Classifications

• • 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
  • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
  • A61L31/08—Materials for coatings
  • A61L31/10—Macromolecular materials
• • 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
  • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
  • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L31/16—Biologically active materials, e.g. therapeutic substances
• • 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
  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
  • A61L2300/114—Nitric oxide, i.e. NO
• • 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
  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
  • A61L2300/25—Peptides having up to 20 amino acids in a defined sequence
• • 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
  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
  • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
  • A61L2300/256—Antibodies, e.g. immunoglobulins, vaccines
• • 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
  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
  • A61L2300/416—Anti-neoplastic or anti-proliferative or anti-restenosis or anti-angiogenic agents, e.g. paclitaxel, sirolimus
• • 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
  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
  • A61L2300/602—Type of release, e.g. controlled, sustained, slow
  • A61L2300/604—Biodegradation
• • 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
  • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
  • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
  • A61L2300/606—Coatings
  • A61L2300/608—Coatings having two or more layers

Definitions

• the invention relates generally to implantable medical devices, and in particular to biodegradable polymer coated implantable stents that promote vascular healing in diabetics.
• the stent itself reduces restenosis in a mechanical way by providing a larger lumen. For example, some stents gradually enlarge over time.
• many stents are implanted in a contracted form mounted on a partially expanded balloon of a balloon catheter and then expanded in situ to contact the lumen wall.
• U. S. Patent No. 5,059,211 discloses an expandable stent for supporting the interior wall of a coronary artery wherein the stent body is made of a porous bioabsorbable material.
• U. S. Patent No. 5,662,960 discloses a friction-reducing coating of commingled hydrogel suitable for application to polymeric plastic, rubber or metallic substrates that can be applied to the surface of a stent.
• agents that affect cell proliferation have been tested as pharmacological treatments for stenosis and restenosis in an attempt to slow or inhibit proliferation of smooth muscle cells.
• These compositions have included heparin, coumarin, aspirin, fish oils, calcium antagonists, steroids, prostacyclin, ultraviolet irradiation, and others.
• Such agents may be systemically applied or may be delivered on a more local basis using a drug delivery catheter or a drug eluting stent.
• biodegradable polymer matrices loaded with a pharmaceutical may be implanted at a treatment site. As the polymer degrades, a medicament is released directly at the treatment site.
• U.S. Patent No. 5,342,348 to Kaplan and U.S. Patent No. 5,419,760 to Norciso are exemplary of this technology.
• U.S. Patent 5,766,710 discloses a stent formed of composite biodegradable polymers of different melting temperatures.
• Porous stents formed from porous polymers or sintered metal particles or fibers have also been used for release of therapeutic drugs within a damaged vessel, as disclosed in U. S. Patent No. 5,843,172.
• tissue surrounding a porous stent tends to infiltrate the pores.
• pores that promote tissue ingrowth are considered to be counterproductive because the growth of neointima can occlude the artery, or other body lumen, into which the stent is being placed.
• U.S. Patent 5,766,584 to Edelman et al. describes a method for inhibiting vascular smooth muscle cell proliferation following injury to the endothelial cell lining by creating a matrix containing endothelial cells and surgically wrapping the matrix about the tunica adventitia.
• the matrix, and especially the endothelial cells attached to the matrix secrete products that diffuse into surrounding tissue, but do not migrate to the endothelial cell lining of the injured blood vessel.
• EDRF endothelium-derived relaxing factor
• thrombin factor Xa operates upon factor Nil to control thrombus formation and, at the same time stimulates production of PARs (Protease Activated Receptors) by pro-inflammatory monocytes and macrophages.
• PARs Protease Activated Receptors
• Nitric oxide produced endogenously by endothelial cells regulates invasion of the proinflammatory monocytes and macrophages. In the lumen of an artery, this two-phase cycle results in influx and proliferation of healing cells through a break in the endothelium.
• Stabilization of the vascular smooth muscle cell population by this naturally counterbalanced process is required to prevent neointimal proliferation leading to restenosis.
• the absence or scarcity of endogenously produced nitric oxide caused by damage to the endothelial layer in the vasculature is thought to be responsible for the proliferation of vascular smooth muscle cells that results in restenosis following vessel injury, for example following angioplasty.
• Nitric oxide dilates blood vessels (Nallance et al., Lancet, 2:997-1000, 1989), inhibits platelet activation and adhesion (Radomski et al., Br. J Pharmacol, 92:181-187, 1987) and, in vitro, nitric oxide limits the proliferation of vascular smooth muscle cells (Garg et al., J. Clin. Invest. 83:1774-1777, 1986). Similarly, in animal models, suppression of platelet-derived mitogens by nitric oxide decreases intimal proliferation (Fems et al., Science, 253:1129-1132, 1991).
• Clinical research has strongly implicated the generally impaired healing of the endothelium in patients who suffer from diabetes mellitus as a major contributor to the diminished therapeutic outcome in these patients when an arterial stent has been implanted.
• Impaired glucose tolerance (IGT) is considered a transitional phase to the development of Type II diabetes and many of the changes in health of endothelium found in Type II diabetics are prefigured in IGT. IGT and diabetes are also independently associated with the occurrence of cardiovascular disease. While Type II diabetic patients make up a significant proportion of those patients who experience such treatment failure, all Type II diabetics do not experience stent failure and the reason why some do and some do not has not hitherto been studied.
• the present invention is based on the discovery that endogenous endothelial healing processes at a site of vascular damage in patients suffering from Type II diabetes can be promoted by coating stents and other implantable devices with biodegradable, bioactive polymers bearing covalently attached bioligands that specifically capture and activate therapeutic progenitors of endothelial cells from the circulating blood of such patients.
• the polymers which biodegrade over time, may also release bioactive agents that re-establish in patients suffering from Type II diabetes the natural endothelial healing process in an artery.
• the bioactive agent(s) attached to the polymers promote endogenous endothelial processes in arteries of diabetics by specifically recruiting to the stent surface progenitors of endothelial cells from circulating blood at the site of stent or device implantation in the vasculature.
• the polymers e.g., the polymer backbone
• the invention provides bioactive implantable stents including a stent structure with a surface coating of a biodegradable, bioactive polymer, and at least one bioligand that specifically binds to an integrin receptor on progenitors of endothelial cells (PECs) in circulating blood.
• the bioligand is covalently bonded to the polymer. This bioligand may itself be bioactive in also activating the PECs, or it may act in conjunction with another bioactive PEC-activating agent.
• the invention provides a kit that includes a biocompatible implantable stent.
• the invention stent has a stent structure with a surface coating of a biodegradable, biocompatible polymer with at least one bioligand or first member of a specific binding pair that binds specifically to an integrin receptor on PECs.
• the bioligand or first member is covalently bound to the biodegradable, biocompatible polymer.
• the invention provides a tubular sheath comprising a biodegradable, bioactive polymer, wherein the polymer comprises at least one bioligand covalently bound to the polymer, wherein the bioligand specifically binds to an integrin receptor on PECs in peripheral blood.
• the invention provides implantable medical devices having a biodegradable, bioactive polymer coated upon at least a portion of a surface. At least one bioligand that specifically binds an integrin receptor on PECs found in peripheral blood is covalently bound to the polymer.
• the invention provides methods for treating damaged arterial endothelium in heart or limb in a patient having Type II diabetes comprising implanting an invention stent to promote natural healing of damaged endothelium in the artery wall of the patient.
• the invention provides methods for using a polymer as a medical device, a pharmaceutical, or as a carrier for covalent immobilization of a bioligand or first member of a specific binding pair that specifically attaches to an integrin receptor in PECs in the circulating blood of a patient with Type II diabetes into which the polymer is implanted.
• the bioligand is a polypeptide that binds specifically to an integrin receptor on PECs in circulating blood; b) the bioligand forms a specific binding pair with an antibody that binds specifically to the integrin receptor; or c) the antibody is tagged with a first member of a specific binding pair and the bioligand comprises a second member of the specific binding pair.
• the invention provides methods for promoting natural healing of endothelium damaged by mechanical intervention in an artery of a subject having Type II diabetes by implanting into the artery following the mechanical intervention an invention stent to promote natural healing of the artery.
• Fig. 1 is a schematic cross-section of an invention multilayered polymer-coated stent.
• Fig. 2 is a flow chart describing the PEC isolation protocol.
• Fig. 3 is a flow chart of the protocol for adhesion assays conducted with ECs and SMCs.
• Fig. 4 is a graph summarizing the results of a representative adhesion assay quantitation based on ATP standard curve. At each time point of the adhesion assay, an ATP assay was done to determine the number of adherent cells.
• Fig. 5 shows the chemical structure of dansyl, an acronym for 5 dimethylamino-1 naphthalenesulfonyl, a reactive fluorescent dye, linked to PEA.
• Figs. 6A-B are flowcharts summarizing surface chemistry optimization protocols.
• Fig. 6 A shows a flowchart of the surface chemistry for conjugation of peptides to the acid version of the polymers (PEA-H).
• Fig. 6B shows a flowchart of the protocol for surface conjugation of peptides, to mixtures of PEA polymers.
• this invention provides stents and methods using such devices to re-establish an endothelial blood/artery barrier in patients suffering from diabetes mellitus, particularly Type II diabetes.
• the invention is also designed to promote endothelial healing at a site of damaged vascular endothelium in patients having impaired glucose tolerance, which is considered a transitional phase to the development of Type II diabetes.
• the invention stents comprise a. biocompatible, resorbable polymeric sheath that encapsulates the stent structure.
• the stent is placed at the conclusion of an angioplasty procedure, or other medical procedure that damages arterial endothelium, without allowing a lapse of time sufficient for infiltration of inflammatory factors from the blood stream into the artery wall.
• the stent is placed at the location of the damage and preferably immediately covers and protects the area of damaged endothelium so as to prevent infiltration of inflammatory factors from the blood stream into the artery wall, while performing its primary function of gathering therapeutic progenitors of endothelial cells from the patient's circulating blood so that the natural processes of endothelial healing can go forward in the patient suffering from Type II diabetes.
• the invention stents perfo ⁇ n as an artificial endothelial layer while promoting the natural cycle of endothelial healing in diabetics as described herein.
• the polymeric sheath may have additional features that contribute to the healing of the artery.
• the invention sheath or covering comprises multiple layers, each of which can perform a distinct function in re-establishing a stable lesion and contributing to healing endothelium of the injured artery wall.
• diabetes and "diabetes mellitus” as used herein mean Type II diabetes as well as impaired glucose tolerance (IGT), which is widely considered a transitional phase to the development of Type II diabetes. Many of the changes in health of endothelium found in Type II diabetics are prefigured in IGT.
• IGT impaired glucose tolerance
• EPCs endothelial progenitor cells
• examples of bioligands suitable for use in capture of PECs from circulating blood are monoclonal antibodies directed against a known or identified surface marker of therapeutic PECs.
• Complementary determinants (CDs) that have been reported to decorate the surface of endothelial cells include CD31, CD34, CD 102, CD105, CD106, CD109, CDwl30, CD141, CD142, CD143, CD144, CDwl45, CD146, CD 147, and CD 166. These cell surface markers can be of varying specificity for a particular cell/developmental type/stage in EC development. CDs 106, 142 and 144 have been reported to mark mature endothelial cells with some specificity.
• CD34 is presently known to be specific for progenitor endothelial cells in non-diabetics and therefore is one of the cell surface markers that is believed to be useful for capturing PECs out of blood circulating in the vessels in a diabetic patient into which the stent is implanted.
• ECM extracellular matrix
• Fibronectin is one of the more ubiquitous members of the ECM. It is a potential ligand for most cell types and is recognized by at least 10 adhesion receptors of the integrin family (Leukemia 1997; 11 :822-829 and Blood 1998; 91(9):3230-3238). In particular, CS5 and REDVDY are both found in the Type III connecting segment of fibronectin.
