EP2553089A1

EP2553089A1 - Cell therapy to improve wound healing and related compositions and methods

Info

Publication number: EP2553089A1
Application number: EP11763511A
Authority: EP
Inventors: Mercedes A. Walton; Julie G. Allickson; Rolando O. Garcia-Morales
Original assignee: Cryo Cell International Inc
Current assignee: Cryo Cell International Inc
Priority date: 2010-04-01
Filing date: 2011-04-01
Publication date: 2013-02-06
Also published as: WO2011123779A1; EP2553089A4; CA2795248A1; JP2013523774A

Abstract

Therapeutic compositions for improving wound healing and related methods of formulation and use are provided. The therapeutic compositions comprise a dosage of MSCs seeded to any one of a biodegradable matrix, a gel, or a solution suitable for subcutaneous injection. The dosage of MSCs may range from about 1X10⁶ MSCs to about 1X10¹⁰ per administration. The administration regime may be daily, weekly, or monthly. The dosage and treatment intervals may be adjusted depending upon a patient's response to treatment. The therapeutic compositions may be co-administered, such as for example, topical application of a seeded matrix or gel combined with simultaneous or other recurring subcutaneous injection of MSCs.

Description

CELL THERAPY TO IMPROVE WOUND HEALING AND RELATED

COMPOSITIONS AND METHODS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority and benefit of U.S. Provisional Patent

Application Serial No. 61/341,652 entitled "Cell Therapy To Improve Wound Healing" filed April 1, 2010, the entirety of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

[0002] Wound healing is a complex process involving a highly-regulated cascade of biochemical and cellular events designed to achieve restoration of tissue integrity following injury. The wound healing process may be separated into three overlapping phases, namely the inflammatory phase, the proliferative phase, and the remodeling phase. Immediately after occurrence of a wound, healing begins with the inflammatory phase that initiates restoration of Hemostasis along with cellular cleansing of the body.

[0003] In the inflammatory phase, the damaged area is filled with plasma, cells, and cell elements including platelets to promote coagulation and formation of a fibrin-rich barrier (clot) to close the wound and to protect against invasion of microorganisms at the wound site. Platelets undergo degranulation and secrete numerous healing mediators including platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-β), epidermal growth factor (EGF), transforming growth factor alpha (TGF-a), vascular endothelial growth factor (VEGF), and adhesive glycoproteins. Vasoactive factors and chemotactic factors are also released during coagulation to recruit inflammatory cells, such as neutrophils and monocytes, to the wound. The first cells to appear at the wound are neutrophils, then 48 to 72 hours later the macrophages become the dominant cells present at the wound site. [0004] The inflammatory cells phagocytize bacteria, cell fragments, and foreign antigens at the wound site. In addition, the inflammatory cells produce growth factors to recruit fibroblasts and endothelial cells to prepare the wound for the proliferative phase. Platelet-derived factors and phagocytosis facilitate monocytes activation for transformation into macrophages. Activated macrophages control tissue repair at the wound site and release chemotactic factors to attract inflammatory cells and prostaglandins for vasodilation. Specifically, activated macrophages produce and secrete many growth factors including PDGF, TGF-a, and VEGF to stimulate healing. Vasodilatation and increased capillary permeability result in local edema in which histamine, kinins, prostaglandins, leukotriens, and endothelial cells product take an important role in promoting healing. After about 5-7 days from wound occurrence, fibroblasts become the predominant cell type at the wound site.

[0005] Overlapping with the inflammatory phase, the proliferative phase begins with the entry of fibroblasts, endothelial cells, and epithelial cells at the wound site with the purpose of closing the wound. This phase includes epithelialization, fibroplasia, and angiogenesis, all of which contribute directly or indirectly to formation of the granulation tissue. The granulation tissue serves as the temporary connective tissue that replaces the fibrin tissue of the blood clot to fill a wound. Early in this phase, vascular permeability leaks proteins, cytokines, and cellular elements to support proliferation of endothelial cells at the wound site. Angiogenesis occurs and the newly-formed vessels support formation of the granulation tissue and provide nutrients and oxygen to the healing tissue. The migration of endothelial cells and angiogenesis rely on the presence of cells and cytokine expression in addition to production and organization of the extracellular matrix in the granulation tissue and the basal endothelial membrane. The extracellular matrix modulates the release of growth factors to support healing. Epithelial cells migrate to the wound site and proliferate to grow skin until the wound closes and it enters into the remodeling phase.

[0006] During the remodeling phase, the tissue at the wound site is reconfigured to recover typical tissue structure. Collagen is continually replaced and remodeled by degradative enzymes. The extracellular matrix matures into a definitive matrix, and fibroblasts are transformed into myofibroblasts to function as contractile tissue. As a result of this final phase, vessels, fibroblasts, and inflammatory cells disappear from the wound site by way of migration, apoptosis, or other mechanisms.

