MEDICAMENT COMPRISING A COMBINATION OF AMNIOTIC MEMBRANE AND UMBILICAL CORD BLOOD SERUM FOR WOUND HEALING

Abstract

The invention is directed at a medicament comprising a combination of dehydrated or cryopreserved amniotic membrane isolated from human or other mammalian placenta, wherein the amniotic membrane is in a form of a sheet or a sheet composite comprising multiple overlain sheets of amniotic membrane; and umbilical cord blood serum, isolated from umbilical cord blood of a human or other mammal, separately of each other, for combined use in a method of treating a tissue wound in a human or other mammalian subject by applying the amniotic membrane to injured tissue of the wound to contact injured tissue of the wound and contacting the amniotic membrane with the umbilical cord blood serum.

Claims

1-9. (canceled)

10. A method of treating a tissue wound in a human or other mammalian subject comprising the steps of: applying an amniotic membrane to an injured tissue of the wound to contact the injured tissue of the wound; and contacting an amniotic membrane with an umbilical cord blood serum; wherein the amniotic membrane is isolated from human or other mammalian placenta; wherein the amniotic membrane is dehydrated or cryopreserved and is in a form selected from a sheet of amniotic membrane, or an amniotic membrane sheet composite, comprising multiple overlain sheets of amniotic membrane; and wherein the umbilical cord blood serum is isolated from umbilical cord blood of a human or other mammal separately of each other.

11. The method according to claim 10, wherein the sheet of amniotic membrane comprises an amniotic membrane that has been formed into a predetermined shape and size.

12. The method according to claim 10, wherein the umbilical cord blood serum is selected to include growth factors and cytokines that include one or more or all of epidermal growth factor, hepatocyte growth factor, macrophage migration inhibitory factor (MIF), and stem cell growth factor beta (SCGF-β) that promote wound healing activity initiated by the amniotic membrane.

13. The method according to claim 10, wherein the umbilical cord blood serum is selected to include growth factors and cytokines that include one or more or all of IL-1a, IL-3, M-CSF, IL-12 p40, IL-16, MIF, IFN-a2, IL-18, MIG, MCP-3, CTACK, SCF, TNF-β, GROa, SCGF-β, b-NGF, HGF, SDF-1a, IL-2Ra, LIF, and TRAIL.

14. The method according to claim 13, wherein the umbilical cord blood serum is selected to include growth factors and cytokines that include one or more or all of IL-3, M-CSF, IL-16, MIF, IL-18, MIG, MCP-3, CTACK, SCF, GROa, SCGF-β, HGF, SDF-1a, IL-2Ra, LIF, and TRAIL.

15. The method according to claim 10, wherein the umbilical cord blood serum includes growth factors and cytokines at a level in a range of from 1 pg/ml to 50000 pg/ml.

16. The method according to claim 10, wherein the umbilical cord blood serum is provided as a liquid, a suspension, a solution, drops, or a dried powder.

17. The method according to claim 10, wherein the tissue wound is a soft tissue wound, a burn, an ulcer, a surgical wound, or a chronic wound that has not healed fully in response to an earlier treatment.

18. A method of treating a wound in a human or other mammalian subject, the method comprising applying a sheet of dehydrated or cryopreserved amniotic membrane to the wound and contacting the amniotic membrane with umbilical cord blood serum, wherein the amniotic membrane was isolated from human or other mammalian placenta.

Description

EXAMPLES

[0082] The invention will now be described in more detail with reference to the examples hereunder, and the accompanying drawings.

[0083] In the drawings

[0084] FIG. 1 shows, for Example 1, concentrations of cytokines and growth factors in cord blood serum;

[0085] FIG. 2 shows, for Example 1, concentrations of MIF and SCGF-beta in cord blood serum;

[0086] FIG. 3 shows, for Example 1, concentrations of SCGF-beta in cord blood serum;

[0087] FIG. 4 shows, for Example 1, concentrations of HGF in cord blood serum;

[0088] FIG. 5 shows, for Example 1, concentrations of CTACK, MIG and SDF-1 in cord blood serum;

[0089] FIG. 6 shows, for Example 2, treatment of a spider bite wound with (A) the wound before treatment, (B) four days post treatment, (C) five weeks post treatment, (D) fifteen weeks post treatment; and

[0090] FIG. 7 shows, for Example 3, treatment of a partial burn wound using dehydrated prepared amniotic membrane.

