by reacting with inorganic acids such as hydrochloric acid, sulfuric acid,
  • hydrobromic acid phosphoric acid, etc.
  • organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, and toluenesulfonic acids.
  • Described herein is a method for treating a disease or disorder comprising a stimuli-induced delivery of a therapeutic agent.
  • the methods are not limited to the treatment or prevention of any particular disease or disorder. Rather, the methods encompass the treatment of any disease or disorder treatable the activity of a particular therapeutic agent.
  • the compositions described elsewhere herein may be designed and constructed to comprise any prodrug comprising at least one therapeutic domain and at least one cleavage domain.
  • the cleavage of the cleavage domain induces the release of a therapeutic agent.
  • the cleavage of the cleavage domain is dependent upon a particular stimulus associated with the disease or disorder to be treated.
  • the stimulus is present specifically at the treatment site and at the time in which the therapeutic agent is needed.
  • a method for stimuli-induced treatments is provided herein.
  • a method for promoting vascularization at a treatment site comprising the stimuli-induced release of a pro-angiogenic agent.
  • a method for treating a disease or disorder associated with reduced vascularization or blood flow is provided.
  • Exemplary disorders treatable by the method includes, but is not limited to, cardiac ischemia, coronary heart failure, peripheral vascular ischemia, peripheral arterial disease, ischemic bowel disease, bone fracture healing, and diabetic ulcers.
  • the controlled and local delivery of pro- angiogenic therapeutic agents by way of the method is used to promote the
  • the method could also be used to enhance vascularization of transplanted cells and tissues, increasing viability of the tissue and the therapeutic efficacy of the treatment.
  • the method can be used to promote angiogenesis or vascularization of a tissue engineered construct ex vivo prior to implantation of the construct.
  • the method may be used to promote angiogenesis or vascularization of a tissue engineered construct in vivo during or after implantation.
  • tissue engineering and regenerative medicine strategies of tissues could benefit from the angiogenic therapy provided by the methods described herein, to promote host vascularization, because simple diffusion is insufficient to deliver nutrients and remove waste from these constructs.
  • a method for treating cancer in a subject comprising the stimuli-induced release of an anti-cancer or anti-tumor agent.
  • MMPs or other cleavage enzymes are upregulated in the tumor microenvironment.
  • the method provides for the stimuli-induced release of an anti-cancer agent from a matrix in order to treat cancer.
  • Exemplary cancers treatable by way of the method include, but are not limited to carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas.
  • sarcomas e.g., sarcomas, carcinomas, and melanomas.
  • adults tumors/cancers and pediatric tumors/cancers are also included.
  • a method for treating inflammation in a subject comprising the stimuli-reduced release of an anti-inflammatory agent.
  • the method may be used to treat acute or chronic inflammation, an autoimmune disease, or other inflammatory disorders.
  • the method comprises forming a matrix described herein.
  • the method comprises polymerizing matrix materials, including for example a plurality of synthetic monomers and a plurality of prodrugs, into a matrix.
  • the polymerization of the matrix comprises the use of thiol-norbornene chemistry to crosslink the matrix.
  • polymerization of the matrix is induced by administration of UV-light. Matrix formation may be performed in an ex vivo or in vivo environment. The relative amount or concentration of the prodrug may be varied depending on the particular application, treatment site, disease severity, and the like.
  • the ratio of prodrug to synthetic monomer in the composition is greater than about 1 : 1. In one embodiment, the ratio of prodrug to synthetic monomer in the composition is greater than about 2: 1. In one embodiment, the ratio of prodrug to synthetic monomer in the composition is greater than about 4: 1. In one embodiment, the ratio of prodrug to synthetic monomer in the composition is greater than about 8: 1. In one embodiment, the ratio of prodrug to synthetic monomer in the composition is greater than about 16: 1.
  • the method comprises administering a hydrogel comprising a polymerized matrix to a treatment site. In one embodiment, the method comprises forming a suitable hydrogel ex vivo and then administering the hydrogel to a desired location in a patient or subject in need. In another embodiment, the method comprises administering a solution comprising hydrogel matrix material or precursors to a location within the patient or subject, followed by inducing the polymerization of the hydrogel in vivo.
  • the hydrogel composition is administered to an ischemic treatment site, or a site in need of angiogenesis or vascularization.
  • the expression, activity, or both, of MMPs are enhanced, which cleave the cleavage domain and release the therapeutic agent from the hydrogel.
  • the therapeutic agent is released only in regions of ischemia and is thereby able to promote angiogenesis at the treatment site.
  • the hydrogel may also be embedded with one or more additional factors that are released from the hydrogel upon either hydrogel degradation or prodrug cleavage.
  • the hydrogel is embedded with cells, including for example, endothelial cells, progenitor cells, and the like, that would aid promoting angiogenesis at the site.
  • the hydrogel may be administered to a patient or subject in need in a wide variety of ways. Modes of administration include intravenous, intravascular,
  • the methods described herein result in localized administration of the therapeutic agent comprising hydrogel to the site or sites in need of treatment. Any administration may be a single application of a composition or multiple applications. Administrations may be to single site or to more than one site in the individual to be treated. Multiple administrations may occur essentially at the same time or separated in time.
  • compositions provided herein are principally directed to compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.
  • compositions and methods are further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the compositions and methods should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
  • DIEA diisopropylethylamme
  • NMP NMP was used as the activator base
  • Qk(DL), SPARCn 3 (DL), and SPARC 3X (DL) where 0.5 M diisopropylcarbodiimide (DIC, Chem-Impex International) in DMF was used as the activator and 1 M hydroxybenzotriazole (HOBt, Advanced ChemTech) in DMF used as the activator base.