[0007] Many human conditions, diseases, treatments, and disorders disrupt the complex cascade of events during wound repair, such as age, malnutrition, dehydration, infection, anemia, diabetes, various degrees of burns, chronic kidney disease, and medication, among many others. These conditions, diseases, treatments, and disorders prolong or suspend wound healing in the inflammatory phase of the healing process, disrupt the normal clotting mechanisms, disrupt normal leukocyte function, or block angiogenesis or formation of granulation tissue. As a result, patients may experience pain, tenderness, swelling, pyrexia, delayed healing, fragility of wound healing tissues (granulation tissue), odor, wound breakdown, infection, ulcers (venous, arterial, diabetic, or pressure), or any form of chronic wounds. Patient suffering caused by wound healing problems can be exacerbated with high costs for treatment, long treatment periods, inconvenience, and related psychological effects.

[0008] Over 100 known physiologic factors contribute to wound healing deficiencies in individuals with diabetes. These include decreased or impaired growth factor production, angiogenic response, macrophage function, collagen accumulation, epidermal barrier function, quantity of granulation tissue, and keratinocyte and fibroblast migration and proliferation, among others. For example, diabetic foot wounds are frequently chronic by definition and can result in decreased angiogenic response, neuropathy, trauma, and foot deformity.

[0009] Thus, there is a present need for therapeutic compositions and cell therapies to address the aforementioned problems relating to diseases and conditions in the area of wound healing and specifically for healing chronic wounds.

SUMMARY OF THE INVENTION

[0010] The present invention provides therapeutic compositions for improving wound healing and methods of manufacture and use thereof. The therapeutic compositions comprise menses-derived stem-like cells seeded in a vehicle for delivering a therapeutically-effective amount of the cells to a wound to improve healing. The menses-derived stem-like cells are isolated from menses and comprise any one or combination of a substantially heterogeneous population of cells, a substantially homogenous population of cells, or a population of cells that express CD-I 17.

[0011] In an embodiment, the menses-derived stem-like cells are seeded in the vehicle in a range from about lxlO⁶ cells to about lxlO¹⁰ cells. In another embodiment, the menses-derived stem-like cells are seeded at more than about lxl 0¹⁰ cells per vehicle or less than about lxlO⁶ cells depending upon patient tolerance and progress of wound healing during treatment.

[0012] The vehicle may be any one of a biodegradable matrix, a gel, or a pharmaceutically-acceptable excipient suitable for subcutaneous injection of the menses-derived stem-like cells.

[0013] A method for preparing a composition to improve wound healing is provided. The method comprises seeding a therapeutically-effective amount of menses-derived stem-like cells in a vehicle for administering the cells to a wound. The seeding comprises adding menses- derived stem-like cells in a range of about lxl 0⁶ cells to about lxlO¹⁰ cells to the vehicle. In another embodiment, the menses-derived stem-like cells are seeded at more than lxlO¹⁰ cells or less than about lxlO⁶ cells per vehicle.

[0014] The menses-derived stem-like cells and may be any one or more of a substantially heterogeneous population of cells, a substantially homogenous population of cells, or a population of cells that express CD-I 17.

[0015] A method for improving wound healing is also provided. The method comprises administering a dosage of a therapeutically-effective amount of menses-derived stem-like cells seeded in a vehicle at least once to a wound. In an embodiment, the method comprises substantially debriding the wound before administering the vehicle seeded with the menses- derived stem-like cells to the wound. The dosage of the menses-derived stem-like cells may be seeded in a range of about lxlO⁶ cells to about lxlO¹⁰ cells per vehicle. The dosage of menses- derived stem-like cells may be seeded at more than lxlO¹⁰ cells or less than lxlO⁶ cells per vehicle.

[0016] In an alternative embodiment, the method comprises administering at least two forms of the vehicle seeded with a dosage of menses-derived stem-like cells to a wound.

[0017] A biodegradable matrix or gel seeded with the therapeutically effective amount of pluripotent cells isolated from menses is administered to the wound at least once per day, week, or month for a treatment duration of one month up to 6 months or more depending upon patient tolerance and progress of wound healing during treatment.

[0018] The pharmaceutically-acceptable excipient seeded with the therapeutically effective dosage of menses-derived stem-like cells may also be co-administered to the wound on a daily, weekly, monthly basis by subcutaneous injection for a treatment duration of one month up to 6 months or more depending upon patient tolerance and progress of wound healing during treatment.

[0019] In an embodiment, the pharmaceutically-acceptable excipient seeded with the therapeutically effective amount of menses-derived stem-like cells is administered to a wound weekly by subcutaneous injection for a treatment duration of one month up to 6 months or more depending upon patient tolerance and progress of wound healing during treatment.

DESCRIPTION OF THE FIGURES

[0020] FIG. 1 shows a wound assessment and treatment flow diagram.

