CA2147508A1 - A method of promoting wound healing and compositions useful for same - Google Patents
A method of promoting wound healing and compositions useful for sameInfo
- Publication number
- CA2147508A1 CA2147508A1 CA002147508A CA2147508A CA2147508A1 CA 2147508 A1 CA2147508 A1 CA 2147508A1 CA 002147508 A CA002147508 A CA 002147508A CA 2147508 A CA2147508 A CA 2147508A CA 2147508 A1 CA2147508 A1 CA 2147508A1
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- Canada
- Prior art keywords
- wound
- topical composition
- composition according
- vbhf
- tissue
- 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.)
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- 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
- A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
Abstract
The present invention relates to a method of inducing, stimulating, enhancing, accelerating or otherwise promoting wound healing by the application of vitreous from mammalian ocular tissue or an extract on one or more components of the vitreous. The present invention is also directed to a topical composition comprising vitreous or an extract or one or more isolated components thereof capable of differentially stimulating growth and proliferation of granulation tissue but with substantially no effect on skin tissue.
Description
WO 94/09800 PCr/AUs3/00554 21~7~
A METHOD OF PROMOTING WOUND HEALING
AND COMPOSITIONS USEFUL FOR SAME
The present invention relates to a method of ind~ ng, stim~ ting, enh~n~ing, accelerating or otherwise promoting wound healing by the appli~tiolt of vitreousfrom m~mm~liAn ocular tissue or an extract or one or more components of the vitreous. The present invention is also directed to a topical colll~osition comprising 10 vitreous or an extract or one or more isolated components thereof capable of differentially stimulating growth and proliferation of gr~nlll~tion tissue but with sllhst~nti~lly no effect on skin tissue.
The term "vitreous" is used in the subject spel~ific~tion as a short hand notation for 15 the "vitreous humour" or "vitreous body" which adopts the noment~l~h-re proposed by Balazs and Denlinger (1984). The viLlcous is conciclered to int~ le the cQnnective tissue ~ulloul~ded by the lens, ciliary body and retina For the purposes of describing the pl csell~ invention, the skin is considered to consist 20 of an outer epidermal layer and an inner dlo-rm~l layer. These two layers rest on a third subcutaneous tissue layer; from which gr~m-l~ti~n tissue is derived.
Reference herein to "skin tissue" is taken as lcfelcnce to the epidermal and dermal layers but not the subcllt~neous/gr~n~ tion.
25 There are three overlapping phases of tissue lespollse to injury inflAmm~tiQn;
gr~nnl~tion tissue production; and matrix form~tion and remo-l~lling T~ese phases are shown in Figure 1.
The infl~mm~tory phase is ~csori~tecl with disruption of blood vessels. This results 30 in ~ll~v~C~tion of blood cells and plasma conl~onents into ~ulloullding tissue and the clotting process which also involves pl~telet release of chemoattractants and mitogens. This in turn leads to further invasion of the wounded tissue by blood cells WO 94/09800 PCr/AUs3/00554 i4~
A METHOD OF PROMOTING WOUND HEALING
AND COMPOSITIONS USEFUL FOR SAME
The present invention relates to a method of ind~ ng, stim~ ting, enh~n~ing, accelerating or otherwise promoting wound healing by the appli~tiolt of vitreousfrom m~mm~liAn ocular tissue or an extract or one or more components of the vitreous. The present invention is also directed to a topical colll~osition comprising 10 vitreous or an extract or one or more isolated components thereof capable of differentially stimulating growth and proliferation of gr~nlll~tion tissue but with sllhst~nti~lly no effect on skin tissue.
The term "vitreous" is used in the subject spel~ific~tion as a short hand notation for 15 the "vitreous humour" or "vitreous body" which adopts the noment~l~h-re proposed by Balazs and Denlinger (1984). The viLlcous is conciclered to int~ le the cQnnective tissue ~ulloul~ded by the lens, ciliary body and retina For the purposes of describing the pl csell~ invention, the skin is considered to consist 20 of an outer epidermal layer and an inner dlo-rm~l layer. These two layers rest on a third subcutaneous tissue layer; from which gr~m-l~ti~n tissue is derived.
Reference herein to "skin tissue" is taken as lcfelcnce to the epidermal and dermal layers but not the subcllt~neous/gr~n~ tion.
25 There are three overlapping phases of tissue lespollse to injury inflAmm~tiQn;
gr~nnl~tion tissue production; and matrix form~tion and remo-l~lling T~ese phases are shown in Figure 1.
The infl~mm~tory phase is ~csori~tecl with disruption of blood vessels. This results 30 in ~ll~v~C~tion of blood cells and plasma conl~onents into ~ulloullding tissue and the clotting process which also involves pl~telet release of chemoattractants and mitogens. This in turn leads to further invasion of the wounded tissue by blood cells WO 94/09800 PCr/AUs3/00554 i4~
such as neutrophils and monocytes which, like platelets, are crucial to the next phase of wound healing (Clarke, 1988).
Gr~n~ tion tissue consists of a mixture of fibroblasts, monocyte-derived and tissue 5 macrophages and new blood vessels. The term "gr~nlll~tion tissue" derives from its granular appearance which is histoiogically due to the numerous newly formed blood vessels. Gr~n~ tion tissue may be found at sites of ~,vound repair such as in chronic non-healing wounds or at sites of bone fracture where the gr~mll~tion tissue is co~ osed largely of specialised myofibroblast referred to hereinafter as "osteoblasts".