• the sequence for the CS5 peptide is: Gly-Glu-Glu- Ile-Gln-Ile-Glv-His-Ile-Pro--4rg-C7/z.--4 ⁇ - ' ⁇ /- ⁇ -ryr-His-Leu-Tvr-Pro (SEQ ID NO:l), which contains REDVDY (underlined) (SEQ ID NO:2). It has been discovered that CS5 and REDVDY peptides bind specifically to integrin receptors on PECs.
• REDV The minimal active cell binding amino acid sequence, REDV , is somewhat i related to the RGDs, a major active site in the central cell binding domain of fibronectin.
• REDV is novel in its cell type selectivity.
• the integrin ⁇ 4 ⁇ l is known to bind to the REDV sequence and is found on ECs but not on SMCs (JBC (1991) 266(6):3579- 35S5; Am. J of Pathology (1994) 145:1070-1081; and Blood ( 1998) 91(9):3230-32384). This becomes even more important in recruiting PECs versus smooth muscle progenitor cells (SPCs) in peripheral blood.
• SPCs smooth muscle progenitor cells
• Bioligands e.g., peptides and polypeptides
• a biodegradable polymer as described herein for coating at least a portion of the surface of an interventional implantable device, such as a vascular stent, to endow the coating with the property of preferential and specific recruitment of a subpopulation of PECs from the circulating bloodstream of a diabetic patient into which the device is implanted.
• the resulting localized concentration of PECs throughout the stent will enhance endothelial wound healing of the arterial wall of the diabetic patient.
• the bioligand is an antibody, such as a monoclonal antibody, and is specific for an integrin receptor identified on PECs as described above.
• a stent having a polymer coating to which the capture antibody is bound will, when implanted in a Type II diabetic, in turn bind to and hold captured PECs near the polymer surface for activation and subsequent migration.
• an antibody is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies.
• An antibody useful in a method of the invention, or an antigen-binding fragment thereof, is characterized, for example, by having specific binding activity for an epitope of a target molecule.
• the antibody for example, includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof.
• non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains (see Huse et al., Science 246:1275-1281 (1989)).
• These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol.
• Monoclonal antibodies suitable for use as bioligands may also be obtained from a number of commercial sources. Such commercial antibodies are available against a wide variety of targets.
• Antibody probes can be conjugated to molecular backbones using standard chemistries, as discussed below.
• the term "binds specifically" or "specific binding activity,” when used in reference to an antibody means that an interaction of the antibody and a particular epitope has a dissociation constant of at least about 1 x 10 "6 , generally at least about 1 x 10 "7 , usually at least about 1 x 10 "8 , and particularly at least about 1 x 10 "9 or 1 x 10 "10 or less.
• Fab, F(ab') 2 , Fd and Fv fragments of an antibody that retain specific binding activity for an epitope of an antigen are included within the definition of an antibody.
• a pair of biocompatible specific binding partners can be used to specifically capture PECs from the circulating blood of Type II diabetics.
• one of the specific binding pair acts as the bioligand covalently attached to the polymer coating of the stent or other implantable device.
• the other member of the pair of specific binding partners is attached or allowed to attach to an integrin receptor on the PECs of the diabetic patient to be treated (either ex vivo or in vivo by administration to the blood of the patient).
• a Mab that binds specifically to a PEC cell surface marker such as CD 144
• molecule A a PEC cell surface marker
• streptavidin molecule B
• a Mab that binds specifically to a PEC cell surface marker such as CD 144
• molecule A a PEC cell surface marker
• streptavidin molecule B
• a Mab that binds specifically to a PEC cell surface marker such as CD 144
• the roles of the specific binding partners, A and B can be reversed, with biotin, for example, being attached to the polymer of the stent and streptavidin being attached to a monoclonal antibody administered to the patient for specific attachment to the integrin receptor on the patient's PECs.
• Mab-A conjugates are added to the patient's blood either in vivo (e.g., parenterally) or ex vivo (e.g., by extracorporeal circulation of the patient's blood) either prior to, contemporaneously with, or immediately following installation of the stent or other therapeutic device.
• circulating therapeutic EPC-Mab-A complexes are preferentially recruited to binding partner B, streptavidin, which is covalently attached to the device coating, enhancing the local concentration of therapeutic PECs at the site of intervention and injury.
• a monoclonal antibody administered to the blood of a human is preferably a "humanized monoclonal antibody" and suitable antigen-binding fragments can be commissioned commercially or can readily be produced recombinantly using well known techniques.
• this aspect of the invention is illustrated by reference to specific binding partners biotin and streptavidin, any biocompatible pair of specific binding partners can be used in an analogous way.
• the biocompatible bioligand can further comprise one member of a specific binding pair, such as a biotin-streptavidin, and the other member of the specific binding pair can be pre-attached to the polymer.
• a specific binding pair such as a biotin-streptavidin
• the bioligand is administered to the patient's blood stream, either in vivo or ex vivo, and allowed to bind to its specific target on therapeutic PECs therein, via a specific binding pair bridge. If the bioligand is administered to the patient's blood stream in vivo (e.g., parenterally), the PECs in the blood stream become bound to the polymer in vivo via the bioligand-specific binding pair-polymer complex.
• small proteinaceous motifs such as the B domain of bacterial Protein A and the functionally equivalent region of Protein G, are known to form a specific binding pair with, and thereby capture Fc-containing antibodies.
• the antibody administered to the diabetic patient's blood is an Fc- containing antibody that is specific for an integrin receptor on PECs in blood and the bioligand attached to the polymer of the stent is a "sticky" peptide or polypeptide, such as Protein A and Protein G, which will capture the antibody and hold it near to the polymer surface of the stent to aid in recruiting PECs to the area of endothelium damage.
• these "sticky" peptides or polypeptides may also capture other circulating, Fc- containing, native antibodies, thereby reducing specificity of the reaction for the therapeutic purposes.
• Protein A is a constituent of staphylococcus A bacteria that binds the Fc region of particular antibodies or immunoglobulin molecules.
• the Protein A bioligand can be or contain the amino acid sequence:
• MTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVD GVWTYDDATKTFTVTE SEQ ID NO:3
• a functionally equivalent peptidic derivative thereof such as, by way of an example, the functionally equivalent peptide or polypeptide having the amino acid sequence:
• Protein G is a constituent of group G streptococci bacteria, and displays similar activity to Protein A, namely binding the Fc region of particular antibody or immunoglobulin molecules.
• the Protein G bioligand can be, or contain Protein G having an amino acid sequence:
• Such Protein A and Protein G molecules can be covalently attached as bioligands to the bioactive polymer coatings on the stent structure (e.g., the inner layer of a multilayered stent as described herein) and will act as bioligands to capture out of the patient's circulating blood stream Fc-containing antibodies that have been complexed with the patients' therapeutic PECs.
• Bioligands are selected and conjugated to the polymer backbone while avoiding steric hindrance to binding of the ligand to its biological target.
• bioactive agents that activate the progenitor endothelial cells and are contemplated for attachment to the polymer backbone in the polymer coatings covering the invention medical devices include the bradykinins.
• Bradykinins are vasoactive nonapeptides formed by the action of proteases on kininogens, to produce the decapeptide kallidin (KRPPGFSPFR) (SEQ ID NO:7), which can undergo further C-terminal proteolytic cleavage to yield the bradykinin 1 nonapeptide: (KRPPGFSPF) (SEQ ID NO: 8), or N- terminal proteolytic cleavage to yield the bradykinin 2 nonapeptide: (RPPGFSPFR) (SEQ ID NO: 9).
• KRPPGFSPFR decapeptide kallidin
• Bradykinins 1 and 2 are functionally distinct as agonists of specific • bradykinin cell surface receptors Bl and B2 respectively: both kallidin and bradykinin 2 are natural bioligands for the B2 receptor; whereas their C-terminal metabolites (bradykinin 1 and the octapeptide RPPGFSPF (SEQ ID NO: 10) respectively) are bioligands for the Bl receptor.
• a portion of circulating bradykinin peptides can be subject to a further post-translational modification: hydroxylation of the second proline residue in the sequence (Pro3 to Hyp3 in the bradykinin 2 amino acid numbering). Bradykinins are very potent vasodilators, increasing permeability of post-capillary venules, and acting on endothelial cells to activate calmodulin and thereby nitric oxide synthase.
• Bradykinin peptides are incorporated into the bioactive polymers used in the invention stents by attachment at one end of the peptide.
• the unattached end of the bradykinin extends freely from the polymer as a bioligand to contact endothelial cells in the vessel wall as well as progenitor endothelial cells captured from the blood in the vessel into which the stent is implanted. Thereby the endothelial cells with which contact is made become activated.
• the bioactive agent can be a nucleoside, such as adenosine, which is also known to be a potent activator of endothelial cells to produce nitric oxide endogenously.
• Endothelial cells activated in this way activate further progenitor endothelial cells with which they come into contact.
• a cascade of endothelial cell activation at the site of the injury is caused to result in endogenous production of nitric oxide and development of an endothelial lining on the surface of the stent that contacts blood.
• the invention stent has a multilayered polymer covering that encapsulates a stent structure.
• Fig. 1 shows a schematic cross-section of an example of an invention stent 11 with stent struts 10 and a multilayered sheath or covering.
• the outer layer 16 of the stent covering or sheath lies directly next to the artery wall.
• Bioactive agents and additional bioactive agents, as described herein, are incorporated into the outer layer of the stent covering or sheath to promote healing of the epithelium.
• An optional diffusion barrier layer 14 can be placed between and in contact with outer layer 16 and inner layer 12.
• the inner layer 12 of the multilayered stent covering is exposed to the circulating blood with its PECs and has bioligands for recruitment of PECs covalently attached thereto.
• a biocompatible polymer of the type specifically described herein e.g., having a chemical structure described by structures I and III herein
• One or more bioligands that bind specifically to PECs such as those having an amino acid sequence as set forth in SEQ ID NOS:l, 2, or 11, or a member of a specific binding pair for which the other member is contained within or conjugated with a specifically binding bioligand, are covalently attached to the polymer in the inner layer using techniques of covalent attachment described herein.
• streptavidin can be bound to the polymer of the inner layer of the sheath for use with a biotin-tagged antibody that specifically binds the target on PECs in the circulating blood (which biotin-tagged antibody will be administered to the patient's blood stream).
• one or more "bioactive agent,” as described herein, but not “an additional bioactive agent” can also be covalently bound to the polymer in the inner layer of the multilayered stent.
• the bioactive agent is selected to activate PECs attracted to the inner layer of the sheath from the circulating blood of diabetic patients by the bioligands attached to the inner layer of the stent covering.
• the stent takes an active role in the process of re-establishing the natural endothelial cell layer at the site of one or more damaged areas of arterial endothelium.
• the outer layer 16 comprises a polymer layer loaded with a bioactive agent and/or an additional bioactive agent, or combination thereof, specifically including those that limit cellular proliferation or reduce inflammation as disclosed herein.
• a bioactive agent and/or an additional bioactive agent or combination thereof, specifically including those that limit cellular proliferation or reduce inflammation as disclosed herein.
• These cellular proliferation limiting and/or inflammation reducing drugs and bioactive agents can be solubilized in the polymer solid phase and, hence, are preferably not bound to the polymer of the outer layer. Rather such bioactive agents and additional bioactive agents are loaded into the polymer and sequestered there until the stent is put into place. Once implanted, the bioactive agents in the outer layer 16 are eluted and diffuse into the artery wall.
• bioactive agents for incorporation into the outer layer of invention multilayered stents include rapamycin and any of its analogs or derivatives, such aseverolimus (also known as sirolimus), paclitaxel or any of its analogs or derivatives, and statins, such as simvastatin.
• rapamycin and any of its analogs or derivatives, such aseverolimus (also known as sirolimus), paclitaxel or any of its analogs or derivatives, and statins, such as simvastatin.
• non-covalently bound bioactive agents and/or additional bioactive agents can be intermingled with or "loaded into” any biocompatible biodegradable polymer as is known in the art since the outer layer in this embodiment of the invention does not come into contact with blood, except during placement of the stent.