[0021] FIG. 2 shows a graph of the average wound area for a study of 48 adult male

Nude Sprague-Dawley rats (250gm) separated into 3 experimental groups of 16 rats verses day after surgery. The 3 groups were treated as follows: (1) Normal wound with matrix only (acellular dermal matrix, Promogran); (2) Ischemic wound with MSC vehicle (solution); and (3) Ischemic wound with MSC vehicle and MSCs, as described in the Rat Study disclosed hereinafter.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0022] The present invention provides a therapeutic composition for improving wound healing using menses-derived stem-like cells (MSCs) and methods of making and using the same. MSCs may be administered in therapeutic compositions to a subject for the purpose of improving or accelerating wound healing and addressing other problems associated with wound healing. The administration of MSCs to the wound site leads and/or induces MSCs to mimic the body's natural response to a wound. For example, MSCs may differentiate into the other cell types that either comprise and/or support the natural wound healing apparatus, e.g., fibroblasts, inflammatory cells, or cells for angiogenesis, or produce and secrete chemotactic factors, growth factors, or other wound healing mediators. [0023] Menses-derived Stem-like Cells

[0024] The term "Menses-derived stem-like cells", "MSC", or "MSCs" means any population of viable, highly proliferative, multipotent cells, if not, pluripotent cells, isolated from menses according to the methods of U.S. Patent Application Serial No. 12/615,372, which is incorporated herein by reference in its entirety, where the population is a heterogeneous populations of cells or any cell population derived therefrom including, but not limited to, a homogeneous population of cells, a lineage-uncommitted population of cells, a lineage- committed population of cells, a cell population that positively expresses the surface marker CD117 and/or SSEA-4, or any cell population derived therefrom.

[0025] Menses-derived stem-like cells for practicing the invention may be isolated using any suitable technique that produces a sufficient amount of MSCs capable performing the functions and methods set out in the present disclosure. Examples of these methods include, but are not limited to, the methods described in U.S. Patent Application Serial No. 12/615,372. Cell surface markers provide another means for isolating and/or enriching MSCs. For example, surface markers such as CD117 on the surface of MSCs are reactive with certain monoclonal or polyclonal antibodies described in U.S. Patent Application Serial No. 12/615,372. Antibodies can be used as reagents to capture or enrich a MSC cell population.

[0026] MSC Isolation

[0027] As a non-limiting embodiment, MSCs may be isolated from menstrual flow, cleansed with antibiotic treatment, and collected. MSCs may be used as (i) a fresh source of cells, (ii) a culture of a fresh source of cells, (iii) a cell isolate from a culture of fresh cells (i.e., obtained by CD117 selection or other positive or negative selection), (iv) cells that are thawed after cryopreservation, (v) cells that are cultured after being cryopreserved and then thawed, (vi) a cell isolate from a culture of a cryopreserved and thawed sample, or (vii) any other population of cells as disclosed in U.S. Patent Application Serial No. 12/615,372, to seed the therapeutic composition of the present invention.

[0028] In one non-limiting example, MSCs are isolated from menstrual flow collected with intravaginal placement of a collection cup for about 1 to 3 hours. The collected menstrual flow from two separate collections is placed in two collection tubes containing refrigerated media as follows: the first tube has media comprising HBSS with sodium heparin, and antibiotics, and the second tube has media comprising HBSS with sodium heparin and the antibiotics, for example, as disclosed in U.S. Patent Application Serial No. 12/615,372.

[0029] The collection tubes with media and menstrual flow are hermetically sealed and shipped in a Styrofoam cooler with frozen foam bricks for processing within about 24 to about 72 hours of collection. The menstrual flow in media samples undergo an antibiotic wash in an antibiotic cocktail. The final concentrated cocktail comprises Vancomycin (160 ug/ml), Claforan (500 ug/ml), Amikacin (200 ug/ml), Gentamycin (240 ug/ml), and Amphotericin B (5.4 ug/ml) in HBSS, which is the same concentration used for collection.

[0030] In a first tube, 1 ml of stock solution antibiotic cocktail will be added to 19 ml of

HBSS and 0.4 ml of heparin (400 units). In a second tube, 0.2 ml of heparin (200 units) will be added to 10 ml ofDPBS.

[0031] The menstrual flow specimen in the first tube (heparin and DPBS or alternatively

HBSS) undergoes antibiotic wash treatment. The specimen is filtered with a 100 micron filter to a 50 ml conical tube. First collection tube will be washed with 10 ml of DPBS and 0.2 ml of heparin and added to 50 ml conical tube. The 50 ml conical tube will then be centrifuged at 840g for 7 minutes at 4.0 degrees C. Supernatant should be removed being careful not to disturb the pellet. 1 ml of diluted antibiotic cocktail and 0.4 ml of heparin should be added to the remaining pellet along with HBSS to resuspend the pellet. This mixture with cells should be incubated at 2 to 8 degrees C for 20 to 30 hours with sample being placed on a rotator for the first 5 hours of incubation. After incubation, the mixture of cells may be centrifuged again with same centrifugation parameters with the pellet being resuspended in 5 ml of DPBS or HBSS.

[0032] The menstrual flow specimen in the second tube (heparin, HBSS) is used for infectious disease testing.

[0033] Cell count, cell viability, infectious disease testing, and microbiological testing may take place for each of the two menstrual cell samples according to the techniques disclosed in U.S. Patent Application Serial No. 12/615,372.

[0034] The two menstrual cell samples (approximately 5 ml) may then be cryopreserved according to the techniques disclosed in U.S. Patent Application Serial No. 12/615,372. 5 ml of cryopreservation media (20% HSA and 20% DMSO in DPBS) should be added separately to each of the two menstrual cell samples.

[0035] Cryopreserved cells may be thawed according to the techniques disclosed in U.S.