  • Peptides were cleaved from resin in a cleavage cocktail composed of 92.5 vol% trifluoroacetic acid (TFA, Alfa Aesar) and 2.5 vol% each triisopropylsilane (Alfa Aesar), 3,6-Dioxa-l,8-octanedithiol (Alfa Aesar), and distilled, deionized water (ddH 2 0) for 2-3 hours.
  • TFA trifluoroacetic acid
  • Alfa Aesar triisopropylsilane
  • Alfa Aesar 3,6-Dioxa-l,8-octanedithiol
  • ddH 2 0 distilled, deionized water
  • Peptides were dialyzed in ddH 2 0 (1-500 or 1000 MWCO tubing, Spectrum Laboratories) overnight and collected by lyophilization. Final peptide concentrations were assessed via absorbance at 205 nm using an Evolution 300 UV/Vis detector (Thermo Scientific) (Anthis et al, 2013, Protein Sci. 22(6):851-8). Solid peptide and stock peptide solutions in PBS were stored at -20 °C until use. Norbornene functionalization of polyfethylene glycol)
  • HUVECs were cultured in Endothelial Growth Media 2 (EGM-2;
  • EBM-2 Endothelial Basal Media-2
  • EGM-2 SingleQuots EGM-2 SingleQuots
  • Cells were maintained at 37 °C with 5% C0 2 , split 1 :4, and used before passage 10.
  • Reduced growth factor Matrigel (BD Biosciences) was thawed overnight on ice at 4 °C, diluted to 7.8 mg/mL with control media, and polymerized via incubation at 37 °C for 30 minutes (150 ⁇ per well of a 48-well plate).
  • Cells (1.2xl0 5 cells/mL) were suspended in either control media alone, or control media containing peptide drugs as well as negative and positive controls.
  • Cell solutions 200 ⁇ /well were placed on the polymerized Matrigel. Cells were incubated at 37 °C for 8 hours before fluorescent imaging (0.5 ⁇ .
  • HUVECs were suspended in control media at 1.0-2.0xl0 4 cells/mL. 0.5 mL of the cell solution was seeded in each well of a 24-well plate. To achieve uniform cell adhesion, cells were allowed to adhere at room temperature for 1 hour before being transferred to the incubator (Ryan JA. 2012 Corning guide for identifying and correcting common cell growth problems: Corning Incorporated Life Sciences). Sixteen (16) hours later, cells were washed twice with PBS and treated with 0.5 mL of either control media with or without peptide drugs or controls. A preliminary dose screening study was conducted to identify concentration at which each "N" peptide induced proliferation, and that concentration was used for both the "N" and "2T" treatments.
  • Peptide property predictions Peptide structure was predicted using the Pep fold 1.5 de novo structure prediction server, and displayed in cartoon mode color-coded by group (Thevenet et al, 2012, Nucleic Acids Res. 40(W1):W288-W93).
  • Peptide characteristics were calculated using classifications from Lehninger Principles of Biochemistry (Lehninger et al., 2000, Lehninger principles of biochemistry. 3rd ed. New York: Worth Publishers), as well as the Kyte-Doolittle (Kyte et al, 1982, J Mol Biol. 157(l):105-32) and Hopp-Woods (Hopp et al, 1981, P Natl Acad Sci-Biol.
  • Peptide-crosslinked PEG hydrogels were formed via thiol-ene photopolymerizations.
  • Cysteine-terminated peptides and 10 kDa PEGN (4-arm or 8-arm) were dissolved in PBS, with the exception of Qk(DL), which was dissolved in a 50/50 mixture of ddH 2 0 and acetonitrile, in a 1 : 1 thiol :ene ratio to form a precursor solution containing 10 wt% PEG and 0.05 wt% lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, synthesized as previously described (Fairbanks et al, 2009, Biomaterials.
  • LiAP lithium phenyl-2,4,6-trimethylbenzoylphosphinate
  • Hydrogels were swollen in buffer (50 mM Tricine (Acros Organics), 50 mM NaCl, 10 mM CaCl 2 , 50 ⁇ ZnCl 2 (Alfa Aesar), and 0.05 wt% Brij35 (Alfa Aesar) in ddH 2 0, pH 7.4) at 37 °C for 24 hours to achieve equilibrium swelling before characterization. Hydrogel images were collected using a Canon EOS Rebel T2i digital camera. Mass swelling ratio was determined by measuring equilibrium swelling and dry gel mass after lyophilization. The efficiency of peptide incorporation into the hydrogels was measured by collecting the buffer solution and quantifying the amount of peptide released into buffer via absorbance at 205 nm.
  • buffer 50 mM Tricine (Acros Organics), 50 mM NaCl, 10 mM CaCl 2 , 50 ⁇ ZnCl 2 (Alfa Aesar), and 0.05 wt% Brij35 (Alfa Ae
  • the amount of peptide incorporated into the gel but not fully crosslinked was quantified by incubating gels in 1 mL of 0.35 mg/mL Ellman's reagent in PBS for 30 minutes, then measuring absorbance at 405 nm on a Tecan infiniteM200 microplate reader, and comparing to a standard curve generated using peptide alone (Ellman GL. 1959, Arch Biochem Biophys. 82(l):70-7).
  • hydrogels were stored in buffer 1 mL buffer (10 mM CaC12, 50 mM NaCl, 50 ⁇ ZnC12 (Alfa Aesar), 50 mM Tricine (Acros Organics), and 0.05 wt% Brij35 (Alfa Aesar) in ddH20, pH 7.4) at 37 °C for 24 hours, at which point solutions were changed to either fresh buffer, or buffer containing 10 nM recombinant human MMP2 (PeproTech). As MMP2 inactivates over time (Patterson et al., 2010, Biomaterials. 31(30):7836-45), MMP2 solutions were collected and replaced with fresh MMP2 every 48 hours.
