Activated leukocyte conditioned supernatant and uses for wound healing

• US 9,168,287 B2
• Filed: 09/08/2011
• Issued: 10/27/2015
• Est. Priority Date: 09/09/2010
• Status: Expired due to Fees

• Current Assignee: Marcrocure, Ltd.
• Original Assignee: Macrocure, Ltd. (Leap Therapeutics, Inc.)
• Inventors: Shirvan, Mitchell, Bain, Eilat, Bubis, Marina, Ginis, Irene
• Primary Examiner(s): Gamett, Daniel C
• Application Number:
  US13/821,676
• Publication Number:
  US 20130224148A1
• Time in Patent Office: 1,510 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 35/15 Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells presenting a specific antigen A61K39/00; therapeutic combinations of antibodies, or fragments thereof, and blood-derived cells A61K39/00
• CPC Class Codes: A61K 35/16 Blood plasma; Blood serum umbilical cord blood A61K35/51
• CPC Class Codes: A61K 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• CPC Class Codes: A61K 38/191 Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
• CPC Class Codes: A61K 38/204 IL-6
• CPC Class Codes: A61K 38/2053 IL-8
• CPC Class Codes: A61L 15/32 Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin A61L15/225 takes precedence
• CPC Class Codes: A61L 15/40 containing ingredients of undetermined constitution or reaction products thereof , e.g. plant or animal extracts
• CPC Class Codes: A61L 15/42 Use of materials characterised by their function or physical properties liquid bandages A61L26/00
• CPC Class Codes: A61L 15/44 Medicaments
• CPC Class Codes: A61L 2300/41 Anti-inflammatory agents, e.g. NSAIDs
• CPC Class Codes: A61L 2300/412 Tissue-regenerating or healing or proliferative agents
• CPC Class Codes: A61L 2300/414 Growth factors
• CPC Class Codes: A61L 2300/45 Mixtures of two or more drugs, e.g. synergistic mixtures
• CPC Class Codes: A61L 2300/64 Animal cells
• CPC Class Codes: A61L 26/0057 Ingredients of undetermined constitution or reaction products thereof
• CPC Class Codes: A61L 26/0066 Medicaments; Biocides
• CPC Class Codes: A61L 27/3804 characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• CPC Class Codes: A61L 27/54 Biologically active materials, e.g. therapeutic substances A61L27/227 takes precedence
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/04 : for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 41/00 : Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
• CPC Class Codes: A61P 9/14 : Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers

Abstract

Disclosed are therapeutic, activated leukocyte conditioned supernatants, methods of making them, and methods of using the conditioned supernatants to repair or promote healing of wounds.

Claims

1. A substantially cell-free activated leukocyte-conditioned supernatant (ALCS), wherein the ALCS is made by:

incubating human leukocytes under conditions of time and temperature to activate the leukocytes, wherein the activated leukocytes comprise activated granulocytes, activated monocytes, and activated lymphocytes;

subjecting the activated leukocytes to hypo-osmotic shock;

adding to the activated leukocytes subjected to the hypo-osmotic shock to a physiologically acceptable solution containing inorganic ions, organic osmolytes, or a combination thereof in an amount which restores isotonicity;

separating the activated leukocytes from the resulting isotonic solution;

mixing the separated activated leukocytes with serum to form an incubation composition;

incubating the incubation composition ii) at room temperature for 8 to 72 hours;

or (ii) at about 37°

C. for 3 to 5 hours; and

separating the activated leukocytes and other cellular matter from the incubation composition to produce a substantially cell-free ALCS.

2. The substantially cell-free ALCS of claim 1, wherein the ALCS comprises at least about 1000 pg/mL human IL-8, at least about 10-20 pg/mL human IL-6, and at least about 20 pg/mL human TNFalpha.

3. An article of manufacture comprising the ALCS of claim 1 or 2, and a dressing, scaffold, or matrix.

4. The article of manufacture of claim 3, wherein:

(i) the dressing is gauze, a bandage, a non-adhesive mesh, a membrane, foils, foam, or a tissue adhesive;

(ii) the dressing is a moisture-keeping barrier;

(iii) the dressing is a moisture keeping barrier selected from the group comprising a paste, a cream, an ointment, a nonpermeable or semi-permeable membrane or foil, a hydrocolloid, a hydrogel, and combinations thereof;

(iii) the dressing is an antimicrobial dressing;

(iv) the scaffold or matrix is a solid before implantation; and

/or

(v) the scaffold or matrix is a gel that solidifies following implantation.

