WO2001008716A1

WO2001008716A1 - Method of preservation of amnion and uses thereof

Info

Publication number: WO2001008716A1
Application number: PCT/AU2000/000913
Authority: WO
Inventors: Ann Pinfold
Original assignee: Ann Pinfold
Priority date: 1999-08-02
Filing date: 2000-08-02
Publication date: 2001-02-08
Also published as: AUPQ196799A0

Abstract

This invention relates to a method of preservation of amnion such that one or more of its biological properties, such as pain relief, anti-inflammatory activity, swelling-reduction, inhibition of bacterial activity, control of exuberant granulation, wound healing or scar reduction, are substantially retained. The invention also relates to a method of preparing a wound dressing using amnion, uses of amnion for the manufacture of a medicament, packaged amnion, and methods of treating a wound or treating pain, inflammation and/or swelling in an animal using amnion.

Description

METHOD OF PRESERVATION OF AMNION AND USES THEREOF

TECHNICAL FIELD

This invention relates to a method of preservation and in particular to a method of preservation of amnion. uses of amnion and methods of treatment using amnion. BACKGROUND

Amnion, including human amnion, has been used sporadically since the beginning of this century to aid in the wound-healing process. The benefits of amnion as a wound dressing have been well-documented since the late 1970's.

Amnion is the membrane that completely lines the uterine cavity, in which the foetus develops and grows. It is initially composed of a single layer of cells, formed when the ovum is fertilised and the cells begin to divide. The developing foetus is enclosed within the amnion and is spared from destruction by the maternal immune system. While not completely understood, the unique feature which makes amnion an impermeable immunological barrier between mother and embryo is its lack of expression of surface antigens which normally mediate an immune response, for example major histocompatibility complex antigens (MHC). Taken a step further, amnion may be used as skin grafts without the need to tissue type donors to hosts.

Amnion dressings harvested from one species have proven beneficial in treating injuries in other species. For example, fresh amnion dressings harvested from equine species, while extremely beneficial in treating equine injuries, have also proven to be beneficial in treating injuries in avian species. Previous literature indicates that amnion dressings offer pain relief, inhibit bacterial activity, control exuberant granulation tissue, speed wound closure and reduce scarring.

Dried, ground and otherwise stored amnion appears to offer no significant improvements over conventional sterile dressings, while fresh or frozen amnion has been shown to aid in the rapid healing of wounds. However, the use of fresh/frozen amnion has been limited to trials and non-commercial applications due to various limitations.

The storage limit of frozen amnion is unknown but the literature suggests a maximum of

12 months. Transportation of frozen amnion dressings presents problems with regard to thawing and resultant spoilage of the product. Packaging has been another problematic area, with multiple dressings being frozen in sterile specimen vials containing saline.

Unused thawed dressings must be discarded after 3 weeks refrigeration.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. SUMMARY OF THE INVENTION

Lyophilisation (vacuum freeze-drying) is a method by which amnion may be preserved which provides an extended shelf-life of more than 2 years if stored under the correct conditions. Lyophilisation is a well-known dehydration technique and includes vaporisation of the frozen moisture content of tissue under conditions of vacuum. Because dehydration is carried out at very low temperatures, the method has the potential to preserve temperature-sensitive molecules such as the biological proteins and enzymes found within amniotic tissue. Some advantages of the lyophilisation procedures used in the present invention over other methods of preservation are: reduced damage to the essential organic material, relatively rapid reconstitution upon rehydration with saline, retention of beneficial wound-healing characteristics of fresh amnion.

According to a first aspect, the invention provides a method of preserving amnion, or part thereof, such that one or more of the biological properties of the amnion are substantially retained, including the step of lyophilising the amnion, or part thereof.

Preferably, the biological property retained is a wound-healing property although the retention of one or more of the other biological properties of amnion, such as pain relief, anti-inflammatory activity, swelling reduction, inhibition of bacterial activity, control of exuberant granulation, wound-healing and scar reduction is also contemplated.

