WO2002017880A2 - Materiaux hydrogels produisant du monoxyde d'azote - Google Patents

Materiaux hydrogels produisant du monoxyde d'azote Download PDF

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WO2002017880A2
WO2002017880A2 PCT/US2001/027414 US0127414W WO0217880A2 WO 2002017880 A2 WO2002017880 A2 WO 2002017880A2 US 0127414 W US0127414 W US 0127414W WO 0217880 A2 WO0217880 A2 WO 0217880A2
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macromer
hydrogels
composition
peg
region
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PCT/US2001/027414
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WO2002017880A3 (fr
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Jennifer L. Hill-West
Kristyn Simcha Masters
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Rice University
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Priority claimed from US09/653,406 external-priority patent/US7279176B1/en
Application filed by Rice University filed Critical Rice University
Priority to US10/129,418 priority Critical patent/US7052711B2/en
Priority to EP01968448A priority patent/EP1315476A2/fr
Priority to AU2001288694A priority patent/AU2001288694A1/en
Publication of WO2002017880A2 publication Critical patent/WO2002017880A2/fr
Publication of WO2002017880A3 publication Critical patent/WO2002017880A3/fr
Priority to US11/281,242 priority patent/US7651697B2/en

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    • 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
    • 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
    • A61K47/58—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 by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to polymerizable hydrogel materials that produce physiologically relevant amounts of nitric oxide (NO).
  • Endothelial cells normally present as a monolayer in the intimal layer of the arterial wall, are believed to play an important role in the regulation of smooth muscle cell (SMC) proliferation in vivo. Endothelial cells are seriously disrupted by most forms of vascular injury, including that caused by percutaneous transluminal coronary angioplasty and similar procedures. Approximately 35-50% of patients treated by percutaneous transluminal coronary angioplasty experience clinically significant renarrowing of the artery, or restenosis, within six months of the initial treatment.
  • SMC smooth muscle cell
  • Restenosis is due, at least in part, to migration and proliferation of smooth muscle cells in the arterial wall along with increases in secretion of matrix proteins to form an obstructive neointimal layer within the arterial wall. Similar issues limit the performance of vascular grafts.
  • the processes that regulate arterial wound healing following vascular injury, such as that caused by angioplasty, are as yet poorly understood, but are believed to involve a complex cascade of blood and vessel wall-derived factors. Numerous factors that stimulate intimal thickening and restenosis have been identified through administration of exogenous proteins, genetic alteration of cells, or through the blockade of certain signals using antibodies or other specific growth factor inhibitors.
  • Endothelial cells produce a number of substances known to down-regulate smooth muscle cell proliferation, including heparin sulfate, prostacyclin (PG12), and NO.
  • NO is an endothelium-derived target molecule useful for the prevention of restenosis because, in addition to limiting the proliferation of smooth muscle cells (Garg et al., (1989) J. Clin. Invest, 83:1774-7), NO reduces platelet aggregation (de Graaf et al., (1992) Circulation, 85:2284-90; Radomski et al., (1987) Br. J. Pharmacol., 92:181-7), increases endothelial cell proliferation (Ziche et al., (1993) Biochem. Biophys. Res.
  • NO cyclic guanosine monophosphate
  • cGMP cyclic guanosine monophosphate
  • the effects of NO can often be mimicked by the administration of cGMP or more stable derivatives of cGMP (Garg et al, (1989) J. Clin. Invest, 83:1774-7).
  • NO has been found to inhibit ribonucleotide reductase, an enzyme that converts ribonucleotides into deoxy ribonucleotides, thus significantly impacting DNA synthesis (Lepoivre et al., (1991) Biochem. Biophys. Res. Comm., 179:442-8; Kwon et al., (1991) J. Exp. Med., 174:761-7), as well as several enzymes involved in cellular respiration (Stuehr et al, (1989) J. Exp. Med., 169:1543-55).
  • NO donor molecules A number of molecules that produce NO under physiological conditions (NO donors) have been identified and evaluated both in vitro and in vivo. NO donor molecules exert biological effects mimicking those of NO and include S-nitrosothiols (Diodati et al, (1993) Thromb. Haem., 70:654-8; Lefer et al., (1993) Circulation, 88:2337-50; DeMeyer et al., (1995) J. Cardiovasc. Pharmacol, 26:272-9), organic nitrates (Ignarro et al., (1981) J. Pharmacol. Exp.
  • L-arginine is often thought of as a NO donor, as L-arginine is a substrate for NO synthase, and thus administration of L- arginine increases endogenous NO production and elicits responses similar to those caused by NO donors in most cases (Cooke et al., (1992) J. Clin. Invest, 90:1168-72).
  • NO/nucleophile complexes has been reported by Smith et al., (1996) J. Med. Chem., 39:1148-56. These materials were capable of releasing NO for as long as five weeks in vitro and were able to limit smooth muscle cell proliferation in culture and to reduce platelet adherence to vascular graft materials in an arterio-venous shunt model. These materials show promise for numerous clinical applications where localized NO production would be desired, such as anti-thrombotic coating materials for catheters, but probably will not be useful for the direct treatment of tissues in vivo as these materials suffer from a number of disadvantages.
  • polymers may be produced as films, powders, or microspheres, but they cannot be formed in situ in direct contact with cells and tissues, thus making it difficult to strictly localize NO treatment to a tissue and potentially causing issues with the retention of the polymer at the site of application.
  • the formulation issues will also make local administration during laparoscopic or catheter-based procedures difficult or impossible.
  • biocompatibility of the base polymer is a serious issue for implantable, NO-releasing polymers, especially those intended for long-term use, as inflammatory and thrombotic responses may develop after the cessation of NO release.
  • approaches that are common today are typically based on simple wound care regimens involving debridement, cleaning, and application of moist dressings (Thomas S, Leigh (1998).
  • WOUND DRESSINGS BIOLOGY AND MANAGEMENT, D. Leaper and K. Harding. New York, NY, Oxford University Press. More advanced dressings such as topical gels containing growth factors have resulted in enhanced healing rates in some clinical studies (Wieman T, Smiell J, Su Y. Efficacy and safety of a topical gel formulation of recombinant human platelet-derived growth factor-BB (beclapermin) in patients with nonhealing diabetic ulcers: a phase III randomized, placebo-controlled, double-blind study. Diabetes Care 1998; 21: 822-827; Wieman TJ and the Beclapermin Gel Studies Group.
  • endothelialization of blood-contacting implants may significantly improve device performance by decreasing thrombogenicity and smooth muscle cell proliferation.
  • NO releasing compounds or compounds modulating NO levels could be administered solely to the site in need of treatment, and in some cases, reduce or eliminate side effects due to systemic administration of the agents, particularly over prolonged time periods.
  • Biocompatible polymeric materials releasing or producing physiological amounts of nitric oxide (NO) for prolonged periods of time are described herein.
  • the biocompatible polymeric materials are applied to sites on or in a patient in need of treatment thereof for disorders such as restenosis, thrombosis, asthma, wound healing, arthritis, penile erectile dysfunction or other conditions where NO plays a significant role.
  • the polymeric materials can be formed into films, coatings, or microparticles for application to medical devices, such as stents, vascular grafts and catheters.
  • the polymeric materials can also be applied directly to biological tissues and can be polymerized in situ.
  • the polymers are formed of macromers, which may include biodegradable regions, and have bound thereto groups that are released in situ to elevate or otherwise modulate NO levels at the site where treatment is needed.
  • the macromers can form a homo or hetero-dispersion or solution, which is polymerized to form a polymeric material, that in the latter case can be a semi-interpenetrating network or interpenetrating network.
  • Compounds to be released can be physically entrapped, covalently or ionically bound to macromer, or actually form a part of the polymeric material.