  • buffer solutions were collected and replaced every 48 hours as well.
  • hydrogel bathing solutions were collected and gels were removed from solution and wet mass measured. Gels were then frozen, lyophilized, and the dry mass measured. Hydrogel swelling ratio was calculated by dividing hydrogel wet mass by dry mass Hydrogel incubation solutions were stored at -80 °C for subsequent peptide release quantification. Gels were inspected daily until complete degradation occurred, for a maximum of 10 days.
  • the amount of "2T" peptide released into solution was quantified by High Performance Liquid Chromatography (HPLC, Shimadzu Prominence) with a Kromasil Eternity CI 8 column (4.6 x 50 mm). Water and acetonitrile containing 1% TFA were used as the mobile phases and samples were run using gradients from 5% to 95% acetonitrile at 0.5 mL/minute. Peptide elution was monitored using a UVTVis detector (SPA-20AV, Shimadzu Prominance) at 214 nm, and concentrations were determined by integrating peak area and comparing to standard curves generated using the "2T" form of the peptides. The amount of peptide incorporated into the hydrogels was determined by fully degrading the gels in 1 M NaOH prior to HPLC analysis. Formation and hydrogel subcutaneous implantation
  • mice 6-8 week old female BALB/c mice were obtained from Taconic (Hudson, NY). Mice were given 1.6 mg/mL acetaminophen in water from 1 day pre- to 3 days post-surgery as analgesia. Anesthesia (60 mg/kg ketamine and 4 mg/kg xylazine) was administered via intraperitoneal injection. Two subcutaneous pockets were formed on each side of the mouse dorsal flank, and one hydrogel implant placed in each pocket with the open circular face of the reactor in contact with the underlying tissue. After various time points, mice were sacrificed by C0 2 inhalation, and the reactors dissected from surrounding tissue for analysis. Hemoglobin quantification
  • Hydrogel material and invading tissue was removed from explanted reactors and weighed. Implants were manually homogenized and sonicated in 1 mL Drabkin's reagent (RICAA chemical). Samples were then centrifuged at 14,000 g for 20 minutes and the supernatant filtered through 0.45 ⁇ polyvinylidene fluoride (PVDF) filters (PerkinElmer), to remove particulates. The hemoglobin concentration in each sample was determined by measure absorbance at 540 nm, and compared to hemoglobin standards (Alfa Aesar) (Barcelos et al., 2004, Inflamm Res, 53: 576-584).
  • VDF polyvinylidene fluoride
  • Example 1 Hydrogels designed to provide sustained, stimuli-responsive release of pro- angiogenic peptides.
  • peptides significantly increased both proliferation and tube formation in both their native form and in the form they would be released from the hydrogel networks: Qk (mimicking VEGF), SPARC 113 , and SPARCng (mimicking the secreted protein acidic and rich in cysteine, SPARC).
  • SPARCns was then incorporated into poly(ethylene glycol) hydrogel networks via MMP-degradable tethers, and enzymatically-responsive hydrogel degradation and peptide release was
  • Poly ethylene glycol was chosen as the basis for this biomaterial as its hydrophilic nature makes PEG resistant to protein adsorption, providing it with its inert, biocompatible nature. Additionally, as PEG hydrogels are synthetic rather than naturally derived, they can be easily modified to control properties such as stiffness and degradation rate. Moreover, hydrogels are commonly used for regenerative medicine applications. While there are a number of chemistries which can be used to crosslink PEG hydrogels, a thiol-norbornene chemistry was chosen. This allows for rapid formation of hydrogels under cytocompatible conditions. This crosslinking occurs via step-growth polymerization, creating homogeneous hydrogel networks, and allows for easy incorporation of peptides through the use of thiol-containing cysteine amino acids.
  • the constructs designed and developed herein are used to deliver low molecular weight peptides that mimic the angiogenic potential of these proteins. This allows higher concentrations of more stable angiogenic factors to be delivered to the ischemic tissue.
  • a stimuli-responsive tether to link the peptides into the hydrogel allows for the extended, controlled release of the peptides to host tissue. This is important as extended delivery of biomolecules has been shown critical in the formation of mature, stable vessels in vivo. Additionally, while the hydrogel provides initial structural support to the infarcted tissue, hydrogel degradation is ultimately desired as even a relatively inert material like PEG can elicit a foreign body response if allowed to remain in the body indefinitely.
  • IPESLRAG SEQ ID NO: 1
  • SEQ ID NO: 2 IPES (SEQ ID NO: 2).
  • SEQ ID NO: 3 IPES (SEQ ID NO: 3) fragment.
  • This sequence has been optimized for cleavage by MMP-14, but is also susceptible to MMPs 1, 2, 3, 7 & 9. It should be noted that the sequence is cleaved in the center; this means that when the angiogenic peptides are released from the hydrogel, residual amino acids will remain on either side of the pro- angiogenic peptides.
  • the use of the tether facilitates peptide delivery in an ischemia-dependent manner.
  • Hydrogels were formed as follows. First, poly (ethylene glycol) (PEG) was functionalized with terminal norbornene groups (PEGN) as previously described (Fairbanks et al, 2009, Adv Mater, 21(48): 5005-5010). PEG was reacted with 4-
  • hydrogels were formed using thiol-ene photopolymerization (Fairbanks et al, 2009, Adv Mater, 21(48): 5005-5010). 10 wt% PEGN was combined with crosslinking peptides in a 1 : 1 thiol :ene ratio to form a precursor solution also containing 0.05 wt% lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) photoinitiator. 40 of hydrogel precursor solution was then injected into a cylindrical mold and exposed to 365 nm UV light for 10 minutes.