Most preferably, the biological property is a wound-healing property.

Preferably the amnion, or part thereof, is derived from an equine source although one skilled in the art will recognise that it may be from any other mammalian source eg. bovine or alpaca sources. Preferably the amnion, or part thereof, is placed in approximately 2% povidon iodine/saline solution within approximately 24 hours after birth of an offspring. Preferably the amnion, or part thereof, is cut into sections suitable for use as dressings. Most preferably the sections are approximately 5 cms by approximately 10 cms.

According to a second aspect, the invention provides a method of preserving an amnion, or part thereof including the steps of a) treating the amnion, or part thereof, with a sterilising solution; b) rinsing the amnion, or part thereof, in sterile saline and acetic acid; and c) lyophilising the amnion, or part thereof. According to a third aspect, the invention provides a method of preparing a wound dressing including the steps of a) treating amnion, or part thereof, in a sterilising solution; b) rinsing the amnion, or part thereof, in sterile saline and acetic acid; c) lyophilising the amnion, or part thereof; and d) rehydrating the lyophilised amnion, or part thereof, as required.

Preferably the sterilising solution is a povidon iodine/saline solution. Most preferably the sterilising solution is a solution of approximately 2% povidon iodine in saline.

Preferably the sterile saline and acetic acid solution of step (b) contains approximately 0.25% v/v acetic acid.

Preferably rehydration is carried out in saline solution.

According to a fourth aspect, the invention provides amnion, or part thereof, prepared by the method of the first or second aspects.

According to a fifth aspect, the invention provides a wound dressing prepared by the method of the third aspect.

According to a sixth aspect, the invention provides a method of treating a wound in an animal including the application of the amnion of the fourth aspect and/or the wound dressing of the fifth aspect to the site of the wound.

Preferably the amnion, or part thereof, is rehydrated. Preferably it is rehydrated by exposure to saline solution. Preferably the amnion, or part thereof, and/or wound dressing is applied to the wound under paraffin impregnated gauze dressings. Preferably the animal is a mammal and most preferably it is an equine species.

However, the method is also useful for the treatment of humans and any other animals to which such amnion, or part thereof, could conceivably be applied.

According to a seventh aspect the invention provides use of the amnion, or part thereof, of the fourth aspect and/or the wound dressing of the fifth aspect for the manufacture of a medicament for the treatment of a wound in an animal.

According to an eighth aspect, the invention provides lyophilised amnion.

According to a ninth aspect, the invention provides packaged amnion, or part thereof, wherein the amnion, or part thereof, is preserved according to the first or second aspects and further packaged.

According to a tenth aspect, the invention provides a packaged wound dressing, wherein the dressing is prepared according to the third aspect and further packaged.

Preferably the packaged amnion, or part thereof, or wound dressing is packaged in heat-sealed, laminated pouches. Preferably the pouches prevent moisture penetration and contamination of the packaged amnion, or part thereof, or the packaged wound dressing.

According to an eleventh aspect, the invention provides a method of treating pain, inflammation and/or swelling in an animal including the application of (a) amnion, or part thereof, and/or (b) a wound dressing according to the invention, to the site of the pain, inflammation and/or swelling.

Preferably, the amnion, or part thereof, is rehydrated. More preferably, the amnion, or part thereof is rehydrated by exposure to saline solution. Preferably, the animal is a mammal. More preferably, the mammal is an equine species. However, it will be clear to the skilled addressee that the mammal may also be a human and that the method may be applied to any other animals to which such amnion, or part thereof, and/or the wound dressing, could conceivably be applied. According to a twelfth aspect, the invention provides use of (a) amnion, or part thereof, and/or (b) a wound dressing according to the invention for the manufacture of a medicament for the treatment of pain, inflammation and/or swelling in an animal.

Preferably, the animal is a mammal. More preferably, the mammal is an equine species. However, it will be clear to the skilled addressee that the mammal may also be a human and that the method may be applied to any other animals to which such amnion, or part thereof, and/or the wound dressing, could conceivably be applied.