  • Hydrogels can be formed by ionic and/or covalent crosslinking.
  • Other active agents including therapeutic, prophylactic, or diagnostic agents, can also be included within the polymeric material.
  • Figure 1 is a schematic of the synthesis of S-nitrosocysteine hydrogels (Acryloyl-PEG-Cys-NO).
  • Figure 2 is a schematic of the synthesis of acryloyl-PEG-Lysine 5 NO- nucleophile complex hydrogels.
  • Figure 3 is a schematic of the synthesis of acryloyl-PEG-DETA-NO- nucleophile complex hydrogels.
  • Figure 4 is a graph showing the temporal release (%NO released over time in days) of NO from acryloyl-PEG-Lys 5 -NO hydrogels at pH 7.4 (circles) andpH 3 (squares).
  • Figure 5 is a graph showing the temporal release (%NO released over time in hours) of NO from acryloyl-PEG-DETA-NO hydrogels at pH 7.4 (circles) and pH 2 (squares).
  • Figure 6 is a graph showing the temporal release (%NO released over time in hours) of NO from PEG-Cys-NO hydrogels at pH 7.4 (circles) and pH 2 (squares).
  • Figure 7A is a graph showing the temporal release ( ⁇ mol NO released per gram of polymer over time in hours) of NO from PVA-NO-bFGF hydrogels at pH 7.4, 37°C.
  • Figure 7B is a graph showing the temporal release (% of theoretical bFGF released per gram of gel over time in hours) from
  • PVA-NO-bFGF hydrogels at pH 7.4, 37°C.
  • Figures 8A and 8B are graphs showing that acryloyl-PEG-Lysine-NO hydrogels inhibit the proliferation of smooth muscle cells.
  • Figure 8A % of control cell number, hydrogel formulation.
  • Figure 8B % of control cell number, soluble polymer.
  • Figures 9A and 9B are graphs showing the inhibition of SMC proliferation by NO released from acryloyl-PEG-DETA-NO hydrogels ( Figure 9A) and soluble polymer ( Figure 9B), as a percentage of the control.
  • Figures 10A and 10B are graphs showing inhibition of SMC proliferation by NO released from acryloyl-PEG-Cys-NO hydrogels ( Figure 10A) and soluble polymer ( Figure 10B), as a percentage of controls.
  • Figure 11 is a graph comparing the degree of inhibition of smooth muscle cell growth by NO released from hydrogels: acryloyl-PEG-Lys-NO, acryloyl-PEG-DETA-NO, and acryloyl-PEG-Cys-NO, compared to control hydrogel with NO.
  • the percent inhibition of smooth muscle cell growth is determined by comparing the cell growth for each NO-releasing hydrogel to a control PEG-diacrylate hydrogel.
  • Figure 12 is a graph of endothelial cell proliferation, which was stimulated when cultured in the presence of NO-releasing PEG hydrogels with varying NO release kinetics. PEG-diacrylate hydrogels were used as a control. The last three bars represent a statistical variance of p ⁇ 0.01 compared to control.
  • Figure 13 is a graph of endothelial cell proliferation, which was stimulated when cultured on NO-releasing hydrogels that contained the cell adhesion peptide sequence RGDS. The last bar represents a statistical variance of p ⁇ 0.02 versus either RGDS or DETA-NO hydrogels alone.
  • Figure 14 is a graph of endothelial cell proliferation, which was stimulated when cultured on NO-releasing hydrogels that contained the cell adhesion peptide sequence REDV. The last bar represents a statistical variance of p ⁇ 0.02 versus either REDV or DETA-NO hydrogels alone.
  • Figure 15 is a graph of the proliferation of HDFs cultured in the presence of NO-releasing PVA hydrogels by cell counts. No significant changes in proliferation were observed with exposure to PVA-NO materials.
  • Figure 16A and 16B are graphs of the matrix production by fibroblasts cultured in the presence of NO-releasing PVA hydrogels. 16A shows the release of NO from PVA hydrogels increased the production of collagen by HDFs (p ⁇ 0.01 versus control) while 16B shows only slightly increased total matrix produced per cell (p > 0.05).
  • Figure 17A and 17B are graphs comparing wound area and perimeter over time. At the time of each dressing change, pictures of the wounds were taken to assess wound area (17A) and wound perimeter (17B) using image analysis software.
  • Figure 18 is a graph of the granulation tissue thickness of wounds by examination of histological sections from wounds treated with PVA or PVA- NO hydrogels. A trend of increasing granulation tissue thickness with increasing NO concentration was observed.
  • Figure 19 is a graph reflecting wound collagen synthesis. Histological sections from animals at day 29 revealed that wound collagen synthesis was significantly increased through treatment with NO-releasing hydrogels. The second bar represents a statistical variance of p ⁇ 0.001 versus control.
  • Biocompatible polymeric materials releasing or producing physiological amounts of nitric oxide (NO) and methods of use for the treatment of disorders such as restenosis, thrombosis, asthma, wound healing, arthritis, penile erectile dysfunction, or other conditions where NO plays a significant role, are provided herein.
  • the polymeric materials are biocompatible and release or produce NO.
  • the polymers are also biodegradable, form hydrogels, polymerize in situ and are tissue adherent.
  • the polymeric materials can also be formed into films, coatings, or microparticles for application to medical devices, such as stents, vascular grafts and catheters. These properties are conferred by the selection of the macromer components as well as addition of various groups to the components.
  • polymerizable means that the regions have the capacity to form additional covalent bonds resulting in macromer interlinking, for example, carbon-carbon double bonds of acrylate-type molecules. Such polymerization is characteristically initiated by free-radical formation resulting from photon abso ⁇ tion of certain dyes and chemical compounds to ultimately produce free-radicals, although polymerization can be obtained using other methods and reagents known to those skilled in the art.
  • the polymeric materials described herein must be biocompatible, i.e., not eliciting a significant or unacceptable toxic or immunogenic response following administration to or implantation into an individual.
  • polymeric materials which are biocompatible, including both natural and synthetic polymers.
  • examples include proteins (of the same origin as the recipient), polysaccharides such as chondroitin sulfate and hyaluronic acid, polyurethanes, polyesters, polyamides, and acrylates.
  • Polymers can be degradable or non-degradable.
  • the preferred polymeric materials will be selected based on a combination of properties conferred by the various components, which may include at least one water soluble region, such as polyethylene glycol (PEG) or polyvinyl alcohol (PVA), at least one biodegradable region such as regions that degrade hydrolytically, and at least one group that can be used to polymerize the macromers in situ.
  • at least one water soluble region such as polyethylene glycol (PEG) or polyvinyl alcohol (PVA)
  • PEG polyethylene glycol
  • PVA polyvinyl alcohol
  • biodegradable region such as regions that degrade hydrolytically
  • group that can be used to polymerize the macromers in situ at least one group that can be used to polymerize the macromers in situ.
  • hydrogels described herein are the ability to covalently attach a variety of bioactive molecules.
  • cell adhesion peptide sequences also referred to herein as cell adhesion ligands
  • RGD the letters indicate the single letter amino acid nomenclature known to those skilled in the art
  • REDV the letters indicate the single letter amino acid nomenclature known to those skilled in the art
  • the cell adhesion ligands are used to specifically target the adhesion, proliferation, and migration of certain cells.
  • the cell adhesion ligand may be a peptide, protein, carbohydrate, or other type of moiety that will assist in seeding cells onto devices.
  • other bioactive molecules such as growth factors have also been shown to retain their efficacy when covalently attached to PEG (Mann B, Schmedlen R, West J. Tethered-TGF-beta increases extracellular matrix production of vascular smooth muscle cells. Biomaterials 2001; 22: 439-444).
  • PEG Mann B, Schmedlen R, West J. Tethered-TGF-beta increases extracellular matrix production of vascular smooth muscle cells. Biomaterials 2001; 22: 439-444.