Patent Application Serial No. 12/615,372. Alternatively, each of the cell samples may be further processed to isolate cells expressing specific cell markers, for example, CD117.

[0036] Cell selection may be performed on as little as 2.5 million cells and up to 100 million cells and according to the techniques disclosed in U.S. Patent Application Serial No.

12/615,372.

[0037] Fresh cells or thawed cells should be placed on ice for at least 15 minutes before cell selection. Cells should be centrifuged at 300 g for 10 minutes at 4 degrees C. The supernatant should be removed and the pellet resuspended in 300 ml of working buffer (as disclosed in U.S. Patent Application Serial No. 12/615,372) with about one drop of DNase Pulmozyme for every 10 ml of working buffer, 100 ul of FeR blocking reagent, and 100 ul of CD117 microbeads. This mixture should be chilled on ice for 20 to 25 minutes. 2 ml of working buffer should be added. The tubes containing the mixtures should be centrifuged for 10 minutes at 300 g at 4 degrees C. Supernatant should be removed and the pellet resuspended in 500 ul of working buffer.

[0038] The resuspended cells should then be run through a MS column (wet with about

500 ul of working buffer) on a magnetic stand. The MS column should be washed three times with 500 ul of working buffer. Non-labeled cells that washed through the column should be removed and analyzed for cell count, cell viability, etc. The MS column should then be removed from the magnetic stand and 1 ml of working buffer should be run through the MS column to remove labeled cells. The collected labeled cells may be run through a separate MS column to concentrate the labeled cells. In this case, the labeled cells are CD117 cells.

[0039] The aforementioned processing should take place according to aseptic techniques in a biological safety cabinet with universal precautions taken.

[0040] The isolated CD 117+ MSCs may then be cryopreserved according to the techniques disclosed in U.S. Patent Application Serial No. 12/615,372. Cryopreserved or fresh cells may then be prepared for seeding matrix, gel, or pharmaceutically-acceptable excipient according to the present invention. CD117 selected cells are thawed (if cryopreserved). Prior to the use of the cells the preparation will be washed 5X to remove antibiotics from the cells. The cells are washed with 10ml of DPBS and 0.2ml Heparin (preservative-free). The sample is vortexed lightly and centrifuged at 840g for 7 minutes at 4.0°C. Supernatant is removed and the pellet rewashed 5X before preparing cells for treatment. Thawed or fresh CD117 cells are then plated for expansion in 15% Chang's complete media or Human Serum DMEM complete media (or equivalent).

[0041] After sufficient expansion, cells may optionally be selected for CD117 and then expanded again. The goal of a single expansion or double expansion of CD117 selected cells is to obtain the desired number of cells for the application. These expanded CD 117+ cells may be used immediately or may be cryopreserved and then later thawed for seeding.

[0042] Formulation

[0043] The present invention provides therapeutic compositions comprising MSCs and a pharmaceutically-acceptable excipient or matrix or gel. A subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient or matrix or gel and MSCs, either alone or in combination with one or more bioactive agents (such as, for example, growth factors), at a strength effective for administration by various means to a patient wound to improve healing. Any preparation of therapeutic compositions with bioactive agents is an alternative aspect of the invention. In addition, if desired for treatment, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, or pH buffering agents which enhance the effectiveness of MSCs.

[0044] Matrices into which MSCs can be incorporated or embedded include matrices which are biocompatible, recipient-compatible and which degrade into products which are not harmful to the recipient. These matrices provide support and protection for MSCs. As a non- limiting example, to prepare the biomatrix seeded with MSCs, any of the following matrices may be used: Integra Bilayer Matrix Wound Dressing, Promogran Matrix Wound Dressing, or equivalent. In an embodiment, a whole wound dressing may be used or it may be cut into smaller pieces. In another embodiment, four equal pieces may be used. The wound dressing is placed inside two T75 flasks and two Petri dishes for seeding with desired dosage of MSCs. MSCs may be plated in 15% Chang's media (325ml MEMa, 75ml FBS, 90ml Chang B, 10ml Chang C, 5ml Penstrep, & 5ml L-glutamine.) Cell dosage is selected. MSCs are then seeded in the matrix by slowly applying the admixture of Chang's media and desired dosage of MSCs to the selected matrices. Specifically, MSCs were seeded by expelling them slowly from an automated pipette directly onto the matrix so the cells would absorb onto the material.

[0045] Non-Limiting Example of Seeding Matrix with MSCs

[0046] In an embodiment, a method for culturing a monolayer cell line of CD117+ MSCs within a matrix is provided.

[0047] A cryopreserved sample of MSCs (M29156RA CD117 Positive Fraction) is thawed in a 37-40°C water bath without letting cells thaw completely. The partially thawed cells are transferred to chilled 15% Changs cell media with DNase (1 drop per 10 mL) and mixed gently by inversion. For a 5 mL cell preparation, use 25 mL of chilled media. Centrifuge the preparation at 120 g for 5 minutes. Pipette off supernatant and re-suspend cells in complete 15% Changs media without DNase by gentle inversion. Centrifuge again at 120 g for 5 minutes. Pipette off supernatant and plate cells in flasks at 5,000 cells per cm² to harvest the following day (ex 1,000,000 cells in 1 T175 flask); plate 1,000 cells per cm² to harvest in 3-5 days.