  • LAP lithium phenyl-2,4,6- trimethylbenzoylphosphinate
  • Hydrogels were synthesized using these three PEG precursors ( Figure 3) and a non-degradable crosslinking peptide. All hydrogels were produced at 10 wt% PEG, with a 1 : 1 thiokene ratio, and allowed to swell in PBS for 48 hours before mechanical testing. Mechanical testing demonstrated that by increasing the number of arms while keeping arm length constant, the hydrogel stiffness was increased from 1.4 to 9.4 kPa. Further halving the length of each PEG arm further increased the hydrogel stiffness to 17.6 kPa, within the target range for cardiac tissue (Figure 3). The next step in producing these stimuli-responsive hydrogels was to identify peptides for incorporation. Ten pro-angiogenic peptides were identified as being angiogenic in vitro, in vivo, or both (Table 1). These peptides are derived from a variety of sources, including angiogenic proteins and extracellular matrix molecules.
  • Peptides were synthesized in both their "native” (as-published) form, and in their “two-tailed” form (2T) - that is, the form they will be released from the hydrogel in, with the residual LRAG amino acids on the N terminus, and IPES amino acids on the C terminus. A scrambled control was also synthesized. Table 2 depicts several of the angiogenic peptides in their 2T form, as well as in a degradable (containing the MMP- sensitive cleavage sequence at both ends), and in a nondegradable control sequence. Peptides were synthesized in house using standard microwave-assisted solid phase peptide synthesis, and correct synthesis confirmed using mass spectrometry. Figure 4 depicts the MALDI-ToF plot demonstrating the correct synthesis of the 2T version of the QK peptide.
  • Table 1 Ten angiogenic peptides used, in their native form.
  • the peptides used during the present studies comprise a c-terminal glycine residue, which is product of peptide synthesis.
  • Table 2 Angiogenic peptides investigated. Standard amino acid abbreviations are used: Alanine A, Arginine R, Asparagine N, Aspartic Acid D, Cysteine C, Glutamine Q, Glutamic Acid E, Glycine G, Histidine H, Isoleucine I, Leucine L, Lysine K, Methionine M, Phenylalanine F, Proline P, Serine S, Threonine T, Tryptophan W, Tyrosine Y, Valine V. Functionally distinction portions of the peptide are separated by spaces, with the pro- angiogenic portion indicated in italics.
  • (S) indicates serine amino acids used in place of cysteine to prevent tethering of the angiogenic sequence to hydrogel networks.
  • (N) indicates peptides in their "naked” (as-published) form; (2T) indicates peptides containing the residual amino acid "tails" that will be present upon their release from hydrogel networks;
  • (DL) indicates peptides containing the MMP-responsive sequence and
  • (NDL) indicates peptides containing a control non-degradable sequence.
  • the pro-angiogenic efficacy of these peptides was then assessed using two in vitro models of angiogenesis: the human umbilical vein endothelial cell (HUVEC) proliferation and tube formation assay.
  • HUVEC human umbilical vein endothelial cell
  • HUVECs are seeded in basal media (EBM-2) at 10,000 cells/well in 24-well plates and allowed to adhere overnight. Cells are then washed with PBS, and treated with either basal media alone, or basal media
  • VEGF vascular endothelial growth factor
  • chemotherapeutic drug VEGF and the chemotherapeutic drug
  • Figure 5 depicts the results of the proliferation assay for all ten of the angiogenic peptides, in their native form, listed in Table 1 , while Figure 6 and depicts the results for the ten peptides in their 2T form. It is observed that all peptides, except for T7, increase proliferation in the naked form
  • Figure 7 depicts the results of peptides Qk, SPARC 113 , SPARCng, and
  • Pepl2 both in their naked and 2T forms, along with positive and negative controls. Treatment with Qk, SPARC 113 , SPARCng, and Pepl2 in their naked forms caused statistically significant increases in proliferation over control media alone. While slight changes in bioactivity were observed upon inclusion of MMP-responsive peptide remnants, all four peptides significantly increased proliferation in their two-tailed form in at least one concentration investigated.
  • Figure 6 and Figure 7 depict the results of the HUVEC proliferation assay with cells treated with the naked and 2T versions of
  • the peptides were next evaluated in the HUVEC tube formation assay.
  • Reduced growth- factor Matrigel was polymerized in 48-well plates. 2.4 x 10 5 cells/well in control media or media containing various treatments were seeded onto Matrigel. Cells were incubated for 8 hours before imaging at 4x using a Nikon Eclipse Ti inverted light microscope. Fluorescent images were obtained using the live cell stain Calcein AM and quantified using the image analysis program Angioquant.
  • Figure 8 depicts the results of the tube formation assay for all ten of the angiogenic peptides, in their native form, listed in Table 1
  • Figure 9 depicts the results for the ten peptides in their 2T form. It is observed that all peptides, except Ten 2, increase HUVEC tube formation in the naked form ( Figure 8), while only Qk, SPARC 113 , and SPARCng significantly increase HUVEC tube formation in the 2T form ( Figure 9).
  • Figure 10 depicts images of tube formation in cells treated with either the naked or 2T forms of QK or SPARCng.
  • Figure 11 depicts the quantification of tube length in cells treated with either the native or 2T forms of Qk, SPARC 113 , SPARCns, and Pep 12.