EXAMPLE 1

Materials and Methods

Umbilical Cord Blood Serum Composition

Placental Donation and Umbilical Cord Blood Collection

[0091] Placentas were collected from human female subjects. These mothers had uncomplicated pregnancies and gave their informed consent for the retrieval of umbilical cord blood and amniotic membrane from the placenta for therapeutic and research purposes. Samples of the mother’s blood were taken and tested for HIV, Hepatitis B and C, HTLV I and II, CMV and syphilis. Umbilical cord blood was collected by direct cannulation of cord blood into a component blood transfer bag/ 50 ml centrifuge/falcon tube, with no anti-coagulants or preservatives, taking all sterile precautions after the infant was removed and the umbilical cord clamped. The blood was drained from the placenta by gravitation.

Laboratory Processing

[0092] The blood was stored at 4° C. for 2 to 24 hours. The clotted blood was centrifuged for 60 minutes at 3500 rpm, at room temperature. The serum fraction (supernatant) was removed from the clotted pellet and transferred to a sterile 50 ml falcon tube, using aseptic techniques. The serum was diluted with Balanced Salt Solution (BSS) to make a 20% dilution. The diluted cord blood serum was centrifuged for 10 minutes at 3500 rpm, at room temperature following which it was filter sterilized through a 0.20 .Math.m filter.

[0093] After filter sterilization 4.5 ml aliquots of the diluted serum were transferred to 5.0 ml dropper bottles. The pH and ABO blood grouping of the serum was tested using standard procedures.

[0094] Umbilical cord blood serum may be freeze-dried and processed to a powder form which is stable at room temperature.

[0095] Umbilical cord blood serum is stored at -80° C. for long term storage. For use, it is thawed and stored at 4-8° C.

Analysis of Contents of Umbilical Cord Blood Serum

[0096] In order to gain insight into the mechanism whereby umbilical cord blood serum (as found in the Next Biosciences OptiSerum™ product) mediates its regenerative effect, the umbilical cord serum from 14 donors was analyzed for the presence of 21 cytokines and growth factors.

[0097] A Bio-Plex Pro Human Cytokine Standard 21-plex (BioRad; Group II) and a BioPlex 200 multiplex system was utilized to assess cytokine levels in umbilical cord blood from 14 donors. Dilutions of standards were as directed by the manufacturer, while all dilutions of cord blood serum (using the manufacturer’s sample diluent) were at 20% (as per OptiSerum™). The concentration of the following cytokines and growth factors in umbilical cord serum (IL-1α, IL-3, M-CSF, IL-12 p40, IL-16, MIF, IFN-α2, IL-18, MIG, MCP-3, CTACK, SCF, TNF-β, HGF, SDF-1α, IL-2Rα, LlF, TRAIL) were determined as per the manufacturer’s instructions. Eighteen of these were found to be present in the samples tested. The average levels of 16 cytokines and growth factors range from 10 - 50 000 pg/ml (many of these cytokines are either immune modulators or stem cell factors), while two are detected between 0.4 and 2 pg/ml. Three pro-inflammatory cytokines (IL-1α, IL-12 p40; IFN-α2) are not detectable in umbilical cord serum.

Amniotic Membrane

Mechanical Removal of the Amniotic Membrane from the Chorion and Other Placental Tissue

[0098] Using aseptic techniques, placenta donated by human female subjects who had uncomplicated pregnancies were transferred from sterile transport bags to the stainless steel dish with 500 ml cold saline. The placenta was rinsed to remove any blood. The amniotic membrane was mechanically removed from the chorion by pulling the amniotic membrane off by hand towards the umbilical cord and the membrane was cut loose from the cord. The membrane was rinsed in 200 ml saline.

[0099] The chorionic membrane was wiped from the amniotic membrane with sterile gauze and saline. The membrane was rinsed repeatedly with saline, to remove all residual tissue and to keep the membrane wet. The membrane was transferred to 1% antibiotic/antimycotic : DMEM solution and incubated at 2 - 8° C. for 48 - 72 hrs.

Dehydrated Amniotic Membrane

[0100] The amniotic membrane was wiped to remove all excess chorion and antibiotic/antimycotic solution and rinsed in sterile saline. The membrane was incubated in Trypsin/EDTA solution for 2 hours 30 minutes. The amniotic membrane was removed from the Trypsin/EDTA solution and washed with DMEM. Gauze wipes were used to remove the amniotic epithelial cell layer following which the membrane was rinsed repeatedly with saline to remove all residual tissue and to keep the membrane wet. The amniotic membrane was stretched out on a clean dry surface in a Bio-Flow cabinet and left until completely dry and then packaged and sterilized using Gamma irradiation. Dehydrated amniotic membrane is stored at room temperature.