10 Proliferation of fibroblasts and the growth of new blood vessels into the wound occur ~imnlt~nçously and interdepenrlently and are stimnl~ted by chemoattractants and growth factors from macrophages and pl~t~let~. Such chemo~ttractants and growth factors inc~ le thrombin, fibroblast growth factor (FGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) (Leibovich and Ross, 1975). Re-15 vascularization occurs from c~rill~ries s~,oulil,gfrom ~ ent bloodvessels followedlater by smo~th muscle recmit~nentl l~is stage is an iln~ t part of wound repair since it supplies oxygen and essential nutrients to the proli~l ~lillg repair tissue.
In deep wounds l~ in~ skin, fibroblasts migrate into the wound space from the 20 sub~lt~n~oous layer and lay down large amounts of loose extr~ r matrix, initially rich in fibron~ctin and hyaluronic acid (Kull~ine~l et al., 1980). Within the wound itself, the fibroblasts undergo a phenotypic mo~nl~ti(7n to become the so-called~ yori~L~lasts". The myofibroblasts have an increased mobility and contractile capacity without losing matrix synthetic function. Myofibroblasts are the most 25 numerous cells in gr~nlll~ti~n tissue. They are actin-rich and becomc ~ligned within the wound and their contraction is res~ sible for wound closure (Gabbiani et aL,1972).
Wo 94/09800 214 7 ~ ~ ~ PCr/AU93/00554 Re-epithç~ tion occurs co~currently wit_ fibroplasia and is an important mef h ~ni~m to prevent bacterial infection and loss of tissue fluid. The epithçli~l cells undergo marked phenotypic alteration con~omit~nt with migration that result in S increased cell mobility. Like many other cell types, migration of epitheli~l cells does not depend on cell proliferation. Once re-epith~ li7~tion is comrlete, the cellsrevert to their original phenotype.
The third overlapping phase of wound repair is matrix formation and remod~olling.
10 The composilion and structure of the extr~ellnl~r matrix of gran~ tion tissuechanges conlil-uously from the time it is first deposited and is controlled by cytokines such as Lla~rolllling growth factor-,B [TGF-,B] (Roberts et aL, 1986). During the formation of gr~nlll~tion tissue, fibronectin provides a su~k~ for the migrationof cells, a linkage for myofibroblasts to effect wound contraction, and ~flhçsion sites 15 for collagen fibril formation. Collagen types I, III and V appear and provide tensile strength for the wound and proteoglycans provide resilience to deform~tion of the tissue.
Wounds requiring healing by secondary intel tion are wounds with edges which are20 widely separated such that no epidermis or dermis is present. As a consequence, healing progresses from the base u~w~ds as well as from the edge ill~V~`iS. The dermis shows little wound h~lin~ activity in this respect and the majority of gr~nlll~tion tissue which eventually fills the wound site comes from the subcutaneous fat at the wound edge and from the subcutaneous floor of the wound. As noted 25 above, gramll~tion tissue is formed by the proliferation and migration of the~ulrou~ding co~nective tissue elem~.nt of the sub~ t~l-euus tissue; it is col,lposed in the first in~t~nce of c~rill~ry loops and fibroblasts together with a variable number of infl~mm~tQry cells.
30 The clinical need to provide pro~ , filnction~l coverage and to prevent scar formation in deep, extensive secondary indication wounds and the need to initiate and accelerate healing in chronic non-healing secon~ry in~iic~tion wounds, has led WO 94/09800 PCr/AU93/005~4 2~
to the experiment~l use of a vast number of materials in human trials.
These materials are generally ill-defined combin~tion~ of growth factors, which have been derived from a number of sources (see Van Brunt and l~ lsner, 1988; Buckley5 et al, 1985).
Platelet Derived Wound Healing Formula (PDWHF ) has been used, topically~ in an attempt to accelerate wound he~ling. It is considered likely t_at PDWHF stim-ll~t,os repair of chronically non-he~ling cutaneous wounds by a combination of growth 10 factors present in the formula. One study showed that wounds of dirr~l-ellt etiologies responded equally well to PDWHF (Knighton et al, 1988).
In an extensive study in human subjects, Carter et al. (1988) used three crude al-ations: autologous serum, porcine lysate (obtained from ~lci~lc keratinocytes) 1~ and bovine platelet lysate. All three showed limite-l success in trials on p~ti~.ntc with non-he~ling, deep-ulcerated wounds (Carter et al, 1988).
~xtracts from m~mm~ n eyes have also studied for their potential wound healing properties. In GB 1 342 761, a whole eye extract was shown to enh~nce cicatrisation 20 of corneal vounds. Furthermore, GB 1 603 034 ~ oses an aqueous solution of atissue regenerating extract such as from villeous which ~ Leci regeneraticn of cells of the epidermal layer. A similar effect on epidermal cells was disclosed in US
Patent No. 4,670,257 using an aqueous salt extract of ocular tissue, such as from vitreous.
There is a need to identify and develop s~lbst~nces capable of accelerating wound healing by selectively tar~,e~ g gr~n~ tion tissue but not cells of the epidermal and dermal layers of the skin. Such substances will provide a major advance in woundhealing management especially for secondary intention wounds which lack epidermis 30 and clerrni~. These snhst~nces would also be useful for promoting gr~mll~tinn tissue during repair of fractured bones.