  • Figure 10 depicts images of tube formation of HUVECs treated with naked or 2T SPARCng, compared to positive control (VEGF) and negative control (Basal media).
  • Treatment with the positive control VEGF caused the formation of smooth, fully connected honeycomb like tube networks, as compared to the basal media alone, where minimal tube network formation is seen, and the networks are punctuated and disconnected.
  • Treatment with SPARCng in both the naked and 2T form caused the formation of smooth tube networks more similar to that of the positive control than basal media alone.
  • Quantification as depicted in Figure 11, reflected the qualitative observations of the tube networks: the positive control VEGF significantly increased tube length, as did SPARCng in both its naked and two-tailed form. Treatment with the scrambled peptide did not significantly affect tube network formation, while the inhibitor of angiogenesis sulforaphane significantly decreased tube length.
  • hydrogels made from 8- arm PEG were formed with either the MMP-degradable peptide SPARCn 8 (2DL) or the non-degradable SPARCn 8 (2NDL). Gels were then incubated at 37 °C in either PBS alone, or PBS containing 250 ⁇ g/mL collagenase. Hydrogels were removed, frozen at -80 °C, and lyophilized to track hydrogel dry mass over time. As shown in Figure 13 A, when SPARC 118 was tethered to the hydrogel via the non-degradable linker and incubated in either PBS alone or the collagenase solution, no significant change in hydrogel mass was observed.
  • hydrogels formed with SPARCng tethered via the MMP-degradable sequence were stable when incubated in PBS alone. It was only when the hydrogels produced with the MMP-degradable sequence were incubated in the collagenase solution that rapid degradation of the hydrogels was observed, with hydrogels fully degrading in 24 hours. This confirms that enzymatically -responsive hydrogel degradation can be achieved using the MMP-responsive, peptide-releasing sequence. In order to confirm that peptide degradation, the hydrogel-bathing solutions were also collected and stored at -80 °C until peptide quantification by High Performance Liquid Chromatography. As can be seen in Figure 13B, release of the pro- angiogenic peptide SPARCng mirrored hydrogel degradation, with only the
  • SPARCiig(2DL) hydrogels in collagenase solution resulting in release of peptide While full peptide release was not observed, this was not unexpected as only peptide released in its two-tailed form was included in quantification, and it is likely that some peptides remained tethered at one end to the multi-arm PEG macromers upon release.
  • pro-angiogenic peptides can be screened in common angiogenic assays: HUVEC proliferation and tube formation. Based on these assays, it is shown herein that Qk, SPARC 113 , and SPARCng all retain their pro-angiogenic activity in their two-tail form, the form comprising additional amino acid at its N-terminus and C-terminus corresponding to the cleaved portions of the MMP- sensitive cleavable peptide. Further, it is shown that SPARCng can be incorporated into the hydrogel network via the MMP-degradable peptide sequence IPESLRAG (SEQ ID NO: 1), and stimuli-responsive hydrogel degradation and peptide release was achieved. The present data demonstrates that stimuli-responsive delivery of pro-angiogenic peptides can be achieved from PEG hydrogel networks. Experiments are conducted to examine the efficacy of hydrogel released peptides in both in vitro and in vivo models.
  • Example 2 Development of enzymatically-responsive polyfethylene glycol) hydrogels for the delivery of therapeutic peptides
  • hydrogel-based platform technology that controls and sustains peptide drug release via matrix metalloproteinase (MMP) activity.
  • MMP matrix metalloproteinase
  • enzymatically-responsive PEG hydrogels upon treatment with MMP2 showed hydrogels containing Qk, SPARCn 3 , and SPARCng degraded in ⁇ 6.7, ⁇ 6 and ⁇ 1 days, and released ⁇ 5, ⁇ 8 and ⁇ 19% of peptide, respectively.
  • Peptide drug size controlled hydrogel swelling and degradation rate, while hydrophobicity impacted peptide release. While pro-angiogenic peptides were the focus of this study, the design parameters detailed allow for adaptation of hydrogels to control peptide release for a variety of therapeutic applications.
  • VEC tube formation and proliferation assays.
  • Three peptide drugs retained bioactivity in released forms.
  • These and two additional peptides encompassing a range of peptide sizes and hydrophobicities were incorporated into PEG hydrogels via
  • IPESLRAG enzymatically responsive linkers
  • IPESLRAG enzymatically responsive sequence
  • SEQ ID NO: 1 was utilized as it is susceptible to matrix metalloproteinases (MMPs) 1, 2, 3, 7, 9 & 14 (Cantley et al., 2001, Nat Biotechnol. 19(7):661-7), many of which are expressed at increased levels in diseased or regenerating tissues.
  • MMPs matrix metalloproteinases
  • the resulting hydrogels were characterized, and enzymatically-responsive hydrogel degradation and "2T" peptide release upon treatment of gels with MMP2 was investigated.
  • Bioactive peptides were selected from literature to determine the effect of the residual peptide "tails" left on peptides upon enzymatically-responsive hydrogel release. Bioactive peptide selection was restricted to pro-angiogenic peptides to allow for objective comparison of the impact of the "tails” on all peptides using well-established in vitro assays (Auerbach et al, 2003, Clin Chem. 49(l):32-40). Peptides were chosen with a variety of sizes and hydrophobicity (Table 4), to investigate if these characteristics provided predictive power for the effect of "tails" on bioactivity.
  • SmPho KLVPL A (SEQ ID NO : Model of small, hydrophobic peptide
  • VTVEGLEPG SEQ ID Fibrillin 1 and Fibronectin Ill-like domain NO: 20
  • the peptides used for the present studies may have a C-terminal glycine residue, which is product of peptide synthesis. No additional Gly added to C-termini of Combl (N) or Scrambled. No additional He added to C-temini of Qk(2T).
