EP2553089A1 - Thérapie cellulaire pour améliorer la cicatrisation des plaies et compositions et méthodes associées - Google Patents

Thérapie cellulaire pour améliorer la cicatrisation des plaies et compositions et méthodes associées

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Publication number
EP2553089A1
EP2553089A1 EP11763511A EP11763511A EP2553089A1 EP 2553089 A1 EP2553089 A1 EP 2553089A1 EP 11763511 A EP11763511 A EP 11763511A EP 11763511 A EP11763511 A EP 11763511A EP 2553089 A1 EP2553089 A1 EP 2553089A1
Authority
EP
European Patent Office
Prior art keywords
cells
wound
menses
derived stem
seeded
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP11763511A
Other languages
German (de)
English (en)
Other versions
EP2553089A4 (fr
Inventor
Mercedes A. Walton
Julie G. Allickson
Rolando O. Garcia-Morales
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cryo Cell International Inc
Original Assignee
Cryo Cell International Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cryo Cell International Inc filed Critical Cryo Cell International Inc
Publication of EP2553089A1 publication Critical patent/EP2553089A1/fr
Publication of EP2553089A4 publication Critical patent/EP2553089A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K35/48—Reproductive organs
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
    • C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells

Definitions

  • 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.
  • healing begins with the inflammatory phase that initiates restoration of Hemostasis along with cellular cleansing of the body.
  • 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- ⁇ ), epidermal growth factor (EGF), transforming growth factor alpha (TGF-a), vascular endothelial growth factor (VEGF), and adhesive glycoproteins.
  • PDGF platelet-derived growth factor
  • TGF- ⁇ transforming growth factor beta
  • EGF-a epidermal growth factor
  • TGF-a transforming growth factor alpha
  • VEGF vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • 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.
  • the inflammatory cells phagocytize bacteria, cell fragments, and foreign antigens at the wound site.
  • 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.
  • 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.
  • fibroblasts become the predominant cell type at the wound site.
  • 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.
  • 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.
  • 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.
  • 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.
  • vessels, fibroblasts, and inflammatory cells disappear from the wound site by way of migration, apoptosis, or other mechanisms.
  • 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.
  • the menses-derived stem-like cells are seeded in the vehicle in a range from about lxlO 6 cells to about lxlO 10 cells. In another embodiment, the menses-derived stem-like cells are seeded at more than about lxl 0 10 cells per vehicle or less than about lxlO 6 cells depending upon patient tolerance and progress of wound healing during treatment.
  • 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.
  • a method for preparing a composition to improve wound healing 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 0 6 cells to about lxlO 10 cells to the vehicle.
  • the menses-derived stem-like cells are seeded at more than lxlO 10 cells or less than about lxlO 6 cells per vehicle.
  • the menses-derived stem-like cells 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.
  • a method for improving wound healing 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.
  • 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 lxlO 6 cells to about lxlO 10 cells per vehicle.
  • the dosage of menses- derived stem-like cells may be seeded at more than lxlO 10 cells or less than lxlO 6 cells per vehicle.
  • the method comprises administering at least two forms of the vehicle seeded with a dosage of menses-derived stem-like cells to a wound.
  • 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.
  • 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.
  • 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.
  • FIG. 1 shows a wound assessment and treatment flow diagram.
  • 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.
  • 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.
  • 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.
  • Menses-derived Stem-like Cells 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.
  • Menses-derived stem-like cells 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HBSS HBSS and 0.4 ml of heparin (400 units).
  • 0.2 ml of heparin (200 units) will be added to 10 ml ofDPBS.
  • 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.
  • the menstrual flow specimen in the second tube (heparin, HBSS) is used for infectious disease testing.
  • the two menstrual cell samples 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.
  • Cryopreserved cells may be thawed according to the techniques disclosed in U.S.
  • each of the cell samples may be further processed to isolate cells expressing specific cell markers, for example, CD117.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • bioactive agents such as, for example, growth factors
  • Any preparation of therapeutic compositions with bioactive agents is an alternative aspect of the invention.
  • 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.
  • 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.
  • any of the following matrices may be used: Integra Bilayer Matrix Wound Dressing, Promogran Matrix Wound Dressing, or equivalent.
  • 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.
  • Chang's media 325ml MEMa, 75ml FBS, 90ml Chang B, 10ml Chang C, 5ml Penstrep, & 5ml L-glutamine.
  • a method for culturing a monolayer cell line of CD117+ MSCs within a matrix is provided.
  • 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 cm 2 to harvest the following day (ex 1,000,000 cells in 1 T175 flask); plate 1,000 cells per cm 2 to harvest in 3-5 days.
  • 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.
  • 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.
  • 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.
  • MSCs may also be combined with a pharmaceutically acceptable carrier or excipient for delivery by a device, e.g., a syringe, by subcutaneous injection.
  • a pharmaceutically acceptable carrier or excipient for example, vehicles, adjuvants, carriers or diluents, are readily available to the public.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a wound Prior to administration, a wound may be debrided by physical or chemical means.
  • a matrix seeded with MSCs is applied to a wound.
  • MSCs may be sprayed or applied topically in admixture with a bioabsorbable gel or solution to a wound.
  • a matrix may be applied to the wound site after topical application of MSCs and bandaged in place.
  • MSCs are injected subcutaneously at a wound site.
  • 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.
  • dosages of the therapeutic composition may range from about lxlO 6 cells to about lxlO 10 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 lxlO 10 cells or less than lxlO 6 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.
  • MSCs may range from about lxlO 6 cells to about
  • Suitable dosages may also include more than lxl 0 10 cells or less than lxl 0 6 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.
  • MSCs may range from about lxlO 6 cells to about lxlO 10 cells seeded in a gel. Suitable dosages may also include more than lxlO 10 cells or less than lxlO 6 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.
  • MSCs may range from about lxl 0 6 cells to about lxlO 10 cells subcutaneously injected adjacent to, or within, the wound site. Suitable dosages may also include more than lxl 0 10 cells or less than l lO 6 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.
  • 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.
  • MSCs may be administered in any combination of seeded matrices, gels, and/or subcutaneous injections at sufficient dosages for sufficient treatment durations.
  • a biodegradable matrix seeded with MSCs in a dosage range of about 1X10 6 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.
  • 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.
  • Treatment may be supplemented by local subcutaneous injection of about 1X10 6 to about 1X10 10 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.
  • a gel seeded with MSCs in a dosage range of about 1X10 6 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.
  • 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.
  • the treatment may be supplemented by local subcutaneous injection of about
  • 1X10 6 to about lX10 10 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.
  • MSCs suspended in a pharmaceutically-acceptable excipient in a dosage range of about 1X10 6 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.
  • 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.
  • blood samples will be collected for the determination of plasma levels of stem cells products, cytokines, and growth factors.
  • serum samples will be collected for future PK analysis (if needed).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HE hematoxylin and eosin
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • HGF hepatocyte growth factor
  • wound areas were assessed for statistically significant differences using one-way ANOVA with Tukey's HSD post-hoc test.
  • the average wound area for ischemic wounds treated with the MSCs in the acellular matrix vehicle 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.
  • the rate of healing in ischemic wounds was significantly slower than healing in both normal wounds and ischemic wounds treated with MSCs.
  • the wounds in all three groups were approaching complete healing, and the average wound areas were not significantly different.
  • all wounds appeared visually to be healed.