• a diffusion barrier layer 14 of resorbable polymer that acts as a diffusion barrier to the bioactive agent or additional bioactive agent contained in the outer layer.
• the purpose of this diffusion barrier is to direct elution of the drug/biologic into the artery wall to prevent proliferation of smooth muscle cells, while limiting or preventing passage of the drug/biologic into the inner layer.
• the diffusion barrier layer 14 can accomplish its purpose of partitioning of the drug through hydrophobic/hydrophilic interaction related to the solubility of the bioactive agent in the polymer solid phase.
• the polymer barrier layer is selected to be less hydrophobic than the agent(s), and if the bioactive agent or additional bioactive agent in the outer layer is hydrophilic, the barrier layer is selected to be hydrophobic.
• the barrier layer can be selected from such polymers as polyester, poly(amino acid), poly(ester amide), poly(ester urethane), polyurethane, polylactone, poly(ester ether), or copolymers thereof, whose charge properties are well known by those of skill in the art.
• the barrier layer is considered optional because the inner layer of the stent may itself prove an effective diffusion barrier, depending upon the properties of the polymers and various active agents contained in the inner and outer layers of the stent.
• the stent structure used in manufacture of the invention multilayered stent as well as the stents comprising a single layer of polymer covering described herein is made of a biodegradable and absorbable material with sufficient strength and stiffness to replace a conventional stent structure, such as a stainless steel or wire mesh stent structure.
• a cross-linked poly(ester amide), polycaprolactone, or poly(ester urethane) as described herein can be used for this purpose so that the stent structure as well as its covering(s) is completely bioabsorbable, for example, over a period of three months to years.
• each of the layers, and the stent structure as well will be re-absorbed by the body through natural processes, including enzymatic action, allowing the re-established endothelial cell layer to resume its dual function of acting as a blood/artery barrier and providing natural control and stabilization of the intra-cellular matrix within the artery wall through the production of nitric oxide.
• biodegradable means that at least the polymer coating of the invention stent is capable of being broken down into innocuous and biocompatible products in the normal functioning of the body.
• the entire stent, including the stent structure is biodegradable.
• the preferred biodegradable, biocompatible polymers have hydrolyzable ester and/or amide linkages, which provide the biodegradability, and are typically chain terminated with carboxyl or capping groups.
• Biodegradable, blood compatible polymers suitable for use in the practice of the invention of the type specifically described herein bear functionalities that allow for facile covalent attachment of bioactive agents to the polymer.
• a polymer bearing carboxyl groups can readily react with a bioactive agent having an amino moiety, thereby covalently bonding the bioactive agent to the polymer via the resulting amide group.
• the biodegradable, biocompatible polymer and the bioligands and bioactive agents can contain numerous complementary functional groups that can be used to covalently attach the bioactive agent to the biodegradable, biocompatible polymer.
• bioactive agent means agents that play an active role in the endogenous healing processes at a site of stent implantation by holding bioligands or members of a specific binding pair, and/or releasing a bioactive or therapeutic agent during biodegradation of the polymer.
• Bioactive agents include those specifically described herein as having properties that capture (i.e., "bioligands"), attract and activate captured circulating PECs, and are contemplated for covalent attachment to the polymers used in coating the invention stents.
• bioactive agents include, but are not limited to, agents that, when freed from the polymer backbone during polymer degradation, promote endogenous production of a therapeutic natural wound healing agent, such as nitric oxide endogenously produced by endothelial cells.
• a therapeutic natural wound healing agent such as nitric oxide endogenously produced by endothelial cells.
• the "bioactive agents" released from the polymers during degradation may be directly active in promoting natural wound healing processes by endothelial cells while controlling proliferation of smooth muscle cells in the vessel at the locus of the damage.
• These bioactive agents can include any agent that donates, transfers, or releases nitric oxide, elevates endogenous levels of nitric oxide, stimulates endogenous synthesis of nitric oxide, or serves as a substrate for nitric oxide synthase or that inhibits proliferation of smooth muscle cells.
• Such agents include, for example, aminoxyls, furoxans, nitrosothiols, nitrates and anthocyanins; nucleosides such as adenosine, and nucleotides such as adenosine diphosphate (ADP) and adenosine triphosphate (ATP); neurotransmitter/neuromodulators such as acetylcholine and 5-hydroxytryptamine (serotonin/5 -HT); histamine and catecholamines such as adrenalin and noradrenalin; lipid molecules such as sphingosine-1 -phosphate and lysophosphatidic acid; amino acids such as arginine and lysine; peptides such as the bradykinins, substance P and calcium gene- related peptide (CGRP), and proteins such as insulin, vascular endothelial growth factor (VEGF), and thrombin.
• nucleosides such as adenosine, and nu
• bioactive agents are optionally covalently attached to the bioactive polymers used in the coverings of the invention stents and devices.
• Aminoxyls contemplated for use as bioactive agents have the structure:
• aminoxyls include the following compounds:
• Furoxans contemplated for use as bioactive agents have the structure:
• An exemplary furoxan is 4-phenyl-3-furoxancarbonitrile, as set forth below:
• Anthocyanins are also contemplated for use as bioactive agents.
• Anthocyanins are glycosylated anthocyanidins and have the structure:
• Anthocyanins are known to stimulate NO production in vivo and therefore are suitable for use as bioactive agents in the practice of the invention.
• Bioactive agents for dispersion into and release from the surface coverings of the invention stents and medical devices also include anti-proliferants, rapamycin and any of its analogs or derivatives, paclitaxel or any of its taxene analogs or derivatives, everolimus, Sirolimus, tacrolimus, or any of its -limus named family of drugs, and statins such as simvastatin, atorvastatin, fluvastatin, pravastatin, lovastatin, rosuvastatin, geldanamycins, such as 17AAG (17-allylamino-17-demethoxygeldanamycin); Epothilone D and other epothilones, 17-dimethylaminoethylamino-17-demethoxy-geldanamycin and other polyketide inhibitors of heat shock protein 90 (Hsp90), Cilostazol, and the like.
• statins such as simvastatin, atorvastatin, fluvastat
• Polymers contemplated for use in forming the blood-compatible, hydrophilic coating or inner layer in the invention multilayered stents include polyesters, poly(amino acids), polyester amides, polyurethanes, or copolymers thereof.
• biodegradable polyesters include poly( ⁇ -hydroxy -C 5 alkyl carboxylic acids), e.g., polyglycolic acids, poly-L-lactides, and poly-D,L-lactides; poly-3-hydroxy butyrate; polyhydroxyvalerate; polycaprolactones, e.g., poly( ⁇ -caprolactone); and modified poly( ⁇ - hydroxyacid)homopolymers, e.g., homopolymers of the cyclic diester monomer, 3- (S)[alkyloxycarbonyl)methyl]-l,4-dioxane-2,5-dione which has the formula 4 where R is lower alkyl, depicted in Kimura, Y., "Biocompatible Polymers” in Biomedical Applications of Polymeric Materials, Tsuruta, T., et al, eds., CRC Press, 1993 at page 179.
• the glycolide-lactide copolymers include poly(glycolide-L-lactide) copolymers formed utilizing a monomer mole ratio of glycolic acid to L-lactic acid ranging from 5:95 to 95:5 and preferably a monomer mole ratio of glycolic acid to L-lactic acid ranging from 45:65 to 95:5.
• the glycolide-caprolactone copolymers include glycolide and ⁇ -caprolactone block copolymer, e.g., Monocryl or Poliglecaprone.
• the biodegradable polymers useful in forming the coatings for the invention biocompatible polymer coated stents and medical devices also include those comprising at least one amino acid conjugated to at least one non-amino acid moiety per repeat unit.
• non-amino acid moiety includes various chemical moieties, but specifically excludes amino acid derivatives and peptidomimetics as described herein.
• the polymers containing at least one amino acid are not contemplated to include poly(amino acid) segments, including naturally occurring polypeptides, unless specifically described as such.
• the non-amino acid is placed between two adjacent amino acids in the repeat unit.
• the polymers may comprise at least two different amino acids per repeat unit.
• Preferred for use in forming the biocompatible polymer surface coverings of the invention stents and medical devices (and the inner layers of invention multilayered stents) are polyester amides (PEAs) and polyester urethanes (PEURs) that have built-in functional groups on PEA or PEUR side chains, and these built-in functional groups can react with other chemicals and lead to the incorporation of additional functional groups to expand the functionality of PEA or PEUR further. Therefore, such polymers used in the invention compositions and methods are ready for reaction with other chemicals having a hydrophilic structure to increase water solubility and with bioactive agents and additional bioactive agents, without the necessity of prior modification.
• polymers used in the invention polymer coated stents and medical devices display minimal hydrolytic degradation when tested in a saline (PBS) medium, but in an enzymatic solution, such as chymotrypsin or CT, a uniform erosive behavior has been observed.
• PBS saline
• PEAs and PEURs have a chemical formula described by the general structural formula (I):
• H H ff ?, H — PEUR— is O— R 4 -0-C-C-N-C-0— R 4 -O-C-CH- -islI 1 R 3 R, and wherein n ranges from about 50 to about 150, m ranges about 0.1 to about 0.9: p ranges from about 0.9 to about 0.1; wherein R .
• R 2 is hydrogen or (C 6 -C_o)aryl (C ⁇ -C 6 ) alkyl or t-butyl or other protecting group
• R 3 is selected from the group consisting of hydrogen, (Ci-C ⁇ ) alkyl, (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl and (C 6 -C 10 ) aryl (C ⁇ -C 6 ) alkyl
• R- t is selected from the group consisting of (C 2 -C 20 ) alkyl ene, (C 2 -C 20 ) alkenylene or alkyloxy, and bicyclic- fragments of l,4:3,6-dianhydrohexitols of general formula (II):
• R ⁇ and R 4 are selected from (C 2 -C 0 ) alkyl ene and (C 2 -C 20 ) alkenylene; wherein at least one of R . and R 4 is (C 2 -C 20 ) alkenylene; n is about 5 to about 150; each R 2 is independently hydrogen, or (C ⁇ -C ⁇ aryKC C ⁇ alkyl; and each R 3 is independently hydrogen, ( -C ⁇ alkyl, (C 2 - C 6 )alkenyl, (C 2 -C 6 )alkynyl, or (C 6 -C 10 )aryl(C 1 -C 6 )alkyl.
• R 3 is CH 2 Ph and the alpha amino acid used in synthesis is L- phenylalanine.
• R 3 is CH 2 -CH(CH 3 )
• the polymer contains the alpha- amino acid, leucine.
• the polymer molecules may also have the active agent attached thereto, optionally via a linker or incorporated into a crosslinker between molecules.
• the polymer is contained in a polymer-bioactive agent conjugate having the structural formula (III):
• R 5 is selected from the group consisting of
• R 8 is H or (C1-C8) alkyl; and R 6 is a bioactive agent.
• two molecules of the polymer can be crosslinked to provide an -R 5 -R 6 -R 5 - conjugate.
• the bioactive agent is covalently linked to one molecule of the polymer through the -R 5 -R 6 - R 5 - conjugate and R 5 is independently selected from the group consisting of-O-, -S-, and -NR 8 -, wherein R 8 is H or (Cj-C 8 ) alkyl.
• a linker, -X-Y- can be inserted between R 5 and bioactive agent R 6 in the molecule of structural formula III, wherein X is selected from the group consisting of (CrC 18 ) alkylene, substituted alkylene, (C 3 -C 8 ) cyclo alkylene, substituted cycloalkylene, 5-6 membered heterocyclic system containing 1-3 heteroatoms selected from the group O, N, and S, substituted heterocyclic, (C 2 -C ⁇ 8 ) alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, C 6 and C 10 aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkynyl, substituted arylalkynyl, arylalkenyl, substituted arylalkenyl, arylalky
• one molecule of the polymer is covalently linked to a bioactive agent through an -R 5 -R 6 -Y-X- R 5 - bridge (Formula VI).