  • the core water soluble region can consist of poly(ethylene glycol), referred to herein as "PEG", poly(ethylene oxide), poly(vinyl acetate), poly(vinyl alcohol), referred to herein as "PVA", poly(vmylpyrrolidone), poly(ethyloxazoline), poly(ethylene oxide)-co-poly(propyleneoxide) block copolymers, polysaccharides or carbohydrates such as hyaluronic acid, dextran, heparin sulfate, chondroitin sulfate, heparin, or alginate, or proteins such as gelatin, collagen, albumin, or ovalbumin.
  • Hydrophilic regions will generally be tissue adhesive. Both hydrophobic and hydrophilic polymers which include a large number of exposed carboxylic groups are tissue adhesive or bioadhesive. Ligands such as RGD peptides and lectins which bind to carbohydrate molecules on cells can also be bound to the polymer to increase tissue adhesiveness.
  • Polyesters Holland et al, 1986 Controlled Release, 4:155-180
  • ⁇ - hydroxy acids viz., lactic acid, glycolic acid
  • closure devices closure devices
  • drug delivery systems U.S. Patent No. 4,741,337 to Smith et al; Spilizewski et al, 1985 J. Control. Rel 2:197-203.
  • poly(hydroxy acids) In addition to the poly(hydroxy acids), several other polymers are known to biodegrade, including polyanhydrides and polyorthoesters, which take advantage of labile backbone linkages, as reported by Domb et al, 1989 Macromolecules, 22:3200; Heller et al, 1990 Biodegradable Polymers as Drug Delivery Systems, Chasin, M. and Langer, R., Eds., Dekker, New York, 121-161. Polyaminoacids have also been synthesized since it is desirable to have polymers that degrade into naturally occurring materials for in vivo use.
  • the time required for a polymer to degrade can be tailored by selecting appropriate monomers. Differences in crystallinity also alter degradation rates. Due to the relatively hydrophobic nature of these polymers, actual mass loss only begins when the oligomeric fragments are small enough to be water soluble. Hence, initial polymer molecular weight influences the degradation rate.
  • the biodegradable region is preferably hydrolyzable under in vivo conditions.
  • Hydrolyzable groups may be polymers and oligomers of glycolide, lactide, ⁇ -caprolactone, other ⁇ -hydroxy acids, and other biologically degradable polymers that yield materials that are non-toxic or present as normal metabolites in the body.
  • Preferred poly( ⁇ -hydroxy acid)s are poly(glycolic acid), poly(DL-lactic acid) and poly(L-lactic acid).
  • Other useful materials include poly(amino acids), poly(anhydrides), poly(orthoesters), and poly(phosphoesters).
  • Polylactones such as poly( ⁇ - caprolactone), poly( ⁇ -caprolactone), poly( ⁇ -valerolactone) and poly(gamma- butyrolactone), for example, are also useful.
  • Biodegradable regions can also be constructed from polymers or monomers using linkages susceptible to biodegradation by enzymes, such as ester, peptide, anhydride, orthoester, and phosphoester bonds.
  • Degradable materials of biological origin are well known, for example, crosslinked gelatin.
  • Hyaluronic acid has been crosslinked and used as a degradable swelling polymer for biomedical applications (U.S. Patent No. 4,987,744 to della Valle et al., U.S. Patent 4,957,744 to Delia Valle et al. (1991) Polym. Mater. Sci. Eng., 62:731-735).
  • Biodegradable Hydrogels A number of polymers have been described which include both water soluble regions and biodegradable regions. Sawhney et al., (1990) J. Biomed. Mater. Res. 24:1397-1411, copolymerized lactide, glycolide and ⁇ - caprolactone with PEG to increase its hydrophilicity and degradation rate.
  • U.S. Patent No. 5,410,016 issued on April 25, 1995 to Hubbell, et al. describes materials which are based on polyethylene glycol (PEG), because of its high biocompatible and thromboresistant nature, with short polylactide extensions to impart biodegradation and acrylate termini to allow rapid photopolymerization without observable heat production. These materials are readily modified to produce hydrogels which release or produce NO.
  • the polymerizable regions are separated by at least one degradable region to facilitate uniform degradation in vivo. There are several variations of these polymers.
  • the polymerizable regions can be attached directly to degradable extensions or indirectly via water soluble nondegradable sections so long as the polymerizable regions are separated by a degradable section.
  • the macromer composition contains a simple water soluble region coupled to a degradable region
  • one polymerizable region may be attached to the water soluble region and the other attached to the degradable extension or region.
  • the water soluble region forms the central core of the macromer composition and has at least two degradable regions attached to the core. At least two polymerizable regions are attached to the degradable regions so that, upon degradation, the polymerizable regions, particularly in the polymerized gel form, are separated.
  • the central core of the macromer composition is formed by a degradable region, at least two water soluble regions can be attached to the core and polymerizable regions can be attached to each water soluble region. The net result will be the same after gel formation and exposure to in vivo degradation conditions.
  • the macromer composition has a water soluble backbone region and a degradable region affixed to the macromer backbone. At least two polymerizable regions are attached to the degradable regions, so that they are separated upon degradation, resulting in gel product dissolution.
  • the macromer backbone is formed of a nondegradable backbone having water soluble regions as branches or grafts attached to the degradable backbone. Two or more polymerizable regions are attached to the water soluble branches or grafts.
  • the backbone may be star shaped, which may include a water soluble region, a biodegradable region or a water soluble region which is also biodegradable. In this general embodiment, the star region contains either water soluble or biodegradable branches or grafts with polymerizable regions attached thereto. Again, the polymerizable regions must be separated at some point by a degradable region.
  • the polymerizable regions may be polymerizable by photoinitiation by free radical generation, most preferably in the visible or long wavelength ultraviolet radiation.
  • the preferred polymerizable regions are acrylates, diacrylates, oligoacrylates, dimethacrylates, oligomethoacrylates, or other biologically acceptable photopolymerizable groups.
  • a preferred tertiary amine is triethanol amine.
  • Useful photoinitiators are those which can be used to initiate by free radical generation polymerization of the macromers without cytotoxicity and within a short time frame, minutes at most and most preferably seconds.
  • Preferred dyes as initiators of choice for long wavelength ultraviolet (LWUV) light initiation are ethyl eosin, 2,2-dimethoxy-2-phenyl acetophenone, other acetophenone derivatives, other eosin derivatives, such as eosin Y, and camphorquinone.
  • crosslinking and polymerization are initiated among copolymers by a light-activated free-radical polymerization initiator such as 2,2-dimethoxy-2-phenylacetophenone or a combination of ethyl eosin (lO ⁇ -lO 2 mM) and triethanolamine (0.001 to 0.1 M), for example.
  • a light-activated free-radical polymerization initiator such as 2,2-dimethoxy-2-phenylacetophenone or a combination of ethyl eosin (lO ⁇ -lO 2 mM) and triethanolamine (0.001 to 0.1 M), for example.
  • the choice of the photoinitiator is largely dependent on the photopolymerizable regions.
  • the macromer includes at least one carbon-carbon double bond
  • light abso ⁇ tion by the dye causes the dye to assume a triplet state, the triplet state subsequently reacting with the amine to form a free radical which initiates polymerization.
  • Preferred dyes for use with these materials include eosin dye and initiators such as 2,2- dimethyl-2-phenylacetophenone, 2-methoxy-2-phenylacetophenone, and camphorquinone.
  • copolymers may be polymerized in situ by long wavelength ultraviolet light or by laser light of about 514 nm, for example.
  • Initiation of polymerization is accomplished by irradiation with light at a wavelength of between about 200-700 nm, most preferably in the long wavelength ultraviolet range or visible range, 320 nm or higher, most preferably about 514 nm or 365 nm.