  • Hydrophobic amino acids are G, A, V, L, I, M, F, Y, W
  • SPARC 3x was also inactivated by the "tails", with the "N” form significantly increasing tube length ⁇ 1.5-fold, and the "2T” form resulting in a statistically insignificant ⁇ 1.4-fold increase.
  • KRX-725(N) significantly increased tube length ⁇ 1.2- fold, while KRX-725(2T) resulted in a statistically insignificant ⁇ 1.1 -fold increase.
  • the scrambled peptide did not significantly affect relative tube length ( ⁇ 0.9-fold), indicating that the observed results are due to specific peptide drugs.
  • KRX-725 completely lost bioactivity upon inclusion of the "2T", with KRX-725(N) significantly increasing proliferation ⁇ 26%, and KRX-725(2T) decreasing proliferation ⁇ 3% below that of control media, both at 1 nM.
  • the scrambled peptide did not affect proliferation ( ⁇ 3%), again indicating that the observed results are sequence-specific. Based on these results, three peptides were identified that retained bioactivities in released form from
  • Figure 16 illustrates the predicted peptide structures for all five peptide -releasing sequences investigated, as well as the degradable linker
  • IPESLRAG (SEQ ID NO: 1).
  • the degradable linker was predicted to form an a-helix with - 1.5 turns, a structure maintained in all five of the peptide -releasing sequences. Sequences designed to release large peptides (SPARC 3 x, SPARC 113 , Scrambled, and Qk) all exhibited increased number or length of a-helixes above those contributed by the "DL"s alone ( Figure 16D - Figure 16G), while the smaller peptide drugs (SmPho and SPARC us) had either equal or reduced a-helix length (Figure 16B and Figure 16C).
  • the central region of Qk(DL) and SPARCn 3 (DL) was predicted to have - 1.5 and ⁇ 0.5 turns, respectively.
  • SPARC 3 x(DL) was predicted to have a longer, ⁇ 3.5 -turn C-terminal a- helix, but no central a-helix. Scrambled(DL) similarly had an extended ⁇ 2.5 turn N- terminal a-helix, and both the N- and C- terminal a-helixes on Qk(DL) were also extended to ⁇ 2.5 turns. SmPho(DL) and SPARC 118 (DL) were not predicted to have any additional a-helixes, and SPARC 118 , had a slightly shorter N-termini a-helix.
  • the Qk(DL) peptide initially designed (C IPESLRAG KLTWQELYQLKYKGI PESLRAG C(SEQ ID NO: 13)) was not sufficiently soluble in aqueous solution to allow for incorporation into PEG hydrogels. Therefore, the sequence was modified to enhance solubility by inclusion of four additional hydrophilic Glu (E) amino acids on both ends (Table 3). Even with these additional hydrophilic amino acids, the peptide required the use of a water/acetonitrile co-solvent to form hydrogels. All other (DL) peptides were soluble in buffer at the necessary concentrations for hydrogel formation.
  • Hydrogels described here were constructed from 10%wt 4-arm lOkDa PEG. Macroscopically, the Qk(DL) gels were somewhat opaque, while all other gels were transparent (Figure 17A).
  • SPARCii 8 (DL), SmPho(DL), SPARC 3X (DL), SPARCn 3 (DL), and Scrambled(DL) hydrogels showed singular peaks at the expected "2T" peptide molecular weight, further confirming that the MMP is cleaving the peptides at the expected sites and that peptides are not nonspecifically degraded upon release from the hydrogels (Figure 23).
  • the HUVEC tube formation assay was used to assess the pro-angiogenic potential of degraded hydrogel products and released peptide drugs.
  • Filtered, degraded hydrogel solutions were diluted in media and assessed for bioactivity using the tube formation assay such that the concentration of SPARCn 8 (2T) present was 100 nM, the concentration utilized in the efficacy screening study, equating to ⁇ 1/7, 000 th gel/well.
  • Therapeutic peptides are often delivered via injection (Hardy et al, 2008, Biochem Pharm. 75(4):891-9; Hardy et al, 2007, Peptides. 28(3):691-701), osmotic pumps (Santulli et al, 2009, J of Trans Med. 7:41) or diffusional release of from polymeric particles (Ben-Sasson et al, 2003, Blood. 102(6):2099-107; Failla et al, 2008, Blood. 111(7):3479-88) or gels (Santulli et al, 2009, J of Trans Med. 7:41; Choi et al, 2004, Pharm Res.
  • the swelling ratio of the gels is inversely related to sequence length, with smaller peptides (SPARC ng and SmPho) having larger swelling ratios than gels formed with larger peptides (Qk, SPARCn 3 , Scrambled, and SPARC 3X , Figure 17B).
  • SPARC ng and SmPho smaller peptides
  • Qk, SPARCn 3 Small peptides
  • SPARCn 3 Small peptides
  • SPARC 3X SPARC 3X
  • IPESLRAG (SEQ ID NO: 1) into pro-angiogenic peptides resulted in hydrogels that were degraded by MMP2.
  • the mesh size of these gels varied from 30 ⁇ 4 nm (Qk(DL)) to 63 ⁇ 2 nm (SmPho(DL)) (Zustiak et al, 2010, Biomacromolecules. 11(5): 1348-57), but should have minimal hindrance to diffusion of MMP2 ( ⁇ 2.6 nm) within all gels (Erickson et al, 2009, Biol Proced Online. 11(1):32-51), indicating that the observed differences in degradation rate are not due to diffusional limitations.
