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WO2009002811A2 - Compositions thérapeutique plaquette et procédés - Google Patents

Compositions thérapeutique plaquette et procédés Download PDF

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Publication number
WO2009002811A2
WO2009002811A2 PCT/US2008/067592 US2008067592W WO2009002811A2 WO 2009002811 A2 WO2009002811 A2 WO 2009002811A2 US 2008067592 W US2008067592 W US 2008067592W WO 2009002811 A2 WO2009002811 A2 WO 2009002811A2
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Prior art keywords
platelets
wound
factor
angiogenesis
platelet
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WO2009002811A3 (fr
Inventor
Dennis Orgill
Giorgio Pietramaggiori
Giannoula Klement
David Cervi
Judah Folkman
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Brigham and Womens Hospital Inc
Boston Childrens Hospital
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Brigham and Womens Hospital Inc
Boston Childrens Hospital
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/19—Platelets; Megacaryocytes
    • 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
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0057—Ingredients of undetermined constitution or reaction products thereof
    • 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
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/0066—Medicaments; Biocides
    • 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
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/008—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors
    • 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
    • A61L2300/64—Animal cells

Definitions

  • angiogenesis is essential to initiate and support the repair process. New, small blood vessels form in the granulation tissue, and are a hallmark of tissue healing. Once healing has ceased, endothelial cells in the wound bed revert to a quiescent phenotype, demonstrating that timely switching between pro- and anti-angiogenesis is necessary to initiate and terminate the healing process, respectively 1 .
  • Circulating blood components such as platelets and leukocytes interact with activated endothelium and exposed collagen matrix; environments present in any typical wound. It is in this conducive environment that platelets are found in high concentrations to orchestrate wound healing.
  • 5"8 Recently, a biochemical 'cross-talk' between platelets and tumor cells has been described whereby cancer cells have the capacity to alter the levels of platelet consituitents, 5 primarily angiogenenic factors. Overall, tumor angiogenesis and meta formation may be governed, in part, by the presentation of factors by platelets.
  • angiogenesis regulating proteins carried by platelets are present at different times during wound healing, and have been shown to be stored in separate (pro- and antiangiogenic) alpha-granule compartments in the platelets cytoplasm.
  • Evidence indicates that a protease activated receptors facilitates a selective release of pro- or anti-angiogenic factors from the platelets.
  • the separate release of inhibitors and stimulators of angiogenesis is thought to a timely and orchestrated release of individual proteins needed for angiogenesis, rather than a single bolus delivery of all proteins contained in platelets as would be consistent with platelet degranulation. Clotted or frozen platelets do not have this same effect.
  • platelet levels of growth factors and cytokines are also variable 46"49 .
  • the concentration of some angiogenic growth factors, such as VEGF, bFGF, PDGF and PF-4, in platelets (but not in plasma) changes as a function of the stage of tumor growth 46 ' 48> 49 .
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • HGF hepatocyte growth factor
  • ANG-I angiopoietin-1
  • IGF- 1 insulin-like growth factor- 1
  • EGF epidermal growth factor
  • PDGF platelet derived growth factor
  • 11 VEGF is one of the most studied angiogenic factors. Depending on the particular isoform present in the blood stream, which governs the degree of heparin-binding affinity, VEGF may be presented or concentrated at the tumor site via platelets.
  • Platelets also harbor inhibitors of angiogenesis, including platelet factor-4 (PF-4), thrombospondin-1 (TSP-I), transforming growth factor beta-1 (TGF-betal), plasminogen activator inhibitor type-1 (tPA), alpha 2 - antiplasmin and alpha 2 -macroglobulin. All have been shown to participate in wound healing in healing-impaired animal models and in human, chronic wounds. 14 ' 13> 14 Thus, the use of platelet concentrates as a treatment option of recalcitrant wounds has become common practice in surgery.
  • PF-4 platelet factor-4
  • TSP-I thrombospondin-1
  • TGF-betal transforming growth factor beta-1
  • tPA plasminogen activator inhibitor type-1
  • alpha 2 - antiplasmin alpha 2 -macroglobulin.
  • platelet concentrate includes autologous platelet gels (APG), or platelet preparations prepared from the patient's own blood (see, e.g., Horn et al., 2007, Arch. Facial Plast. Surg. 9: 174-183).
  • APG autologous platelet gels
  • platelet preparations prepared from the patient's own blood see, e.g., Horn et al., 2007, Arch. Facial Plast. Surg. 9: 174-183.
  • Pro- and anti-angiogenic growth factors and cytokines are required to modulate vascular growth, but they may not be sufficient for sustaining angiogenesis.
  • platelets were made dysfunctional growing blood vessels exhibited moderate maturation and functionality.
  • previous work corroborates this finding, because the use of recombinant growth factors or sonicated platelets on experimental full-thickness diabetic wounds did not match up with the pro-angiogenic properties of intact platelets. Platelet open canallicular system and the integrity of the membranes may be the gate controlling uptake and delivery of the various proteins that regulate angiogenesis.
  • Ma et al. describe the involvement of protease activated receptors PAR-I and PAR-4 in the counter-regulation of the release of VEGF and endostatin from human platelets (Ma et al., 2005, Proc. Natl. Acad. Sci. U.S.A. 102: 216-220). Specifically, Ma et al. teach that human platelets contain endostatin, and that its release can be triggered by activation of PAR- 4 but not PAR-I. Ma et al. also teach that PAR-I activation leads to the suppression of endostatin release but also to stimulation of the release of the proangiogenic factor VEGF, while PAR-4 activation stimulates endostatin release and suppresses release of VEGF. Ma et al. conclude that PAR-I and PAR-4 appear to act in a counter-regulatory manner to modulate release of factors regulating angiogenesis.
  • platelets can be used to deliver exogenously added angiogenesis regulating factors directly to a wound site to promote healing at that site.
  • a composition for promoting wound healing comprising platelets comprising an exogenous angiogenesis promoting factor and a pharmaceutically acceptable carrier.
  • the platelets are autologous to the individual.
  • the angiogenesis regulating factor is selected, for example, from the group consisting of agents that activate the VEGF pathway, agents that activate the neuropilin 1 & 2 pathways, VEGF-A and C, bFGF, HGF, angiopoietin-1, insulin-like growth factor- 1, epidermal growth factor, platelet derived growth factor, platelet factor 4, thrombospondin-1, TGF-beta-1, plasminogen activator inhibitor type-1 (PAI-I, alpha2-antiplasmin and alpha2-macroglobulin VEGFRl (flt- 1), VEGFR2 (flk-2), VEGFR3 (flt-4), heparin sulfate proteoglycan, VEGF121, VEGF145, VEGF165, VEGF168, VEGF
  • the wound is selected, for example, from a burn, an incision, a laceration, an abrasion, an ulcer, a diabetic wound, a venous stasis wound, a vascular wound, a radiation wound, a steroid wound or a bony defect.
  • the wound is a diabetic ulcer or a decubitus ulcer.
  • the method further comprises the step of loading the platelets with the exogenous angiogenesis regulating factor.
  • the loading comprises contacting platelets with the factor.
  • the platelets comprise at least two angiogenesis promoting factors.
  • the composition comprises a platelet gel comprising the platelets and the angiogenesis regulating factor.
  • the gel can be formed by contacting the platelets with calcium and a platelet activating agent.
  • the platelet activating agent can be selected, for example, from the group consisting of thrombin, collagen, serotonin, ADP, acetylcholine and combinations thereof.
  • the angiogenesis promoting factor comprises a PAR-I agonist.
  • the PAR-I agonist can be selected, for example, from the group of TFLLR-NH 2 ; TFLLRNPNDK-NH 2 ; SFLLRNPNDKYEPF-NH 2 ; and SFLLRN-NH 2 [0019]
  • the angiogenesis promoting factor comprises a PAR-4 antagonist.
  • the PAR-4 antagonist can be selected, for example, from the group of transcinnamoyl- YPGKF-NH 2 (tcY-NH 2 ; Ma et al.) and YD-3, a non-peptide PAR4 antagonist nonpeptide PAR-4 antagonist, YD-3 (ethyl 4-(l- benzyl-lH-indazol-3-yl)benzoate) (2006, Eur. J. Pharmacol. 546: 142-147).
  • the platelets further comprise an exogenous angiogenesis inhibiting factor.
  • compositions as described herein comprising platelets comprising an exogenous angiogenesis regulating factor and a pharmaceutically acceptable carrier for promotion of wound healing.