WO 94/09800 pcr/Au93/oo5s4 2147~Q~
Acconlillgly, one aspect of the present invention provides a vitreous formnlation from m~mm~ n ocular tissue which is capable of ~lefelGl~lially accelerating growth of gr~nl-l~ti-)n tissue colllp~ed to skin tissue. The vitreous form-~l~tion may cc,~ lise i~o~ted vitreous, vitreous extract or one or more colllyol~ents of a villeolls S having the desired activity. A vitreous extract may be an aqueous extract or a salt extracted preparation.
The vitreous formulation is generally ~pted or in a form suitable for topical ~rlmini~ration. Accolding to this aspect of the present invention there is provided 10 a topical composition colnplising vitreous or an extract thereof from m~mm~ nocular tissue wherein said vitreous or its extract ~rGrel elltially accelerates growth of granulation tissue colllpared to epidermal and dermal tissue.
For ccllvellience reference to an "extract" of villeous in~hlrles lerGlellce to one or lS more active conl~ollents isolated and/or purified thcl-er~om. Furthermore, by way of shorthand notation, the vitreous form-~l~tion~ and topical co..-l~silions of the present invention are hereinafter referred to as a vit.cous based he~lin~ form~ tion (VBHF). The term "topical cc~l"l O~ilio~ is used herein in its broadest sense and incl~ es the applic~tion of the coni~osilion to one or more layers of subcllts~neQus 20 tissue and directly at the site of bone fracture.
The G~ression "epiderm~l tissue" is used in its broadest sense and is particularly directed to keratinocytes. 'Dermal tissue" is also used in its broadest sense and in~ s skin fibroblasts. Sub-~t~neous/gr~n~ tion tissue is also used in its 25 broadest sense and inchl~es vascular smooth muscle cells and myofibroblasts. The present invention is predicated in part on the ~ ing discovery by the hlv~ o~
that t_e VBHF ~tim~ tPs proliferation of vascular smooth muscle andmyofibroblasts but sllbst~nti~lly not skin fibroblasts and keratillocytes.
WO 94/09800 pcr/Au93/oo5s4 2~ S~ - 6 -Acco-di-lg to ~ ~ref~;lled aspect of the present invention, there is provided a topical composition colll~lisi-lgvitreous or an extract thereof from m~mm~ n ocular tissue wherein said vitreous or its extract prefcl-elltially stim~ t~s growth of vascular 5 smooth muscle cells and/or myofibroblasts while subst~ntiAlly not stim~ tin~ growth of skin fibroblasts and/or keratinocytes. The ~lefele~tial effect is conveniently and particularily observed and monitored in vitro.
Reference herein to a "vitreous extract" includes vitreous i~ol~tecl away from the 10 m~mm~ n ocular tissue, vitreous following at least one step of purification or fractionation or any active components therein. The m~mmAl is generally alivestock animal such as a bovine, ovine, equine or porcine animal or a goat or a laboratory test animal such as a murine ~nim~l, rabbit or guinea pig. The most preferred source of the vitreous of the present invention is from a bovine ~nim~
In another embo.iimçnt, the vi~leous is fr~ior~ted and one or more components or a ll~Lule of cc,ll.~o--ents i~ol~tecl and purified and ~vhich have a similar differential effect on gr~n~ ti-)n and skin tissue as does the vitreous or extract thereof.
Accordil.g to this aspect of the present invention, there is provided a component i~ol~te~hle from m~mm~ n vitreous or extract thereof, said cc,m~o..ent capable of accelerating or promoting growth of gr~mll~tion tissue such as vascular smooth muscle cells or myofibroblasts while having a minim~l prolifelalive effect on skin 25 cells such as k~lalillocytes and dermal derivedfibroblasts. Preferablythe component is in isolated and purified form me~nin~ that a CC.ll~ osition of matter comprises at least 20æ, more ~lerelably at least 35%, even more l,rererably at least 45%, still more plefelably at least 55-~5% and even still more pleft;lably at least 75-90~ of the co.ll~onent as determined by weight, activity, antibody binding or other 30 co..vel ient means.
Wo 94/09800 PCr/AUs3/00554 2~ 7~
The VBHF of the present invention may further co,l,~lice one or more exogenous factors added to the composition and which may also assist the wound healing process. Such exogenous factors conte.mpl~ted in this aspect of the present invention s inc~ e but are not limiteci to PDWHF, FGF, EGF or insulin-like growth factor (IGF), one or more antibiotics or other ~ntimi~robial agents or vit~minc such asvitamin E, D and/or K. The VBHF may aLo com~ise one or more pharm~. elltir~lly acceptable carriers andlor ciilll~ntc.
10 The VBHF may be topically applied directly or after incorporation into a medicated foam, gel, cream or liquid. Alternatively, it may be inco~ ated into a solid matrix such as a bandage, dressing, gauze or sutures. The VBHF may be in icol~te-l and/or purified form or may be frozen or lyophili7erl in which case it is reconctitlltecl prior to use.
15 Reference can conveniently be made to the British Pharmacopoeia (1980) for a description of collve~ on~l agents which may be usefillly incorporated into VBHF.
T~e VBHF of the present invention is co.~le...~ ted to be particularly usefu inwound healing by seconrl~ry intçntion of wounds which require accelerated growth20 of gr~n~ tion tissue such as ulcers which maybe associatedwith diabetes, peripheral vascular clice~ce or plCS~llC sores or bed sores; dermal tissue damage such as following various degrees of burns; post operative skin lesions l~king skin where accelerated growth of subcl~t~neous/gr~m-l~tion tissue is lc~uired; acceleratedhealing of gr~mll~tion tissue in bone fractures; accelerated growth of 25 subc~lt~n~ouslgr~nlll~tion tissue prior to aprlic~tinn of a skin graft.