  • hydrolytic degradation observed for the SPARCiig(DL) and SmPho(DL) gels is likely a minor contributor to accelerated enzymatic degradation, as there is a substantial difference in timescales ( ⁇ 1 versus ⁇ 10 days) of hydrolytic and enzymatic degradation for SmPho(DL) gels.
  • the hydrogels hydro lyrically degraded due to the presence of ester bonds between PEG and norbornene groups (Roberts et al, 2013, Biomaterials. 34(38):9969-79; Shih et al, 2012, Biomacromolecules. 13(7):2003-12).
  • Modifications to the present platform could employ an alternate chemistry for norbornene functionalization of PEG where esters are replaced with amide bonds, preventing hydrolytic degradation (Roberts et al, 2013, Biomaterials. 34(38):9969-79).
  • Hydrophobicity is strongly related with “2T” release ( Figure 19), implying that once the first "DL” is cleaved, hydrophobic peptides decrease the cleavage rate of the second "DL". Differences in peptide structure may also impact the rate of "DL” cleavage, as the more slowly degrading gels have a larger proportion of a-helices ( Figure 16).
  • the degradation and release data here provide valuable insight for development of similar enzymatically-controlled peptide release systems: large, hydrophilic peptide drugs are predicted to produce gels that slowly degrade and fully release a large fraction of the peptide, while small, hydrophobic peptide drugs are expected to produce gels that degrade slowly and release only a modest amount of peptide drug.
  • linear relationships were found between two measures of peptide size (molecular weight and sequence length) and hydrogel degradation, and between three measures of peptide hydrophobicity (% hydrophobic amino acids, Kyte-Doolittle average, and Hopp-Woods average) and "2T" peptide release, it is likely these underlying peptide drug characteristics control hydrogel behavior.
  • Degraded SPARCn 8 (DL) and SPARCn 3 (DL) hydrogels significantly increased HUVEC tube length 2.8 and 1.7-fold, demonstrating that these hydrogels release bioactive components upon MMP mediated degradation.
  • the degraded Qk(DL) gels significantly increased HUVEC tube length 3.1 -fold control media, despite releasing substantially less peptide in "2T" form than the SPARCn 8 (DL) and SPARCn 3 (DL) hydrogels.
  • degraded Qk(DL) hydrogels induced tube formation at lower "2T" levels (4.5 nM) than those previously investigated in the screening study (100 nM).
  • SPARCii 3 (DL), and Qk(DL) hydrogels release bioactive components upon MMP- mediated degradation.
  • hydrogels investigated here contain - 0.8 ⁇ peptide per 40 gel.
  • SPARCii 3 (DL) and SPARCn 8 (DL) gels and in vitro efficacy at 100 nM one gel should release enough "2T” drug to reach target concentrations in ⁇ 1.5 L of tissue.
  • the decreased “2T” release from the Qk(DL) gels means these gels only release enough "2T” drug to reach 100 nM in ⁇ 0.4 L of tissue.
  • SPARCii 8 (DL), SPARCn 3 (DL), and Qk(DL) gels were all able to induce tube formation at the same gel dilution ratios. This indicates that the all three gels should be able to reach therapeutic levels in similar volumes of target tissue. Only one concentration of degraded gel was assessed for in vitro bioactivity, and it is possible that the enzymatically- responsive gels developed here are bioactive at even lower concentrations and could achieve therapeutically relevant concentrations in even larger volumes of tissue.
  • Degradation time could also be altered by changing the specific degradable substrate used (Patterson et al, 2010, Biomaterials. 31(30):7836-45; Cantley et al, 2001, Nat Biotechnol. 19(7):661-7); however, this would also alter the "tails" left on the peptide, likely affecting bioactivity based on the data observed here.
  • peptide drugs investigated here were restricted to pro-angiogenic peptides; however, controlled release any therapeutic peptide is feasible, including antiinflammatory (Akeson et al, 1996, J Biol Chem. 271(48):30517-23; Schultz et al, 2005, Biomaterials 26(15):2621-30) and chemotherapeutic (Yang et al., 2003, Cancer Res.
  • release of multiple peptide drugs could be achieved by using a mixture of crosslinking peptides during gel formation or, as illustrated here, the differences in release among peptide drugs could be exploited to deliver two drugs over different timeframes using one delivery system.
  • sequential delivery of pro-angiogenic and pro-maturation growth factors improves vessel formation and stability in vivo (Brudno et al, 2013, Biomaterials. 34(36):9201-9).
  • the peptide drug delivered affects the rate of hydrogel degradation and peptide release, achieving well-controlled sequential release of multiple factors is non-trivial.
  • peptides with varying properties were incorporated into PEG hydrogels via the enzymatically-responsive linker IPESLRAG (SEQ ID NO: 1), achieving enzymatically-responsive hydrogel degradation and peptide release.
  • Linear regression analysis indicated peptide drug size controls the rate of hydrogel degradation, while peptide drug hydrophobicity affects the amount of peptide fully released from the PEG macromers.
  • This represents a novel method to controllably deliver peptide drugs in an enzymatically-responsive manner, with potential applications to aid in development of tissue engineered constructs, or for the treatment of tissue disorders such as ischemia, chronic inflammation, and tumors.
  • SPARC 113 and SPARCiis This illustrates that differences in peptide release are due to differences in rates of cleavage of the degradable linker, and not due to differences in the rate of diffusion of the peptides out of the gel after release from the network.
  • Tube network formation was assessed upon treatment with both the "N" and "2T” forms of Qk, SPARC 113 , and SPARCng in a dose escalation study. Data is shown in Figure 26 - Figure 28, and statistical analyses are summarized in Table 5.