  • the platelets are autologous to the individual.
  • the angiogenesis regulating factor is selected, for example, from the group consisting of agents that activate the VEGF pathway, agents that activate the neuropilin 1 & 2 pathways, VEGF-A and C, bFGF, HGF, angiopoietin-1, insulin-like growth factor- 1, epidermal growth factor, platelet derived growth factor, platelet factor 4, thrombospondin-1, TGF-beta-1, plasminogen activator inhibitor type-1 (PAI-I, alpha2- antiplasmin and alpha2-macroglobulinVEGFRl (flt-1), VEGFR2 (flk-2), VEGFR3 (flt-4), heparin sulfate proteoglycan, VEGF121, VEGF145, VEGF165, VEGF168, VEGF189, VEGF -B and -D, PLGF 1, PLGF2, HIV-I TAT, Sema-E, Sema-III, Se
  • the wound is selected, for example, from a burn, an incision, a laceration, an abrasion, an ulcer, a diabetic wound, a venous stasis wound, a vascular wound, a radiation wound, a steroid wound or a bony defect.
  • the wound is a diabetic ulcer.
  • the wound is a decubitus ulcer.
  • the platelets are loaded with an exogenous angiogenesis regulating factor.
  • loading comprises contacting platelets with a factor.
  • the platelets comprise at least two angiogenesis promoting factors.
  • the composition comprises a platelet gel comprising platelets and an angiogenesis regulating factor.
  • the gel is formed by contacting the platelets with calcium and a platelet activating agent.
  • the platelet activating agent is selected, for example, from the group consisting of thrombin, collagen, serotonin, ADP, acetylcholine and combinations thereof.
  • the angiogenesis regulating factor comprises a PAR-I agonist.
  • the PAR-I agonist is selected, for example, from the group consisting Of TFLLR-NH 2 , TFLLRNPND K -N H 2 , SFLLRNPNDKYEPF-NH 2 , and SFLLRN- NH 2 .
  • the angiogenesis promoting factor comprises a PAR-4 antagonist.
  • the PAR-4 antagonist is selected, for example, from the group consisting of transcinnamoyl- YPGKF-NH 2 , and ethyl 4-(l -benzyl- lH-indazol-3- yl)benzoate.
  • the platelets further comprise an exogenous angiogenesis inhibiting factor.
  • a platelet composition comprising an exogenous angiogenesis regulating factor in the preparation of a medicament for the promotion of wound healing.
  • the wound is selected, for example, from a burn, an incision, a laceration, an abrasion, an ulcer, a diabetic wound, a venous stasis wound, a vascular wound, a radiation wound, a steroid wound or a bony defect.
  • the wound is a diabetic ulcer.
  • the wound is a decubitus ulcer.
  • the platelets are loaded with an exogenous angiogenesis regulating factor.
  • loading comprises contacting platelets with a factor.
  • the platelets comprise at least two angiogenesis promoting factors.
  • the composition comprises a platelet gel comprising platelets and an angiogenesis regulating factor.
  • the gel is formed by contacting platelets with calcium and a platelet activating agent.
  • the platelet activating agent is selected, for example, from the group consisting of thrombin, collagen, serotonin, ADP, acetylcholine and combinations thereof.
  • the angiogenesis regulating factor comprises a PAR-I agonist.
  • the PAR-I agonist is selected, for example, from the group consisting Of TFLLR-NH 2 , TFLLRNPNDK-NH.., SFLLRNPNDKYEPF-NH 2 , and SFLLRN- NH 2 .
  • the angiogenesis promoting factor comprises a PAR-4 antagonist.
  • the PAR-4 antagonist is selected, for example, from the group consisting of transcinnamoyl- YPGKF-NH 2 , and ethyl 4-(l -benzyl- lH-indazol-3-yl) benzoate.
  • the platelets further comprise an exogenous angiogenesis inhibiting factor.
  • Another aspect described herein relates to a method of promoting wound healing in an individual in need thereof, the method comprising the step of contacting a wound with a composition as described herein comprising isolated platelets comprising an exogenous angiogenesis regulating factor, wherein the contacting promotes healing of the wound.
  • a method of preparing a wound healing composition comprising contacting platelets with an exogenous angiogenesis regulating factor.
  • the angiogenesis regulating factor is selected, for example, from the group consisting of agents that activate the VEGF pathway, agents that activate the neuropilin 1 & 2 pathways, VEGF-A and C, bFGF, HGF, angiopoietin-1, insulin-like growth factor- 1, epidermal growth factor, platelet derived growth factor, platelet factor 4, thrombospondin-1, TGF-beta-1, plasminogen activator inhibitor type-1 (PAI-I, alpha2-antiplasmin and alpha2-macroglobulin VEGFRl (flt- 1), VEGFR2 (flk-2), VEGFR3 (flt-4), heparin sulfate proteoglycan, VEGF121, VEGF145, VEGF165, VEGF168, VEGF189, VEGF -B and -D, PLGF 1, PLGF2, HIV-I TAT, Sem
  • the method further comprises contacting the platelets that have been contacted with an angiogenesis regulating factor with a platelet activating agent.
  • the platelet activating agent can be selected, for example, from the group of thrombin, collagen, serotonin, ADP, acetylcholine and combinations thereof.
  • kits for the preparation of a wound healing composition comprising an angiogenesis regulating factor and a platelet preparation.
  • kits for the preparation of a wound healing composition comprising an angiogenesis regulating factor and a container for holding and/or incubating platelets.
  • Kits as described herein can further comprise packaging materials therefor and, optionally, instructions for the preparation and/or use of a platelet wound healing composition. Definitions:
  • angiogenesis refers to any alteration of an existing vascular bed or the formation of new vasculature which benefits tissue perfusion. This includes the formation of new vessels by sprouting of endothelial cells from existing blood vessels or the remodeling of existing vessels to alter size, maturity, direction or flow properties to improve blood perfusion of tissues.
  • angiogenesis regulating factor refers to an agent that modulates angiogenesis.
  • An “angiogenesis regulating factor” regulates the angiogenic process, including but not limited to the following phases of the process: the degradation of the extracellular matrix; cell proliferation; cell migration and structural organization (see, e.g., Kumar et al, 1998, Int. J. Oncology 12:749-757; Bussolino et al., 1997, Trends in Biochem, 22:251-256).
  • Such a factor thus influences the rate or progress of angiogenesis, e.g., initiating angiogenesis, accelerating angiogenesis, or inhibiting angiogenesis.
  • an "angiogenesis regulating factor” positively or negatively influences angiogenesis in a chick chorioallantoic membrane ("CAM") assay performed, for example, as described by Iruela-Arispe et al., 1999, Circulation 100: 1423-1431, which is incorporated herein by reference.
  • CAM chick chorioallantoic membrane
  • positively or negatively influences is meant an at least 10% difference in angiogenesis relative to the absence of that factor. Further detail of this assay is provided herein below.
  • angiogenesis regulating factor encompasses both those factors that occur naturally in vivo and participate in the angiogenic regulatory pathways in vivo, as well as factors or compounds, either derived from naturally-occurring polypeptides or molecules (including, but not limited to fragments of naturally occurring polypeptides, e.g., anti- angiogenic fragments of collagen polypeptides) or otherwise generated or identified, that have angiogenesis-regulating activities.
  • an angiogenesis regulating factor is an angiogenesis-promoting factor (also referred to as a proangiogenesis factor).
  • An angiogenesis-promoting factor is one that causes an increase in angiogenic activity, e.g., at least a 10% increase in angiogenic activity as measured by the CAM assay as described by Iruela-Arispe et al., 1999, Circulation 100: 1423-1431, relative to the angiogenic activity observed in the absence of that factor.
  • an angiogenesis regulating factor is an angiogenesis-inhibiting factor (also referred to as an antiangiogenesis factor).
  • An angiogenesis-inhibiting factor is one that causes a decrease in angiogenic activity, e.g., at least a 10% decrease in angiogenic activity as measured by the CAM assay as described by Iruela-Arispe et al., 1999, Circulation 100: 1423-1431, relative to the angiogenic activity observed in the absence of that factor.
  • the term "promotes healing” means that treatment with a given agent decreases the time required for 90% wound closure of a full-thickness skin wound by at least 1 day relative to a control wound not treated with that agent.
  • a treatment that promotes healing will decrease the time necessary to heal a full thickness wound by 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or even 7 days (one week) or more.