Cryopreserved Amniotic Membrane

[0101] The amniotic membrane was wiped to remove all excess chorion and antibiotic/antimycotic solution and rinsed in sterile saline. The membrane was incubated in Trypsin/EDTA solution for 2 hours 30 minutes. The membrane was removed from the Trypsin/EDTA solution and washed with DMEM. The amniotic membrane was stretched out on a nitrocellulose membrane carrier with the stromal side of the membrane facing onto the nitrocellulose paper. DMEM: 87% Glycerol (1:1 ratio (v/v)) was added to the amniotic membrane before freezing at -80° C. Cryopreserved amniotic membrane is stored at -20° C. For use, it is thawed at room temperature and washed in saline to remove the glycerol cryoprotectant.

Treatment of Soft Tissue Wounds

[0102] An appropriately sized dehydrated or cryopreserved amniotic membrane dressing was applied so that it covers the enter wound. Following this, 4 drops of umbilical cord blood serum was applied to the wound, directly onto the amniotic membrane wound dressing. A further 4 drops of umbilical cord blood serum were placed on the wound every 4 days until the wound was fully healed. If required a new amniotic membrane dressing was placed on the wound every 7 days. The dehydrated membrane was found to attach easily to the wound surface and to absorb fluids from the wound.

Results

Analysis of Umbilical Cord Blood Serum

[0103] The product umbilical blood cord serum (OptiSerum™) was analyzed to determine the concentration of various growth factors and cytokines. Values shown in the results take the dilution of umbilical cord blood serum into account and represent the levels in the original umbilical cord blood serum. The cytokines successfully tested are shown in Table 1 below. ICAM-1 and VCAM-1 standards were not successfully detected.

[0103] TABLE-US-00001 List of growth factors and cytokines included in the BioPlex Group II Human Cytokine Assay IL-1α
interleukin-1 alpha IL-3
interleukin-3 M-CSF
macrophage colony stimulating factor IL-12 p40
interleukin-12 subunit p40 IL-16
interleukin-16 MIF
macrophage migration inhibitory factor IFN-α2
interferon-alpha 2 IL-18
interleukin-18 MIG
monokine induced by gamma interferon MCP-3
monocyte chemotactic protein 3 CTACK
cutaneous T-cell attracting chemokine SCF
Skp, Cullin, F-box containing complex TNF-P
tumour necrosis factor-beta GROα
growth-regulated oncogene-alpha SCGF-β
stem cell growth factor-beta b-NGF
nerve growth factor-beta HGF
hepatocyte growth factor SDF-1α
stromal cell-derived factor-1 alpha IL-2Rα
interleukin-2 receptor alpha LIF
leukemia inhibitory factor TRAIL
tumor necrosis factor (TNF)-related apoptosis inducing ligand

[0104] Sixteen growth factors and cytokines (out of the 21 tested) are present at significant levels in the cord blood samples tested (FIG. 1). Average levels range from 10 - 50 000 pg/ml. Many of these cytokines are either immune modulators or stem cell factors. Detectable levels of the remaining five cytokines (IL-1α; IL-12 p40; IFN-α2; TNF-β; β-NGF) are negligible.

[0105] Within the group of 21 cytokines, 3 are present at very high levels and specifically at concentrations greater than 1000 pg/ml (in other words, in the ng/ml range). These are Macrophage migration inhibitory factor (MIF), Stem cell growth factor beta (SCGF-β) and Hepatocyte growth factor (HGF). The concentration of these factors in each of the cord blood samples is shown in FIGS. 2-4. The concentration of each protein varies considerably between the 14 samples tested i.e. 2000-25 000 pg/ml (MIF); 10 000 -50 000 pg/ml (SCGF-beta); 500 - 2000 pg/ml (HGF).

[0106] Three additional cytokines are expressed at high levels (FIG. 5), namely Cutaneous T-cell attracting chemokine (CTACK), Monokine induced by gamma IFN (MIG) and Stromal cell-derived factor 1 (SDF-1). The concentration of these factors generally ranges between 100 pg/ml and 1000 pg/ml, with one sample (NC/AM/15/000097) containing 2000 pg/ml of MIG (FIG. 5). It is understood that epidermal growth factor (EGF), hepatocyte growth factor (HGF), Macrophage migration inhibitory factor (MIF) and Stem cell growth factor beta (SCGF-β) would be present at high levels in umbilical cord blood serum as these growth factors are associated with stem cell growth and development.