5. A method for treating a wound or inhibiting the onset of infection in a wound comprising administering to the wound the ALCS of any one of claim 1 or 2.

6. The method of claim 5, wherein:

(i) the wound is a decubital ulcer, a pressure ulcer, a lower extremity ulcer, tendonitis, a deep sternal wound, a post-operative wound, a refractory post-operative wound of the trunk area, a wound to the great saphenous vein following harvesting of the great saphenous vein, a venous ulcer, a burn wound, a battlefield wound, or an anal fissure;

(ii) the wound is a lower extremity ulcer in a diabetic patient;

(iii) the wound is a venous ulcer, pressure ulcer, or post-operative ulcer;

(iv) the wound is caused by trauma;

(v) the wound is caused by surgery;

(vi) the ALCS is injected into the wound;

(vii) the ALCS is injected into tissue surrounding the wound;

(viii) the wound is an open wound and the ALCS is applied into the open wound;

(ix) the wound is an open wound and the ALCS is applied to the open wound via a dressing,

(x) the wound is an open wound and the ALCS is applied to the open wound via a dry dressing, a barrier dressing, or a bioactive dressing;

(xi) the ALCS is derived from an autologous blood sample; and

/or

(xii) the ALCS is derived from an allogeneic blood sample.

9. The method of claim 4, wherein the subjecting the leukocytes to hypo-osmotic shock comprises contacting the leukocytes with water or a hypotonic salt solution for about 25 to about 45 seconds.

7. A method of making a substantially cell-free ALCS activated leukocyte-conditioned supernatant (ALCS), comprising:

incubating human leukocytes under conditions of time and temperature to activate the leukocytes;

subjecting the leukocytes to hypo-osmotic shock;

adding to the leukocytes subjected to the hypo-osmotic shock to a physiologically acceptable solution containing inorganic ions, organic osmolytes, or a combination thereof in an amount which restores isotonicity;

separating the leukocytes from the resulting isotonic solution;

mixing the separated leukocytes with serum to form an incubation composition;

incubating the incubation composition (i) at room temperature for 8 to 72 hours;

or (ii) at about 37°

C. for 3 to 5 hours; and

separating the leukocytes and other cellular matter from the incubation composition to produce a substantially cell-free ALCS.

8. The method of claim 7, wherein the incubation of the human leukocytes to activate the leukocytes:

(i) occurs at a temperature of about 12°

C. to about 28°

C. for a time ranging from about 8 to about 20 hours;

(ii) occurs at a temperature of about 18°

C. to about 24°

C. for a time ranging from about 8 to about 12 hours;

(iii) occurs at a temperature of 12°

C. to about 28°

C. for a time ranging from about 90 minutes upwards of 2, 3, 4, 5, 6, 7, 8, or 12 to about 20 hours;

or

(iv) occurs at a temperature up to about 37°

C. and for a time ranging from 5 hours to about 24 hours.

10. The method of claim 7, wherein:

(i) the leukocytes and the serum are obtained from the same donor;

(ii) the leukocytes and the serum are obtained from different donors;

(iii) the serum of is obtained by contacting a separate sample of human plasma with a coagulating agent and removing coagulated solids;

(iv) the serum is obtained by contacting a separate sample of human plasma with a coagulating agent and removing coagulated solids, wherein;

(a) the plasma and the leukocytes are obtained from the same donor,

(b) the plasma and the leukocytes are obtained from different donors,

(c) the contacting of the human plasma with the coagulating agent is conducted substantially contemporaneously with the incubating of the human leukocytes under conditions of time and temperature to activate the leukocytes,

(e) the plasma is obtained from an AB+ donor, and/or

(f) the coagulating agent is CaCl₂;

(v) the serum is frozen prior to contacting leukocytes; and

/or

(vi) the method further comprises freezing, lyophilizing, or freeze-drying the ALCS.

11. The method of claim 7, further comprising mixing and the leukocytes separated from the incubation composition with a second serum to form a second incubation composition;

incubating the second incubation composition under conditions of time and temperature to increase the concentration of at least one cytokine or growth factor in the incubation composition; and

separating the leukocytes and other cellular matter from the second incubation composition, so as to produce a second sample of the substantially cell-free ALCS.

12. The method of claim 7, wherein the incubation of the human leukocytes to activate the leukocytes is conducted in a container to which leukocytes adhere and/or which contains a scaffold comprising leukocyte agonists or adhesion molecules.