  • An alternative measure of wound healing is measurement of cell proliferation at a given time after treatment (see the Examples herein below). Such measurement generally requires a biopsy (i.e., another wound), however, which is clearly not preferred for the evaluation of treatment efficacy in human subjects outside of, for example, a clinical trial setting. Thus, measurements based on cell proliferation at the wound site are useful for evaluating whether a given treatment is effective in an experimental setting but is not favored for a normal clinical setting.
  • the term "isolated" when used in reference, for example, to platelets means that the platelets are present at an enriched concentration relative to their concentration in circulating blood in vivo. Normal human blood has a platelet count of 150,000 to 400,000 per microliter. A sample is enriched if it has 800,000 platelets per microliter or more.
  • the term encompasses platelet-rich plasma (PRP) with a concentration of 1 x 10 6 platelets per microliter or more, including, for example, 1 x 10 7 per microliter, 1 x 10 8 per microliter, 2 x 10 8 per microliter, or 3 x 10 8 per microliter or more.
  • PRP platelet-rich plasma
  • the term "loaded" when used in reference to platelets means the platelets comprise an exogenous angiogenesis regulating factor.
  • an "exogenous" angiogenesis regulating factor refers to an angiogenesis regulating factor that has been exogenously introduced to the platelets.
  • exogenously added factors can include factors normally carried by platelets in vivo, e.g., VEGF, or factors not normally carried by platelets in vivo.
  • the term “exogenous” as used in this context does not mean that the factor is not normally carried by platelets, but rather that the particular molecules of the factor were exogenously added to the platelets by those preparing a platelet preparation as described herein. It necessarily follows that platelets that have not been exposed to an exogenous source of angiogenesis regulating factor have not been "loaded” with that factor.
  • platelet activating agent refers to an agent that stimulates platelets to release factors involved in the coagulation cascade and to secrete cytokines involved in wound healing and inflammation.
  • Platelet activating agents include, but are not limited to collagen (which is exposed when the endothelial blood vessel lining is damaged), thrombin, (primarily through PAR-I), ADP, ADP receptors (P2Y1 and P2Y12) expressed on platelets, a negatively charged surface (e.g., glass), serotonin, acetylcholine and combinations thereof.
  • PAR-I agonist refers to an agent that activates Protease Activated Receptor- 1 (thrombin receptor) signaling activity.
  • PAR-I on platelets is activated by a mechanism involving proteolytic cleavage of a portion of the extracellular domain to generate a new N-terminus which then acts as a tethered or intramolecular ligand (agonist) for the receptor.
  • the hexapeptide SFLLRN-NH2 comprising the new N-terminus after cleavage is referred to as the Thrombin Receptor Activating Peptide, or "TRAP.”
  • Peptide constructs based on this peptide sequence have strong PAR-I agonist activity.
  • PAR-I agonists thus include, for example, TFLLR-NH 2 (Ma et al., 2004, PNAS 102: 216-220); TFLLRNPNDK; SFLLRNPNDKYEPF; and SFLLRN-NH 2.
  • agents that interfere with the binding of this peptide to the receptor tend to have antagonist activity.
  • PAR-4 antagonist refers to an agent that antagonizes the activity of Protease Activated Receptor-4 (also a thrombin receptor) signaling activity.
  • PAR- 4 on platelets is activated by a mechanism involving proteolytic cleavage of a portion of the extracellular domain to generate a new N-terminus which then acts as a tethered or intramolecular ligand (agonist) for the receptor.
  • PAR-4 antagonists include transcinnamoyl- YPGKF-NH 2 , (tcY-NH 2 ; Ma et al., 2005, Proc. Natl. Acad. Sci. U.S.A.
  • Figure 1 shows the results of treatment of full thickness cutaneous wounds of diabetic mice with platelet preparations from healthy ("wild-type") or tumor-bearing animals. The figure shows the clinical aspects of wounds on day 10 post wound.
  • Panel A shows full-thickness wounds excised on the dorsum of db/db mice and folio wed-up twice a week for four weeks.
  • Tumor Conditioned PRP 89% wound closure
  • wounds left to heal spontaneously (49%).
  • Scale bar 0.5 cm.
  • Panel B shows time to 90% wound Closure.
  • Tumor conditioned PRP treated wounds achieved 90% wound closure in a shorter time (11.1 days) when compared to both wild type PRP (16.7) and wounds left to heal spontaneously (20.6).
  • ** p ⁇ 0.01 compared to wt PRP and NT, *p ⁇ 0.01 compared to NT.
  • NT Non-treated wounds
  • WT PRP wild type platelet rich plasma treated wound (lOO ⁇ l)
  • T PRP tumor conditioned platelet rich plasma treated wounds (100 ⁇ l).
  • FIG. 2 shows cell proliferation in wounds treated with wild-type PRP and PRP from tumor bearing animals (TPRP).
  • Cross sectional wound tissues were harvested on day 10, stained for Ki67and photographs were taken from the middle of the wound bed (data not shown). Positively (e.g., brown) stained cells are actively proliferating and were quantified by counting (Figure T).
  • Tumor conditioned PRP treated wounds showed on day 10 higher levels of proliferation (46.2%) when compared to both wild type PRP (23.2) and wounds left to heal spontaneously (20.2).
  • * p ⁇ 0.01 compared to wt PRP and NT.
  • NT Non-treated wounds
  • WT PRP wild type platelet rich plasma treated wound (lOO ⁇ l)
  • T PRP tumor conditioned platelet rich plasma treated wounds (100 ⁇ l).
  • Figure 3 shows quantified results of staining wound tissues for CD31.
  • Cross sectional wound tissues were harvested on day 10. stained for CD31 (PECAM-I), and photographs were taken from the middle of the wound bed (data not shown).
  • Microphotographs were also obtained from cross sectional wound tissues stained for phosphorylated VEGF Rec2/3 on day 10 (data not shown). Both PRP treatments induced activation of the VEGF pathway, as opposed to wounds left to heal spontaneously which did not show any staining (data not shown).
  • Three pictures were taken in from each wound tissue (one in the middle and two from the edges of the wounds; data not shown) and blood vessels were quantified.
  • NT Non-treated wounds
  • WT PRP wild type platelet rich plasma treated wound (lOO ⁇ l)
  • T PRP tumor conditioned platelet rich plasma treated wounds (100 ⁇ l).
  • Figure 4 shows a characterization of the proteome of platelets (A) and plasma (B) derived from tumor bearing and healthy mice.
  • the angiogenic proteome was analyzed, showing a characteristic distribution between platelets and plasma. The majority of the angiogenic factors analyzed were found to have comparable levels in healthy and tumor conditioned samples.
  • Figure 5 shows the healing staging system; wound tissues on day 10 were staged using a two dimensional plot representing vascularity on the X-axis and cell proliferation on the Y-axis.
  • NT was set to 1.
  • T PRP induced the highest histological stimulation of the wound tissues when compared to both WT PRP and NT.
  • Previously frozen TPRP and PRP failed to acheive the same levels of angiogenic stimulation of TPRP and PRP. Frozen TPRP kept some proliferative potential.
  • Methods and compositions for enhanced wound healing are provided herein.
  • the methods and compositions take advantage of the ability of platelets to deliver angiogenesis regulating factors.
  • the methods and compositions described herein take advantage of the ability of platelets to deliver exogenously added angiogenesis-regulating factors that aid the healing of wounds.
  • This ability to deliver angiogenesis-regulating factors represents an advance in wound healing approaches because while the benefits of angiogenic factors for wound healing may be recognized, in many instances simply injecting angiogenic factors into a wound or into the circulation does not result in a therapeutic benefit.
  • the proangiogenic factor VEGF for example, is rapidly cleared from the circulation.
  • the delivery of VEGF or other angiogenic factors to a wound site by platelets circumvents this problem and can provide a high local concentration of the factors directly to wound tissues.
  • Activated platelets have long been known to release granules containing factors involved in the coagulation cascade.
  • the inventors have discovered, however, that platelets achieve the delivery of angiogenic factors not by simply secreting or “releasing" the factors, but rather, by extending processes from the platelet cell surface that contact the target endothelial cell. By doing so, there is no release of the factor to the general circulation, and thus little chance for the factors to be diluted out or otherwise cleared before they contact cell surface receptors on the target cell.
  • the release of exogenously added factors by this mechanism from platelets placed at the wound site provides for the effective delivery of such factors directly to the wound site.
  • Platelets for use in the methods and compositions described herein can be isolated from a donor or from the individual to be treated with the composition. That is, platelets can be non-autologous or autologous. However, autologous platelets are preferred, primarily because they avoid possible infectious disease issues encountered with non-autologous donor blood products. Methods for the isolation of platelets are well known in the art.