  • photooxidizable and photoreducible dyes that may be used to initiate polymerization. These include acridine dyes, for example, acriblarine; thiazine dyes, for example, thionine; xanthine dyes, for example, rose bengal; and phenazine dyes, for example, methylene blue. These are used with cocatalysts such as amines, for example, triethanolamine; sulphur compounds, for example, RSO 2 Rj; heterocycles, for example, imidazole; enolates; organometallics; and other compounds, such as N-phenyl glycine. Other initiators include camphorquinones and acetophenone derivatives.
  • Thermal polymerization initiator systems may also be used. Such systems that are unstable at 37°C and would initiate free radical polymerization at physiological temperatures include, for example, potassium persulfate, with or without tetramethyl ethylenediamine; benzoylperoxide, with or without triethanolamine; and ammonium persulfate with sodium bisulfite.
  • initiation chemistries may be used besides photoinitiation. These include, for example, water and amine initiation schemes with isocyanate or isothiocyanate containing macromers used as the polymerizable regions. Preferred Embodiments
  • the polymeric materials in the macromer composition are polymerizable and at least substantially water soluble.
  • a first macromer includes at least one water soluble region, at least one NO carrying region, and at least one free radical-polymerizable region.
  • a second macromer includes at least one water soluble region and at least two free radical polymerizable regions. The regions can, in some embodiments, be both water soluble and biodegradable.
  • the macromer composition is polymerized by exposure of the polymerizable regions to free radicals generated, for example, by photosensitive chemicals and dyes.
  • Examples of these macromers are PVA or PEG.
  • end caps permit rapid polymerization and gelation.
  • Acrylates are preferred because they can be polymerized using several initiating systems, e.g., an eosin dye, by brief exposure to ultraviolet or visible light.
  • a PEG central structural unit (core) is preferred on the basis of its high hydrophilicity and water solubility, accompanied by excellent biocompatibility.
  • a short oligo or poly( ⁇ -hydroxy acid), such as polyglycolic acid, can be used as a biodegradable chain extension because it rapidly degrades by hydrolysis of the ester linkage into glycolic acid, a harmless metabolite.
  • polyglycolic acid Although highly crystalline polyglycolic acid is insoluble in water and most common organic solvents, the entire macromer composition is water-soluble and can be rapidly gelled into a biodegradable network while in contact with aqueous tissue fluids.
  • Such networks can be used to entrap and homogeneously disperse water-soluble drugs and enzymes and to deliver them at a controlled rate. Further, they may be used to entrap particulate suspensions of water-insoluble drugs.
  • Other preferred chain extensions are polylactic acid, polycaprolactone, polyorthoesters, and polyanhydrides. Polypeptides may also be used.
  • Such "polymeric" blocks should be understood to included dimeric, trimeric, and oligomeric blocks.
  • PVA contains many pendant hydroxyl groups.
  • PVA polyvinyl alcohol
  • the macromers disclosed in U.S. Patent No. 5,508,317 are PVA prepolymers modified with pendant crosslinkable groups, such as acrylamide groups containing crosslinkable olefinically unsarurated groups. These macromers can be polymerized by photopolymerization or redox free radical polymerization, for example.
  • Several embodiments of the macromers of the invention are disclosed herein describing formulations for photopolymerizable macromers. However, one of skill in the art, after studying this disclosure, would know how to make and use macromers formulated for other methods of polymerization.
  • the starting polymers are, in particular, derivatives of polyvinyl alcohol or copolymers of vinyl alcohol that contain, for example, a 1,3-diol skeleton.
  • the crosslinkable group or the further modifier can be bonded to the starting polymer skeleton in various ways, for example through a certain percentage of the 1,3-diol units being modified to give a 1,3-dioxane, which contains a crosslinkable radical, or a further modifier in the 2-position.
  • Another possibility is for a certain percentage of hydroxyl groups in the starting polymer to be esterified by means of an unsarurated organic acid, these ester-bonded radicals containing a crosslinkable group.
  • the hydrophobicity of these macromers can be increased by substituting some of the pendant hydroxyl groups with more hydrophobic substiruents.
  • the properties of the macromers can also be modified by inco ⁇ orating a co-monomer in the macromer backbone.
  • the macromers can also be formed having pendant groups crosslinkable by other means.
  • NO donors A number of molecules that produce NO under physiological conditions (NO donors) have been identified and evaluated both in vitro and in vivo, including S-nitrosothiols, organic nitrates, and complexes of NO with nucleophiles.
  • L-arginine is a NO donor, since L-arginine is a substrate for NO synthase, and thus administration of L-arg ⁇ ie increases endogenous NO production and elicits responses similar to those caused by NO donors in most cases.
  • Other NO donors include molsidomine, CAS754, SPM-5185, and SIN- 1.
  • Other compounds capable of producing and/or donating NO may also be used. These include organic nitrates, nitrosylating compounds, nitrosoesters, and L-arginine.
  • the molecules which produce NO, or release or generate NO are preferably attached to regions containing nucleophiles and/or thiols such as S- nitrosothiols capable of forming a complex with NO.
  • nucleophiles and/or thiols such as S- nitrosothiols capable of forming a complex with NO.
  • the polymeric materials can also be used for drug delivery, preferably localized release of prophylactic, therapeutic or diagnostic agents at the site where the materials are needed, although the polymeric materials can be loaded with agent to be released systemically.
  • agents include proteins or peptides, polysaccharides, nucleic acid molecules, and simple orgamc molecules, both natural and synthetic.
  • Representative materials include antibiotics, antivirals, and antifungal drugs, anti-inflammatories (steroidal or non-steroidal), hormones, growth factors, cytokines, neuroactive agents, vasoconstrictors and other molecules involved in the cardiovascular responses, enzymes, antineoplastic agents, local anesthetics, antiangiogenic agents, antibodies, drugs affecting reproductive organs, and oligonucleotides such as antisense oligonucleotides. Diagnostic materials may be radioactive, bound to or cleave a chromogenic substrate, or detectable by ultrasound, x- ray, MRI, or other standard imaging means.
  • agents can be mixed with macromer prior to polymerization, applied into or onto the polymer, or bound to the macromer prior to or at the time of polymerization, either covalently or ionically, so that the agent is released by degradation (enzymatic or hydrolytic) or diffusion at the site where the polymer is applied.
  • polymeric materials described herein can be used in cell culture, on cell culture substrates, or as coatings on medical implants or devices such as stents, vascular grafts, or catheters, or formed using standard techniques into microparticles or other types of formulations which may be used in or administered to a patient.
  • coatings on medical devices or implants may be used when the proliferation of endothelial cells over the medical device would prolong the use and safety of the device.
  • One of skill in the art may envision many such uses. An example is found in dialysis. The coating would allow endothelial proliferation over the graft used during a dialysis session, thereby prolonging the usable time for each graft, thus delaying the need for a kidney transplant. Coatings may provide other advantages seen upon local delivery of NO such as decreased proliferation of smooth muscle cells and decreased platelet aggregation.
  • Polymeric materials capable of releasing physiological amounts of NO for prolonged periods of time can be applied to sites on or in a patient in need of treatment thereof.
  • Representative disorders or conditions that can be treated with NO include restenosis, thrombosis, asthma, wound healing, arthritis, and penile or female erectile dysfunction.
  • the material can be applied as a macromer solution and polymerized in situ or polymerization can be initiated prior to application.
  • the polymeric materials can also be coated onto medical devices. Wound Healing
  • the formulations are particularly useful for treatment of all types of wounds, including burns, surgical wounds, and open leg and foot wounds.