[0048] Cells are ready to trypsinize when 75%-80% confluent. Prior to trypsinization, warm adequate amounts of TrypLE (Trypsin-like enzyme), Changs Media, and DPBS at approximately 36.0-38.0°C. Aspirate the media from the flask. Wash T-25, T-75 or T-175 flasks with 5, 10 and 25 mL of pre-warmed DPBS without calcium or magnesium, respectively. For T- 25, T-75 or T-175 add 1.5 mL, 3 mL and 6 mL of pre-warmed TrypLE respectively. Agitate and incubate the flask for about 5 minutes in a C0₂ Incubator at 36.0-38.0°C. Gently dislodge the cells and dilute the contents of the flask with Changs media volume equal to that of previously added TrypLE. Transfer contents of the flask to a 50 mL centrifuge tube. Wash T-25, T-75 or T- 175 non-tissue culture treated flasks with an additional 5, 10 and 25 mL of DPBS without calcium or magnesium, respectively. Add wash to the 50 mL centrifuge tube containing contents of the flask. Centrifuge the tube at 120 g for 5 minutes. Discard the supernatant after centrifugation. Record volume of the cell pellet and remove 20 to perform cell count.

[0049] Seeding the matrix is performed by first cutting the matrix to a desired size using sterile scissors and place into the appropriate size Petri dish. Add media to the dish and allow the matrix to absorb it. Plate 2,000,000 cells (or other cell dosage) slowly onto the matrix starting from the center and spiraling out to the edges. Allow the cells to settle into the matrix. Punch out the desired matrix size and transfer to a wound with the cell side facing the wound. Residual MSCs may be plated onto another matrix or concentrated and cryopreserved according to the methods disclosed in U.S. Patent Application Serial No. 12/615,372.

[0050] In another example, MSCs may be combined in an admixture with a bioabsorbable gel, which supports growth and/or differentiation of MSCs. Cell dosage is selected. MSCs are then gently seeded in the gel by slowly applying the admixture of Chang's media and desired dosage of MSCs to the gel. Specifically, MSCs may be seeded by expelling them slowly from the automated pipette directly onto the gel so the cells could be gently mixed into the gel. By way of non-limiting examples, Dermagran-B 30z Wound Gel (Tube), Dermagran-B Hydrophilic Wound Gel manufactured by Derma Sciences. Other suitable gels may comprise a hydrophilic dressing that provides either a primary cover or filler for chronic or acute wounds. Gel may contain zinc-nutrient dressing formulation and balanced pH technology, providing a moist, mildly acidic environment conductive to wound healing along with Vitamin A, Vitamin B6, Calcium, and Magnesium.

[0051] MSCs may also be combined with a pharmaceutically acceptable carrier or excipient for delivery by a device, e.g., a syringe, by subcutaneous injection. The pharmaceutically-acceptable carrier or excipient described herein, for example, vehicles, adjuvants, carriers or diluents, are readily available to the public. Generally, the pharmaceutically acceptable carrier or excipient is one which is chemically inert to the cells and one which has no detrimental side effects or toxicity under the conditions of use. Examples include, but are not limited to, saline, solvents, dispersion media, cell culture media, aqueous buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants. Any carrier or excipient should be sterile and fluid to the extent for easy delivery via syringe.

[0052] This aspect of the invention can be prepared by incorporating MSCs, in a pharmaceutically acceptable carrier or diluent and, as required, other ingredients enumerated above. In this example, MSCs may be combined in an admixture with a solution, which supports cell viability of the MSCs. Cell dosage is selected. MSCs are then gently seeded in the solution by slowly applying the admixture of Chang's media and desired dosage of MSCs to the selected solution. Specifically, MSCs may be seeded by expelling them slowly from an automated pipette directly onto the solution so the cells could be gently mixed into the gel. A conventional syringe can be used so long as the needle lumen or bore is of sufficient diameter (e.g. 30 gauge or larger) that shear forces will not damage the cells during subcutaneous delivery.

[0053] Treatment

[0054] The therapeutic composition with MSCs are to be administered within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit/risk ratio.

[0055] MSCs may be administered in therapeutic compositions to subjects for the purpose of improving or accelerating wound healing. The administration of MSCs to the wound site leads the cells to mimic the body's natural response to a wound. For example, MSCs function to spontaneously differentiate into the other cell types that comprise and/or support the natural wound healing apparatus, e.g., fibroblasts, inflammatory cells or cells for angiogenesis.

[0056] Prior to administration, a wound may be debrided by physical or chemical means.

[0057] In an embodiment, a matrix seeded with MSCs is applied to a wound.

[0058] In another embodiment, MSCs may be sprayed or applied topically in admixture with a bioabsorbable gel or solution to a wound. Optionally, a matrix may be applied to the wound site after topical application of MSCs and bandaged in place.

[0059] In yet another example, MSCs are injected subcutaneously at a wound site.

