JPS5888316A - Wound treating composition, manufacture and use - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides compositions useful for promoting wound healing, 4I
K relates to a method for producing amniotic membranes, such as human amniotic membranes, of such compositions.

Wound healing is adversely affected by poor general health and several local factors such as inadequate blood supply and chronic infection. If direct closure of the wound is in fact carried out, a wound bed that produces a healthy internal wound that will close either in the surrounding epithelialization area or in the autologous trough is desirable; The polycythemia of creams, powders, solutions and bandages used to promote the healing of polycythemia shows the incomplete state of knowledge on this subject.

Human amniotic membrane has been occasionally used since at least 1110 to promote the production of granulation tissue, and more recently as a biological dressing (dreamimg) for open wounds, including burns and chronic ulcers on the feet. came 0 e.g. R, N
, Mattkewa et al., Erltiah J.
Plastic Surgery, 3 4,
7@~711 (1981) discloses the use of human amnion for the treatment of severe head burns.

The problem with using amniotic membrane in promoting wound healing is that
The supply of sterile amniotic membrane is commonly
αn) Because it is obtained from childbirth, it is necessarily limited. If stored and dried membranes have not been found to provide better protection than regular sterile bandages, amniotic membranes should be freshly applied or preserved in normal saline when applied to other tissues. It appears to offer nine advantages compared to bandages.

H, Bt & Degom and W, Page Fa
sLk, Br1tishJ, 0bstatrica
and Gysaaaology, 1 g
.

294-30G (1981) discloses a method for maintaining human amnion in culture with consideration for establishing amniotic membrane banks for therapeutic use.

Attempts have also been made to obtain cell-containing extracts with the ability to stimulate blood vessel proliferation (angiogenesis) in model systems. For example, Tel & RT et al.
27 & 871 studied a number of normal human cell lineages for the production of angiogenic factors, but many such cell lines have been studied, e.g. by Amgrbaek et al., Dow.
el, Biol, 41.

391-194 (1974) and FolkgMss.

Ca5ear Ram, 84, fl 1011
It is disclosed to have poor angiogenic activity as determined by chorioallantoic evaluation of ~11 and 36° 110-114 (11176).

However, the mm lineage of human foreskin fibroblasts was discovered to synthesize the desired angiogenic factors in small children. T. &rt et al. describe a method for obtaining extracts containing cells containing such factors by culturing and harvesting the cells and then mechanically disrupting the harvested cells. .

Now, it has been discovered that amniotic membranes can be cultured in @% medium to release growth-promoting factors into the culture medium, and these factors are useful in aiding wound healing. As mentioned above, Bu
Gos and Fa values kK, the literature never suggests that the amniotic growth medium used obtained as a proboscis in that method can be used for wound treatment, also US Pat. No. 41! ? The examples in '871 do not imply that the medium used to culture prepuce line blastoblasts can be a useful source of extracellular angiogenic factors. Indeed, it clearly states that the culture medium is to be discarded.

According to the present invention, there is therefore provided a round, substantially amniotic membrane-free composition for use in the treatment of wounds comprising or derived from a culture medium in which amniotic membrane, preferably human amniotic membrane, has been cultured.

It has now been discovered that the amniotic membrane-free composition proboscis according to the invention is surprisingly stable to freeze-drying and to long-term storage in the freeze-dried state. This stability makes the amniotic composition % compared to intact amnion, in that such compositions can be conveniently and stored for very long periods of time.
If it is useful. Furthermore, the method of manufacturing such compositions is much simpler than methods involving cell disruption such as those described in US Pat. No. 42-871.

Preferably, the amniotic membrane is e.g.
ia%) in the form of sterile amniotic membrane obtained from parturition.

The amniotic membrane can be cultured in a suitable tissue culture medium buffered to pH 7.2 to 7.4. Examples of suitable tissue culture media include H. J. Morton, Is Vitr.
a...

111-108(11?O)K is 40 described. Such media include known essential azanoic acids, mineral salts,
Contain carbon sources such as carbohydrates (eg dulcose) and bitanes in various combinations.

The amniotic membrane contains oxygen, preferably also in relatively low concentrations: 1. The culture is preferably carried out in an atmosphere containing carbon dioxide at 6% or less, for example 25%.

The membrane can be cultured in one or more culture media, e.g. it is first cultured in a first medium such as medium A (as described below) for 4-6 days, followed by a second medium such as medium B ( It can be cultured in the following standard υ). Since contaminants from the amniotic membrane can be washed out by the culture medium during the first few days of culture, subsequent culture medium changes are preferred for use in wound treatment. Similarly, when using 8 types of media,
Preferably for use in accordance with the present invention is a snowfalling medium.

After separation from intact amnion, the culture medium is preferably subjected to centrifugation to remove free cells and then preferably lyophilized for ease of storage.

The lyophilizate can be sterilized, for example by irradiation with cesium I (rII). If necessary for use, the lyophilizate in dry powder or substantially dry powder form can be sterilized by addition of sterile distilled water. It can be reassembled by

Preparations that are 41 active in the above-mentioned chorion assay are those prepared using relatively large pore size molecular sieves, such as Saphag.
Lyophilizate to be reconstituted on ryl 300
It has been discovered that filtration can be produced by Todarafi.