  • ulcers There are generally three types of open leg wounds, termed ulcers: venous stasis ulcers, generally seen in sedentary elderly people when blood flow to the leg becomes sluggish; decubitus ulcers, also termed pressure sores or bed sores, which occurs most often in people who are bedridden and are unable to frequently change position; and diabetic foot ulcers, caused by poor blood circulation to the feet. Due to the aging of the population, there will likely be a greater demand for effective and user friendly wound treatments in the near future.
  • wound refers to all types of tissue injuries, including those inflicted by surgery and trauma, including burns, as well as injuries from chronic or acute medical conditions, such as atherosclerosis or diabetes.
  • Example 13 shows that exogenous NO released from hydrogel wound dressings may enhance wound healing of chronic wounds.
  • In vivo results examining effects of NO in the diabetic wound model suggest that the most useful parameters for assessing efficacy of wound healing are granulation tissue thickness and matrix production. Similar findings have been reported in studies examining the effect of growth factors on wound healing in animals (Greenhalgh D. The role of growth factors, in wound healing. J Trauma 1996; 41: 159-167).
  • a review of multiple animal models for assessing efficacy of PDGF concluded that epithelialization and wound contraction were not significantly altered, whereas in most models, including the diabetic mouse model, granulation tissue thickness was consistently increased following application of the growth factor (LeGrand EK. Preclinical promise of Becaplermin (rhPDGF-BB) in wound healing. Am J Surg 1998; 176:48S-54S).
  • Hydrogels may be modified to covalently attach growth factors, while maintaining the bioactivity of the growth factor (Mann B, Schmedlen R, West J. Tethered-TGF-beta increases extracellular matrix production of vascular smooth muscle cells. Biomaterials 2001; 22: 439-444). Thus, it is possible to develop hydrogels that provide combined NO and growth factor therapy to further enhance the healing of chronic wounds.
  • the materials described herein overcome some of the disadvantages of existing wound treatments by allowing the formation of hydrogel coatings through in situ polymerization. This technology may simplify the often difficult application of wound dressings to areas such as foot ulcers. Additionally, a multitude of factors to promote wound healing may be inco ⁇ orated into these dressings through simple modification of the hydrogel material. Treatment of Restenosis
  • a preferred application is a method of reducing the effects of restenosis on post-surgical patients.
  • One embodiment of the method includes coating the surface within an artery with an aqueous solution of light-sensitive free radical polymerizable initiator and a number of macromers.
  • the coated artery is subjected to a Xenon arc laser inducing polymerization of the macromers.
  • the physiological conditions within the artery will induce the release of NO. This release will be strictly localized for prolonged periods of time.
  • a stent coated with the NO-releasing hydrogel is implanted in an artery.
  • a preferred application is a method of reducing formation of adhesions after a surgical procedure in a patient.
  • the method includes coating damaged tissue surfaces in a patient with an aqueous solution of a light-sensitive free-radical polymerization initiator and a macromer solution as described above.
  • the coated tissue surfaces are exposed to light sufficient to polymerize the macromer.
  • the light-sensitive free-radical polymerization initiator may be a single compound (e.g., 2,2-dimethoxy-2- phenyl acetophenone) or a combination of a dye and a cocatalyst (e.g., ethyl eosin and triethanol amine).
  • the macromer is mixed with a photoinitiator or photoinitiator/cocatalyst mixture to form an aqueous mixture and the mixture is applied to a tissue surface to which tissue adhesion is desired.
  • the tissue surface is contacted with the tissue with which adhesion is desired, fonriing a tissue junction.
  • the tissue junction is then irradiated until the macromers are polymerized. Tissue Coatings.
  • an ultrathin coating is applied to the surface of a tissue, most preferably the lumen of a tissue such as a blood vessel.
  • a tissue most preferably the lumen of a tissue such as a blood vessel.
  • An initiator is applied to the surface of the tissue, allowed to react, adsorb or bond to tissue, the unbound initiator is removed by dilution or rinsing, and the macromer solution is applied and polymerized. This method is capable of creating uniform polymeric coating of between one and 500 microns in thickness, most preferably about twenty microns, which does not evoke thrombosis or localized inflammation.
  • the polymeric materials can also be used to create tissue supports by fo ⁇ ning shaped articles within the body to serve a mechanical function.
  • Such supports include, for example, sealants for bleeding organs, sealants for bone defects and space-fillers for vascular aneurisms. Further, such supports can include strictures to hold organs, vessels or tubes in a particular position for a controlled period of time.
  • the polymeric materials can be use as carriers for biologically active materials such as therapeutic, prophylactic or diagnostic agents, including hormones, enzymes, antibiotics, antineoplastic agents, and cell suspensions.
  • the polymeric material may be used to temporarily preserve functional properties of an agent to be released, as well as provide prolonged, controlled release of the agent into local tissues or systemic circulation.
  • the macromers are polymerized with the biologically active materials to form microspheres or nanoparticles containing the biologically active material.
  • the macromer, photoinitiator, and agent to be encapsulated are mixed in an aqueous mixture. Particles of the mixture are formed using standard techniques, for example, by mixing in oil to form an emulsion, forming droplets in oil using a nozzle, or forming droplets in air using a nozzle.
  • the suspension or droplets are irradiated with a light suitable for photopolymerization of the macromer.
  • These materials are particularly useful for controlled drug delivery of hydrophilic materials, since the water soluble regions of the polymer enable access of water to the materials entrapped within the polymer. Moreover, it is possible to polymerize the macromer composition containing the material to be entrapped without exposing the material to organic solvents. Release may occur by diffusion of the material from the polymer prior to degradation and/or by diffusion of the material from the polymer as it degrades, depending upon the characteristic pore sizes within the polymer, which is controlled by the molecular weight between crosslinks and the crosslink density. Deactivation of the entrapped material is reduced due to the immobilizing and protective effect of the gel and catastrophic burst effects associated with other controlled-release systems are avoided.
  • the enzyme can be exposed to substrate while the enzyme is entrapped, provided the gel proportions are chosen to allow the substrate to permeate the gel. Degradation of the polymer facilitates eventual controlled release of free macromolecules in vivo by gradual hydrolysis of the terminal ester linkages.
  • the materials include BAB block copolymers of polyethylene glycol (A) with polycysteine (B) that are subsequently reacted with NaNO 2 to form S-nitrosothiols, BAB block copolymers of polyethylene glycol (“PEG”) (A) and diethylenefriamine (“DETA”) (B) that are subsequently reacted with NO gas to form nucleophile/NO complexes, and BAB block copolymers of polyethylene glycol (A) and polylysine (B) that are subsequently reacted with NO gas to form nucleophile/NO complexes.
  • Blended compounds may also be prepared for providing biphasic release of profiles, such as PEG-Cys-DETA-NO. All polymers are further terminated with reactive acrylate groups to allow rapid polymerization in situ.
  • Such materials would be expected to have good biocompatibility, provided that a water soluble, biocompatible polymer such as PEG comprises the bulk of the material and has a sufficiently high molecular weight, and to slowly biodegrade due to the presence of two ester bonds and two amide bonds in each polymer chain.
  • a water soluble, biocompatible polymer such as PEG comprises the bulk of the material and has a sufficiently high molecular weight, and to slowly biodegrade due to the presence of two ester bonds and two amide bonds in each polymer chain.
  • PEG polyethylene glycol
  • PVA hydrogels As demonstrated by examples 4 and 5 below, three types of PVA hydrogels were made and demonstrated release of NO and inco ⁇ orated drug (bFGF): PVA-NH 2 -NO hydrogels; PVA-Cys-NO hydrogels; PVA-NO-bFGF hydrogels. The results are similar to those for the PEG based hydrogels.
  • EXAMPLE 1 SYNTHESIS OF PEG-CYS-NO MACROMERS AND HYDROGELS.