[0060] The therapeutic compositions of the present invention are administered in a manner compatible with the dosage formulation of MSCs and in a therapeutically-effective amount. The quantity of MSCs to be administered depends, for instance, on the subject to be treated, capacity of the subject's organ, cellular and immune system to accommodate the therapeutic composition, and the nature of the cell or tissue therapy, etc. Amount and frequency of dosage of the therapeutic composition required to be administered depends on the judgment of the practitioner and is specific to each individual. Suitable regimens for initial administration and follow on administration are also variable, but can include an initial dosage administration followed by repeated doses at one or more intervals as desired or indicated (e.g. hours, days, weeks, months, or years) by a subsequent administration of a matrix, gel, or injection alone or in some combination.

[0061] In an embodiment, dosages of the therapeutic composition may range from about lxlO⁶ cells to about lxlO¹⁰ cells per wound site and may also dependent on the route of administration and the size of the wound site. Dosages may also include more than lxlO¹⁰ cells or less than lxlO⁶ cells per administration at a wound site. Treatments may involve administration of dosages may occur daily, weekly, or monthly for a month up to 6 months or more. Dosage and treatment depends in part on patient tolerance and progress of treatment.

[0062] In a non-limiting example, MSCs may range from about lxlO⁶ cells to about

1x10¹⁰ cells seeded in a matrix. The seeded matrices is applied topically to a wound site. Suitable dosages may also include more than lxl 0¹⁰ cells or less than lxl 0⁶ cells per administration at a wound site. Treatments may involve administration of dosages may occur daily, weekly, or monthly for a month up to 6 months or more. If after one week scaffold is not proficient, then local subcutaneous injection of same number of cells will be applied. Dosages may be increased or decreased as needed.

[0063] In a non-limiting example, MSCs may range from about lxlO⁶ cells to about lxlO¹⁰ cells seeded in a gel. Suitable dosages may also include more than lxlO¹⁰ cells or less than lxlO⁶ cells per administration at a wound site. The admixture is applied topically to a wound site. Treatments may involve administration of dosages daily, weekly, or monthly for a month up to 6 months or more. Dosages may be increased or decreased as needed.

[0064] In another non-limiting example, MSCs may range from about lxl 0⁶ cells to about lxlO¹⁰ cells subcutaneously injected adjacent to, or within, the wound site. Suitable dosages may also include more than lxl 0¹⁰ cells or less than l lO⁶ cells per administration at a wound site. Treatments may involve administration of dosages daily, weekly, or monthly for a month up to 6 months or more.

[0065] In another embodiment, dosage administration may involve administering a matrix or gel seeded with a dosage of MSCs coupled with follow on administrations of subcutaneous injections of MSCs either concurrently with administration of the matrix or gel in repeated dosage intervals or at alternative intervals.

[0066] In yet further embodiments, MSCs may be administered in any combination of seeded matrices, gels, and/or subcutaneous injections at sufficient dosages for sufficient treatment durations.

[0067] Administration with Matrix

[0068] In this example, a biodegradable matrix seeded with MSCs in a dosage range of about 1X10⁶ cells is prepared according to the formulation methods and either applied to a wound or stored under refrigerated conditions (28°C) prior to administration to a wound. The wound may be debribed prior to administration of the seeded biodegradable matrix as needed. The seeded biodegradable matrix is administered by topical application to a wound. The matrix should be bandaged to maintain contact between the matrix and the wound to allow for MSC migration to the wound site. [0069] Administration is repeated weekly for up to 6 months depending upon progress of wound healing. The cell dosages and frequency of administration may be increased or decreased accordingly to patient tolerance and progress of treatment.

[0070] Treatment may be supplemented by local subcutaneous injection of about 1X10⁶ to about 1X10¹⁰ MSCs according to the formulations of the invention and depending upon wound condition. Subcutaneous injection may occur daily, weekly, or monthly depending upon the severity of the wound, healing progress and tolerance.

[0071] Administration With Gel

[0072] In this example, a gel seeded with MSCs in a dosage range of about 1X10⁶ cells is prepared and either applied to a wound or stored under refrigerated conditions (28°C) prior to administration. The wound may be debribed prior to administration of the seeded gel. The seeded gel is administered topically by spraying or direct application to a wound. The gel should be bandaged to maintain contact between the gel and MSCs to the wound to allow for migration of MSCs to the wound site.

[0073] Administration may be repeated weekly for up to 6 months depending upon progress of wound healing. Dosages and frequency of administration may be increased or decreased according to the severity of the wound, progress of the treatment, and tolerance.

[0074] The treatment may be supplemented by local subcutaneous injection of about

1X10⁶ to about lX10¹⁰MSCs according to the formulations of the invention and depending upon wound condition. Subcutaneous injection may occur daily, weekly, or monthly depending upon the severity of the wound, healing progress, and tolerance.

[0075] Administration With Subcutaneous Injection [0076] In this example, MSCs suspended in a pharmaceutically-acceptable excipient in a dosage range of about 1X10⁶ cells is prepared and either applied to a wound or stored under refrigerated conditions (28°C) prior to administration. The wound may be debribed prior to subcutaneous injection. Administration may be repeated weekly for up to 6 months depending upon progress of wound healing. Dosages and frequency of administration may be increased or decreased according to the severity of the wound, progress of the treatment, and tolerance.