• X is selected from the group consisting of (C ⁇ -C 18 ) alkylene, substituted alkylene, (C -C 8 ) cycloalkylene, substituted cycloalkylene, 5-6 membered heterocyclic system containing 1-3 heteroatoms selected from the group O, N, and S, substituted heterocyclic, (C 2 -C ⁇ 8 ) alkenyl 1, substituted alkenyl, alkynyl, substituted alkynyl, C 6 and
• Cio aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkynyl, substituted arylalkynyl, arylalkenyl, substituted arylalkenyl, arylalkynyl, substituted arylkynyl, wherein the substituents are selected from the group consisting of H, F, Cl, Br, I, (C C 6 ) alkyl, -CN, -NO 2 , -OH, -O(C C 4 alkyl), -S(Q-
• the polymer coating on the invention stents and medical devices comprises four partially crosslinked molecules of the polymer of structural formula (III), except that only two of the four molecules omit R 6 and are crosslinked to provide a single -R 5 -X-R 5 - conjugate, wherein X is selected from the group consisting of (d-C 18 ) alkylene, substituted alkylene, (C -C 8 ) cycloalkylene, substituted cycloalkylene, 5-6 membered heterocyclic system containing 1-3 heteroatoms selected from the group consisting of O, N, and S, substituted heterocyclic, (C 2 -C 18 ) alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, C and Cio aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkynyl, substituted arylalkynyl,
• R 9 and R 10 are independently H or (d-C 6 ) alkyl).
• aryl is used with reference to structural formulae herein to denote a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. In certain embodiments, one or more of the ring atoms can be substituted with one or more of nitro, cyano, halo, trifluoromethyl, or trifluoromethoxy. Examples of aryl include, but are not limited to, phenyl, naphthyl, and nitrophenyl.
• alkenylene is used with reference to structural formulae herein to mean a divalent branched or unbranched hydrocarbon chain containing at least one unsaturated bond in the main chain or in a side chain.
• the molecular weights and polydisperities herein are determined by gel permeation chromatograph using polystyrene standards. More particularly, number and weight average molecular weights (M n and M w ) are determined, for example, using a Model 510 gel permeation chromatograph (Water Associates, Inc., Milford, MA) equipped with a high-pressure liquid chromatographic pump, a Waters 486 UV detector and a Waters 2410 differential refractive index detector. Tetrahydrofuran (THF) is used as the eluent (1.0 mL/min).
• THF Tetrahydrofuran
• the polystyrene standards have a narrow molecular weight distribution.
• the bts- ⁇ -amino acid is entered into a polycondensation reaction with a di-acid such as sebacic acid, to obtain the final polymer having both ester and amide bonds.
• a di-acid such as sebacic acid
• di-acid chloride can also be used.
• R 4 in (I) is -C 4 H 8 - or -C 6 H 12 -.
• R 1 in (I) is -C 4 H 8 - or-C 8 H 16 -.
• the unsaturated PEAs can be prepared by solution polycondensation of either (1) di-p-toluene sulfonic acid salt of diester of alpha-amino acid and unsaturated diol and di-p-nitrophenyl ester of saturated dicarboxylic acid or (2) di-p-toluene sulfonic acid salt of alpha-amino acid and saturated diol and di-nitrophenyl ester of unsaturated dicarboxylic acid or (3) di-p-toluene sulfonic acid salt of diester of alpha-amino acid and unsaturated diol and di-nitrophenyl ester of unsaturated dicarboxylic acid.
• the aryl sulfonic acid salts are used instead of the free amine base because the aryl sulfonic acid group is a very good leaving group which can promote the condensation reaction to move to the right of the reaction equation so product is obtained in high yield and because the p-toluene sulfonic acid salts are known for use in synthesizing polymers containing amino acid residues.
• the di-p-nitrophenyl esters of unsaturated dicarboxylic acid can be synthesized from p-nitrophenyl and unsaturated dicarboxylic acid chloride, e.g., by dissolving triethylamine and p-nitrophenyl in acetone and adding unsaturated dicarboxylic acid chloride drop wise with stirring at -78°C and pouring into water to precipitate product.
• Suitable acid chlorides included acrylic methacrylic, crotonic, isocrotonic, angelic, tiglic, sorbic, cinnamic, allocinnamic, phenylpropiolic, fumaric, maleic, mesaconic, citraconic, glutaconic, itaconic, ethenyl-butane dioic and 2-propenyl-butanedioic acid chlorides.
• Additional compounds that can be used in the place of di-p-nitrophenyl esters of unsaturated dicarboxylic acid include those having structural formula (VIII): o o II II R 5 -O-C-O-R O-C-O-R 5 (N ⁇ i) wherein each R 5 is independently (d -C ⁇ o)aryl optionally substituted with one or more nitro, cyano, halo, trifluoromethyl, or trifluoromethoxy; and Rj is independently (C 2 - C 20 )alkylene or (C 2 -C 8 )alkyloxy(C 2 -C 20 )alkylene.
• the di-aryl sulfonic acid salts of diesters of alpha-amino acid and unsaturated diol can be prepared by admixing alpha-amino acid, e.g., p-aryl sulfonic acid monohydrate and saturated or unsaturated diol in toluene, heating to reflux temperature, until water evolution is minimal, then cooling.
• alpha-amino acid e.g., p-aryl sulfonic acid monohydrate
• saturated or unsaturated diol in toluene
• the unsaturated diols include, for example, 2-butene-l,3-diol and l,18-octadec-9-en-diol.
• R 4 of (III) of 6,503,535 and/or R 1 of (V) of 6,503,538 is C 2 -C 20 alkenylene as described above.
• the reaction is carried out, for example, by adding dry triethylamine to a mixture of said (III) and (IN) of 6,503,538 and said (N) in dry ⁇ , ⁇ -dimethylacetamide, at room temperature, then increasing the temperature to 80°C and stirring for 16 hours, then cooling the reaction solution to room temperature, diluting with ethanol, pouring into water, separating polymer, washing separated polymer with water, drying to about 30°C under reduced pressure and then purifying up to negative test on p-nitrophenyl and p-toluene sulfonic acid.
• a preferred reactant (IV) is p-toluene sulfonic acid salt of benzyl ester
• the benzyl ester protecting group is preferably removed from (II) to confer biodegradability, but it should not be removed by hydrogenolysis as in Example 22 of U.S. Patent No. 6,503,538 because hydrogenolysis would saturate the desired double bonds; rather the benzyl ester group should be converted to an acid group by a method that would preserve unsaturation, e.g., by treatment with fluoroacetic acid or gaseous HF.
• the lysine reactant (IV) can be protected by a protecting group different from benzyl which can be readily removed in the finished product while preserving unsaturation, e.g., the lysine reactant can be protected with t-butyl (i.e., the reactant can be t-butyl ester of lysine) and the t-butyl can be converted to H while preserving unsaturation by treatment of the product (II) with dilute acid.
• a working example of the compound having structural formula (II) is provided by substituting p-toluene sulfonic acid salt of L-phenylalanine 2-butene-l,4-diester for (III) in Example 1 of 6,503,538 or by substituting di-p-nitrophenyl fumarate for (V) in Example 1 of 6,503,538 or by substituting p-toluene sulfonic acid salt of L-phenylalanine 2-butene- 1,3 -diester for III in Example 1 of 6,503,538 and also substituting de-p- nitrophenyl fumarate for (V) in Example 1 of 6,503,538.
• Aminoxyl radical e.g., 4-amino TEMPO
• carbonyldiimidazol as a condensing agent
• Bioactive agents as described herein, can be attached via the double bond functionality.
• Hydrophilicity can be imparted by bonding to poly(ethylene glycol) diacrylate.
• polymers contemplated for use in forming the invention polymer coated stents and medical devices include those set forth in U.S. Patent Nos. 5,516, 881; 6,338,047; 6,476,204; 6,503,538; and in U.S. Application Nos. 10/096,435; 10/101,408; 10/143,572; and 10/194,965; the entire contents of each of which is incorporated herein by reference.
• the PEA/PEUR polymers described herein may contain up to two amino acids per monomer and preferably have weight average molecular weights ranging from 10,000 to 125,000; these polymers and copolymers typically have inherent viscosities at 25 °C, determined by standard viscosimetric methods, ranging from 0.3 to 4.0, preferably ranging from 0.5 to 3.5.
• tributyltin (IV) catalysts are commonly used to form polyesters such as poly(caprolactone), poly(glycolide), poly(lactide), and the like.
• a wide variety of catalysts can be used to form polymers suitable for use in the practice of the invention.
• poly(caprolactones) contemplated for use have an exemplary structural formula (IX) as follows:
• the first step involves the copolymerization of lactide and ⁇ -caprolactone in the presence of benzyl alcohol using stannous octoate as the catalyst to form a polymer of structural formula (XII). .
• hydroxy terminated polymer chains can then be capped with maleic anhydride to form polymer chains having structural formula (XIII):
• the PEA/PEUR polymers described herein can be fabricated in a variety of molecular weights, and the appropriate molecular weight for use with a given bioactive agent is readily determined by one of skill in the art. Thus, e.g., a suitable molecular weight will be on the order of about 5,000 to about 300,000, for example about 5,000 to about 250,000, or about 75,000 to about 200,000, or about 100,000 to about 150,000.
• Polymers useful in the making the invention polymer coated stents and medical devices such as PEA PEUR polymers, biodegrade by enzymatic action at the surface. Therefore, the polymers administer the bioactive agent to the subject at a controlled release rate, which is specific and constant over a prolonged period. Additionally, since PEA/PEUR polymers break down in vivo via hydrolytic enzymes without production of adverse side products, the polymer coatings on the invention stents and medical devices are substantially non-inflammatory.
• dispensersed means at least one bioactive agent as disclosed herein is dispersed, mixed, dissolved, homogenized, and/or covalently bound (“dispersed") in a polymer, for example attached to the surface of the polymer or polymer coating.
• bioactive agents can be dispersed within the polymer matrix without chemical linkage to the polymer carrier, it is also contemplated that the bioactive agent or additional bioactive agent can be covalently bound to the biodegradable polymers via a wide variety of suitable functional groups.
• the carboxyl group chain end can be used to react with a complimentary moiety on the bioactive agent or additional bioactive agent, such as hydroxy, amino, thio, and the like.
• a complimentary moiety on the bioactive agent or additional bioactive agent such as hydroxy, amino, thio, and the like.
• a bioactive agent can be linked to any of the polymers of structures (I)-(NII) through an amide, ester, ether, amino, ketone, thioether, sulfmyl, sulfonyl, or disulfide linkage.
• a linkage can be formed from suitably functionalized starting materials using synthetic procedures that are known in the art.
• a polymer can be linked to the bioactive agent or additional bioactive agent via a carboxyl group (e.g., COOH) of the polymer.
• a carboxyl group e.g., COOH
• a compound of structures (I) and (III) can react with an amino functional group or a hydroxyl functional group of a bioactive agent to provide a biodegradable polymer having the bioactive agent attached via an amide linkage or carboxylic ester linkage, respectively.
• the carboxyl group of the polymer can be benzylated or transformed into an acyl halide, acyl anhydride/"mixed" anhydride, or active ester.
• the free - ⁇ H 2 ends of the polymer molecule can be acylated to assure that the bioactive agent will attach only via a carboxyl group of the polymer and not to the free ends of the polymer.
• the bioactive agent or additional bioactive agent can be attached to the polymer via a linker molecule, for example, as described in structural formulae (V - VII).
• a linker may be utilized to indirectly attach the bioactive agent and/or adjuvant to the biodegradable polymer.
• the linker compounds include poly(ethylene glycol) having a molecular weight (MW) of about 44 to about 10,000, preferably 44 to 2000; amino acids, such as serine; polypeptides with repeat number from 1 to 100; and any other suitable low molecular weight polymers.
• the linker typically separates the bioactive agent from the polymer by about 5 angstroms up to about 200 angstroms.
• alkyl refers to a straight or branched chain hydrocarbon group including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, and the like.