  • an acryloyl-PEG-Cys-NO polymer was formed by first reacting polyethylene glycol N-hydroxysuccinimide monoacrylate (ACRL-PEG-NHS, MW 3400, commercially available from Shearwater Polymers, Huntington, AL) with L-cysteine at an 1:2 molar ratio in 50 mM sodium bicarbonate buffer (pH 8.5) for 2 hours; the product was then dialyzed in a cellulose ester membrane (Molecular weight cutoff 500, Spectrum Labs, Website, Georgia Hills, CA) in diH 2 O, and lyophilized.
  • ACRL-PEG-NHS polyethylene glycol N-hydroxysuccinimide monoacrylate
  • L-cysteine at an 1:2 molar ratio in 50 mM sodium bicarbonate buffer (pH 8.5) for 2 hours
  • a cellulose ester membrane Molecular weight cutoff 500
  • acryloyl-PEG- Cys copolymer was performed using gel permeation chromatography (GPC) with an evaporative light scattering detector and a UV detector at 260 nm (Polymer Laboratories, Amherst, MA). Successful synthesis of acryloyl- PEG-Cys was determined by a shift in the position of the peak from the evaporative light scattering detector. The copolymer was then reacted with an equimolar amount of NaNO 2 at pH 2 and 37°C for 20 minutes to form S- nitrosocysteine.
  • GPC gel permeation chromatography
  • N- vinylpyrrolidone 0.15% N- vinylpyrrolidone was present in this mixture as it was used as a solvent for the photoinitiator. Exposure to UV light (365 nm, 10 mW/cm 2 ) was used to crosslink the polymer, resulting in conversion to a hydrogel (Sawhney et al., (1993) Macromol 26:581-7).
  • EXAMPLE 2 SYNTHESIS OF PEG-LYS 5 -NO MACROMERS AND HYDROGELS.
  • a copolymer of ACRL-PEG-NHS MW 3400, Shearwater Polymers
  • the resultant copolymer was analyzed via GPC, then dissolved in water and reacted with NO gas in an evacuated vessel, thus forming NO-nucleophile complexes with the amine groups on the lysine side groups.
  • Poly(vinyl alcohol) (Hoechst, Mowiol 4-88) was dissolved in diH 2 0 and warmed to 95°C in a round bottom flask under continuous stirring. After one hour, the solution was cooled to room temperature, and a crosslinkable acetal group, methacrylamidoacetaldehyde dimethyl acetal (NAAADA) was added. The amine acetal, gamma-aminobutyraldehyde diethyl acetal, was also added, and the mixture was acidified using glacial acetic acid and 37% hydrochloric acid. The mixture was allowed to stir at room temperature for nine hours, after which the pH was adjusted to pH 3.6 using triethylamine. In order to purify the polymer, the solution was then diafiltered through a MW
  • the neutralized amine-modified polymer was placed in a round bottom flask with stopcock. The flask was evacuated and filled with nitric oxide gas until the desired conversion of amines to NO nucleophile complexes was achieved.
  • Photocrosslinked hydrogels were formed from the PVA-NH 2 -NO by adding 0.1% IRGACURETM 2959 (Ciba-Geigy) photoinitiator (based on total solution volume) and then exposing to UV light (2 mW/cm 2 , 365 nm) for 30 seconds. Addition of the photoinitiator brings the final polymer concentration to 20% w/v.
  • EXAMPLE 5 SYNTHESIS OF PVA-CYS-NO MACROMERS AND HYDROGELS.
  • PVA-NH 2 was synthesized as described above. The amine terminus of cysteine was acetylated using acetic anhydride, and the carboxyl end of the cysteine was coupled to the PVA-NH 2 using water-soluble ED AC chemistry. The resulting PVA-Cys was then purified using diafiltration and brought to a concentration of 22% w/v. PVA-Cys-NO was formed by adding sodium nitrite at an equimolar amount to cysteine residues, adjusting the pH to 2, and incubating at 37°C for 15 minutes.
  • EXAMPLE 6 SYNTHESIS OF PVA-NO-BFGF HYDROGELS.
  • PVA-NO-bFGF hydrogels For PVA-NO-bFGF hydrogels, the above procedure was used to make the PVA-NO polymer. Immediately prior to exposure to UV light, 25 ⁇ g/ml bFGF was added to the polymer solution and mixed well. Gels were crosslinked as described earlier and stored in HEPES buffered saline, pH 7.4, 37°C. EXAMPLE 7: NO RELEASE FROM HYDROGELS.
  • the hydrogels were weighed and stored in HEPES buffered saline, pH 7.4, at 37°C. Aliquots of the buffer were removed at each time point and replaced with fresh buffer. The samples from each time point were then analyzed for nitrite content using a colorimetric assay based on the Griess reaction.
  • NO release from acryloyl-PEG-Lys 5 -NO hydrogels is shown in Figure 4.
  • NO release from acryloyl -PEG-DETA-NO hydrogels is shown in Figure 5.
  • NO release from acryloyl-PEG-Cys-NO hydrogels is shown in Figure 6.
  • EXAMPLE 8 NO AND BFGF RELEASE FROM PVA-NO-BFGF HYDROGELS.
  • the release of NO release from PVA-NO-bFGF hydrogels prepared as described in Example 6 was determined in the same manner as Example 7 and is shown in Figure 7 A. Release of bFGF was quantified using that BCA assay (Pierce Chemicals) and is shown in Figure 7B. Release of NO continues for well over 12 hours, while the growth factor is completely released within the first 5 hours.
  • EXAMPLE 9 EFFECTS OF NO-RELEAS ⁇ G MACROMERS ON CULTURED SMOOTH MUSCLE CELLS: PROLIFERATION AND VIABILITY.
  • smooth muscle cells were grown in the presence of NO-releasing materials, and the effects of those materials on the cells evaluated.
  • Smooth muscle cells isolated from Wistar-Kyoto rats (passage 11-15, provided by T. Scott-Burden) were cultured in Minimum Essential Medium supplemented with 10% FBS, 2 mM L-glutamine, 500 units penicillin, and 100 mg/L streptomycin, at 37°C in a 5% CO 2 environment.
  • the cells were seeded into 24-well tissue culture plates (Becton Dickinson, Franklin Lakes, NJ) at a density of 10,000 cells/cm 2 .
  • NO donors in either soluble or hydrogel form were added to the media in the wells one day after seeding.
  • cell numbers were determined by preparing single cell suspensions with trypsin and counting three samples from each group using a Coulter counter (Multisizer #0646, Coulter Electronics, Hialeah, FL).
  • the effects of NO donors in solution on the proliferation of smooth muscle cells were first investigated by performing a NO dose response curve, whereupon cells were cultured with a range of NO donor concentrations (1 ⁇ M - 10 mM) in order to identify appropriate dosages for hydrogel studies.
  • NO-nucleophile complexes (Lys-NO and DETA-NO) were formed by reacting either L-lysine or DETA with NO gas in water for 24 hours.
  • Soluble Cys-NO was synthesized by reacting an equimolar amount of L-cysteine with NaNO 2 at pH 2 and 37°C for 20 minutes. All NO donor solutions were adjusted to pH 7.4 prior to addition to cell cultures. Smooth muscle cell proliferation in the presence of NO-producing and control hydrogels was then investigated using the optimal NO dose determined above.