Acc~lding to this aspect of the present invention there is co~-te~--.l~te-1 a method of stimlll~tin~, enhancing, accelerating or olhclwise promoting h~ n~ of a wound onan ~nim~l, said method COnl~JLiSillg cont~cting said wound with an effective amount 30 of VBHF as hereinbefore clçfinec~ for a time and under conclitions sufficient for wound healing to occur.
WO 94/09800 Pcr/Au93/oo554 Such wounds.are generally healed by secQnfl~ry intention as d~fin.o-l above. Theeffective amount is the amount of VBHF effective to stim~ te, enh~n~e, accelerate or otherwise promote wound he~ling which is cc l~v~;niently ~cc~ccerl by stiml~ tion S of vascular smooth muscle cells or myofibroblasts with sllbst~nti~lly no stimlll~ting effect on keratinocytes or skin fibroblasts. Reference above to a "wound" in~lu~5 refelence to a bone fracture.
The animal to be treated may be a hnm ~n, livestock anim al as hereinbefore ~l.ofin~
10 a laboratoly test animal as hereinbefore defin~-l, a companion animal (e.g dog or cat) or a captive wild ~nim~l Most pl~,relably, ho~ er, the ~nimal is a hnm~n The wound he.~l in~ protocol may ~n~l ~e a single aprli~tion of VBHF or a regime involving mllltiple applir~tion~ of VBHF on an hourly, twice daily, daily, mlll~iple 15 daily, weekly, multirle weekly or monthly basis, depen-ling on the wou~d to be treated, the extent of the wound and the healing capacity of the p~ti--nt The method may also Colll~l~SC further tre~tment wit_ one or more other agents such as antibiotics or other ~ntimi~robial agents, ~n~esthetics or other wound healing promotents. VBHF may be used as i~ol~tecl~ or may require cc~nce~ ation or 20 dilution. Dilutions contemrl~ted herein are in the order of 1:2 to about 1:10although dilutions of the order 1:2 to 1:5 are ~r~r~.l e~ If the VBHF is cc).~cenLI ated this may be done by any number of procedures inrln~ling lyophili~tio~ or filtration and extend to a concell~ ion factor of 2 to 10.
25 In ~nr-ther embo-liment, the VBHF is used in a non-liquid form such as following lyophili~tio~ Such a form may be particulary useful for the applic~tion to a solid matrix such as a bandage, dressing, gauze or sutures.
WO 94/09800 pcr/Au93/oos54 21~7~
g The present invention is further described byreÇele,lce to the following non-limiting Figures and/or F~Ample~c.
S In the Figures:
Figure 1 is a graphical represe-nt~tion showing t_e phases of wound repair.
Figure 2 is a gr~phic~l represent~tion of the effect of VBHF on tritiated - thymidine 10 incorporation into vascular smooth muscle cells.
Figure 3 is a graphical representation showing the effect of VBHF on tritiated -thymidine incol~olation into myofibroblasts.
lS Figure 4 is a graphical representation showing the effect of VBHF on tritiated thymidine incol~ration into osteoblasts.
FSgure S is a gr~phiç~l repr~.sent~tion of the effect of VBHF on tritiated - thymidine incolporation into skin fibroblasts.
Figure 6 is a gr~phic~l represçnt~tion showing the effect of VBHF on tritiated -thymidine h~col~uralion into keratinocytes.
Figure 7 is a grap_ical rc~lese.~ ;on of the effect of VBHF on tritiated - thymidine 25 i~lcol~lalion into vascular endothçlinm Figure 8 is a gr~rhi~l lc~lese~ t;on showing the effect of VBHF on proliferationof vascular smooth muscle ce~ls.
30 Figure 9 is a gr~phi~l repr~o~entation showing the effect of VBHF on proliferation of myofibroblasts.
WO 94/09800 PCr/AU93/00554 ~ 4~
Figure 10 is a gr~phiç~l repres~ont~tion showing the effect of VBHF on proliferation of skin fibroblasts.
Figure 11 is a graphical repr~sent~tion showing the effect of VBHF on proliferation 5 of keratinocytes.
Figure 12 is a graphical re~lesent~tion showing the effect of VBHF on proliferation of vascular endothelil-m 10 Figure 13 is a gr~phic~l represe-nt~tion showing the effect of VBHF on migration of vascular smooth muscle cells.
Figure 14 is a graphical repr~s~.nt~tion showing the effect of VBHF on migrationof myofibroblasts.
Figure 15 is a gr~rhic ~l repre~ent~tion showing the effect of VBHF on migrationof skin fibroblasts.
Figure 16 is a gr~rhic~l repr~-~çnt~tion showing the effect of VBHF on migration20 of ~eratil~ocytes.
Figure 17 is a photographic repr~se-nt~tion showing the effect of VBHF on morphology of vascular smooth muscle cells. In the presence of: A. DMEM to 0.5%
v/v PCS; B. DMEM to 0.5% v/v FCS and 1:2 diluted VBHF; C. DMEM to 0.5%
25 v/v FCS to 1:4 diluted VBHF.
Figure 18 is a photographic represe-nt~tion showing the effect of VBHF on morphology of myofibroblasts. In the presence of: A. DMEM to 0.5% v/v FCS; B.
DMEM to 0.5% v/v FCS and 1:2 diluted VBHF; C. DMEM to 0.5% v/v FCS to 1:4 30 diluted VBHF.