According to the snowfall aspect of the present invention, a medical bandage (asrgie@l areaa4mg) containing a substantially amnion-free growth promoting factor derived from amniotic membrane 0jlI nutrition.
For example, a surgical bandage is provided with the substantially amniotic cal composition described above. Such bandages can be impregnated with the composition in liquid medium form and then allowed to dry. Alternatively, for example, the composition may be absorbed into the wound-facing layer of a surgical dressing; the dressing may include an antiseptic.

According to a further aspect of the invention, there is provided an ointment, such as a water-based cream, comprising an amnion-free growth promoting factor derived from the culture of amniotic membrane. Soft ζ is Br
1tish Pharttstxaopaaia.

1180.891-02, and may be, for example, an emulsifying ointment, a macrogel ointment, or a paraffin ointment, which may be formulated as water #I, e.g. -Hydroxyethyl acrylate, gelatin or British Patent No. L! $248
It can also be absorbed into hydrophilic liquids based on the polymers described in No. 99.

The ointment may contain other active ingredients, such as disinfectants. Including a disinfectant in the ointment can reduce the amount of r-ray radiation required to manufacture and maintain the sterility of the ointment.

Soft dressings should be implanted with a non-adhesive wound dressing, preferably a non-adhesive wound dressing, such as a knee rough-in dressing, which can be applied directly to the wound bed. If the non-adhesive dressing selected is not absorbent, it may be desirable to coat it with a conventional absorbent material, such as heptalulose. This can then be covered with a conventional occlusive film. This occlusive film may be held in place by adhesive tape.

A dressing comprising an ointment, an absorbent layer and a non-adhesive layer impregnated with an occlusive film is prepared using a sterile ointment.
valopa) K can be pre-filled.

Further in accordance with the invention there is provided a method of treating a wound as an aid to wound healing comprising applying to the wound a composition substantially free of amniotic membrane according to the invention.
The compositions can be applied, for example, alone or in or on a suitable carrier.

Examples of suitable carriers are bandages and the soft tissue described above.

Other wounds include Weataby et al., Annals R,C,
S.

The composition may be washed continuously or intermittently with the composition in aqueous form, as in methods similar to those described in 8% g, ss, ass~858 (1.at).

Also according to the invention, a composition for healing a wound comprising culturing amnion in a culture medium, isolating any free amniotic cells from the medium and/or lyophilizing the medium. A manufacturing method is provided.

The composition according to the invention and its manufacturing method will be illustrated by way of examples.

EXAMPLE Human amniotic membranes were collected aseptically from selectively exposed areas of normal women at term. The membrane was supplemented with 5% fetal serum (FC8) or newborn calf serum (NBC8) and containing nine bank salts (Hgsk'm 5alt) and IIEP.
The samples were transferred to water-cooled minimal essential media buffered at pH 7, 4K at E and obtained in the laboratory 1-11 hours after collection.

These were mixed with pH 1, BsO merpetsco phosphate (D%lb
Immediately wash with 4 changes of saline buffered with a phosphor (getto'a phospho@tg) to wash off the thrombus, and wash off the decidua remaining on the first surface of the chorion II with a gauze rod and forceps that hold the lung. .

Gently handled to prevent the amniotic membrane from detaching from the attached chorion. Large piece of amniotic membrane (tioxl 20x −
240X! 40m), cut and remove, 24.1X
Spread the villi 1[K-supported amnion in 4 243x1s Listerene dishes with culture medium lio-go.
Place in a bowl, supplemented with 10% FC8 or NBC8, containing fE/s salt and at pH 7.2-7.4 with 94 mAf sodium bicarbonate and 20 rnM HEPI Kg.
Tissue culture medium 199 (medium A) mixed with
Used for 6 days, it was subsequently replaced with Earle's salt (lowJl/sodium bicarbonate and 20mM HEPES).
1: arlaiaalt) (medium B). Antibiotics (Venicilia G I OO units/wt and streptomycin 10 Gμf/−) were added and the @911 snout was incubated at α5 against media containing Henk and Earle salts, respectively.
% and in air containing 25% COl,
The temperature was kept at 37 tl' in a humid atmosphere (98% humidity).

These OCO* flkltld medium t jl H7
, 2 to 7.4 Km. Change medium for 24 hours and then twice a week.

The removed medium B was collected and then centrifuged at 4C to 3,000t to remove free amniotic cells. The clear supernatant was then lyophilized and the rl/gN from the Cesium-90 source was
Then, sterilize it by spraying.

The lyophilizate was stored at -20C until required for use. If necessary, IIK was reconstituted by adding sterile distilled water.

BRIEF DESCRIPTION OF THE DRAWINGS The method of treating hot water and surgical dressings for use in treating such wounds according to the present invention will now be described with reference to the accompanying drawings.

Figure 1 is a plan view of the bandage) FIG. 2 is a sectional view taken along line A-A in FIG. 1, and FIG. 3 is a cross-sectional view of another bandage.