  • Hydrogels containing acryloyl-PEG-Lys 5 -NO, acryloyl- PEG-DETA-NO, and acryloyl-PEG-Cys-NO were formed as described above, except that the gel solutions were sterile filtered through 0.2 ⁇ m syringe filters (Gelman Sciences, Ann Arbor, MI) prior to adding 2,2-dimethoxy-2-phenyl acetophenone. PEG-diacrylate hydrogels containing no NO donors were used as a control. The hydrogels were photopolymerized in cell culture inserts (8 ⁇ m pore size, Becton Dickinson, Frariklin Lakes, NJ) and placed in the media over the cultured cells. All three hydrogel NO donors significantly inhibited SMC growth (p ⁇
  • hydrogels were removed and the blood was then incubated with the collagen-coated glass slides (two per group) for 20 minutes at 37°C and then rinsed with HBS. Platelet counts per field of view at 40x were counted under a fluorescent microscope (Zeiss Axiovert 135, Thornwood, NY) in four randomly chosen areas per slide. Photos of platelets which had been exposed to control PEG-diacrylate or acryloyl-PEG-Cys-NO hydrogels demonstrate that exposure to the NO- releasing hydrogels inhibits platelet adhesion to thrombogenic surfaces. Glass slides coated with collagen were used as a thrombogenic surface to which platelets would normally adhere.
  • Bovine aortic endothelial cells (BAECs, passage 5-10, Clonetics) were cultured in Dulbecco's Modified Eagle Medium with identical supplements and culture conditions to the smooth muscle cells. The viability of endothelial cells exposed to NO-releasing PEG gels was examined through the use of a Live/Dead staining kit (Molecular Probes, Eugene, OR). BAECs were seeded into 24- well plates at a concentration of 10,000 cells/cm 2 , and hydrogels were added as described above. After two days in culture, cell viability was assessed.
  • a 4 ⁇ M solution of ethidium bromide causes dead cells to fluoresce red due to their increased permeability
  • a 2 ⁇ M solution of calcein AM causes viable cells to fluoresce green due to esterase activity.
  • Cells were examined under a fluorescence microscope (Zeiss Axiovert 135, Thomwood, NY), and photomicrographs were taken using a digital camera (Sony).
  • Endothelial cell proliferation in the presence of NO-producing and control hydrogels was then investigated using the optimal NO dose deteixnined above. After four days in culture with the hydrogels, cell numbers were determined by preparing single cell suspensions with trypsin and counting three samples from each group using a Coulter Counter as described above.
  • Endothelial cell proliferation on NO-releasing hydrogels In order for these hydrogels to effectively prevent restenosis, re- endothelialization must occur not only in areas surrounding the hydrogel, but also upon the hydrogel itself.
  • a cell adhesion ligand was first covalently inco ⁇ orated into these hydrogels, as cells will not attach to PEG unless the polymer is modified with an adhesive sequence (Hern D, Hubbell J. Inco ⁇ oration of adhesion peptides into nonadhesive hydrogels useful for tissue resurfacing, J Biomed Mater Res 1998; 39: 266-276).
  • hydrogels containing the adhesive peptide sequence RGDS (Arginine- Glycine-Aspartic acid-Serine) and the NO donor DETA-NO were synthesized.
  • RGDS was covalently bound to PEG by reaction with ACRL- PEG-NHS in a ratio of 1:2 (RGDS:polymer) in 50 mM sodium bicarbonate buffer (pH 8.5) for two hours at room temperature. The solution was then dialyzed and lyophilized to obtain ACRL-PEG-RGDS.
  • ACRL-PEG-DETA- NO was prepared as described above.
  • BAECs were immediately seeded upon the hydrogels at a density of 7500 cells/cm 2 .
  • Controls consisted of hydrogels with the NO donor alone or RGDS alone. Two days after cell seeding, cells were trypsinized and cell number was assessed by counting on a Coulter Counter.
  • Hydrogels containing the cell adhesive ligand REDV were also prepared. Synthesis of acryloyl-PEG-REDV was identical to the synthesis of acryloyl-PEG-RGDS except that the concentration of REDV in the hydrogels was 14 ⁇ mol/ml. Endothelial cell seeding experiments were also identical. Results: Cell proliferation and viability
  • Hydrogels containing the cell adhesive ligand REDV were also prepared.
  • Synthesis of acryloyl-PEG-REDV was identical to the synthesis of acryloyl-PEG-RGDS except that the concentration of REDV in the hydrogels was 14 ⁇ mol/ml and the concentration of RGDS was 1.4 ⁇ mol/ml.
  • Endothelial cell seeding experiments were also identical.
  • Fig. 14 shows the average cell number data for acryloyl-PEG-REDV.
  • EXAMPLE 12 NO-PRODUCING PEG DERIVATIVES COMBINED WITH PEG-DIACRYLATE. The monoacrylate NO-producing PEG derivatives have been combined with PEG-diacrylate to allow crosslinking into hydrogels.
  • Biodegradable PEG-diacrylate derivatives such as copolymers with ⁇ -hydroxy acids (Sawhney AS, Pathak CP, Hubbell JA. Bioerodible hydrogels based on photopolymerized poly(ethylene glycol)-co-poly(alpha-hydroxy acid) diacrylate macromers. Macromol 1993; 26: 581-587) or proteolytically degradable peptides (West JL, Hubbell JA. Polymeric biomaterials with degradation sites for proteases, involved in cell migration. Macromol 1999; 32: 241-244) could be substituted for PEG-diacrylate to create biodegradable, NO-producing hydrogels. This allows separate determination of NO production kinetics and biodegradation characteristics. Flexibility in the duration of NO release may also prove useful in the extension of this therapy to applications other than thrombosis and restenosis.
  • EXAMPLE 13 EVALUATION OF HYDROGELS FOR ENHANCEMENT OF WOUND HEALING In vitro evaluation of effects of PVA-NO on fibroblast viability and proliferation
  • HDFs Human dermal fibroblasts (HDFs, passage 5-11, Clonetics) were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% FBS, 2 mM L-glutamine, 500 units penicillin, and 100 mg/L streptomycin, at 37°C in a 5% CO 2 environment.
  • NO-releasing hydrogels containing 0.1 ⁇ mol to 5 ⁇ mol of the NO donor(0.1 mM to 5 mM NO donor in 1 ml cell culture media) were polymerized and suspended in the cell culture media in transwell inserts 24 hours following cell seeding.
  • Extracellular matrix (ECM) production was assessed in fibroblast culture through inco ⁇ oration of 3 H-glycine into glycoprotein, elastin, and collagen portions of the ECM as determined by sequential enzyme digestion (TEC assay; Scott-Burden T, Resink T, B ⁇ rgin M, B ⁇ hler F. Extracellular matrix: Differential influence on growth and biosynthesis patterns of vascular smooth muscle cells from SHR and WKY rats. J Cell Physiol 1989; 141: 267-274). HDFs were seeded at 8000 cells/cm 2 in 24-well tissue culture polystyrene plates, and PVA-NO hydrogels were formed in transwell inserts and added to the cell culture media 24 hours following cell seeding.
  • the media was supplemented with l ⁇ Ci/ml 3 H-glycine at this time.
  • Two days following the addition of the hydrogels the cells in non-radioactive wells were trypsinized and counted on a Coulter Counter.
  • the cells in the remaining wells were lysed in a solution of 25 mM ammonium hydroxide for 30 minutes, and the plate was then dehydrated. A sequential digestion of extracellular matrix was performed in order to digest glycoproteins, elastin, and collagen.
  • Radioactivity in samples from each digestion step was determined by scintigraphy (Minaxi ⁇ Tri-Carb 4000, Packard Instrument Co., Meridien, CT).
  • a circular piece of sterile PVA-NO or control hydrogel was cut to match the size of the wounds using a sterile circular cork borer, and applied to the wound.
  • the wounds were covered with a transparent semi-occlusive secondary dressing (Tegaderm, 3M), adhered to the area surrounding the wound using tincture of Benzoin.
  • wounds were redressed while the mice were under isoflurane inhalation anesthesia.
  • the secondary dressing and the hydrogel were removed and the wounds were flushed with sterile saline to remove debris and to clean the wound area.
  • a digital planimetric image of the wound was recorded using a Pixera video camera.