  • Qk(N) significantly increased relative tube length to ⁇ 2.2-, ⁇ 2.3-, ⁇ 2.9-, ⁇ 2.6-, and ⁇ 2.9-fold that of control media at 0.01, 0.1, 1, 10, and 100 ⁇ , respectively
  • Qk(2T) significantly increased relative tube length ⁇ 2.5-, ⁇ 3.1-, ⁇ 2.8-, and ⁇ 2.5-fold that of control media at 0.01, 0.1, 1, and 10 ⁇ , respectively.
  • SPARC 118 significantly increased tube length at every concentration in its "N” form (-1.8-, -2.0-, -1.9-, -2.0-, and ⁇ 2.2-fold at 0.01 , 0.1, 1, 10, and 100 ⁇ , respectively), but only at 0.01 ⁇ in the "2T" form ( ⁇ 1.9 fold, Figure 26).
  • the ability of Qk to induce tube network formation was significantly affected by peptide concentration, but not by the presence of amino acid "tails" ("N” vs "2T”).
  • SPARC 113 was significantly affected both by concentration and by the amino acid "tails", and there was a significant interaction between these two factors.
  • the ability of SPARCns to induce tube network formation was significantly affected by peptide concentration and the presence of the amino acid "tails", but no interaction existed between these two factors (Table 5).
  • Table 5 Results of statistical analysis performed for Figure 26.
  • Results for the three pro- angiogenic peptides were analyzed using a two-way ANOVA to determine the effect of the peptide dose and presence of amino acid "tails" on the peptide's ability to induce tube network formation.
  • Results for the scrambled peptide were analyzed using a one-way ANVOA. ns p>0.05; * p ⁇ 0.05; # p ⁇ 0.0001.
  • SPARC 113 The ability of SPARC 113 to induce tube network formation was significantly affected by the incorporation of "2T", as well as the concentration of peptide used, with a significant interaction occurring between these two factors (Figure 26, Table 5).
  • SPARCii 3 (N) exhibited increasing tube lengths from 0.01 to 1 ⁇ , before decreasing and losing statistical significance as concentration was increased to 100 ⁇ .
  • SPARCii 3 (2T) exhibited an increase in relative tube length from 0.01 to 100 ⁇ , and only reached significance at the highest concentration investigated.
  • SPARC 113 has previously been shown to induce cord formation of bovine aortic endothelial (BAE) cells after 7-10 days of in vitro culture at 125 ⁇ (Lane et al., 1994, J Cell Biol, 125 : 929-943), and to increase vessel formation in the chicken chorioallantoic membrane (CAM) assay at 10-500 ⁇ , with a maximum increase in capillary density occurring at 50 ⁇ (Iruelaarispe et al, 1995, Mol Cel Biol, 6: 327-343). While the concentration ranges and assays differ from those investigated here, the observed trend of increasing then decreasing response with increasing doses is consistent with previous results.
  • BAE bovine aortic endothelial
  • SPARC 118 (2T) was only able to increase relative tube length at the lowest concentration, in contrast to the "N" version of the peptide which significantly increased tube length at every concentration investigated ( Figure 26). While both the presence of the tails and the concentration of peptide used affected tube length, there was no interaction between these two factors (Table 5). However, when tube formation was investigated using SPARCng(DL), degraded from gels, it was observed that SPARCng significantly increased tube length at only the highest gel fraction (1/70 ⁇ ; Figure 27) corresponding to the highest released peptide concentration (10 ⁇ ; Figure 28).
  • the similar peptide SPARC 119 (KGHK rather than K GHK) causes dose-dependent increases in capillary density in the CAM assay as peptide concentration is increased from 10 to 5,000 ⁇ (Iruelaarispe et al, 1995, Mol Cel Biol, 6: 327-343), similar to the relatively steady increase in tube length upon treatment with increasing concentrations of SPARCng(N) observed here.
  • SPARC 119 also induces cord formation of BAE cells when used at 125 ⁇ (Lane et al., 1994, J Cell Biol, 125: 929-943). Increases in capillary density (Iruelaarispe et al, 1995, Mol Cel Biol, 6: 327-343) and the increase in tube length with increasing dose observed here suggest that if higher concentrations of SPARC ng(N) and (2T) been investigated, larger increases in relative tube length may have been observed.
  • the disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety.

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Abstract

L'invention concerne des compositions et des procédés pour un traitement en réponse à des stimuli. Par exemple, dans certains modes de réalisation, les compositions et les procédés permettent une libération en réponse à des stimuli d'un agent thérapeutique, la libération dépendant de l'environnement local d'un site de traitement. Dans un mode de réalisation, la composition comprend un promédicament comprenant au moins un domaine thérapeutique et au moins un domaine de clivage, de telle sorte qu'un clivage induit par des stimuli libère un agent thérapeutique.
PCT/US2014/049774 2013-08-05 2014-08-05 Compositions et procédés pour la libération en réponse à des stimuli d'un agent thérapeutique WO2015021044A1 (fr)

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WO2021112772A1 (fr) * 2019-12-06 2021-06-10 Nanyang Technological University Hydrogels anti-inflammatoires sensibles à une inflammation

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Publication number Priority date Publication date Assignee Title
WO2021112772A1 (fr) * 2019-12-06 2021-06-10 Nanyang Technological University Hydrogels anti-inflammatoires sensibles à une inflammation
CN115038465A (zh) * 2019-12-06 2022-09-09 南洋理工大学 炎症反应性抗炎水凝胶
EP4069310A4 (fr) * 2019-12-06 2023-12-13 Nanyang Technological University Hydrogels anti-inflammatoires sensibles à une inflammation

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