[0077] During any treatment with MSCs, laboratory evaluations include hematocrit, Hgb, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), platelet count, red cell count, white blood cell count (WBC) and differential, prothrombin time (PT) and International normalized ratio (INR). Clinical chemistry includes analysis of parameters such as calcium, sodium, potassium, chloride, bicarbonate, albumin, total and direct bilirubin, ALT, AST, triglycerides, cholesterol, blood glucose, BUN, ALP, serum creatinine, cardiac troponin, CK, serum amylase and pancreatic lipase. Elevations in serum amylase or CK will trigger analysis of pancreatic amylase and CK isoenzymes (CK-MM, CK-MB, CK-BB), respectively. Urinalysis include analysis of specific gravity, pH, protein, glucose, ketones, occult blood, bilirubin, urobilinogen, and nitrite. Microscopy (identification of cells, casts and crystals) should be performed if urinalysis is abnormal.

[0078] During treatment, blood samples will be collected for the determination of plasma levels of stem cells products, cytokines, and growth factors. In addition, serum samples will be collected for future PK analysis (if needed).

[0079] The primary endpoints for any treatment include serious adverse events, dose limiting toxicity, or Grade 1-4 laboratory abnormalities. Secondary endpoints for treatment include any one of a change in dosage from baseline to day 28, patient not affected by treatment, rapid wound healing in response to treatment, rapid MSC growth (i.e. 1 log increase from nadir on two consecutive measurements while still in treatment), or significant change in laboratory tests from day one through day 28 of treatment.

[0080] Rat Study

[0081] A rat study was performed to determine efficacy of treatment with the therapeutic composition comprising a matrix and MSCs in rats. The matrix was an ORC Promogran Matrix having a 45% oxidized regenerated cellulose and 55% collagen, packaged under sterile conditions and freeze dried.

[0082] For the study, 48 adult male Nude Sprague-Dawley rats (250gm) separated into 3 experimental groups of 16 rats. The 3 groups were treated as follows: (1) Normal wound with matrix only (acellular dermal matrix, Promogran); (2) Ischemic wound with MSC vehicle (solution); and (3) Ischemic wound with MSC vehicle and MSCs. Each rat was anesthetized, weighed and administered a subcutaneous injection of buprenorphine (0.01-0.05 mg/kg SQ given at 8 to 12 hour intervals) for pain. The backs of the rats were clipped and the skin disinfected with 3 alternating scrubs of 10% povidone-iodine solution scrub (Betadine) followed by 70% ethanol wash with the third 70% isopropyl alcohol rinse followed by an application of povidone- iodine solution to the surgical site. During surgery, rats were kept at a temperature that did not exceed 39°C. The rats underwent a bipedicle "skin flap" surgery as described in Chen C et al. Wound Repair Regen 1999;7:486-94. A rectangular template for the flap measuring 2.5 X 11 cm was traced on the back of the rat. The rostral base of the template was at the inferior angle of the scapulas and extend caudally to the level of the ischial tuberosities, which was the caudal base. Two pairs of 6 mm diameter punch wounds were created along the flap at 3.6 cm and 7.2 cm from the base of the template and centered on each side of the midline of the template. The biopsies served as day 0 samples and were frozen at -80°C until analyzed.

[0083] For the ischemic skin flap group, the lateral lines of the template were incised through the panniculus camosus into the relatively avascular plane of facial tissue that underlies the panniculus camosus muscle layer to create the flap. The skin flap was raised by blunt dissection through the plane of loose connective tissue that overlies the skeletal muscle, then repositioned and re-attached with 9 mm stainless steel staples along the incisions.

[0084] On the day of surgery (day 0) then every other day (days 2, 4, 6, 8, and 10), rats were anesthetized with isoflurane, weighed, the wound dressing removed, the wound surface area traced on an acetate sheet with a felt tip pen, the test agent reapplied topically to the wound, and the wounds redressed. After recovery, rats were returned to their cage. Wounds were treated at the time of surgery with Promogran Matrix vehicle and MSCs or the Promogran Matrix vehicle alone, and dressed with a non-adherent dressing (Tegaderm, 3M). For the group receiving MSCs and not scheduled for sacrifice, MSCs at a dosage of 100,000 MSCs per 100 microliters were injected into the matrix bandaged to a wound on days 2, 4, 6, and 8. Acetate traces of each wound were digitized using a high-resolution, HP Scan Jet 3C Scanner. The size of each wound was calculated using the NIH ImageJ (http://rsbweb.nih.gov/ij/). Mean wound areas and standard error of the mean were calculated.

[0085] On days 2, 6, 8, and 10, four rats from each group were sacrificed for analysis of the wounds. For euthanasia groups of rats were anesthetized by isoflurane, then sacrificed. The wound sites were excised with an 8 mm punch, and the tissues specimens were frozen at -80°C for analysis. [0086] The primary endpoint was the rate of wound healing as assessed by wound size measurement every other day.

[0087] Histology of the wounds from days 0, 2, 6, 8, and 10 was assessed with hematoxylin and eosin (HE) staining and immunohistochemical or immunofluorescent staining of paraffin sections. Immunohistochemical staining was used to assess for human cells within the wounds using a human major histocompatibility antigen.