Abstract

La présente invention a pour objet des compositions thérapeutiques permettant d'améliorer la cicatrisation des plaies, des procédés de formulation associés et des méthodes d'utilisation associées. Les compositions thérapeutiques comprennent un dosage de MSC ensemencées,au choix, sur une matrice biodégradable, un gel, ou une solution convenant pour une injection sous-cutanée. Le dosage des MSC peut être compris dans une plage d'environ 1 X 106 MSC à environ 1 X 1010 par administration. Le régime d'administration peut être quotidien, hebdomadaire ou mensuel. Le dosage et les intervalles de traitement peuvent être ajustés en fonction de la réponse d'un patient au traitement. Les compositions thérapeutiques peuvent être co-administrées, par exemple, une application topique d'une matrice ou d'un gel ensemencé combinée à une injection sous-cutanée simultanée ou autrement périodique de MSC.
EP11763511.0A 2010-04-01 2011-04-01 Thérapie cellulaire pour améliorer la cicatrisation des plaies et compositions et méthodes associées Withdrawn EP2553089A4 (fr)

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WO2016036322A1 (fr) 2014-09-04 2016-03-10 Kemijski inštitut Dispositif à base de cellules pour traitement local utilisant des protéines thérapeutiques
CN106176813A (zh) * 2016-07-28 2016-12-07 广州赛莱拉干细胞科技股份有限公司 一种修复皮肤溃疡的组合物及其制备方法
CN106074605A (zh) * 2016-07-28 2016-11-09 广州赛莱拉干细胞科技股份有限公司 一种修复皮肤溃疡的组合物及其制备方法
CN110693911A (zh) * 2019-10-30 2020-01-17 南通大学 经血源性子宫内膜干细胞制剂及其制备方法和应用

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JP2006230316A (ja) * 2005-02-25 2006-09-07 Japan Health Science Foundation 瘢痕のない創傷治癒能を有する細胞およびその調製方法
US20100040588A1 (en) * 2007-03-01 2010-02-18 Cryo-Cell International, Inc. Procurement, Isolation, and Cryopreservation of Endometrial/Menstrual Cells
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