Wo 94/09800 PCr/AU93/00554 21~7~08 Figure 19 is a photographic represent~tion showing the effect of VBHF on morphology of skin fibroblasts. In the presence of: ~ DMEM to 0.5% v/v FCS;
B. DMEM to 0.5~o v/v FCS and 1:2 diluted VBHF; C. DMEM to 0.5% v/v FCS to 5 1:4 diluted VBHF.
Figure 20 is a photographic represent~tion showing the effect of VBHF on morphology of keratinocytes. In the presence of: A. DMEM to 0.5% v/v FCS; B.
DMEM to 0.5% v/v FCS and 1:2 diluted VBHF; C. DMEM to 0.5% v/v FCS to 1:4 10 cliluted VBHF.
EX,~MPLE 1 Isolation of vitreous 15 The vitreous is removed from 2 freshly sl~ htered bovine's eye in the following m~nner. Pr~rerably, all procedures outlined below are carried out in a l~min~r flow hood.
(i) the outside of the eye is rinsed in 70% v/v eth~nol (ii) a syringe fitted with a 19G needle is inserted into the cavity cont~iningthe aqueous humour and the aqueous humour is collectecl (iii) an in~ ion is made around the cornea with a scalpel and the cornea is removed.
(iv) using a pair of forceps, the lens is removed.
(v) the vitreous is then collected in a 10ml sterile syringe.
(vi) the syringe and its contents are then frozen at -70 degrees ~e WO 94/09800 PCr/AU93/00554 Preparation of vitreous based h~z-l;ng forrmll~*~n S The VBHF is used as icol~te~ according to the method of FY~mple 1 or additional conl~ollcnts added such as one or more pharma~e~ltie~lly acceptable carriers and/or dilll-ontc, emnlcifying com~onents such as Be~lometh~cone or Cetrimide or components s~lecte~l from saline, protein, antibiotic, carbohydrates. Alternatively, or in addition to, the vitreous is fractionated to remove ullw~lted com~llents or to 10 isolate and purify desired active components.
Once the VBH~ is ~le~aled, it is then stored or transported as is or in a frozen or lyophilised condition.
Wound Tl~l..- -,' (i) The wound is debrided and if infection is present it is removed by the appli~tion of a suitable antibiotic or ~ntimi~robial agent.
(ii) The wound is washed in 0.9% w/v sterile saline.
(iii) An aliquot of lml of the VBHF is applied from a syringe and dispersed evenly over the wound.
(iv) A piece of plastic backed gauze (cut to the same size of the wound) is moictçnecl with 0.9% w/v sterile saline and laid over the wound.
(v) Plastic wrap is placed over the gauze.
(vi) Tre~tment is repeated every 12hrs.
WO 94/09800 2 14 7 ~1~ 8 pcr/Al)93/ooss4 E~MPLE 4 tion for Skin G~
(i) Steps (i) to (v) of FY~mrle 3 are carried out until a suitable bed of gr~n~ tion tissue is present.
(ii) A skin graft can then be applied to the growing dermal tissue.
(iii) Treatment with VBHF can be contin--ed if required.
EX~MPLE S
T~ of Isch~ Ulcer 15 A clinical trial was carried out in a hospital e~lvho~ ent. A patient was a male 72 years of age in good health with the exception of a Scm x 3cm ulcer on the lower left leg The ulcer had failed to respond to various proprietary dressings which had been applied over a period of six month~. In ~ litioll during this tre~tment period, skin grafting had also been carried out and as a result, the patient required frequent 20 hospit~ tioJl All of these l,c~ cnt regimes were lm~uccec~ful. The wound lacked epiderm~l and dermal tissue, secor~ ry inte-nti~n he~ling by ~le~ive granulation tissue growth was required.
The patient was started on a tre~tm~nt regime which involved an appliç~tion, every 25 12 hours, of the VBHF, as outlined in Fy~mrle 3. As a result of this tre~tment regime, the patient's ulcer healed completely in 14 days with no recurrence for the period of the test which was 18 months.
WO 94/09800 PCr/AU93/00554 Q~ - 14-EX~MPLE 6 Tre~ nt of Diabetic Ulcer S A ~ linic~l test was carried out in a hospital ellvi,o~lent. A patient was a male 76 years of age in good health with the exception of a chronic ulcer me~cl~ring 7cm x 4cm on lower right leg. The ulcer had been re~;u"ent over 25 years during which time he~ling had never been complete using various proprietary dressings and skin grafting techniques. Accelerated growth of gr~n~ tion tissue was required to 10 sll~ceccfully heal the wound.
The patient was commenced on a tre~tment regime using a topical appli--~tion of the VBHF as outlined in Fx~mple 2. As a result of this tre~tment regime, the patient's ulcer was 90% healed after 12 dayc and comrletely healed after 22 days, with no 15 recurrence of the ulcer for the time of the test which was 17 monthc.
Tr ~ of ro~L L~ c Ulcer 20 A rlinic~l trial was carried out initially in a hospit~l setting and later by the patient at home. The patient was a female 48 years of age with a post tr~llm~tic non-healing ulcer.
The ulcer measured Scm x Scm and was sit l~ted on the lower left leg.
No progress had been made in he~ling the ulcer using various dressing techniques25 and there was no evidence of new skin growth. In ~ lition~ over the S year history of the ulcer, repeated skin grafting reslllted in short term coverage of the wound, but each skin graft eventually degenerated. The wound lacked epicl~rmic tissue int ~ ing both epithei~l and dermal tissue and required gr~nlll~qtion tissue growth for sllcceccf wound healing.