[0088] Biochemical analysis of wound excisions from days 0, 2, 6, 8, and 10 were performed using commercial ELIS A assays for vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF).

[0089] As shown in Table 1, wound areas were assessed for statistically significant differences using one-way ANOVA with Tukey's HSD post-hoc test. On Day 2 after injury, the average wound area for ischemic wounds treated with the MSCs in the acellular matrix vehicle (Promogran) was significantly less than the average wound areas for ischemic wounds treated with acellular matrix vehicle only or in normal, non-ischemic wounds treated with acellular matrix vehicle. MSCs provided an early positive effect on wound healing in ischemic skin that exceeded healing in untreated wounds. On Day 4 and Day 6, the rate of healing in ischemic wounds was significantly slower than healing in both normal wounds and ischemic wounds treated with MSCs. On Day 8, the wounds in all three groups were approaching complete healing, and the average wound areas were not significantly different. On day 10 all wounds appeared visually to be healed.

[0090] TABLE 1 : Statistical Analysis of Wound Area (Significance (PO.05).

[0091] The graphs of the average wound area for the three treatment groups verses day after surgery are presented in FIG. 2.

[0092] While preferred embodiments of the present invention have been shown and described, it will be apparent to those skilled in the art that many changes and modifications may be made without departing from the invention in its broader aspects. The appended claims are intended to cover, therefore, all such changes and modifications as fall within the true spirit and scope of the invention.

Claims

The invention claimed is:

1. A composition for improving wound healing, the composition comprising, menses-derived stem-like cells, and

a vehicle for delivering a therapeutically effective amount of the menses-derived stemlike cells to a wound to improve healing, wherein the vehicle is seeded with the therapeutically effective amount of the menses-derived stem-like cells.

2. The composition of claim 1, wherein the menses-derived stem-like cells comprise any one or more of a substantially heterogeneous population of cells, a substantially homogenous population of cells, or a population of cells that express CD117.

3. The composition of claim 1, wherein the menses-derived stem-like cells seeded in the vehicle range from about lxlO⁶ cells to about lxlO¹⁰ cells.

4. The composition of claim 1, wherein the vehicle comprises any one of a biodegradable matrix, a gel, or a pharmaceutically acceptable excipient suitable for subcutaneous injection of the menses-derived stem-like cells.

5. A method for preparing a composition to improve wound healing comprising seeding a pharmaceutically effective amount of menses-derived stem-like cells in a vehicle for administering the cells to a wound.

6. The method of claim 5, wherein the seeding of the menses-derived stem-like cells comprises adding cells in a range of about lxlO⁶ cells to about lxlO¹⁰ cells to the vehicle.

7. The method of claim 5, wherein the menses-derived stem-like cells comprise any one or more of a substantially heterogeneous population of cells, a substantially homogenous population of cells, or a population of cells that express CD117.

8. The method of claim 5, wherein the vehicle comprises any one of a biodegradable matrix, a gel, or a pharmaceutically acceptable excipient suitable for subcutaneous injection of the menses-derived stem-like cells.

9. A method for improving wound healing, the method comprising administering a dosage of a therapeutically effective amount of menses-derived stem-like cells and seeded in a vehicle at least once to a wound.

10. The method of claim 9, wherein the dosage of the menses-derived stem-like cells is seeded in a range of about lxlO⁶ cells to about lxl 0¹⁰ cells per vehicle.

11. The method of claim 9, wherein the dosage of the menses-derived stem-like cells is seeded at more than lxl 0¹⁰ cells per vehicle.

12. The method of claim 9, wherein the menses-derived stem-like cells comprise any one of a substantially heterogeneous population of cells, a substantially homogenous population of cells, or a population of cells that express CD117.

13. The method of claim 9, wherein the vehicle comprises any one of a biodegradable matrix, a gel, or a pharmaceutically-acceptable excipient suitable for subcutaneous injection of the menses-derived stem-like cells.

14. The method of claim 13, wherein the method comprises administering at least two forms of the vehicle seeded with a dosage of the menses-derived stem-like cells to the wound.

15. The method of claim 14, wherein the method comprises substantially debriding the wound before administering the vehicle seeded with the menses-derived stem-like cells to the wound.

16. The method of claim 9, wherein the method comprises substantially debriding the wound before each instance of administering the vehicle seeded with the menses-derived stemlike cells to the wound.

17. The method of claim 13, wherein a biodegradable matrix or gel seeded with the therapeutically effective amount of the menses-derived stem-like cells is administered to the wound at least once per month for a range of 1 month to 6 months.

18. The method of claim 17, wherein the pharmaceutically-acceptable excipient seeded with the therapeutically effective amount of the menses-derived stem-like cells is also administered to the wound weekly by subcutaneous injection for a range of about 1 month to about 6 months.

19. The method of claim 13, wherein the pharmaceutically-acceptable excipient seeded with the therapeutically effective amount of the menses-derived stem-like cells is administered to the wound weekly by subcutaneous injection for a range of 1 month to 6 months.

20. The method of claim 9, wherein the method comprises substantially debriding the wound before administering the vehicle seeded with the menses-derived stem-like cells to the wound.