• alkenyl refers to straight or branched chain hydrocarbyl group's having one or more carbon-carbon double bonds.
• alkynyl refers to straight or branched chain hydrocarbyl groups having at least one carbon-carbon triple bond.
• aryl refers to aromatic groups having in the range of 6 up to 14 carbon atoms.
• the linker may be a polypeptide having from about 2 up to about 25 amino acids.
• Suitable peptides contemplated for use include poly-L- glycine, poly-L-lysine, poly-L-glutamic acid, poly-L-aspartic acid, poly-L-histidine, poly- L-ornithine, poly-L-serine, poly-L-threonine, poly-L-tyrosine, poly-L-leucine, poly-L- lysine-L-phenylalanine, poly-L-arginine, poly-L-lysine-L-tyrosine, and the like.
• the bioactive agent can covalently crosslink the polymer, i.e. the bioactive agent is bound to more than one polymer molecule. This covalent crosslinking can be done with or without additional polymer-bioactive agent linker.
• the bioactive agent molecule can also be incorporated into an intramolecular bridge by covalent attachment between two polymer molecules.
• a linear polymer polypeptide conjugate is made by protecting the potential nucleophiles on the polypeptide backbone and leaving only one reactive group to be bound to the polymer or polymer linker construct. Deprotection is performed according to methods well known in the art for deprotection of peptides (Boc and Fmoc chemistry for example).
• a polypeptide bioactive agent is presented as retro-inverso or partial retro-inverso peptide.
• the terms "peptide” and "polypeptide,” as used herein, include peptides, wholly peptide derivatives (such as branched peptides) and covalent hetero- (such as glyco- and lipo- and glycolipo-) derivatives of peptides.
• the peptides described herein can be synthesized using any technique as is known in the art.
• the peptides and polypeptides can also include "peptide mimetics.”
• Peptide analogs are commonly used in the pharmaceutical industry as non-peptide bioactive agents with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics.” Fauchere, J. (1986) Adv. Bioactive agent Res., 15:29; Veber and Freidinger (1985) TINS p. 392; and Evans et al. (1987) J. Med. Chem., 30:1229; and are usually developed with the aid of computerized molecular modeling.
• Such peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
• substitution of one or more amino acids within a peptide or polypeptide may be used to generate more stable peptides and peptides resistant to endogenous proteases.
• the synthetic peptide or polypeptide e.g., covalently bound to the biodegradable polymer, can also be prepared from D-amino acids, referred to as inverso peptides.
• inverso peptides When a peptide is assembled in the opposite direction of the native peptide sequence, it is referred to as a retro peptide.
• peptides prepared from D-amino acids are very stable to enzymatic hydrolysis.
• the linker can be attached first to the polymer or to the bioactive agent or additional bioactive agent.
• the linker can be either in unprotected form or protected form, using a variety of protecting groups well known to those skilled in the art.
• the unprotected end of the linker can first be attached to the polymer or the bioactive agent or additional bioactive agent.
• the protecting group can then be de-protected using Pd/H 2 hydrogenation, mild acid or base hydrolysis, or any other common de-protection method that js known in the art.
• the de- protected linker can then be attached to the bioactive agent or additional bioactive agent, or to the polymer
• a polyester can be reacted with an aminoxyl, e.g., 4-amino-2,2,6,6-tetramethylpiperidine-l-oxy, in the presence of N,N'-carbonyl diimidazole to replace the hydroxyl moiety in the carboxyl group at the chain end of the polyester with imino linked to aminoxyl-containing radical, so that the imino moiety covalently bonds to the carbon of the carbonyl residue of the carboxyl group.
• an aminoxyl e.g., 4-amino-2,2,6,6-tetramethylpiperidine-l-oxy
• the N,N'-carbonyl diimidazole converts the hydroxyl moiety in the carboxyl group at the chain end of the polyester into an intermediate product moiety which will react with the aminoxyl, e.g., 4-amino-2,2,6,6-tetramethylpiperidine-l-oxy.
• the aminoxyl reactant is typically used in a mole ratio of reactant to polyester ranging from 1:1 to 100:1.
• the mole ratio of N,N'-carbonyl diimidazole to aminoxyl is preferably about 1 :1.
• a typical reaction is as follows.
• a polyester is dissolved in a reaction solvent and reaction is readily carried out at the temperature utilized for the dissolving.
• the reaction solvent may be any in which the polyester will dissolve; this information is normally available from the manufacturer of the polyester.
• the polyester is a polyglycolic acid or a poly(glycolide-L-lactide) (having a monomer mole ratio of glycolic acid to L-lactic acid greater than 50:50), highly refined (99.9+% pure) dimethyl sulfoxide at 115 °C to 130 °C or DMSO at room temperature suitably dissolves the polyester.
• polyester is a poly-L-lactic acid
• a poly-DL-lactic acid or a poly(glycolide-L- lactide) having a monomer mole ratio of glycolic acid to L-lactic acid 50:50 or less than 50:50
• tetrahydrofuran tetrahydrofuran
• dichloromethane DCM
• chloroform at room temperature to 40 ⁇ 50 °C suitably dissolve the polyester.
• the polymers used to make the surface covering for the invention stents and other medical devices as described herein have one or more bioactive agent directly linked to the polymer.
• the residues of the polymer can be linked to the residues of the one or more bioactive agents.
• one residue of the polymer can be directly linked to one residue of the bioactive agent.
• the polymer and the bioactive agent can each have one open valence.
• bioactive agent more than one bioactive agent, multiple bioactive agents, or a mixture of bioactive agents and additional bioactive agents having different therapeutic or palliative activity can be directly linked to the polymer.
• residue of each bioactive agent can be linked to a corresponding residue of the polymer
• the number of residues of the one or more bioactive agents can correspond to the number of open valences on the residue of the polymer.
• a "residue of a polymer” refers to a radical of a polymer having one or more open valences.
• any synthetically feasible atom, atoms, or functional group of the polymer (e.g., on the polymer backbone or pendant group) of the present invention can be removed to provide the open valence, provided bioactivity is substantially retained when the radical is attached to a residue of a bioactive agent.
• any synthetically feasible functional group e.g., carboxyl
• any synthetically feasible functional group can be created on the polymer (e.g., on the polymer backbone or pendant group) to provide the open valence, provided bioactivity is substantially retained when the radical is attached to a residue of a bioactive agent.
• those skilled in the art can select suitably functionalized starting materials that can be derived from the polymer of the present invention using procedures that are known in the art.
• a "residue of a compound of structural formula (*)” refers to a radical of a compound of polymer formulas (I - VII) as described herein having one or more open valences. Any synthetically feasible atom, atoms, or functional group of the compound (e.g., on the polymer backbone or pendant group) can be removed to provide the open valence, provided bioactivity is substantially retained when the radical is attached to a residue of a bioactive agent.
• any synthetically feasible functional group e.g., carboxyl
• any synthetically feasible functional group can be created on the compound of formulas (I - VII) (e.g., on the polymer backbone or pendant group) to provide the open valance, provided bioactivity is substantially retained when the radical is attached to a residue of a bioactive agent.
• those skilled in the art can select suitably functionalized starting materials that can be derived from the compound of formulas (I - VII) using procedures that are known in the art.
• Such a linkage can be formed from suitably functionalized starting materials using synthetic procedures that are known in the art. Based on the linkage that is desired, those skilled in the art can select suitably functional starting material that can be derived from a residue of a compound of structural formula (I - VII) and from a given residue of a bioactive agent or adjuvant using procedures that are known in the art. The residue of the bioactive agent or adjuvant can be linked to any synthetically feasible position on the residue of a compound of structural formula (I - VII). Additionally, the invention also provides compounds having more than one residue of a bioactive agent or adjuvant bioactive agent directly linked to a compound of structural formula (I - VII).
• bioactive agents that can be linked to the polymer molecule can typically depend upon the molecular weight of the polymer. For example, for a compound of structural formula (I), wherein n is about 5 to about 150, preferably about 5 to about 70, up to about 150 bioactive agent molecules (i.e., residues thereof) can be directly linked to the polymer (i.e., residue thereof) by reacting the bioactive agent with side groups of the polymer. In unsaturated polymers, the bioactive agents can also be reacted with double (or triple) bonds in the polymer.
• Stents according to the invention are typically cylindrical in shape.
• the walls of the stent structure can be formed of metal or polymer with openings therein, e.g., a mesh.
• the stent is implanted into a body lumen, such as a blood vessel, where it stays permanently or biodegrades, to keep the vessel open and to improve blood flow to the heart muscle and promote natural wound healing processes at a location of damaged endothelium.
• Stents can also be positioned in vasculature in other parts of the body, such as the kidneys or the brain. The stenting procedure is fairly common, and various types of stents have been developed and used as is known in the art.
• the polymers described herein can be coated onto the surface of a porous stent structure or other medical device as described here in many ways, such as dip-coating, spray-coating, ionic deposition, and the like, as is well known in the art.
• care must be taken not to occlude the pores in the stent structure, which are needed to allow access and migration from the interior of the vessel to the vessel wall of blood borne progenitor endothelial cells and other blood factors that participate in the natural biological process of wound healing.
• the polymer coating on the surface of the stent structure can be a formed as a polymer sheath that is applied over the stent structure.
• the sheath serves as a partial physical barrier to macrophages so that a relatively small number of smooth muscle cells are activated to cause neointimal proliferation.
• the sheath can be laser ablated to form openings in the polymer coating.
• the stent structure can be moved while the laser is held stationary to ablate the structure into a pattern, or alternatively, the laser can be programmed to move along a predetermined pattern by a method known to artisans. A combination of both, i.e. moving both the laser and the structure, is also possible. In the present invention, even a coated stent having a complex stent pattern can be made with high precision.
• the stent structure can be formed of any suitable substance, such as is known in the art, that can be processed (e.g., molded, stamped, woven, etc.) to contain the porous surface features described herein.
• the stent body can be formed from a biocompatible metal, such as stainless steel, tantalum, nitinol, elgiloy, and the like, as well as suitable combinations thereof.
• metal stent structures can be formed of a material comprising metallic fibers uniformly laid to form a three-dimensional non- woven matrix and sintered to form a labyrinth structure exhibiting high porosity, typically in a range from about 50 percent to about 85 percent, preferably at least about 70 percent.
• the metal fibers typically have a diameter in the range from about 1 micron to 25 microns.
• Pores in the stent structure can have an average diameter in the range from about 30 microns to about 65 microns.
• the stent structure should be made of 100% stainless steel, with fully annealed stainless steel being a preferred metal.
• the stent structure can be of the type that is balloon expandable, as is known in the art.
• the stent structure is itself entirely biodegradable, being made of cross-linkable "star structure polymers", or dendrimers, which are well known to those skilled in the art.
• the stent structure is formed from biodegradable cross-linked poly(ester amide), polycaprolactone, or poly(ester urethane) as described herein.
• the stent structure i.e., the "stent struts" is preferably biodegradable and hence are made of such cross-linkable polymers or dendrimers.
• Residues of the polymers described herein can be formed employing any suitable reagents and reaction conditions.
• Suitable reagents and reaction conditions are disclosed, e.g., in Advanced Organic Chemistry, PartB: Reactions and Synthesis, Second Edition, Carey and Sundberg (1983); Advanced Organic Chemistry, Reactions, Mechanisms, and Structure, Second Edition, March (1977); and Comprehensive Organic Transformations, Second Edition, Larock (1999).
• an "additional bioactive agent” refers to a therapeutic or diagnostic agent other than the "bioactive" agents described above that promote the natural wound healing process of re-endothelialization of vessels as disclosed herein.
• additional bioactive agents can also be attached polymer coatings on the surface of the invention stents or to polymers used for coating other types of insertable or implantable medical or therapeutic devices having different treatment aims as are known in the art, wherein contact of the polymer coating with a treatment surface or blood borne cell or factor or release from the polymer coating by biodegradation is desirable.