Wo 94/09800 21~ 7 ~ ~ 8 pcr/Au93/oo554 .
The patient was commenced on a tre~tm~nt regime which involved the topical aprlic~tion of the VBHF as outlined in FY~mrle 3. As a result of this tre~tment, the ulcer was completely healed after 21 days.
S
E~MPLE 8 T~ of Diabetic Ulce~
A clinical trial was carried out in a hospit~l setting on a female 82 year old patient.
10 The patient had 2 ulcers: one on the front of the right foot (Scm x 2cm) and the other at the rear of the right leg (lOcm x 10cm). Both ulcers were the result ofperipheral vascular restriction resulting from diabetes. The ulcers had failed to respond to various proprietary dressings and hyperbaric treatment over S months.The ulcer at the rear had penetrated through the tendon and ~mputation of the limb 15 was likely.
The patient was commen~e~ on a tre~tm~-nt regime for the ulcer on the back of the right leg which involved the topical applic~tion of the VBHF (as o-lLlilled in FY~mrle 3). The ulcer on the front of the right foot was dressed with N-saline as a control.
20 After 14 days the ulcer on the back of the right leg had ~lelsivt; growth of gr~mll~tion tissue, whereas the ulcer on the front of the right foot showed no change.
Tre~tment of the ulcer on the rear of the right leg was then commçnt~e(l as outlined in ry~lnple 4. In both cases rapid growth of gr~n-ll~tinn tissue occurred, the ulcer at the front of the right foot was comrlet~ly healed after æ days whereas the ulcer 25 on the front of the right foot was comrletely healed after 38 days.
EXM~PLE 9 Differen~ ect of VBHF
1. Sources of Cells V~c~ll~r Smnoth Mllcrle ('o.11~
Vascular smooth muscle cells were obtained by enzymatic di~el~ion of 12 week oldrabbit aortae by s~n~l~rd procedures (Campbell and Campbell, 1993). Cells were grown in Dulbecco's Minim~l F.ccçnti~l Medium plus 0.5% v/v foetal calf serurn 10 (DMEM + 0.5% v/v FCS).
Myofibrobl ~cf.c Myofibroblasts were obtained by enzyme dispersion of Selye's gr~nll10m~ pouch in~ ecl in rats by subcllt~nrQus mjection of 1 ml of 1% v/v croton oil in corn oil 15 following a 20 ml bolus of air (Selye, 1953). Cells were grown in DMEM + 0.55'o v/v FCS.
~kin Fihrobl~ct.c Human skin fibroblasts were obtained from Dr Rebecca Mason of the U11iVt;1~ilY of 20 Sydney, New South Wales, Australia and were grown in DMEM + 0.5% v/v FCS.
Ker~tinncyt~.c - Humankeratinocytes (epi(le~nic)were obtainedfromDrRebeccaMason, Ulliv~l~ily of Sydney, New South Wales, Australia together with k~l ati~ocyte growth medium 25 (KGM). For ~~ ent.~, cells were grown in DMEM.
F.n~ fheli~
Endothelial cells were i~ol~terl from bovine aorta by the method of Horrigan et al (1988) grown in me~ lm RPMI + 0.5% v/v FCS.
WO 94/09800 pcr/Au93/oo5s4 214750~-2. VB~
VBHF was prepared according to FY~mrles 1 and 2 in plastic syringes on ice.
The VBHF was stored frozen until required, then serially diluted at the time of use 5 at 1:2, 1:4, 1:8 and 1:16 in either DMEM + 0.5% v/v FCS or RPMI + 0.5% v/v FCS (for endothelial cells). P~nicillin G was usèd to control co..~lnin~tion.
3. Incorporation of 3H-Thymidine V~c~ll~r.~mooth Mnccle ~ell~
10 Cells were seeded at S x 104 cells/well into a 24 well plant in DMEM + 10% v/v FCS. The cells were allowed to attach and flatten for 18 hours, then the cells thoroughly washed in DMEM + 0.5% v/v FCS. The wells were then divided into six groups, four wells per group and exposed to the following con-litions:
15 1. DMEM + 0.5% v/v FCS
2. DMEM + 0.5% v/vFCS + 1:2 VBHF
3. DMEM + 0.5% v/v FCS + 1:4 VBHF
Gr~n~ tion tissue consists of a mixture of fibroblasts, monocyte-derived and tissue 5 macrophages and new blood vessels. The term "gr~nlll~tion tissue" derives from its granular appearance which is histoiogically due to the numerous newly formed blood vessels. Gr~n~ tion tissue may be found at sites of ~,vound repair such as in chronic non-healing wounds or at sites of bone fracture where the gr~mll~tion tissue is co~ osed largely of specialised myofibroblast referred to hereinafter as "osteoblasts".
10 Proliferation of fibroblasts and the growth of new blood vessels into the wound occur ~imnlt~nçously and interdepenrlently and are stimnl~ted by chemoattractants and growth factors from macrophages and pl~t~let~. Such chemo~ttractants and growth factors inc~ le thrombin, fibroblast growth factor (FGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) (Leibovich and Ross, 1975). Re-15 vascularization occurs from c~rill~ries s~,oulil,gfrom ~ ent bloodvessels followedlater by smo~th muscle recmit~nentl l~is stage is an iln~ t part of wound repair since it supplies oxygen and essential nutrients to the proli~l ~lillg repair tissue.