Example 2

Treatment of Spider Bite Wound With Prepared Amniotic Membrane (Amniomatrix™) and Umbilical Cord Blood Serum (Optiserum™)

[0107] The subject is a 24 year old female patient with Spina Bifida suffering from a non-healing wound on her bottom right foot and difficulty walking due to abnormal weight distribution on her feet. In March 2013, the patient developed cellulitis after a suspected spider bite on the base of her right foot. The infection was treated successfully, however, the wound did not heal and instead became necrotic. She had multiple attempts at healing the wound with wound healing care products, surgery and had undergone skin grafts and tendon transplants, none of which were successful.

[0108] A 4×4 cm dehydrated amniotic membrane dressing accompanied by 4 drops of umbilical cord serum was applied to the wound every four days for a 15 week period. From the initial dressing, quite a significant improvement was noted with the wound getting narrower, superficial and clean. Over the weeks, the wound healed remarkably with prominent wound healing stages observed i.e. less callous formation, granulation, keratin formation, epithelialization and a tougher outer surface, thus leading to complete wound closure and healing (FIGS. 6A-D). At 4 days post treatment (FIG. 6B) the wound was narrower, superficial and clean. At 5 weeks post treatment (FIG. 6C), there was good granulation observed and wound contraction. At 15 weeks post treatment (FIG. 6D), there was good epithelialization, a tougher outer surface and wound closure.

Example 3

Treatment of Partial Burn Wound to the Face With Dehydrated Prepared Amniotic Membrane (Amniomatrix™)

[0109] A 32 year old male subject patient presented with partial thickness burns to the face. The injury occurred on 22 Feb. 2016. Dehydrated prepared amniotic membrane was applied to the subject’s forehead. As a comparison, Nanotrix.sup.® (Southern Medical (Pty) Ltd, Southern Implants Office park, 1 Albert Road, Irene, 0062) was applied to the other areas. Nanotrix is a temporary synthetic skin substitute used for treating burns. Simple chloromycetin ointment was applied as an antibiotic over the membranes and there was no sepsis of the wounds. The wound covered by amniotic membrane healed by 25 Feb. 2016 whereas the wound covered by Nanotrix healed by 27 Feb. 2016 (FIG. 7) demonstrating that the amniotic membrane showed improved efficacy over the synthetic Nanotrix product in healing of the burn wound in that the same level of healing was achieved two days earlier with the amniotic membrane than the Nanotrix product. This outcome supported favouring a biologic material over a synthetic material in the healing of wounds, which the present invention inventively provides for with the additional employment of umbilical cord blood serum.

Discussion

[0110] AMNIOTIC MEMBRANE ALLOGRAFTS have been used as a biologic therapy to promote soft tissue healing in ophthalmic and other peripheral wounds. A primary characteristic of amniotic membrane that assists with wound healing is the provision of a scaffold of pre-formed extracellular matrix composed of natural collagen and other proteins. Dehydrated amniotic membrane reduces wound pain and reduces the level of exudate produced by the wound.

[0111] However, it was found by the inventors that many growth factors and cytokines that facilitate the wound healing process through migration of biologically active cells to the wound and stimulation of the proliferation, differentiation and growth of these cells, are not present, due to the processing and storing of the amniotic tissue.

[0112] It was surprisingly found, by the inventors, that this lack of growth factors and cytokines can be effectively be corrected by co-administration of umbilical cord blood serum, in accordance with the invention.

[0113] It is demonstrated, in accordance with the invention, that umbilical cord blood serum contains many growth factors and cytokines that are responsible for cell migration, proliferation and differentiation. Thus, co-administration of amniotic membrane grafts and umbilical cord blood serum was surprisingly found to improve the dysfunction of the inflammation, cellular proliferation, and tissue remodelling stages of the wound healing process and promotes quicker healing of wounds with decreased scarring.

[0114] It was therefore surprisingly found that a combination of umbilical cord blood serum and amniotic membrane, according to the invention, enhances wound healing activity in comparison to the amniotic membrane on its own, including quicker healing of wounds and decreased scarring.

[0115] The co-administration of amniotic membrane and umbilical cord blood serum according to the invention, was therefore found to be particularly used for the treatment of “treat and heal” soft tissue wounds such as spider bites, burns and diabetic ulcers even when these wounds do not respond satisfactorily to other earlier-administered therapies including wound healing care products, surgery, skin grafts and tendon transplants.

[0116] Furthermore, dehydrated amniotic membrane was found to stick to wound surfaces very effectively and also to absorb fluids from the wound. It was further found to reduce pain arising from the wound and to reduce the level of exudate produced by the wound.