  • autologous platelet isolation from whole blood can be performed using a device commercially designed for that purpose, e.g., the Magellan Autologous Platelet Separator (Medtronic Inc., Minneapolis, MN), according to the manufacturer's instructions.
  • the device permits the isolation of platelet-rich plasma (PRP).
  • PRP platelet-rich plasma
  • platelet-rich plasma has a well known meaning in the medical arts, and its method of preparation is well established.
  • autologous platelet-rich plasma is prepared by drawing blood from the individual having the wound, centrifuging the blood, and drawing off the supernatant (platelet-rich plasma), which comprises plasma, white blood cells, and platelets.
  • platelet-rich plasma which comprises plasma, white blood cells, and platelets.
  • the amount of platelet-rich plasma required understandably depends on the size of the wound to be treated. However, 10 mL of platelet-rich plasma is a convenient amount to obtain, requiring approximately 20 mL of blood to be drawn.
  • a stopper top vacuum tube containing sodium citrate to prevent coagulation available, e.g., from BD (Becton, Dickinson and Company) under the trademark "VACUT AINER").
  • the blood is centrifuged at speeds sufficient to produce forces of 135 to 280 g for 3 to 5 minutes at 20 to 37° C.
  • the platelet-rich plasma is then drawn off to be used in the compositions and methods as described herein.
  • Further concentration of PRP can be achieved by additional centrifugation steps.
  • Final concentrations of platelets in PRP preparations can be adjusted, if necessary, using platelet free plasma remaining from the isolation process.
  • a platelet concentration of , for example, 3 x 10 8 platelets per microliter is readily achieved using these methods.
  • Exogenous angiogenic factors can be added to platelets in any of several different ways. Platelets are known to take up material from their surroundings, either actively by endocytosis or passively, e.g., by diffusion. A number of procedures have been suggested for the modulation of the granular content of platelets, but the simple exposure of platelets to media enriched with a specific protein is sufficient for "loading" proteins into platelet alpha- granules. First, one or more factors can be added to platelets by passive diffusion, i.e., the platelets are incubated with the angiogenic factor(s), e.g., at room temperature for 5 minutes or more.
  • the angiogenic factor(s) e.g., at room temperature for 5 minutes or more.
  • the platelet loading approach not include, for example, exposure to liposomes or virus particles.
  • Simple incubation of platelets with the exogenous factor results in the uptake of the factor by the platelets, including uptake into platelet alpha granules.
  • platelets can be loaded with an exogenous angiogenic factor by passing isolated platelets through a network with a diffusion barrier or membrane.
  • a diffusion barrier or membrane on one side of the barrier is conditioned medium from cells that secrete one or more angiogenic factors, which pass through the barrier to where the platelets take them up.
  • the barrier can separate cells expressing the angiogenic factor from the platelets.
  • An apparatus can be set up in which platelets are flowed through a network of passages defined by the barrier. In this manner a steady state flow can be used to generate the platelets loaded with exogenous angiogenic factor.
  • Cells that express angiogenic factor(s) can be, for example, tumor cells known to secrete a given factor or factors, or, alternatively, recombinant cells that express and secrete the factor into their medium.
  • platelets take up and deliver angiogenic factors secreted by tumors.
  • platelets can be flowed by a bed of cultured tumor cells in order to load the platelets with the factors generated by the tumor cells.
  • the tumor cells it is preferable, but not absolutely required, that the tumor cells be cultured in serum-free medium.
  • Additional approaches include, for example, stimulating the exchange of granules by the platelets such that angiogenic factors are taken up, and tagging the angiogenic factor to a moiety that is bound by a platelet cell surface receptor.
  • angiogenic factors there are a wide variety of angiogenic factors that can be used in the wound healing methods and compositions described herein.
  • One of skill in the art can decide which angiogenesis regulator will be used in a given situation. Most often, an exogenous angiogenesis-promoting factor will be used in a platelet preparation used for wound healing.
  • angiogenesis inhibiting factors can be loaded into platelets along with angiogenesis promoting factors.
  • platelets have been shown to release both proangiogenic (e.g., VEGF) and antiangiogenic factors (e.g., endostatin). The release of these factors is apparently selectively controlled through the PAR receptors PAR-I and PAR-4 (see Ma et al., Proc.
  • PAR-I activation causes the release of proangiogenic factors, including VEGF, and the repression of endostatin release.
  • PAR-4 activation causes the release of antiangiogenic factors, including endostatin, and the repression of VEGF release.
  • This counter-regulatory mechanism may be effective in both starting the angiogenesis necessary for the healing process and in avoiding shutting down angiogenesis when the healing process is complete. Therefore, it is contemplated that it can be useful to load platelets with both angiogenesis promoting factors (proangiogenesis factors) and angiogenesis inhibiting factors (antiangiogenesis factors).
  • either the natural milieu of the wound environment would dictate which factors are released from the platelets at what time (e.g., proangiogenic factors early in the healing process, antiangiogenic factors later in the process), or additional factors, e.g., PAR agonists/antagonists, could be administered to the wound site to influence which factors are released by the loaded platelets at what time.
  • factors e.g., proangiogenic factors early in the healing process, antiangiogenic factors later in the process
  • additional factors e.g., PAR agonists/antagonists
  • PAR agonists and antagonists are known in the art.
  • the hexapeptide SFLLRN-NH 2 and peptide constructs based on this peptide sequence have strong PAR-I agonist activity.
  • PAR-I agonists thus include, for example, TFLLR-NH 2 (Ma et al., 2004, PNAS 102: 216-220); TFLLRNPNDK: SFLLRNPNDKYEPF; and SFLLRN-NH 2.
  • agents that interfere with the binding of the SFLLRN-NH 2 peptide to the PAR-I receptor tend to have antagonist activity.
  • PAR-4 antagonists include transcinnamoyl- YPGKF-NH 2 , (tcY-NH 2 ; Ma et al., 2005, Proc. Natl. Acad. Sci. U.S.A. 102: 216-220) and YD-3, a non-peptide PAR4 antagonist (ethyl 4-(l-benzyl-lH-indazol-3-yl)benzoate; see Wu & Teng, 2006, Eur. J. Pharmacol. 546: 142-147).
  • Angiogenesis promoting factors that can be used in compositions and methods described herein include, for example:
  • Angiopoietin-1 Angiopoietin-1; oc2-antiplasmin; aFGF;
  • B61 ligand for Eck receptor tyrosine kinase
  • bFGF ligand for Eck receptor tyrosine kinase
  • G-CSF Granulocyte colony- stimulating factor
  • HGF Hepatocyte growth factor
  • SF Scatter factor
  • Interleukin-8 (IL-8);
  • PD-ECGF Platelet-derived endothelial cell growth factor
  • PDGF-BB Platelet-derived growth factor-BB
  • Plasminogen activator inhibitor- 1 (PAIl);
  • Soluble vascular cell adhesion molecule- 1 Soluble vascular cell adhesion molecule- 1;
  • Tspl Thrombospondin-1
  • VEGF-D and other factors that activate the VEGF signalling pathways (including, but not limited to VEGF121, VEGF145, VEGF165, VEGF168, VEGF189; Agents that activate the neuropilin 1 & 2 pathways; and VEGF receptors, e.g., VEGFRl (flt-1), VEGFR2 (flk-2), VEGFR3 (flt-4);
  • Angiogenesis inhibiting factors that can be used in compositions and methods described herein include, for example:
  • Angiostatin (plasminogen fragment);
  • CDI Cartilage-derived inhibitor
  • Endostatin (collagen XVIII fragment);
  • hCG Human chorionic gonadotropin
  • IP-10 Interferon inducible protein
  • TRIPs Metalloproteinase inhibitors
  • Proliferin-related protein PRP
  • Retinoids Tetrahydrocortisol-S ; Thrombospondin-1 (TSP-I); Transforming growth factor-beta (TGF- ⁇ ); Vasculostatin; and Vasostatin (calreticulin fragment)
  • Combinations of these or other antiangiogenic factors can also be used in the methods and compositions described herein. It is acknowledged that several of the antiangiogenic factors are also listed with the proangio genie factors. This is a reflection of the fact that some factors can participate in both pro- and anti-angiogenic processes, depending upon their context, and indeed, upon when in the healing or angiogenic process they are used. This does not indicate unpredictability in the process, but rather points to the fine-tuned control of the angiogenic process in vivo.