In deep wounds l~ in~ skin, fibroblasts migrate into the wound space from the 20 sub~lt~n~oous layer and lay down large amounts of loose extr~ r matrix, initially rich in fibron~ctin and hyaluronic acid (Kull~ine~l et al., 1980). Within the wound itself, the fibroblasts undergo a phenotypic mo~nl~ti(7n to become the so-called~ yori~L~lasts". The myofibroblasts have an increased mobility and contractile capacity without losing matrix synthetic function. Myofibroblasts are the most 25 numerous cells in gr~nlll~ti~n tissue. They are actin-rich and becomc ~ligned within the wound and their contraction is res~ sible for wound closure (Gabbiani et aL,1972).
Wo 94/09800 214 7 ~ ~ ~ PCr/AU93/00554 Re-epithç~ tion occurs co~currently wit_ fibroplasia and is an important mef h ~ni~m to prevent bacterial infection and loss of tissue fluid. The epithçli~l cells undergo marked phenotypic alteration con~omit~nt with migration that result in S increased cell mobility. Like many other cell types, migration of epitheli~l cells does not depend on cell proliferation. Once re-epith~ li7~tion is comrlete, the cellsrevert to their original phenotype.
The third overlapping phase of wound repair is matrix formation and remod~olling.
10 The composilion and structure of the extr~ellnl~r matrix of gran~ tion tissuechanges conlil-uously from the time it is first deposited and is controlled by cytokines such as Lla~rolllling growth factor-,B [TGF-,B] (Roberts et aL, 1986). During the formation of gr~nlll~tion tissue, fibronectin provides a su~k~ for the migrationof cells, a linkage for myofibroblasts to effect wound contraction, and ~flhçsion sites 15 for collagen fibril formation. Collagen types I, III and V appear and provide tensile strength for the wound and proteoglycans provide resilience to deform~tion of the tissue.
Wounds requiring healing by secondary intel tion are wounds with edges which are20 widely separated such that no epidermis or dermis is present. As a consequence, healing progresses from the base u~w~ds as well as from the edge ill~V~`iS. The dermis shows little wound h~lin~ activity in this respect and the majority of gr~nlll~tion tissue which eventually fills the wound site comes from the subcutaneous fat at the wound edge and from the subcutaneous floor of the wound. As noted 25 above, gramll~tion tissue is formed by the proliferation and migration of the~ulrou~ding co~nective tissue elem~.nt of the sub~ t~l-euus tissue; it is col,lposed in the first in~t~nce of c~rill~ry loops and fibroblasts together with a variable number of infl~mm~tQry cells.
30 The clinical need to provide pro~ , filnction~l coverage and to prevent scar formation in deep, extensive secondary indication wounds and the need to initiate and accelerate healing in chronic non-healing secon~ry in~iic~tion wounds, has led WO 94/09800 PCr/AU93/005~4 2~
to the experiment~l use of a vast number of materials in human trials.
These materials are generally ill-defined combin~tion~ of growth factors, which have been derived from a number of sources (see Van Brunt and l~ lsner, 1988; Buckley5 et al, 1985).
Platelet Derived Wound Healing Formula (PDWHF ) has been used, topically~ in an attempt to accelerate wound he~ling. It is considered likely t_at PDWHF stim-ll~t,os repair of chronically non-he~ling cutaneous wounds by a combination of growth 10 factors present in the formula. One study showed that wounds of dirr~l-ellt etiologies responded equally well to PDWHF (Knighton et al, 1988).
In an extensive study in human subjects, Carter et al. (1988) used three crude al-ations: autologous serum, porcine lysate (obtained from ~lci~lc keratinocytes) 1~ and bovine platelet lysate. All three showed limite-l success in trials on p~ti~.ntc with non-he~ling, deep-ulcerated wounds (Carter et al, 1988).
~xtracts from m~mm~ n eyes have also studied for their potential wound healing properties. In GB 1 342 761, a whole eye extract was shown to enh~nce cicatrisation 20 of corneal vounds. Furthermore, GB 1 603 034 ~ oses an aqueous solution of atissue regenerating extract such as from villeous which ~ Leci regeneraticn of cells of the epidermal layer. A similar effect on epidermal cells was disclosed in US
Patent No. 4,670,257 using an aqueous salt extract of ocular tissue, such as from vitreous.
There is a need to identify and develop s~lbst~nces capable of accelerating wound healing by selectively tar~,e~ g gr~n~ tion tissue but not cells of the epidermal and dermal layers of the skin. Such substances will provide a major advance in woundhealing management especially for secondary intention wounds which lack epidermis 30 and clerrni~. These snhst~nces would also be useful for promoting gr~mll~tinn tissue during repair of fractured bones.
WO 94/09800 pcr/Au93/oo5s4 2147~Q~
Acconlillgly, one aspect of the present invention provides a vitreous formnlation from m~mm~ n ocular tissue which is capable of ~lefelGl~lially accelerating growth of gr~nl-l~ti-)n tissue colllp~ed to skin tissue. The vitreous form-~l~tion may cc,~ lise i~o~ted vitreous, vitreous extract or one or more colllyol~ents of a villeolls S having the desired activity. A vitreous extract may be an aqueous extract or a salt extracted preparation.
The vitreous formulation is generally ~pted or in a form suitable for topical ~rlmini~ration. Accolding to this aspect of the present invention there is provided 10 a topical composition colnplising vitreous or an extract thereof from m~mm~ nocular tissue wherein said vitreous or its extract ~rGrel elltially accelerates growth of granulation tissue colllpared to epidermal and dermal tissue.