In the context of the present invention, the term "biological property" refers to the property of pain relief, anti-inflammatory activity, swelling reduction, inhibition of bacterial activity, control of exuberant granulation, wound-healing, scar reduction and the like.

DESCRIPTION OF THE INVENTION A preferred embodiment of the invention will now be described, by way of example only. Example 1 The following examples make use of equine amnion as a convenient way of exemplifying the invention because equine amnion is easily recoverable and each foaling produces amnion in abundance. The average equine amnion comprises a substantial amount of tissue, averaging 50-100 dressings of 5 x 10 cms. A draught horse amnion produced 190 dressings of varying sizes.

Other reasons why equine amnion was used as an example are that the number of equine births per year is large, often concentrated in large numbers in particular regions. The equine foaling season is generally concentrated between August and January, with most bloodstock born during August and September. The bigger studs, particularly those involved in the bloodstock industry, usually have many foals born on their properties each season. Due to the value of racing stock and the limitations imposed by the production of just one offspring per year, each foaling is normally monitored. The membranes of afterbirth, including the amnion and placenta, are usually collected and inspected to ensure they are "complete" in order to avoid post partum uterine infection in the mare.

Fresh amnion is cleaned, washed with copious amounts of tap water and badly torn or grossly contaminated areas are excised. Amnion is placed in a plastic container, covered with 2% povidon iodine/saline solution and refrigerated for 24-48 hours. The amnion is cut to suitable size for freeze-drying trays. The amnion is sequentially rinsed in 3 sterile baths of saline alone and rinsed in a final bath of saline containing 0.25% v/v acetic acid. The amnion is then layered between aluminium foil and frozen in flat sheets.

The amnion is placed in a chamber, one layer per shelf, and vacuum freeze-dried, over a period of 20-24 hours. The freeze-dried (lyophilised) amnion is removed from the chamber, cut into individual dressings and packaged. Each equine foaling produces viable quantities of amnion, averaging 50-100 dressings of 5 x 10 cms in vapour barrier packs. These packs consist of heat-sealed laminated pouches, constructed to keep the dressings dry and sterile. Failure rate of this type of packaging should be minimal, and is generally due to rough handling causing pinholes or splitting. Storage life is enhanced by reduced handling and storing in cool, dry areas out of direct sunlight. Reconstitution

(or rehydration) of dressings is by immersion in saline for 1-2 minutes. Amnion or amnion dressing treated in this way may be referred to as "reconstituted lyophilised amnion (dressing)". Example 2

Test 1 involved a gelding with an extensive injury to the anterior and lateral aspects of the hock. The wound was grossly contaminated and debri dement of skin flaps was necessary. Conventional treatment over a two-week period resulted in no improvement and granulation tissue had elevated above the skin edges. Copious exudate necessitated bandage changes every day.

Lyophilised amnion dressings were reconstituted and were applied under Vaseline- coated cotton pad dressings, with dressing changes twice per week in the initial stages. After two weeks the dressings and bandages were reapplied once per week. Within the first two weeks the inflammation settled and the granulation tissue reduced to level within the skin edges. No signs of bacterial infection were present and exudate was markedly reduced. Due to the extent of the injury and its location, the wound was treated with amnion dressings for 7 weeks. The amount of disfigurement and scarring was much less than anticipated for this type of injury. This successful outcome led to the search for an improved method of storing amnion. Example 3