Referring to the figure, 792,747 layers were placed on each ulcer bed of a number of patients suffering from chronic ulcers. Surgical gauze layer 3 saturated with reconstituted lyophilizate
The gauze 3 was placed on top and then covered with a sheet of plastic along with a small area of chewing tissue. A tube 7 with many holes 1.1 at its end was introduced between the paraffin 1 and the gauze 3.

The free end 13 of the tube 7 was left protruding from the edge of the sheet of plastic. Next, cut the edge of the glass sheet,
It was secured to the patient's skin by an adhesive tube 15 to form a liquid-proof seal between the plastic and the skin. In this way, the ulcer can be treated with
9. Confined within a rope.

Liquid can only be introduced into this empelog through tube 7, and the reconstituted lyophilizate 50
- into the interior of the Enpelog by means of a syringe 17 attached to the tube 7, the tube then being clamped and sealed, and then every 6 hours for the first 48 hours and then every 12 hours for 3 days, the contents of the Enpelog. The material was removed with a syringe and 50 ml of freshly reconstituted lyophilizate was injected.

Patients in the control group were treated in the same manner except that the reconstituted lyophilizate was cultured in the absence of amniotic membrane and prepared from medium B.

A neovascular (or angiogenic) response was observed when the bandage was removed from patients treated with amniotic media.
Each additional patient then underwent a successful autologous transplant.

Conversely, ulcerated patients in the control group were found to lack sufficient angiogenic tissue to support successful autografting.

A dressing for use in applying a wound healing composition in ointment form is shown in FIG. The wound bed 21 is covered with an ointment 23 comprising the above-mentioned reconstituted lyophilized product in a sustained release matrix. On this ointment 23 is a non-adhesive bandage 25 which is successively covered with a layer of 12 absorbent cellulose bags. The entire bandage is a covered glass film that is adhered to the patient's skin using adhesive tape 31! I9 protects the body from the invasion of infectious agents.

[Brief explanation of drawings]

Figure 1 is a plan view of the bandage; FIG. 2 is a cross-sectional view along @A-A in FIG. 1, and FIG. 3 is a cross-sectional view of another bandage. Figure 2 Figure 3

Claims (1)

Claims: L. A substantially amniotic membrane-free preparation for use in IIO treatment, comprising or derived from a culture medium in which amniotic membranes have been cultured. 2. The preparation according to claim 1, wherein the amniotic membrane is human amniotic membrane. Tortoise Cough feeding medium is passed through a centrifugal valve for removal of free cells.
A preparation according to claim 1 or 2, which is provided for. Table 4. The preparation according to any one of claims 1 to 3, comprising the culture medium in a lyophilized state. & rai1 sterilized by irradiation Claim 4
0vI4 formulation as described in section. & The active fraction is subjected to purification by r-filtration chromatography, and the preparation described in any one of claims 1 to 11 below. 7. t@Nursing medium during culture of the amniotic membrane, pHr, 2-7.
7. The preparation according to any one of claims 1 to 6, which is buffered to a concentration of 4. A preparation according to any of claims 1 to 7, wherein the medium contains carbon dioxide at a concentration of 5% by weight or less during the I@% cultivation of the amniotic membrane. 9. The preparation according to claim 8, wherein the culture medium contains carbon dioxide in a concentration of substantially 25% by weight. 10. A preparation according to any of claims 1 to 9, wherein the amniotic membrane is previously cultured in a preculture medium for 4 to 6 days, and the preparation consists of or is derived from a subsequent culture medium. IL The preparation according to claim 11, wherein the preliminary culture medium is medium A as described below and the subsequent culture medium is medium B as described below. 12% Allowable Wf4 Any of the ranges listed in paragraphs 1-11 of 111m1! Surgical bandages containing agents. Claim 1 impregnated with a preparation in liquid form and then dried! Bandage as described in section. 14. The bandage according to claim 12, wherein the wound-facing layer of the bandage absorbs the preparation. The bandage according to any one of claims 1 to 14, which also contains a bactericidal agent. ointment comprising a preparation according to any of claims 1-11. 17. The ointment is emulsifying ointment, macroglu ointment or paraffin ointment, or it is a hydrophilic ointment as an aqueous solution.
17. The ointment of claim 16 absorbed into a plastic bottle. 1 & 18. An ointment according to claim 16 or 17, which also contains one or more other active ingredients. 19. An ointment according to claim 18, wherein the other active ingredient of IQ, laI or higher consists of or contains a bactericidal agent. 3α culturing the amniotic membrane in a nursing home, separating it from the amniotic membrane as a medium, and then distributing any of the cells to the sheep 1 free from the medium and/or lyophilizing the medium. Method of manufacturing preparations for treating wounds. 21. A method for producing a strawberry preparation according to claim 20 for treating wounds substantially as described below with reference to the Examples. 22 Substantially amnion-free %1fvIil for use in the treatment of wounds as described below with reference to the Examples
1. The preparation according to any one of items 1 to 11. ! & A surgical bandage according to claim 11, substantially as hereinafter described with reference to the accompanying drawings.