  • Functional fragments of known angiogenesis regulating factors are also contemplated for use in the methods and compositions described herein.
  • “functional fragment” is meant a fragment that substantially retains the angiogenesis regulating activity of the full length factor ("substantially retains” means the fragment retains at least 80% of the activity of the full length factor).
  • Guidance as to fragments that will continue to bind a given receptor can be found in the crystal structure of the receptor. A large number of receptors have been crystallized, frequently in complex with corresponding ligands. Such crystal structure information provides guidance as to exactly what regions of a ligand interact with the receptor, and provide information that permit the in silico screening/modeling of compounds expected to bind and activate (or inhibit) the receptor.
  • the crystal structure can provide structure-function correlations that permit one to readily grasp the types of ligands that will have similar binding activity for a given receptor.
  • an angiogenesis-regulating factor influences the well known VEGF-mediated angiogenesis pathway
  • the structure of the VEGF ligand or its receptor(s) involved can be instructive in understanding, for example, what fragments or variants of the ligand would be expected to have similar activity on the receptor.
  • the crystal structure of VEGF at 2.5 A is reported by Muller et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94: 7192-7197.
  • VEGF is a homodimeric member of the cystine knot family of growth factors, with limited sequence homology to platelet-derived growth factor (PDGF) and transforming growth factor p2 (TGF-p). They determined the crystal structure at 2.5 A resolution, and identified its kinase domain receptor (KDR) binding site using mutational analysis.
  • PDGF platelet-derived growth factor
  • TGF-p transforming growth factor p2
  • KDR kinase domain receptor
  • the reference teaches that the VEGF monomer resembles that of PDGF, but its N-terminal segment is helical rather than extended.
  • the dimerization mode of VEGF is taught to be similar to that of PDGF and very different from that of TGF- ⁇ . Mutational analysis of VEGF revealed that symmetrical binding sites for KDR are located at each pole of the VEGF homodimer.
  • Each site was found to contain two functional "hot spots" composed of binding determinants presented across the subunit interface. What were classed as the two most important determinants are located within the largest hot spot on a short, three- stranded sheet that is conserved in PDGF and TGF- ⁇ . Functional analysis of the binding epitopes for two receptor-blocking antibodies reveal different binding determinants near each of the KDR binding hot spots.
  • VEGF vascular endothelial growth factor-like growth factor-receptor
  • VEGF-PDGF super-gene family In this gene family, 8 cysteines are conserved at the same positions. Two of these sites form intermolecular cross linking S-S bonds to form a dimer, while the other six sites form intramolecular S-S bonds to form 3 loop structures.
  • VEGF has at least three important sub-types, of 121, 165 and 189 amino acids, generated by alternative splicing.
  • VEGF 121 and VEGF 16S are representative forms, with VEGF 16S bearing an additional 44 amino acid basic stretch relative to VEGF 121 .
  • the VEGF 165 molecule binds heparin or heparin-like molecules in the matrix and on the cell surface, and can also associate with the cell surface molecule neuropilin-1.
  • the association of VEGF with neuropilin-1 has been reported to increase the affinity of VEGF 16S with one of the VEGF receptors, KDR (VEGFR2), about 10-fold, such that VEGF 165 is the strongest signal transducer of the naturally-occurring VEGF subtypes.
  • the chick chorioallantoic membrane (CAM) assay is frequently used to evaluate the effects of angiogenesis regulating factors because it is relatively easy and provides relatively rapid results.
  • An angiogenesis regulating factor useful in the methods and compositions described herein will modify the number of microvessels in the modified CAM assay described by Iruela-Arispe et al., 1999, Circulation 100: 1423-1431. The method is based on the vertical growth of new capillary vessels into a collagen gel pellet placed on the CAM.
  • the collagen gel is supplemented with an angiogenic factor such as FGF-2 (50 ng/gel) or VEGF (250 ng/gel) in the presence or absence of test proteins/peptides.
  • an angiogenic factor such as FGF-2 (50 ng/gel) or VEGF (250 ng/gel) in the presence or absence of test proteins/peptides.
  • FGF-2 or VEGF in the collagen gel can be omitted or reduced, and replaced or supplemented with the factor under examination.
  • the extent of the angiogenic response is measured using FITC- dextran (50 ⁇ g/mL) (Sigma) injected into the circulation of the CAM.
  • Morphometric analyses are performed, for example, by acquisition of images with a CCD camera. Images are then imported into an analysis package, e.g., NHImage 1.59, and measurements of fluorescence intensity are obtained as positive pixels. Each data point is compared with its own positive and negative controls present in the same CAM and interpreted as a percentage of inhibition, considering the positive control to be 100% (VEGF or FGF-2 alone) and the negative control (vehicle alone) 0%. Similar evaluations are performed for positive regulators of angiogenesis, relative to controls lacking such an agent or factor. Statistical evaluation of the data is performed to check whether groups differ significantly from random, e.g., by analysis of contingency with Yates' correction.
  • Additional angiogenesis assays are known in the art and can be used to evaluate factors for use in the methods and compositions described herein. These include, for example, the corneal micropocket assay, hamster cheek pouch assay, the Matrigel assay, hindlimb ischemia assay, and modifications thereof, and co-culture assays. Donovan et al. describe a comparison of three different in vitro assays developed to evaluate angiogenesis regulators in a human background (Donovan et al., 2001, Angiogenesis 4: 113-121, incorporated herein by reference).
  • the assays examined include: 1) a basic Matrigel assay in which low passage human endothelial cells (Human umbilical vein endothelial cells, HUVEC) are plated in wells coated with Matrigel (Becton Dickinson, Cedex, France) with or without angiogenesis regulator(s); 2) a similar Matrigel assay using "growth factor reduced” or GFR Matrigel; and 3) a co-culture assay in which primary human fibroblasts and HUVEC are co- cultured with or without additional angiogenesis regulator(s) - the fibroblasts produce extracellular matrix and other factors that support HUVEC differentiation and tubule formation.
  • HUVEC Human umbilical vein endothelial cells
  • the co-culture assay provided microvessel networks that most closely resembled microvessel networks in vivo.
  • the basic Matrigel assay and the GFR Matrigel assay can also be used by one of skill in the art to evaluate whether a given factor is an angiogenesis-regulating factor as necessary for the methods and compositions described herein.
  • an in vitro angiogenesis assay kit is marketed by Chemicon (Millipore).
  • the Fibrin Gel In Vitro Angiogenesis Assay Kit is Chemicon Catalog No. ECM630.
  • the treatment methods described herein use platelet wound healing compositions comprising platelets bearing exogenously added angiogenesis regulating factors.
  • the platelets, loaded with exogenous factor(s) can be formulated in high concentration liquid suspension, e.g., at 1-5 x 10 8 platelets per microliter in plasma.
  • This preparation can be overlaid onto a wound and covered with a dressing (preferably a non- absorbent dressing) to enhance the healing of the wound.
  • the platelet compositions useful in the methods and compositions described herein can additionally comprise additional carriers or excipients as known in the art for topical administration.
  • topical administration is used in its broad sense to refer to administration to a wound surface. That surface can be internal or external. It is contemplated that in other particular instances it can be beneficial to administer platelet compositions with a minimum of additional material, i.e., without gelling agent or other excipients.
  • a gel can be prepared using the loaded platelets, and the gel is the form that is administered to the wound surface. Platelet gels, and particularly autologous platelet gels are well known in the art.
  • Loaded platelet preparations as described herein can be formulated as gels by exposing the platelets to calcium and a platelet activating agent, e.g., thrombin, collagen, serotonin, ADP, acetylcholine and combinations thereof.
  • a platelet activating agent e.g., thrombin, collagen, serotonin, ADP, acetylcholine and combinations thereof.
  • thrombin thrombin
  • collagen serotonin
  • ADP acetylcholine
  • acetylcholine acetylcholine
  • Platelet gel formulations are described, for example, in U.S. Patent No. 7,112,342 (which describes, among other things, a platelet gel formulation including platelets, calcium, thrombin and an anti-oxidant, which slows the gel formation and prevents the formation of a hard platelet gel mass), 6,841,170, 6,942,880 and in published U.S. Patent Applications 20020172666, 20030198687 and 20060095121, each of which is incorporated herein by reference. Further, Horn et al. describes the preparation and use of autologous platelet gel on acute human skin wounds (Horn et al., 2007, Arch. Facial Plast. Surg. 9: 174-183, which is incorporated herein by reference).