• additional bioactive agents are not used in the inner layer of the invention multilayered stents, which contain only the bioactive agents that promote the natural would healing process of re-endothelialization of vessels.
• such additional bioactive agent can include, but is not limited to, one or more: polynucleotides, polypeptides, oligonucleotides, gene therapy agents, nucleotide analogs, nucleoside analogs, polynucleic acid decoys, therapeutic antibodies, abciximab, anti-inflammatory agents, blood modifiers, anti-platelet agents, anti- coagulation agents, immune suppressive agents, anti-neoplastic agents, anti-cancer agents, anti-cell proliferation agents, and nitric oxide releasing agents.
• the polynucleotide can include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), double stranded DNA, double stranded RNA, duplex DNA/RNA, antisense polynucleotides, functional RNA or a combination thereof.
• the polynucleotide can be RNA.
• the polynucleotide can be DNA.
• the polynucleotide can be an antisense polynucleotide.
• the polynucleotide can be a sense polynucleotide.
• the polynucleotide can include at least one nucleotide analog.
• the polynucleotide can include a phosphodiester linked 3'-5' and 5'-3' polynucleotide backbone.
• the polynucleotide can include non-phosphodiester linkages, such as phosphotioate type, phosphoramidate and peptide-nucleotide backbones.
• moieties can be linked to the backbone sugars of the polynucleotide. Methods of creating such linkages are well known to those of skill in the art.
• the polynucleotide can be a single-stranded polynucleotide or a double- stranded polynucleotide.
• the polynucleotide can have any suitable length. Specifically, the polynucleotide can be about 2 to about 5,000 nucleotides in length, inclusive; about 2 to about 1000 nucleotides in length, inclusive; about 2 to about 100 nucleotides in length, inclusive; or about 2 to about 10 nucleotides in length, inclusive.
• An antisense polynucleotide is typically a polynucleotide that is complimentary to an mRNA, which encodes a target protein.
• the mRNA can encode a cancer promoting protein i.e., the product of an oncogene.
• the antisense polynucleotide is complimentary to the single-stranded mRNA and will form a duplex and thereby inhibit expression of the target gene, i.e., will inhibit expression of the oncogene.
• the antisense polynucleotides of the invention can form a duplex with the mRNA encoding a target protein and will disallow expression of the target protein.
• a "functional RNA” refers to a ribozyme or other RNA that is not translated.
• a "polynucleic acid decoy” is a polynucleic acid that inhibits the activity of a cellular factor upon binding of the cellular factor to the polynucleic acid decoy.
• the polynucleic acid decoy contains the binding site for the cellular factor.
• cellular factors include, but are not limited to, transcription factors, polymerases and ribosomes.
• An example of a polynucleic acid decoy for use as a transcription factor decoy will be a double-stranded polynucleic acid containing the binding site for the transcription factor.
• the polynucleic acid decoy for a transcription factor can be a single-stranded nucleic acid that hybridizes to itself to form a snap-back duplex containing the binding site for the target transcription factor.
• An example of a transcription factor decoy is the E2F decoy.
• E2F plays a role in transcription of genes that are involved with cell-cycle regulation and that cause cells to proliferate. Controlling E2F allows regulation of cellular proliferation. For example, after injury (e.g., angioplasty, surgery, stenting) smooth muscle cells proliferate in response to the injury. Proliferation may cause restenosis of the treated area (closure of an artery through cellular proliferation).
• modulation of E2F activity allows control of cell proliferation and can be used to decrease proliferation and avoid closure of an artery.
• examples of other such polynucleic acid decoys and target proteins include, but are not limited to, promoter sequences for inhibiting polymerases and ribosome binding sequences for inhibiting ribosomes. It is understood that the invention includes polynucleic acid decoys constructed to inhibit any target cellular factor.
• a "gene therapy agent” refers to an agent that causes expression of a gene product in a target cell through introduction of a gene into the target cell followed by expression.
• a gene therapy agent would be a genetic construct that causes expression of a protein, such as insulin, when introduced into a cell.
• a gene therapy agent can decrease expression of a gene in a target cell.
• An example of such a gene therapy agent would be the introduction of a polynucleic acid segment into a cell that would integrate into a target gene and disrupt expression of the gene. Examples of such agents include viruses and polynucleotides that are able to disrupt a gene through homologous recombination. Methods of introducing and disrupting genes with cells are well known to those of skill in the art.
• An oligonucleotide of the invention can have any suitable length. Specifically, the oligonucleotide can be about 2 to about 100 nucleotides in length, inclusive; up to about 20 nucleotides in length, inclusive; or about 15 to about 30 nucleotides in length, inclusive.
• the oligonucleotide can be single-stranded or double-stranded. In one embodiment, the oligonucleotide can'be single-stranded.
• the oligonucleotide can be DNA or RNA. In one embodiment, the oligonucleotide can be DNA. In one embodiment, the oligonucleotide can be synthesized according to commonly known chemical methods.
• the oligonucleotide can be obtained from a commercial supplier.
• the oligonucleotide can include, but is not limited to, at least one nucleotide analog, such as bromo derivatives, azido derivatives, fluorescent derivatives or a combination thereof. Nucleotide analogs are well known to those of skill in the art.
• the oligonucleotide can include a chain terminator.
• the oligonucleotide can also be used, e.g., as a cross-linking reagent or a fluorescent tag. Many common linkages can be employed to couple an oligonucleotide to another moiety, e.g., phosphate, hydroxyl, etc.
• a moiety may be linked to the oligonucleotide through a nucleotide analog incorporated into the oligonucleotide.
• the oligonucleotide can include a phosphodiester linked 3'-5' and 5'-3' oligonucleotide backbone.
• the oligonucleotide can include non-phosphodiester linkages, such as phosphotioate type, phosphoramidate and peptide-nucleotide backbones.
• moieties can be linked to the backbone sugars of the oligonucleotide. Methods of creating such linkages are well known to those of skill in the art.
• Nucleotide and nucleoside analogues are well known on the art.
• nucleoside analogs include, but are not limited to, Cytovene® (Roche Laboratories), Epivir® (Glaxo Wellcome), Gemzar® (Lilly), Hivid® (Roche Laboratories), Rebetron® (Schering), Videx® (Bristol-Myers Squibb), Zerit® (Bristol-Myers Squibb), and Zovirax® (Glaxo Wellcome). See, Physician 's Desk Reference, 2005 Edition.
• Polypeptides acting as additional bioactive agents attached to the polymers in the invention stent coverings and other medical devices can have any suitable length. Specifically, the polypeptides can be about 2 to about 5,000 amino acids in length, inclusive; about 2 to about 2,000 amino acids in length, inclusive; about 2 to about 1,000 amino acids in length, inclusive; or about 2 to about 100 amino acids in length, inclusive.
• the additional bioactive agent polypeptide attached to the polymer coatings for the invention medical devices can be an antibody.
• the antibody can bind to a cell adhesion molecule, such as a cadherin, integrin or selectin.
• the antibody can bind to an extracellular matrix molecule, such as collagen, elastin, fibronectin or laminin.
• the antibody can bind to a receptor, such as an adrenergic receptor, B-cell receptor, complement receptor, cholinergic receptor, estrogen receptor, insulin receptor, low-density lipoprotein receptor, growth factor receptor or T-cell receptor.
• Antibodies attached to polymers (either directly or by a linker) in the invention medical devices can also bind to platelet aggregation factors (e.g., fibrinogen), cell proliferation factors (e.g., growth factors and cytokines), and blood clotting factors (e.g., fibrinogen).
• an antibody can be conjugated to an active agent, such as a toxin.
• the antibody can be Abciximab (ReoProR)).
• Abciximab is an Fab fragment of a chimeric antibody that binds to beta(3) integrins.
• Abciximab is specific for platelet glycoprotein Ilb/IIIa receptors, e.g., on blood cells.
• Human aortic smooth muscle cells express alpha(v)beta(3) integrins on their surface. Treating beta(3) expressing smooth muscle cells may prohibit adhesion of other cells and decrease cellular migration or proliferation, thus reducing restenosis following percutaneous coronary interventions (CPI) e.g., stenosis, angioplasty, stenting.
• CPI percutaneous coronary interventions
• Abciximab also inhibits aggregation of blood platelets.
• the peptide can be a glycopeptide.
• glycopeptide refers to oligopeptide (e.g. heptapeptide) antibiotics, characterized by a multi-ring peptide core optionally substituted with saccharide groups, such as vancomycin. Examples of glycopeptides included in this definition may be found in "Glycopeptides Classification, Occurrence, and Discovery," by Raymond C. Rao and Louise W. Crandall, ("Bioactive agents and the Pha ⁇ naceutical Sciences” Volume 63, edited by Ramakrishnan Nagarajan, published by Marcal Dekker, Inc.). Additional examples of glycopeptides are disclosed in U.S. Patent Nos.
• glycopeptides include those identified as A477, A35512, A40926, A41030, A42867, A47934, A80407, A82846, A83850, A84575, AB-65, Actaplanin, Actinoidin, Ardacin, Avoparcin, Azureomycin, Balhimyein, Chloroorientiein, Chloropolysporin, Decaplanin, -demethylvancomycin, Eremomycin, Galacardin, Helvecardin, Izupeptin, Kibdelin, LL-AM374, Mannopeptin, MM45289, MM47756, MM47761, MM49721, MM47766, MM55260, MM55266, MM55270, MM56597, MM56598, OA-7653, Orenticin, Parvodicin, Ristocetin, Ristomycin, Synmonicin, Teicoplanin, UK-68597, UD-69542, UK-720
• glycopeptide or "glycopeptide antibiotic” as used herein is also intended to include the general class of glycopeptides disclosed above on which the sugar moiety is absent, i.e. the aglycone series of glycopeptides. For example, removal of the disaccharide moiety appended to the phenol on vancomycin by mild hydrolysis gives vancomycin aglycone. Also included within the scope of the term “glycopeptide antibiotics" are synthetic derivatives of the general class of glycopeptides disclosed above, included alkylated and acylated derivatives.
• glycopeptides that have been further appended with additional saccharide residues, especially aminoglycosides, in a manner similar to vancosamine.
• lipidated glycopeptide refers specifically to those glycopeptide antibiotics that have been synthetically modified to contain a lipid substituent.
• lipid substituent refers to any substituent contains 5 or more carbon atoms, preferably, 10 to 40 carbon atoms.
• the lipid substituent may optionally contain from 1 to 6 heteroatoms selected from halo, oxygen, nitrogen, sulfur, and phosphorous. Lipidated glycopeptide antibiotics are well known in the art. See, for example, in U.S. Patent Nos.
• Anti-inflammatory agents useful for attachment to polymer coatings of the invention stents and other medical devices, or for loading into the outer layer of the invention multilayered stents include, e.g. analgesics (e.g., NSAIDS and salicyclates), antirheumatic agents, gastrointestinal agents, gout preparations, hormones (glucocorticoids), nasal preparations, ophthalmic preparations, otic preparations (e.g., antibiotic and steroid combinations), respiratory agents, and skin & mucous membrane agents. See, Physician 's Desk Reference, 2005 Edition.
• the anti-inflammatory agent can include dexamethasone, which is chemically designated as (110, 16I)-9-fluro- 11,17,21 -trihydroxy- 16-methylpregna- 1 ,4-diene-3 ,20-dione.
• the anti-inflammatory agent can include sirolimus (rapamycin), which is a triene macrolide antibiotic isolated from Steptomyces hygroscopicus.
• Anti-platelet or anti-coagulation agents include, e.g., Coumadin® (DuPont), Fragmin® (Pharmacia & Upjohn), Heparin® (Wyeth-Ayerst), Lovenox®, Normiflo®, Orgaran ® (Organon), Aggrastat® (Merck), Agrylin® (Roberts), Ecotrin® (Smithkline Beecham), Flolan® (Glaxo Wellcome), Halfprin® (Kramer), Integrillin® (COR Therapeutics), Integrillin® (Key), Persantine® (Boehringer Ingelheim), Plavix® (Bristol- Myers Squibb), ReoPro® (Centecor), Ticlid® (Roche), Abbokinase® (Abbott), Activase® (Genentech), Eminase® (Roberts), and Strepase® (Astra). See, Physician 's Desk Reference, 2005 Edition. Specifically,
• Trapidil is chemically designated as N,N-dimethyl-5-methyl-[ 1 ,2,4]triazolo[ 1 ,- 5-a]pyrimidin-7-amine.