Test 2 involved a 26 year old mare with a 4 cm long cut midway along the anterior aspect of a hind cannon. The cut penetrated full-thickness and, due to swelling, the skin edges were not in apposition. The wound did not appear contaminated. It was bathed with an antiseptic solution containing 10%> w/v povidone-iodine (Betadine®) and a reconstituted lyophilised amnion dressing prepared according to the method indicated in Example 1 was applied. This was covered with paraffin impregnated gauze dressing (Jelonet ) and bandaged with a flexible bandaging tape (Vetwrap ) and an elastic, adhesive tape (Conform®). The bandage was removed on day 4 and all swelling was gone. There was no sign of infection; the wound was clean and slightly moist without excessive exudate. Second intention healing by way of granulation tissue was well under way. This new tissue had almost completely filled the wound cavity but was not elevated above the skin edges. The wound was lightly cleaned and a fresh amnion dressing and bandages were applied. This was removed after 4 days and the wound edges had closed by approximately 50 per cent. No further bandaging or treatment was done. The wound completely closed within the next 8 days with minimal scarring and no elevated thickening of the epidermis. The scar was completely hidden under hair and could only be found upon clipping the site. Example 4 Further experiments using reconstituted lyophilised amnion as wound dressings were performed on a variety of distal limb injuries in 12 horses. These included knee, cannon, fetlock, pastern and foot wounds. Wounds were treated on an approximately weekly basis with lyophilised amnion which was reconstituted and applied under paraffin-impregnated gauze dressings.

In many equine distal limb injuries, healing can be delayed (or "stalled") for up to 18 months. This appears to be mostly due to the formation of exuberant granulation tissue preventing skin closure. In other cases, second intention healing stops and does not resume for many months. Of the wounds treated in this example, three were fresh injuries and the other 9 were longer standing wounds varying in duration from 7 days to

5 months prior to treatment. One of the wounds treated was long-standing (5 months) following the removal of a sarcoid. All the above wounds were completely healed after treatment with reconstituted lyophilised amnion. Duration from start of finish of treatment varied from 10 days to

105 days. Eight out of the 12 wounds healed within 35 days.

Fresh Wounds:

Reconstituted lyophilised amnion reduced the amount of fresh wound "breakdown" which usually occurs 3-5 days after injury. Wound "breakdown" includes tissue sloughing accompanied by excessive exudate production. For example, typically during the first 3-5 days of a sutured equine distal limb injury, the wound appears to be closing well. Shortly thereafter, the wound appears to regress; it develops a discharge, bad odour and loss of epithelial tissue, particularly around wound edges. Often sutures tear through or fall out due to sloughing of wound edges, and underlying tissues are revealed. While this wound "breakdown" may still occur, particularly in injuries requiring suturing, the amount of exudate and tissue sloughing was reduced when the reconstituted lyophilised amnion was applied. It was also found that with the use of reconstituted lyophilised amnion on sutured wounds, the removal of sutures at day 3, rather than days 5 - 7, resulted in faster healing times. In one experiment, once sutures were removed there was a marked acceleration in healing, noticeable within 24 hours. On fresh wounds the optimum time between application of reconstituted lyophilised amnion dressings was 4-5 days.

Long Standing Wounds:

Two wounds tested had no granulation tissue but would not close. These wounds responded to reconstituted lyophilised amnion dressings and within a week of treatment epithelialisation had begun. In these cases wound closure was rapid and uneventful.

Three wounds had considerable granulation tissue formation which was elevated well above the skin level, with inflammation of the surrounding tissues. One case was the result of a rope "burn", the second case an injury which had been treated with copper sulphate to try to reduce granulation tissue, and the third resulted from the surgical removal of a sarcoid with subsequent proliferative tissue - whether this was granulation or sarcoid tissue was not determined.

Treatment in each of the above 3 cases resulted in the complete reabsorption of excessive granulation tissue. The typical appearance of granulation tissue changed and became dark, smooth and glossy at around day 10. This reverted to smooth pink tissue by days 15-18. Epithelialisation generally became visible at days 10 to 14. Granulation tissue absorption and epithelialisation remained consistent until final closure. Inflammation in surrounding tissues was reduced. The reconstituted lyophilised amnion dressing resulted in a soothing and pain relieving effect on the wounds, indicated by reduced pain responses (such as flinching and pulling away), after about day 7. New hair growth began about 2 weeks after epithelialisation started. Hair regrowth was usually well established before wound closure.