  • kits for the preparation of platelet gels including autologous platelet gels include, for example, autologous platelet gel products provided by Blood Recovery Systems, Inc., the SymphonyTM II platelet concentrate system from DePuy Orthopaedics, Inc., and the SymphonyTM PCS platelet concentrate system, also from DePuy Orthopaedics, Inc.
  • a platelet preparation of use in the methods and compositions described herein is formed into a semi-solid gel or paste by combination of loaded platelets with, for example, a cellulose or other exogenous gelling agent.
  • Collagen gel for example, would likely form a gel with platelets, in part because exposure of platelets to collagen in vivo activates the platelets.
  • the platelets in order to maintain the regulated release characteristic of platelets in vivo, it may be preferred that the platelets not be activated prior to exposure to the wound. In this instance, it would be preferred that the platelet formulation not comprise collagen or a collagen gel, and it would be preferred that no platelet activating agent be added to the platelet preparation.
  • platelet activation can still occur at the wound site, for example, when the loaded platelets come in contact with wound associated activators, including, e.g., endogenous thrombin, collagen, etc.
  • wound associated activators including, e.g., endogenous thrombin, collagen, etc.
  • many methods have been used to enhance platelet activation (change in temperature, ions, specific molecules, such as thrombin, ADP and N- Acetyl- Glucosamine) 42 ' 43 but it has been found that the simple exposure of affected tissues may provide sufficient stimulus for platelet activity (see the Examples, below).
  • no platelet activation agent is used in platelet preparations as described herein. In other embodiments, it is preferred that no gelling agent is used with the platelet preparation.
  • the platelet compositions described herein can be administered in conjunction with other agents or treatments for enhanced wound healing. These include, but are not limited to, for example, extracellular matrix analogs (e.g., Integra (Integra LifeSciences, Plainsboro, NJ), AlloDerm (Lifecell, Inc., Branchburg, NJ), etc.), V.A.C.TM negative pressure wound therapy approaches (KCI, Inc.), cultured epithelial autografts, tissue engineered products and topical growth factors.
  • extracellular matrix analogs e.g., Integra (Integra LifeSciences, Plainsboro, NJ), AlloDerm (Lifecell, Inc., Branchburg, NJ), etc.
  • V.A.C.TM negative pressure wound therapy approaches KCI, Inc.
  • cultured epithelial autografts tissue engineered products and topical growth factors.
  • Platelet compositions as described herein are useful to enhance or aid in wound healing.
  • the preparations described are placed in contact with a wound surface (including an internal or external wound surface) in order to enhance the healing of the wound.
  • a dressing can be applied to maintain the platelet composition in place.
  • the wound healing platelet composition can include, for example, an occlusive dressing that prevents the composition from diffusing.
  • Such dressings are known to those of skill in the art and include dressings or preparations that are absorbed by the body over time.
  • extracellular matrix products such as IntegraTM can be employed to assist in retaining the platelet composition in the desired location.
  • the methods and compositions described herein can be used to treat a number of different types of wounds.
  • One application of particular importance is enhancing the healing of poorly healing or chronic wounds, such as diabetic wounds and decubitus ulcers, among others.
  • Such wounds have been known to remain for weeks, months or even years, and present an ongoing risk of infection for the entire period in which an open wound remains.
  • wounds that can be treated using the methods and compositions described herein include, for example, any chronic or poorly healing wound, venous stasis wounds, vascular wounds, radiation wounds, steroid wounds, acute surgical wounds, burns, and traumatic lacerations. Additional uses of wound healing compositions as described herein include, for example, sealing down flaps to avoid seromas, bolstering anastamoses of bowel, urinary system and blood vessels, repair of bronchial and tracheal defects, closure of the dura, repair of leaks, e.g., repair of CSF leaks, bile leaks, pancreatic leaks, etc., and repair of bony defects.
  • the ability to manipulate the angiogenic factor profile of platelets is also recognized as providing a method of inhibiting angiogenesis where so desired.
  • antiangiogenic factors can be loaded and the platelets administered for the treatment of, for example, hypertrophic granulation tissues, vascular malformations, pyogenic granuloma and tumors.
  • compositions described herein can be administered in a range of frequencies, that will vary with the type of wound being treated and the exact formulation of the composition.
  • Compositions can be administered, for example, once when an internal wound surface is to be treated, prior to suturing or otherwise closing the external access to the internal wound surface.
  • application can be more frequent, e.g., an initial application, followed by re- application within hours, e.g., 4 hours, 8 hours, 12 hours, etc., or, more likely, followed by re- application once or twice daily, for example, until the wound is closed.
  • any range of re- application that maintains the rate of healing can be used by the ordinarily skilled practitioner.
  • Dosages of platelets bearing exogenously added angiogenesis regulating factors will also vary with the type and size of the wound. Clearly, a larger wound surface will benefit from a larger amount of a wound healing composition, relative to a smaller wound surface.
  • the concentration of platelets in the compositions administered can also vary, but can generally be on the order of 5 x 10 7 platelets per cubic millimeter of the composition or higher, e.g., 1 x 10 8 platelets per mm 3 , 1.5 x 10 8 platelets per mm 3 , 2 x 10 8 platelets per mm 3 , 2.5 x 10 8 platelets per mm 3 , 3 x 10 8 platelets per mm 3 , 3.5 x 10 8 platelets per mm 3 , 4 x 10 8 platelets per mm 3 or higher.
  • Efficacy of treatment can be judged by an ordinarily skilled practitioner. Clearly, where a chronic wound is involved, any healing that leads to closure of the wound involves effective treatment. Alternatively, where the wound is not a chronic wound, e.g., an acute surgical wound, changes in the time required to close the wound (i.e., in the rate of healing) will be apparent to the ordinarily experienced practitioner based on their frequent experience with similar wounds.
  • Efficacy for any given formulation can also be judged using an experimental animal wound healing system, e.g., wild-type mice or rats, or preferably, a diabetic mouse system akin to that described in the Examples herein below.
  • an experimental animal wound healing system e.g., wild-type mice or rats, or preferably, a diabetic mouse system akin to that described in the Examples herein below.
  • efficacy of treatment is evidenced when wound closure for a standardized wound occurs earlier in treated, versus untreated wounds.
  • wound closure occurs at least 5% earlier, but preferably more, e.g., one day earlier, two days earlier, 3 days earlier, or more.
  • kits for the preparation of platelet compositions can include, for example, one or more angiogenesis regulating factors and either a platelet preparation or reagents and containers necessary for the preparation of a platelet preparation.
  • a kit for the preparation of a platelet composition can include an angiogenesis-regulating factor (e.g., VEGF), and vacuum tubes comprising citrate anticoagulant for the isolation of blood to be used for the isolation of platelets.
  • angiogenesis-regulating factor e.g., VEGF
  • Such a kit can additionally comprise buffers or reagents and tubes for the successive centrifuge spins used to concentrate and purify platelets from whole blood and/or a container(s) to hold the platelet composition once prepared.
  • a kit can also comprise a medium for the incubation with exogenous angiogenesis regulating factor (although plasma will often be the medium).
  • a kit can further comprise any agent to be combined with the loaded platelet composition to formulate it for a particular type of administration or delivery - e.g., cellulose or other thickening agent, or calcium and a platelet activating agent, e.g., thrombin.
  • TCPs tumor-conditioned platelets
  • Unprecedented findings were made that tumor-conditioned platelets considerably benefit wound healing.
  • numerous chronic wounds can be amenable to therapies involving such platelets, or platelets engineered to carry exogenous angiogenesis-regulating factors.
  • Wild-type, male, C57BL/6 male mice were injected subcutaneously with 1 x 10 6 Lewis lung carcinoma (LLC) cells in the dorsal flanks or received a sham injection of sterile water. After 4-6 weeks or once the mice began showing signs of wasting and lethargy (nodule was palpated at the injection side), they were euthanized and their blood was collected by a terminal bleed via heart puncture. Platelet-rich plasma (PRP) was obtained through a series of centrifugations as previously described and the platelet concentration was adjusted to 3 x 10 8 / ⁇ l. After collection, 100 ⁇ l of freshly-obtained tumor-conditioned PRP, which is designated henceforth as tumor-conditioned platelets (TCPs), was applied topically as described below in a blinded fashion.
  • LLC Lewis lung carcinoma
  • mice Homozygous, genetically diabetic, 10 week-old Lep/r - db/db male mice (strain C57BL/KsJ-Lepr db ) were used under an approved animal protocol in an association for assessment and accreditation of laboratory animal care international (AAALAC) accredited facility.