• Cilostazol is chemically designated as 6-[4-(l -cyclohexyl-lH-tetrazol-5-yl)- butoxy]-3,4-dihydro-2(lH)-quinolinone.
• Heparin is a glycosaminoglycan with anticoagulant activity; a heterogeneous mixture of variably sulfonated polysaccharide chains composed of repeating units of D- glucosamine and either L-iduronic or D-glucuronic acids.
• Hirudin is an anticoagulant protein extracted from leeches, e.g., Hirudo medicinalis.
• Iloprost is chemically designated as 5-[Hexahydro-5-hydroxy-4-(3-hydroxy-4- methyl- 1 -octen-6-ynyl)-2( 1 H)-pentalenylidene]pentanoic acid.
• the immune suppressive agent can include, e.g., Azathioprine® (Roxane), BayRbo-D® (Bayer Biological), CellCept® (Roche Laboratories), Imuran® (Glaxo Wellcome), MiCRhoGAM® (Ortho-Clinical Diagnostics), Neoran® (Novartis), Orthoclone OKT3® (Ortho Biotech), Prograf® (Fujisawa), PhoGAM® (Ortho-Clinical Diagnostics), Sandimmune® (Novartis), Simulect® (Novartis), and Zenapax® (Roche Laboratories).
• the immune suppressive agent can include rapamycin or thalidomide.
• Rapamycin is a triene macrolide isolated from Streptomyces hygroscopicus.
• Thalidomide is chemically designated as 2-(2,6-dioxo-3-piperidinyl)-lH-iso- indole-l,3(2H)-dione.
• Anti-cancer or anti-cell proliferation agents that can be used as an additional bioactive agent, for example, in the outer layer of the invention multilayered stents include, e.g., nucleotide and nucleoside analogs, such as 2-chloro-deoxyadenosine, adjunct antineoplastic agents, alkylating agents, nitrogen mustards, nitrosoureas, antibiotics, antimetabolites, hormonal agonists/antagonists, androgens, antiandrogens, antiestrogens, estrogen & nitrogen mustard combinations, gonadotropin releasing hormone (GNRH) analogues, progestrins, immunomodulators, miscellaneous antineoplastics, photosensitizing agents, and skin and mucous membrane agents.
• nucleotide and nucleoside analogs such as 2-chloro-deoxyadenosine, adjunct antineoplastic agents, alkylating agents, nitrogen mustards, nitrosoureas, antibiotics, antimetabolites, hormonal
• adjunct antineoplastic agents include Anzemet® (Hoeschst Marion Roussel), Aredia® (Novartis), Didronel® (MGI), Diflucan® (Pfizer), Epogen® (Amgen), Ergamisol® (Janssen), Ethyol® (Alza), Kytril® (SmithKline Beecham), Leucovorin® (hnmunex), Leucovorin® (Glaxo Wellcome), Leucovorin® (Astra), Leukine® (Immunex), Marinol® (Roxane), Mesnex® (Bristol-Myers Squibb Oncology/Immunology), Neupogen (Amgen), Procrit® (Ortho Biotech), Salagen® (MGI), Sandostatin® (Novartis), Zinecard® (Pharmacia and Upjohn), Zofran® (Glaxo Wellcome) and Zyloprim® (
• Suitable miscellaneous alkylating agents include Myleran® (Glaxo Wellcome), Paraplatin® (Bristol-Myers Squibb Oncology/Immunology), Platinol® (Bristol-Myers Squibb Oncology/Immunology) and Thioplex® (Immunex).
• Suitable nitrogen mustards include Alkeran® (Glaxo Wellcome), Cytoxan® (Bristol-Myers Squibb Oncology/Immunology), Ifex® (Bristol-Myers Squibb Oncology/Immunology), Leukeran® (Glaxo Wellcome) and Mustargen® (Merck).
• Suitable nitrosoureas include BiCNU® (Bristol-Myers Squibb Oncology/Immunology), CeeNU® (Bristol-Myers Squibb Oncology/Immunology), Gliadel® (Rhone-Poulenc Rover) and Zanosar® (Pharmacia and Upjohn).
• Suitable antimetabolites include Cytostar-U® (Pharmacia and Upjohn), Fludara® (Berlex), Sterile FUDR® (Roche Laboratories), Leustatin® (Ortho Biotech), Methotrexate® (Immunex), Parinethol® (Glaxo Wellcome), Thioguanine® (Glaxo Wellcome) and Xeloda® (Roche Laboratories).
• Suitable androgens include Nilandron® (Hoechst Marion Roussel) and Teslac® (Bristol-Myers Squibb Oncology/Immunology).
• Suitable antiandrogens include Casodex® (Zeneca) and Eulexin® (Schering).
• Suitable antiestrogens include Arimidex® (Zeneca), Fareston® (Schering), Femara® (Novartis) and Nolvadex® (Zeneca).
• Suitable estrogen and nitrogen mustard combinations include Emcyt® (Pharmacia and Upjohn).
• Suitable estrogens include Estrace® (Bristol-Myers Squibb) and Estrab® (Solvay).
• Suitable gonadotropin releasing hormone (GNRH) analogues include Leupron Depot® (TAP) and Zoladex® (Zeneca).
• Suitable progestins include Depo-Provera® (Pharmacia and Upjohn) and Megace® (Bristol-Myers Squibb Oncology/Immunology).
• Suitable immunomodulators include Erganisol® (Janssen) and Proleukin® (Chiron Corporation).
• Suitable miscellaneous antineoplastics include Camptosar® (Pharmacia and Upjohn), Celestone® (Schering), DTIC-Dome® (Bayer), Elspar® (Merck), Etopophos® (Bristol-Myers Squibb Oncology/Immunology), Etopoxide® (Astra), Gemzar® (Lilly), Hexalen® (U.S.
• Suitable photosensitizing agents include Photofrin® (Sanofi).
• the anti-cancer or anti-cell proliferation agent can include Taxol® (paclitaxol), a nitric oxide-like compound, orNicOX (NCX-4016).
• Taxol® paclitaxol
• NCX-4016 a nitric oxide-like compound
• Taxol® paclitaxol
• NicOX NicOX
• Taxol® is chemically designated as 5 ⁇ ,20-Epoxy-l,2 ⁇ 4,7 ⁇ ,10 ⁇ ,13 ⁇ -hexahydroxytax-l l-en-9-one 4,10-diacetate 2-benzoate 13-ester with (2R,3S)-N-benzoyl-3-phenylisoserine.
• a nitric oxide-like agent includes any bioactive agent that contains a nitric oxide releasing functional group.
• Suitable nitric oxide-like compounds are S-nitrosothiol derivative (adduct) of bovine or human serum albumin and as disclosed, e.g., in U.S. Patent No. 5,650,447. See, e.g., David Marks et al., "Inhibition of neointimal proliferation in rabbits after vascular injury by a single treatment with a protein adduct of nitric oxide," J Clin. Invest.( ⁇ 995) 96:2630-2638.
• NCX-4016 is chemically designated as 2-acetoxy-benzoate 2-(nitroxymethyl)-phenyl ester, and is an antithrombotic agent.
• bioactive agent or additional bioactive agent useful in the present invention is the bioactive substance present in any of the bioactive agents or agents disclosed above.
• Taxol® is typically available as an injectable, slightly yellow viscous solution.
• the bioactive agent is a crystalline powder with the chemical name 5 ⁇ ,20-Epoxy- l,2 ⁇ ,4,7 ⁇ ,10 ⁇ ,13 ⁇ -hexahydroxytax-l l-en-9-one 4,10-diacetate 2-benzoate 13-ester with (2R,3S)-N-benzoyl-3-phenylisoserine. Physician 's Desk Reference (PDR), Medical Economics Company (Montvale, NJ), (53rd Ed.), pp. 1059-1067.
• a "residue of a bioactive agent” or “residue of an additional bioactive agent” is a radical of such bioactive agent as disclosed herein having one or more open valences. Any synthetically feasible atom or atoms of the bioactive agent can be removed to provide the open valence, provided bioactivity is substantially retained when the radical is attached to a residue of a polymer described herein. Based on the linkage that is desired, those skilled in the art can select suitably functionalized starting materials that can be derived from a bioactive agent using procedures that are known in the art. [0186] The residue of a bioactive agent or additional bioactive agent, as described herein, can be formed employing any suitable reagents and reaction conditions.
• Suitable reagents and reaction conditions are disclosed, e.g., in Advanced Organic Chemistry, Part B: Reactions and Synthesis, Second Edition, Carey and Sundberg (1983); Advanced Organic Chemistry, Reactions, Mechanisms and Structure, Second Edition, March (1977); and Comprehensive Organic Transformations, Second Edition, Larock (1999).
• the polymer-bioactive agent linkage can degrade to provide a suitable and effective amount of free bioactive agent.
• the bioactive agent attached to the polymer performs its therapeutic effect while still attached to the polymer, such as is the case with the "sticky" polypeptides Protein A and Protein G, known herein as "bioligands", which function while attached to the polymer to hold a target molecule close to the polymer, and the bradykinins and antibodies, which function by contacting (e.g., bumping into) a receptor on a target molecule.
• bioactive agent can be released and will typically depend, e.g., on the specific polymer, bioactive agent, and polymer/bioactive agent linkage chosen. Typically, up to about 100% of the bioactive agent can be released from the polymer by degradation of the polymer/bioactive agent linkage. Specifically, up to about 90%, up to 75%, up to 50%, or up to 25% of the bioactive agent can be released from the polymer. Factors that typically affect the amount of the bioactive agent that is released from the polymer is the type of polymer/bioactive agent linkage, and the nature and amount of additional substances present in the formulation.
• the polymer-bioactive agent linkage can degrade over a period of time to provide time release of a suitable and effective amount of bioactive agent. Any suitable and effective period of time can be chosen. Typically, the suitable and effective amount of bioactive agent can be released in about twenty-four hours, in about seven days, in about thirty days, in about ninety days, or in about one hundred and twenty days. Factors that typically affect the length of time in which the bioactive agent is released from the polymer-bioactive agent include, e.g., the nature and amount of polymer, the nature and amount of bioactive agent, the nature of the polymer/bioactive agent linkage, and the nature and amount of additional substances present in the formulation.
• a polymer used for coating a medical device or making a sheath for a stent structure as described herein can be physically intermixed with one or more bioactive agents or additional bioactive agents to provide a polymer formulation that is used for coating a medical device or a stent structure.
• mixtureed refers to a polymer of the present invention physically mixed with a bioactive agent or a polymer as described herein that is physically in contact with a bioactive agent.
• a "formulation” refers to a polymer as described herein that is intermixed with one or more bioactive agents or additional bioactive agents.
• the formulation includes such a polymer having one or more bioactive agents present on the surface of the polymer, partially embedded in the polymer, or completely embedded in the polymer. Additionally, the formulation includes a polymer as described herein and a bioactive agent forming a homogeneous composition (i.e., a homogeneous formulation).
• bioactive agents and/or additional bioactive agents can be intermingled with or "loaded into” any biocompatible biodegradable polymer as is known in the art since the outer layer in this embodiment of the invention does not come into contact with blood.
• the inner layer has only bioactive agents covalently attached to a hydrophilic, blood-compatible polymer as described herein.
• any suitable amount of polymer and bioactive agent can be employed to provide the formulation.
• the polymer can be present in about 0.1 wt.% to about 99.9 wt.% of the formulation.
• the polymer can be present above about 25 wt.% of the formulation; above about 50 wt.% of the formulation; above about 75 wt.% % of the formulation; or above about 90 wt.% of the formulation.