Another injury of one week's duration, consisted of an open, contaminated wound with dried and blackened edges, and an overhanging skin flap. There was no granulation tissue in evidence. The flap and skin edges were not debrided. On day 4 the wound tissue was uniformly moist and pink and by day 10 epithelialisation was quite evident. This wound took 33 days to close completely to a fine scar line.

On long standing wounds the optimum time between reconstituted lyophilised amnion dressings was 7 to 10 days.

Cosmetic results were good in that flat wound sites with a good return of hair growth were observed. Scarring in all cases was less than anticipated, resulting only in small, flat, hairless areas at the centre of closure.

The data obtained showed that at least some of the following benefits could be achieved using reconstituted lyophilised amnion wound dressings:

1. Reabsorption of excessive granulation tissue

2. Reduction of underlying and surrounding inflammation

3. No need for debridement of wound edges

4. Reactivation of epithelialisation process in "stalled" wounds 5. Hastened hair regrowth

The above data show that reconstituted lyophilised amnion has anti-inflammatory and swelling reduction properties when applied to wounds. It will, therefore, be clear to the skilled addressee that the reconstituted lyophilised amnion can be used generally as an anti-inflammatory and/or swelling reduction agent even where there is no open wound. Further, it will also be clear to the skilled addressee that in order to do so, it may be necessary to abrade the surface at the site to be treated.

Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of preserving amnion, or part thereof, such that one or more of the biological properties of the amnion are substantially retained, including the step of lyophilising the amnion, or part thereof.

2. A method according to claim 1 wherein the biological property is selected from the group consisting of pain relief, anti-inflammatory activity, swelling reduction, inhibition of bacterial activity, control of exuberant granulation, wound-healing and scar reduction.

3. A method according to claim 2 wherein the biological property is a wound-healing property.

4. A method according to any one of claims 1 to 3 wherein the amnion, or part thereof, is derived from an equine source.

5. A method according to any one of claims 1 to 3 wherein the amnion, or part thereof, is derived from a bovine source.

6. A method according to any one of claims 1 to 3 wherein the amnion, or part thereof, is derived from an alpaca source.

7. A method according to any one of claims 1 to 6 wherein the amnion, or part thereof, is placed in approximately 2% povidon iodine/saline solution within approximately 24 hours after birth of an offspring.

8. A method according to claim 7 wherein the amnion, or part thereof, is cut into sections suitable for use as dressings.

9. A method according to claim 8 wherein the sections are approximately 5 cms by approximately 10 cms.

10. A method of preserving amnion, or part thereof, including the steps of a) treating the amnion, or part thereof, with a sterilising solution; b) rinsing the amnion, or part thereof, in sterile saline and acetic acid; and c) lyophilising the amnion, or part thereof.

11. A method according to claim 10 wherein the sterilising solution is a povidon iodine/saline solution.

12. A method according to claim 11 wherein the sterilising solution is a solution of approximately 2%> povidon iodine in saline.

13. A method according to claim 10 wherein the sterile saline and acetic acid solution of step (b) contains approximately 0.25% v/v acetic acid.

14. A method of preparing a wound dressing including the steps of a) treating amnion, or part thereof, in a sterilising solution; b) rinsing the amnion, or part thereof, in sterile saline and acetic acid; c) lyophilising the amnion, or part thereof; and d) rehydrating the lyophilised amnion, or part thereof, as required.

15. A method according to claim 14 wherein the sterilising solution is a povidon iodine/saline solution.

16. A method according to claim 15 wherein the sterilising solution is a solution of approximately 2% povidon iodine in saline.

17. A method according to claim 14 wherein the sterile saline and acetic acid solution of step (b) contains approximately 0.25%> v/v acetic acid.

18. A method according to claim 14 wherein rehydration is carried out in saline solution.

19. Amnion, or part thereof, preserved by the method of any one of claims 1-13.

20. A wound dressing prepared by the method according to any one of claims 14-18.

21. A method of treating a wound in an animal including the application of amnion, or part thereof, according to claim 19 and/or a wound dressing according to claim 20 to the site of the wound.