  • AALAC laboratory animal care international
  • mice One day prior to surgery, all mice were shaved and depilated (Nair ® , Church & Dwight Co., Princeton, NJ). On the day of the surgery, animals were weighed and anesthetized with 60 mg/kg Nembutal (Pentobarbital, Abbott Laboratories, Chicago, II) and prepared to receive one dorsal wound.
  • Paraffin-embedded sections were re-hydrated followed by antigen retrieval of Ki67 (antigen located on the surface of the chromosomes) (10 min microwave in 10 mM sodium citrate (pH 6.0)) and for platelet endothelial cell adhesion molecule one (PECAM-I) (waterbath at 37 0 C in proteinase K solution (14-22 mg/ml)). Sections were incubated with primary antibodies to PECAM-I (Pharmingen, San Jose, CA) and Ki-67 (Lab Vision, Freemont, Ca) at 4°C overnight and 1 hour at room temperature, respectively. PECAM-I signal was intensified using tyramide amplification system (Perkin Elmer, Boston, MA).
  • Platelet pellets were separated from the corresponding platelet poor plasma (PPP) from PRP and TPRP preparations by an additional centrifugation step at 90Og at room temperature. The two preparates were then processed and analyzed by SELDI-ToF technology (Ciphergen, Fremont, California). Platelet pellets and 20 ⁇ L of PPP from each mouse were lysed using 25 ⁇ L and 40 ⁇ L, respectively, of U9 buffer (2% CHAPS (3-[(3- cholamidopropyl) dimethylammonio]-l-propansulfonate), 50 mM Tris-HCl, pH 9 (Ciphergen) for 1 hr at room temperature.
  • PPP platelet poor plasma
  • Platelet lysates were then centrifuged at 10,000 g for 1 min at 4 0 C. Both platelet extracts and PPP were fractionated by anion-exchange chromatography modified after the Expression Difference Mapping (EDM) Serum Fractionation protocol (Ciphergen, Fremont, CA). The fractionation was performed in a 96- well format filter plate on a Beckman Biomek® 2000 Laboratory Work Station equipped with a DPC® Micromix 5 shaker.
  • EDM Expression Difference Mapping
  • Expression difference mapping (EDM) on ProteinChip arrays was carried out using weak cationic exchange chromatography protein arrays (CMlO ProteinChip arrays; Ciphergen, Fremont, CA) by loading sample fractions onto a 96-well bioprocessor, and equilibrating with 50 mM sodium acetate (Sigma, St. Louis, MO), pH 4. A further dilution of 40 ⁇ L anion exchange chromatography fraction into 100 ⁇ L of the same buffer on each array spot was incubated for 1 hr. Array spots were washed for 3 minutes with 100 ⁇ L 50 mM sodium acetate, pH 4. After rinsing with water, 2 x 1 ⁇ L of sinapinic acid matrix solution was added to each array spot.
  • CCMlO ProteinChip arrays weak cationic exchange chromatography protein arrays
  • Ciphergen Ciphergen, Fremont, CA
  • Wounds were analyzed for cellular proliferation using image analysis of Ki67-stained sections in a manner similar to the method of vessel density quantification.
  • High-powered digital images of Ki67-stained wound sections were used to measure the number of Ki67 + cells relative to the total number of nuclei (Fig. 2).
  • the degree of proliferation was quantified over the entire wound section using 4-6 fields at 2Ox magnification and expressed as a ratio of proliferating nuclei (Ki67 + ) to total nuclei.
  • T and WT PRP modulate Wound-healing kinetics.
  • T PRP treated wounds reached 90% wound closure in 11.1 days, reducing time to 90% closure by 5.6 and 9.5 days when compared to WT PRP and NT wounds, respectively.
  • WT PRP treatment induced faster wound closure when compared to NT (Fig. IB, p ⁇ 0.01).
  • T PRP treated wounds reached higher levels of cell proliferation, as assessed by Ki-67 staining, a marker for proliferating cells. While the average percentage of proliferating cells in NT and WT PRP treated wounds were 20.2, and 23.2, respectively, T PRP treated wounds reached 46.2% (Fig. 2; p ⁇ 0.01) on day 10.
  • Wound bed vascularity was assessed by counting PECAM-I positive (a pan- endothelial marker) blood vessels in high power field. Wounds healing spontaneously (NT group) in the diabetic mouse model, showed lower levels of blood vessel formation on day 9, when compared to both T and WT PRP (Fig. 3). T PRP treatment increased vascularity also when compared to WT PRP (Fig 3). Immunohistochemistry with PECAM-I (a pan- endothelial marker) showed vascularization averaging 61 blood vessels per hpf in TPRP treated wounds, 44 vessels per hpf in PRP treated wounds.
  • Diabetic wounds left untreated showed limited ability to form a collagenous wound matrix, exhibiting a friable and not homogeneous collagen bed.
  • WT PRP treatment increased the amount and density of collagen, resembling an early stage of a scar tissue.
  • T PRP treated wounds induced a unique pattern of distribution of the newly deposited collagen that was found in thick bundles alternating with area of absent collagen and high cellularity.
  • PRP treated wounds exhibited a collagenous matrix fibers parallely oriented and relatively acellular, such as in any typical early scar tissue (data not shown).
  • T PRP wild-type PRP
  • NT left untreated
  • wound healing compositions can be prepared, for example, from the platelets of individuals carrying tumors. While this is the case, given the ethical concerns of using material from tumor-bearing humans for therapy of others, a more palatable and practical approach is to load platelets of non-tumor bearing individuals with angiogenic factors, including angiogenic factors carried by platelets in tumor bearing individuals.
  • NGF nerve growth factor
  • CTAPIII connective tissue activating protein-III
  • PF-4 platelet factor-4
  • bFGF basic fibroblast growth factor
  • PDGF platelet derived growth factor
  • VEGF vascular endothelial growth factor
  • vascularity was plotted against cell proliferation 51 .
  • the data presented in Figure 5 are normalized to the histological stage of NT wounds, which is given an arbitrary value of 1.
  • TPRP treatment of wounds resulted in the highest histological stimulation of tissue repair as compared to NT wounds.
  • TPRP induced over 4-fold increase (p ⁇ 0.01, compared to both PRP and NT) in cell proliferation, while PRP induced a ⁇ 2.5-fold increase (p ⁇ 0.05), compared to NT controls.
  • This Example presents unprecedented data that PRP derived from tumor-bearing animals is capable of accelerating wound closure and healing in a diabetic mouse model.
  • the wound-healing parameters used to stage wound healing appeared markedly improved following a single topical application of TCPs when compared to both wild type derived PRP and spontaneously healing wounds.
  • the wound healing staging system developed in this study was based on the quantification of vasculature and cell proliferation in diabetic wounds 51 .
  • Platelets are the first responder to breaks in vascular integrity but their function is not limited to aggregation and coagulation at the site. It is now appreciated that there are different levels of platelet activation, and that upon adherence to the affected tissues, communicate freely through the open canallicular system. While not wishing to be bound by theory, this discovery that pro- and anti-angiogenic proteins are released in a sequential manner in response to PARl and PAR4 in intact platelets can explain why an unselective destruction, and a simultaneous release of positive and negative stimulators would have counterbalancing effects.
  • angiogenesis is essential to initiate and support the repair process. New, small blood vessels form in the granulation tissue as a consequence of cell- and growth factor mediated events.
  • endothelial cells in the wound bed revert to a quiescent phenotype and start to undergo apoptosis in the early scar tissue, demonstrating that a timely switch between pro- and anti-angiogenesis is necessary to initiate and terminate the healing process, respectively 60 .
  • Such a process is evident herein in an animal model that resembles the phenotype of Type 2 diabetes in humans, wherein wound healing occurs in an accelerated fashion with PRP and T PRP treatments. It was observed that in the later stages of wound healing and by day 21, both PRP and T PRP were also able to modulate inhibition as wounds treated with these preparations demonstrated a down- regulation of angiogenesis while NT wounds showed increased levels of vessels.
  • T PRP T protein kinase
  • Other treatment groups including wild type derived PRP. While not wishing to be bound by theory, altered platelet activation, orchestration and potential redistribution of the growth factors are a likely process that partly governs wound healing.
  • an activated platelet gel can be of use in compositions as described herein, a preferred aspect does not activate the platelets prior to application to a wound surface.
  • VEGF is one of the most studied angiogenic factors; in the blood stream it is transported mainly by platelets 5 and its concentration in the serum may increase with tumor progression in many types of cancers 30"34 .