  • a calibration scale was recorded with each image. Once photographed, fresh dressings were placed on the wounds, and the wounds were covered again with fresh Tegaderm dressings.
  • Wound area was assessed by image analysis using ImagePro Plus 3.0 image analysis software. Using the acquired images and this software, the perimeter of the wound was defined and measured, and the wound areas determined. Means and standard deviations of wound perimeters and areas at each time point were calculated.
  • Tissue surrounding and underlying the wound was sampled from these mice at the time of sacrifice and was fixed in Streck tissue fixative (Zinc- formalin), and embedded in paraffin for histological sectioning. Sections from each wound were stained with Hematoxylin and Eosin and with Masson's Trichrome stains.
  • Granulation tissue thickness was measured at days 8 and 15 and collagen layer thickness was measured in sections from the final time point (29 days). Tissue thickness was measured using ImagePro Plus 3.0 image analysis software on images captured using an Olympus BX50WI microscope and SONY DKC 5000 camera.
  • Control of bias was achieved by assigning a color code to each of the test groups and the control group. Investigators were blinded to the identity of each of the groups and the test and control hydrogels have a similar appearance. All animal experimentation was conducted under appropriate procedures approved by the University of Medicine and Dentistry of New Jersey animal care and use review boards. Characterization of release kinetics As shown previously, release of NO from PVA-NO hydrogels was observed over a period of 48 hours at pH 7.4, as determined by the Griess assay. A slightly acidic pH is often observed in the wound environment, causing us to also evaluate NO release from these hydrogels at pH 6. No inhibition of NO release was observed when hydrogels were exposed to slightly acidic conditions.
  • Hydrogels may be tailored to obtain a range of NO concentrations by blending with unreacted aminated PVA prior to polymerization, allowing easy tailoring of the NO dosage.
  • Control hydrogels for release kinetics studies consisted of aminated PVA which had not been exposed to NO, as well as unmodified PVA (no amine groups) which had been exposed to NO, but did not contain any reactive groups with which to form NO donors. No significant NO release was observed with either control group.
  • FIGS. 16A and 16B show the wound area and perimeter over time. Representative digital images of the wounds at days 10, 17, and 27 were also taken. Images of the wounds were captured every 2-3 days to quantify wound area and perimeter through image processing software. Day 27 was the endpoint of the wound healing study, with animals having closed, epithelialized wounds. Wound area and perimeter were similar in test and control groups.
  • Granulation tissue characterized by proliferating fibroblasts and newly formed microvasculature, was present in the open wound at days 8 and 15 in all groups.
  • Figure 17 shows a graph of the granulation tissue thickness comparing the test and control groups. The results reflect the mean thickness and standard deviation of 3 measurements taken on each of two serial sections (total of 6 measurements) within the region of the open wound. The three measurements were at a central point and 0.5 mm on either side of this point. Granulation tissue tended to be thicker with increasing NO concentration, however this difference was not statistically significant. Representative histological sections of granulation tissue formation at days 8 and 15 in the control group and the 5 mM PVA-NO group.
  • Granulation tissue thickness was assessed through analysis of histological sections stained with hematoxylin and eosin. Representative sections were taken for treatment with a) control hydrogels and b) 5 mM PVA-NO hydrogels at day 8, as well as treatment with c) control hydrogels and d) 5 mM PVA-NO hydrogels at day 15. Increased granulation tissue thickness was observed in wounds treated with NO-releasing hydrogels compared to controls. Collagen deposition was assessed in histological sections stained with

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Abstract

La présente invention concerne des hydrogels dégageant ou produisant du NO, de préférence, des hydrogels polymérisables et biodégradables capables de dégager des quantités physiologiques de NO pour des durées prolongées, que l'on applique à des sites ou chez un patient requérant un traitement à base de NO pour des troubles tels la resténose, la thrombose, l'asthme, la cicatrisation, l'arthrite, la dysérection du pénis ou d'autres pathologies dans lesquelles le NO joue un rôle significatif. Les matériaux polymériques peuvent être préparés sous forme de films, revêtements, ou de microparticules destinés à être appliqués à des dispositifs médicaux, tels des tuteurs, greffes vasculaires et cathéters. Les matériaux polymériques peuvent également être appliqués directement à des tissus biologiques et peuvent être polymérisés in situ. Les hydrogels sont formés de macromères, qui, de préférence, contiennent des zones biodégradables, et présentent des groupes liées à celles-ci qui sont disséminés in situ afin d'accroître ou de moduler autrement les niveaux de NO au site où le traitement est requis. Les macromères peuvent constituer une dispersion ou une solution homogène ou hétérogène, qui est soumise à une polymérisation en vue de former un matériau hydrogel, qui dans ce cas peut constituer un alliage ou un semi-alliage IPN. Des composés destinés à être libérés peuvent être physiquement piégés, en liaison covalente ou ionique au macromère, ou effectivement être incorporés dans le matériau polymérique. L'hydrogel peut être formé par la réticulation ionique ou covalente. D'autres principes actifs, comprenant des agents thérapeutiques, prophylactiques ou diagnostiques, peuvent être incorporés dans le matériau polymérique.
PCT/US2001/027414 1999-09-02 2001-09-04 Materiaux hydrogels produisant du monoxyde d'azote WO2002017880A2 (fr)

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US7651697B2 (en) 1999-09-02 2010-01-26 Rice University Nitric oxide-producing hydrogel materials
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US8591876B2 (en) 2010-12-15 2013-11-26 Novan, Inc. Methods of decreasing sebum production in the skin
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US8981139B2 (en) 2011-02-28 2015-03-17 The University Of North Carolina At Chapel Hill Tertiary S-nitrosothiol-modified nitric—oxide-releasing xerogels and methods of using the same
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US9138462B2 (en) 2009-07-31 2015-09-22 Sanofi-Aventis Deutschland Gmbh Prodrugs comprising an insulin linker conjugate
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US9919072B2 (en) 2009-08-21 2018-03-20 Novan, Inc. Wound dressings, methods of using the same and methods of forming the same
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US8439968B2 (en) 1998-03-30 2013-05-14 Innovational Holdings, Llc Expandable medical device for delivery of beneficial agent
US7819912B2 (en) 1998-03-30 2010-10-26 Innovational Holdings Llc Expandable medical device with beneficial agent delivery mechanism
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US8052735B2 (en) 1998-03-30 2011-11-08 Innovational Holdings, Llc Expandable medical device with ductile hinges
US8623068B2 (en) 1998-03-30 2014-01-07 Conor Medsystems, Inc. Expandable medical device with ductile hinges
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US8187321B2 (en) 2000-10-16 2012-05-29 Innovational Holdings, Llc Expandable medical device for delivery of beneficial agent
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US8349390B2 (en) 2002-09-20 2013-01-08 Conor Medsystems, Inc. Method and apparatus for loading a beneficial agent into an expandable medical device
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US9056126B2 (en) 2004-07-05 2015-06-16 Ascendis Pharma A/S Hydrogel formulations
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US10029015B2 (en) 2004-07-05 2018-07-24 Ascendis Pharma A/S Hydrogel formulations
US7968085B2 (en) 2004-07-05 2011-06-28 Ascendis Pharma A/S Hydrogel formulations
EP1704879A1 (fr) * 2005-03-24 2006-09-27 NOLabs AB Appareil médical intravasculaire, interstitiel ou d'accès à l'intérieur d'un organe comprenant un polymère à élution de monoxide d'azote
US9403851B2 (en) 2005-05-27 2016-08-02 The University Of North Carolina At Chapel Hill Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications
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US8956658B2 (en) 2005-05-27 2015-02-17 The University Of North Carolina At Chapel Hill Nitric oxide-releasing particles for nitric oxide therapeutics and biomedical applications
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