• the bioactive agent can be present in about 0.1 wt.% to about 99.9 wt.% of the formulation.
• the bioactive agent can be present above about 5 wt.% of the formulation; above about 10 wt.% of the formulation; above about 15 wt.% of the formulation; or above about 20 wt.% of the formulation.
• the polymer coating having a bioactive agent dispersed therein can be applied as a polymeric film onto at least a portion of the surface of any medical device to be implanted into a diabetic that is exposed to blood and upon which it is desirable to establish an endothelial layer (e.g., a heart valve, or a synthetic bypass artery).
• the polymeric film can have any suitable thickness on the medical device.
• the thickness of the polymeric film on the medical device can be about 1 to about 50 microns thick or about 5 to about 20 microns thick.
• each of the layers can be from 0.1 micron to 50 microns thick, for example from 0.5 micron to 5 microns in thickness.
• the polymeric film can effectively serve as a bioactive agent-eluting polymeric coating on a medical device, such as a stent structure.
• This bioactive agent eluting polymeric coating can be created on the medical device by any suitable coating process, e.g., dip coating, vacuum depositing, or spray coating the polymeric film, on the medical device.
• the bioactive agent eluting polymer coating system can be applied onto the surface of a stent, a vascular delivery catheter, a delivery balloon, a separate stent cover sheet configuration, or a stent bioactive agent delivery sheath, as described herein to create a type of local bioactive agent delivery system.
• the polymer When the polymer is used as a cover sheet for a stent, the polymer can be processed, for example by extrusion or spinning as is known in the art, to form a woven sheet or mat of fine polymer fibers to which the bioligand is covalently attached, either directly or by means of a linker, as described herein.
• the bioactive agent-eluting polymer coated stents and other medical devices cari be used in conjunction with, e.g., hydrogel-based bioactive agent delivery systems.
• the above-described polymer coated stents and medical devices can be coated with an additional formulation layer applied over the polymer coated stent surface as a sandwich type of configuration to deliver to the blood vessels bioactive agents that promote natural re-endothelialization processes and prevent or reduce in-stent restenosis.
• Such an additional layer of hydrogel-based drug release formulation can comprise various bioactive agents mixed with hydrogels (see, U.S. Patent No.
• any suitable size of polymer and bioactive agent can be employed to provide such a formulation.
• the polymer can have a size of less than about 1 x 10 "4 meters, less than about 1 x 10 "5 meters, less than about 1 x 10 "6 meters, less than about 1 x 10 "7 meters, less than about 1 x 10 "8 meters, or less than about 1 x 10 "9 meters.
• the formulation can degrade to provide a suitable and effective amount of the bioactive agents. Any suitable and effective amount of bioactive agent can be released and will typically depend, e.g., on the specific formulation chosen. Typically, up to about 100% of the bioactive agent can be released from the formulation. Specifically, up to about 90%, up to 75%, up to 50%, or up to 25% of the bioactive agent can be released from the formulation. Factors that typically affect the amount of the bioactive agent that is released from the formulation include, e.g., the nature and amount of polymer, the nature and amount of bioactive agent, and the nature and amount of additional substances present in the formulation.
• the formulation can degrade over a period of time to provide the suitable and effective amount of bioactive agent. Any suitable and effective period of time can be chosen. Typically, the suitable and effective amount of bioactive agent can be released in about twenty-four hours, in about seven days, in about thirty days, in about ninety days, or in about one hundred and twenty days. Factors that typically affect the length of time in which the bioactive agent is released from the formulation include, e.g., the nature and amount of polymer, the nature and amount of bioactive agent, and the nature and amount of additional substances present in the formulation.
• the present invention also provides for an invention stent coated with a formulation that includes a polymer as described herein physically intermixed with one or more bioactive agents.
• the polymer that is present in the formulation can also be linked, either directly or through a linker, to one or more (e.g., 1, 2, 3, or 4) bioactive agents.
• the polymer can be intermixed with one or more (e.g., 1, 2, 3, or 4) bioactive agents and can be linked, either directly or through a linker, to one or more (e.g., 1 , 2, 3, or 4) bioactive agents.
• a polymer used in making an invention stent can include one or more bioactive agents.
• the polymer is physically intermixed with one or more bioactive agents.
• the polymer is linked to one or more bioactive agents, either directly or through a linker.
• the polymer is linked to one or more bioactive agents, either directly or through a linker, and the resulting polymer can also be physically intermixed with one or more bioactive agents.
• a polymer used in making an invention stent whether or not present in a formulation as described herein, whether or not linked to a bioactive agent as described herein, and whether or not intermixed with a bioactive agent as described herein, can also be used in medical therapy or medical diagnosis.
• the polymer can be used in the manufacture of a medical device.
• Suitable medical devices include, e.g., artificial joints, artificial bones, cardiovascular medical devices, stents, shunts, medical devices useful in angioplastic therapy, artificial heart valves, artificial by-passes, sutures, artificial arteries, vascular delivery catheters, drug delivery balloons, separate tubular stent cover sheet configurations (referred to herein as "sheaths”), and stent bioactive agent delivery sleeve types for local bioactive agent delivery systems.
• sheaths separate tubular stent cover sheet configurations
• the invention provides methods for treating a patient suffering from diabetes having a vessel with a damaged endothelium by implanting an invention stent in the vessel at the locus of damage and allowing the stent to interact with blood components within the vessel.
• the invention may further comprise testing a blood sample from the diabetic patient to determine the amount of therapeutic PECs in the sample as compared with a parallel sample of blood from a healthy non- diabetic individual to detect a decrease in the amount of therapeutic PECs in the blood from the diabetic patient. Such testing may be conducted prior to implantation of an invention stent to determine whether the diabetic patient has a decreased amount or concentration of therapeutic PECs as compared with the normal concentration in healthy non-diabetic patients.
• the invention methods for treating diabetics having damaged vasculature may further comprise obtaining therapeutic PECs from the circulating blood of the diabetic, expanding the patient's therapeutic PECs ex vivo, and transfusing the autologous PECs into the circulating blood of the diabetic patient either before or contemporaneously with implantation of the invention stents.
• the PEA-OSu product may be isolated by precipitation, or used without further purification, in which case the PEA-OSu solution is transferred to a round bottom flask, diluted to the desired concentration, and cooled to 0°C.
• a solution of the free amine-containing bioactive agent — the nucleophile, specifically, 4-Amino- Tempo ⁇ in CH 2 C1 2 is added in a single shot at 0°C.
• the nucleophile may be revealed in situ by treating the ammonium salt of the bioactive agent with a hindered base, preferably a tertiary amine, such as like triethylamine or, diisopropylethylamine, in a suitable aprotic solvent, such as dichloromethane (DCM)).
• a hindered base preferably a tertiary amine, such as like triethylamine or, diisopropylethylamine
• a suitable aprotic solvent such as dichloromethane (DCM)
• DCM dichloromethane
• TLC aprotic solvent
• Work-up for the polymer involves customary precipitation of the reaction solution into a mixture of non-solvent, such as hexane/ethyl acetate.
• Solvent is then decanted, polymer residue is resuspended in a suitable solvent, filtered, concentrated by roto-evaporation, cast onto a clean teflon tray, and dried under vacuum to furnish the PEA-bioactive agent conjugate, specifically, PEA-4-Amino-Tempo.
• Aminium/Uronium Salt and Phosphonium Salt Mediated Couplings Two effective catalysts for this type of coupling include: HBTU, O-(benzotriazol-l-yl)-l,l,3,3- teramethyluronium hexafluorophosphate, and BOP, l-benzotriazolyoxytris(dimethyl- amino)phosphonium hexafluorophosphate (Castro's Reagent).
• reagents are employed in the presence of equimolar amounts of the carboxyl group of the polymer and the amino functional group of the bioactive agent (neutral or as the ammonium salt), with a tertiary amine such as diisopropylethylamine, N-methylmorpholine, or dimethyl- substituted pyridines (DMAP), in solvents such as DMF, THF, or acetonitrile.
• a tertiary amine such as diisopropylethylamine, N-methylmorpholine, or dimethyl- substituted pyridines (DMAP)
• solvents such as DMF, THF, or acetonitrile.
• Ester Bond Formation - This example illustrates coupling of a carboxyl group of a polymer with a hydroxyl functional group of the bioactive agent, or equally, coupling of a carboxyl group of the bioactive agent with a hydroxyl functional group of a polymer.
• PEC Isolations To establish the protocol for isolating the progenitor endothelial cells (PECs) from peripheral blood, blood from healthy, normal donors was used.
• a literature review generated multiple PEC isolation protocols (J. C.I. (2000) 105:71-77 ; Ore. (2003) 107:143-149; Ore. (2003) 107:1164-1169; Plast. Reconstruc. Surgr. (2004) 113:284; and Am. J. Physiol. Heart Ore. Physiol. (2004) 286:H1985- HI 993).
• preliminary attempts required modification of the known protocols to ensure successful isolations.
• the flow chart in Fig. 2 presents a modified protocol followed in isolation of PECs.
• Table 1 below indicates the isolation methods and the PEC isolation outcome for PEC isolation from various donors. Initially, both a mononuclear cell Ficoll gradient protocol (designed to isolate human mononuclear cells from peripheral blood) and a CD 133+ magnetic bead purification step were used to ensure the isolation of PECs. It did not appear that the CD 133+ purification step was increasing the isolation of PECs, so this step was omitted from the last two donors.
• Cells were plated either in 12-well or 6-well fibronectin-coated plates and monitored daily, over a span of about 28-30 days.
• the culture media used in the PEC isolations was Endothelial Basal Medium plus SingleQuot Kit (Cambrex Corporation, East Rutherford, New Jersey), a mixture of hydrocortisone, hEGF, FBS, VEGF, hFGF-B, R3-IGF-1, ascorbic acid and heparin. Generally from 10-15 days in culture after the isolation were required before a monolayer became apparent.
• LDL Dil- acetylated-Low Density Lipoprotein
• the human LDL complex delivers cholesterol to cells via receptor-mediated endocytosis.
• the acetylated form of LDL is not taken up by the LDL receptor, but is taken up by macrophages and endothelial cells via a "scavenger" receptor specific for the modified LDL.
• Decreased uptake by endothelial cells as compared with macrophages was determined by microscope and photographed (lOOx magnification). The monolayer remains actively growing for a few months. Cells were replated and reformed the monolayer for several passages (about 30 days in culture) before becoming senescent.
• Fig. 3 shows the flow chart of the protocol followed for this assay.
• the attachment factor in a phosphate buffered saline (PBS) solution, was coated onto a non- tissue culture dish and allowed to adsorb overnight at 4°C. The following day the plate was blocked for 1 hour at room temperature with heat-inactivated, 0.2% bovine serum albumin (BSA) solution (in PBS) to prevent non-specific attachment.
• BSA bovine serum albumin
• a timed adhesion assay was then conducted.
• the assay includes negative control wells coated only with PBS and positive control wells coated with fibronectin. So far, none of the adhesion factors tested has surpassed the cell adhesion and cell spreading induced by fibronectin. In addition to adhesion, spreading is also an important consideration in determining the suitability of a substrate. If the cells are not able to spread, it is unlikely that the cells will proliferate on that surface.
• the biodegradable polymer is a poly(ester amide) (PEA) containing Lysine residues
• the carboxyl groups from the Lysine residues can be used to react with a complementary moiety on the peptide, such as an hydroxy, amino, thio moiety, and the like(5).
• the PEA-H polymer with free COOH reacts with water soluble carbodiimide (WSC) and N-Hydroxysuccinimide (HOSu) to produce an activated ester, which, in turn, reacts with an amino functional group of a peptide to provide an amide linkage (Fig. 6B).
• WSC water soluble carbodiimide
• HOSu N-Hydroxysuccinimide
• Fig. 6B By using a fluorescent dansyl- lysine (Fig. 5), the optimal reaction conditions for activation and conjugation were determined (Fig. 6A).

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