  • High doses of recombinant VEGF have been shown to have beneficial wound healing effects in animal models 35 .
  • attempts have been made to use VEGF as a prognostic factor 35 ' 36 but VEGF presence in platelets appears to have a more functional role.
  • the inventors have demonstrated that platelets are able to present VEGF contained in its granules in a manner that enhances the pro-angiogenic effect of VEGF.
  • platelets In addition to VEGF-A and C, platelets have been documented to contain more than 14 pro-angiogenic growth factors (bFGF, HGF, angiopoietin-1, insulin-like growth factor- 1, epidermal growth factor, platelet derived growth factor), as well as inhibitors of angiogenesis, including platelet factor 4, thrombospondin-1, TGF-beta-1, plasminogen activator inhibitor type-1 (PAI-I), alpha2-antiplasmin and alpha2- macro globulin 37 .
  • pro-angiogenic growth factors bFGF, HGF, angiopoietin-1, insulin-like growth factor- 1, epidermal growth factor, platelet derived growth factor
  • angiogenesis including platelet factor 4, thrombospondin-1, TGF-beta-1, plasminogen activator inhibitor type-1 (PAI-I), alpha2-antiplasmin and alpha2- macro globulin 37 .
  • angiogenesis is initially switched on and subsequently downregulated. This downregulation results in formation of poorly vascularized scar tissue, or may be a consequence of it. It appears that this temporal regulation of angiogenesis is well controlled under physiological conditions and healing and scar formation takes on average 10-14 days. Diabetes results in dysregulation of this process 38 ' 39 and healing is impaired. While some improvement of diabetic ulcer healing is achieved with angiogenic protein delivery 35 , the use of platelet rich plasma is significantly more effective. Platelets of mice bearing tumors, which have been shown to contain significantly more angiogenesis regulators 30 ' 40 ' 41 are even more efficacious in improving wound healing ( Figure 1).
  • platelets in wound healing can offer the advantage of serving as a drug delivery system.
  • the addition of angiogenic factors carried by platelets in tumor-bearing individuals to autologous platelets for wound healing preparations can be particularly advantageous.
  • Platelet-rich fibrin a second- generation platelet concentrate. Part IV: clinical effects on tissue healing. Oral Surg. Oral Med Oral Pathol. Oral Radiol. Endod. 101:e56-e60.
  • Thrombocytes are the major source for soluble vascular endothelial growth factor in peripheral blood. Oncology 58:169-174.
  • Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells.
  • Greenhalgh DG Models of wound healing, J Burn Care Rehabil 2005, 26:293-305
  • Tsuboi R, Rifkin DB Recombinant basic fibroblast growth factor stimulates wound healing in healing-impaired db/db mice, J Exp Med 1990, 172:245-251
  • Geng JG Interaction of vascular endothelial cells with leukocytes, platelets and cancer cells in inflammation, thrombosis and cancer growth and metastasis, Acta Pharmacol Sin 2003, 24:1297-1300

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Abstract

La présente invention concerne des procédés et des compositions destinés à l'administration thérapeutique de facteurs de régulation de l'angiogenèse à des sites in vivo les nécessitant. Les procédés et les compositions portent de manière générale sur l'utilisation de plaquettes portant un ou plusieurs facteurs de régulation exogènes de l'angiogenèse. Selon un aspect, l'invention concerne des procédés d'administration d'un facteur de régulation de l'angiogenèse, le procédé consistant à mettre en contact un tissu avec une composition contenant des plaquettes isolées qui renferment un facteur de régulation exogène de l'angiogenèse. Les procédés peuvent notamment être utilisés pour accélérer la cicatrisation de plaies, ou, dans une variante, pour inhiber la croissance tumorale. Selon un autre aspect, l'invention concerne des compositions thérapeutiques à base de plaquettes et des trousses, les plaquettes portant un ou plusieurs facteurs de régulation exogènes de l'angiogenèse. L'invention concerne également des procédés permettant de moduler la libération de facteurs favorisant et inhibant l'angiogenèse par des plaquettes au moyen d'agonistes et/ou d'antagonistes de récepteurs de plaquette activés par des protéases, à savoir les récepteurs PAR-1 et PAR-4.
PCT/US2008/067592 2007-06-22 2008-06-20 Compositions thérapeutique plaquette et procédés Ceased WO2009002811A2 (fr)

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WO2014055949A1 (fr) * 2012-10-04 2014-04-10 Genesys Research Institute Compositions de plaquettes et utilisations de celles-ci
WO2015109220A1 (fr) * 2014-01-17 2015-07-23 President And Fellows Of Harvard College Leurre plaquettaire et son utilisation
WO2015124570A1 (fr) * 2014-02-18 2015-08-27 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes et composition pharmaceutique pour le traitement de l'infection par le virus de la grippe a
JP2021529770A (ja) * 2018-06-29 2021-11-04 プレートレット バイオジェネシス， インコーポレイテッド 薬物送達のための組成物およびその使用方法
WO2021231990A1 (fr) * 2020-05-15 2021-11-18 Platelet Biogenesis, Inc. Procédés et production améliorés de nouvelles plaquettes
CN115315265A (zh) * 2020-03-20 2022-11-08 台湾粒线体应用技术股份有限公司 线粒体用于促进伤口修复及/或伤口愈合的用途
EP4328299A3 (fr) * 2014-05-16 2024-05-29 Mayo Foundation for Medical Education and Research Compositions de milieux de culture cellulaire pour cellules primaires
US12060576B2 (en) 2018-01-05 2024-08-13 Stellular Bio, Inc. Compositions and methods for producing megakaryocytes
CN119139532A (zh) * 2024-11-20 2024-12-17 昆明医科大学 一种海绵贴片及其制备方法与应用
US12324816B2 (en) 2008-09-16 2025-06-10 Mayo Foundation For Medical Education And Research Compositions containing platelet contents

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US6524568B2 (en) * 1998-06-22 2003-02-25 Cytomedix, Inc. Enriched platelet wound healant
GB2342046B (en) * 1998-06-22 2004-02-18 Autologous Wound Therapy Inc Application for utility patent for improved enriched platelet wound healant

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US12324816B2 (en) 2008-09-16 2025-06-10 Mayo Foundation For Medical Education And Research Compositions containing platelet contents
WO2014055949A1 (fr) * 2012-10-04 2014-04-10 Genesys Research Institute Compositions de plaquettes et utilisations de celles-ci
WO2015109220A1 (fr) * 2014-01-17 2015-07-23 President And Fellows Of Harvard College Leurre plaquettaire et son utilisation
WO2015124570A1 (fr) * 2014-02-18 2015-08-27 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthodes et composition pharmaceutique pour le traitement de l'infection par le virus de la grippe a
US12371657B2 (en) 2014-05-16 2025-07-29 Mayo Foundation For Medical Education And Research Cell culture media compositions for primary cells
US12258576B2 (en) 2014-05-16 2025-03-25 Mayo Foundation For Medical Education And Research Cell culture media compositions for primary cells
EP4328299A3 (fr) * 2014-05-16 2024-05-29 Mayo Foundation for Medical Education and Research Compositions de milieux de culture cellulaire pour cellules primaires
US12060576B2 (en) 2018-01-05 2024-08-13 Stellular Bio, Inc. Compositions and methods for producing megakaryocytes
JP2021529770A (ja) * 2018-06-29 2021-11-04 プレートレット バイオジェネシス， インコーポレイテッド 薬物送達のための組成物およびその使用方法
EP3813853A4 (fr) * 2018-06-29 2022-04-06 Platelet Biogenesis, Inc. Compositions pour l'administration de médicaments et leurs méthodes d'utilisation
US12403161B2 (en) 2018-06-29 2025-09-02 Stellular Bio, Inc. Compositions for drug delivery and methods of use thereof
CN115315265B (zh) * 2020-03-20 2024-05-31 台湾粒线体应用技术股份有限公司 线粒体用于促进伤口修复及/或伤口愈合的用途
CN115315265A (zh) * 2020-03-20 2022-11-08 台湾粒线体应用技术股份有限公司 线粒体用于促进伤口修复及/或伤口愈合的用途
WO2021231990A1 (fr) * 2020-05-15 2021-11-18 Platelet Biogenesis, Inc. Procédés et production améliorés de nouvelles plaquettes
CN119139532A (zh) * 2024-11-20 2024-12-17 昆明医科大学 一种海绵贴片及其制备方法与应用

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