22. A method according to claim 21 wherein the amnion, or part thereof, is rehydrated.

23. A method according to claim 22 wherein the amnion, or part thereof, is rehydrated by exposure to saline solution.

24. A method according to any one of claims 21 to 23 wherein the amnion, or part thereof, and/or the wound dressing is applied to the wound under paraffin-impregnated gauze dressings.

25. A method according to any one of claims 21 to 24 wherein the animal is a mammal.

26. A method according to claim 25 wherein the mammal is an equine species.

27. A method according to claim 25 wherein the mammal is a human.

28. Use of amnion, or part thereof, according to claim 19 and/or a wound dressing according to claim 20 for the manufacture of a medicament for the treatment of a wound in an animal.

29. Use according to claim 28 wherein the animal is a mammal.

30. Use according to claim 29 wherein the mammal is an equine species.

31. Use according to claim 30 wherein the mammal is human.

32. Lyophilised amnion.

33. Packaged amnion, or part thereof, wherein the amnion, or part thereof, is preserved according to the method of any one of claims 1-13 and further packaged.

34. Packaged amnion. or part thereof, according to claim 33 wherein the amnion is packaged in heat-sealed, laminated pouches.

35. Packaged amnion. or part thereof, according to claim 34 wherein the pouches prevent moisture penetration and contamination of the packaged amnion, or part thereof.

36. A packaged wound dressing, wherein the dressing is prepared according to any one of claims 14-18 and further packaged.

37. A packaged wound dressing according to claim 36 wherein the wound dressing is packaged in heat-sealed, laminated pouches.

38. A packaged wound dressing according to claim 37 wherein the pouches prevent moisture penetration and contamination of the packaged wound dressing.

39. A method of treating pain, inflammation and/or swelling in an animal including the application of (a) amnion, or part thereof, and/or (b) a wound dressing according to claim 20, to the site of the pain, inflammation and/or swelling.

40. A method according to claim 39 wherein the amnion, or part thereof, is lyophilised.

41. A method according to claim 40 wherein the amnion, or part thereof, is rehydrated.

42. A method according to claim 41 wherein the amnion, or part thereof, is rehydrated by exposure to saline solution.

43. A method according to any one of claims 39 to 42 wherein the animal is a mammal.

44. A method according to claim 43 wherein the mammal is an equine species.

45. A method according to claim 43 wherein the mammal is a human.

46. A method according to any one of claims 39 to 45 wherein the site of pain, inflammation and/or swelling is abraded before application of the amnion and/or wound dressing.

47. Use of (a) amnion, or part thereof, and/or (b) a wound dressing according to claim 20, for the manufacture of a medicament for the treatment of pain, inflammation and/or swelling in an animal.

48. Use according to claim 47 wherein the amnion, or part thereof, is lyophilised.

49. Use according to claim 47 or claim 48 wherein the animal is a mammal.

50. Use according to claim 49 wherein the mammal is an equine species.

51. Use according to claim 49 wherein the mammal is a human.

52. A method of preserving amnion, or part thereof, substantially as herein described with reference to any one or more of the examples, but excluding comparative examples.

53. A method of preparing a wound dressing, substantially as herein described with reference to any one or more of the examples, but excluding comparative examples.

54. Preserved amnion. or part thereof, substantially as herein described with reference to any one or more of the examples, but excluding comparative examples.

55. A wound dressing, substantially as herein described with reference to any one or more of the examples, but excluding comparative examples.

56. A method of treating a wound in an animal, substantially as herein described with reference to any one or more of the examples, but excluding comparative examples.

57. Use of amnion, or part thereof, substantially as herein described with reference to any one or more of the examples, but excluding comparative examples.

58. Packaged amnion, substantially as herein described with reference to any one or more of the examples, but excluding comparative examples.

59. A packaged wound dressing, substantially as herein described with reference to any one or more of the examples, but excluding comparative examples.

60. A method of treating pain, inflammation and/or swelling in an animal, substantially as herein described with reference to any one or more of the examples, but excluding comparative examples.