For ccllvellience reference to an "extract" of villeous in~hlrles lerGlellce to one or lS more active conl~ollents isolated and/or purified thcl-er~om. Furthermore, by way of shorthand notation, the vitreous form-~l~tion~ and topical co..-l~silions of the present invention are hereinafter referred to as a vit.cous based he~lin~ form~ tion (VBHF). The term "topical cc~l"l O~ilio~ is used herein in its broadest sense and incl~ es the applic~tion of the coni~osilion to one or more layers of subcllts~neQus 20 tissue and directly at the site of bone fracture.
The G~ression "epiderm~l tissue" is used in its broadest sense and is particularly directed to keratinocytes. 'Dermal tissue" is also used in its broadest sense and in~ s skin fibroblasts. Sub-~t~neous/gr~n~ tion tissue is also used in its 25 broadest sense and inchl~es vascular smooth muscle cells and myofibroblasts. The present invention is predicated in part on the ~ ing discovery by the hlv~ o~
that t_e VBHF ~tim~ tPs proliferation of vascular smooth muscle andmyofibroblasts but sllbst~nti~lly not skin fibroblasts and keratillocytes.
WO 94/09800 pcr/Au93/oo5s4 2~ S~ - 6 -Acco-di-lg to ~ ~ref~;lled aspect of the present invention, there is provided a topical composition colll~lisi-lgvitreous or an extract thereof from m~mm~ n ocular tissue wherein said vitreous or its extract prefcl-elltially stim~ t~s growth of vascular 5 smooth muscle cells and/or myofibroblasts while subst~ntiAlly not stim~ tin~ growth of skin fibroblasts and/or keratinocytes. The ~lefele~tial effect is conveniently and particularily observed and monitored in vitro.
Reference herein to a "vitreous extract" includes vitreous i~ol~tecl away from the 10 m~mm~ n ocular tissue, vitreous following at least one step of purification or fractionation or any active components therein. The m~mmAl is generally alivestock animal such as a bovine, ovine, equine or porcine animal or a goat or a laboratory test animal such as a murine ~nim~l, rabbit or guinea pig. The most preferred source of the vitreous of the present invention is from a bovine ~nim~
In another embo.iimçnt, the vi~leous is fr~ior~ted and one or more components or a ll~Lule of cc,ll.~o--ents i~ol~tecl and purified and ~vhich have a similar differential effect on gr~n~ ti-)n and skin tissue as does the vitreous or extract thereof.
Accordil.g to this aspect of the present invention, there is provided a component i~ol~te~hle from m~mm~ n vitreous or extract thereof, said cc,m~o..ent capable of accelerating or promoting growth of gr~mll~tion tissue such as vascular smooth muscle cells or myofibroblasts while having a minim~l prolifelalive effect on skin 25 cells such as k~lalillocytes and dermal derivedfibroblasts. Preferablythe component is in isolated and purified form me~nin~ that a CC.ll~ osition of matter comprises at least 20æ, more ~lerelably at least 35%, even more l,rererably at least 45%, still more plefelably at least 55-~5% and even still more pleft;lably at least 75-90~ of the co.ll~onent as determined by weight, activity, antibody binding or other 30 co..vel ient means.
Wo 94/09800 PCr/AUs3/00554 2~ 7~
The VBHF of the present invention may further co,l,~lice one or more exogenous factors added to the composition and which may also assist the wound healing process. Such exogenous factors conte.mpl~ted in this aspect of the present invention s inc~ e but are not limiteci to PDWHF, FGF, EGF or insulin-like growth factor (IGF), one or more antibiotics or other ~ntimi~robial agents or vit~minc such asvitamin E, D and/or K. The VBHF may aLo com~ise one or more pharm~. elltir~lly acceptable carriers andlor ciilll~ntc.
10 The VBHF may be topically applied directly or after incorporation into a medicated foam, gel, cream or liquid. Alternatively, it may be inco~ ated into a solid matrix such as a bandage, dressing, gauze or sutures. The VBHF may be in icol~te-l and/or purified form or may be frozen or lyophili7erl in which case it is reconctitlltecl prior to use.
15 Reference can conveniently be made to the British Pharmacopoeia (1980) for a description of collve~ on~l agents which may be usefillly incorporated into VBHF.
T~e VBHF of the present invention is co.~le...~ ted to be particularly usefu inwound healing by seconrl~ry intçntion of wounds which require accelerated growth20 of gr~n~ tion tissue such as ulcers which maybe associatedwith diabetes, peripheral vascular clice~ce or plCS~llC sores or bed sores; dermal tissue damage such as following various degrees of burns; post operative skin lesions l~king skin where accelerated growth of subcl~t~neous/gr~m-l~tion tissue is lc~uired; acceleratedhealing of gr~mll~tion tissue in bone fractures; accelerated growth of 25 subc~lt~n~ouslgr~nlll~tion tissue prior to aprlic~tinn of a skin graft.
Acc~lding to this aspect of the present invention there is co~-te~--.l~te-1 a method of stimlll~tin~, enhancing, accelerating or olhclwise promoting h~ n~ of a wound onan ~nim~l, said method COnl~JLiSillg cont~cting said wound with an effective amount 30 of VBHF as hereinbefore clçfinec~ for a time and under conclitions sufficient for wound healing to occur.
WO 94/09800 Pcr/Au93/oo554 Such wounds.are generally healed by secQnfl~ry intention as d~fin.o-l above. Theeffective amount is the amount of VBHF effective to stim~ te, enh~n~e, accelerate or otherwise promote wound he~ling which is cc l~v~;niently ~cc~ccerl by stiml~ tion S of vascular smooth muscle cells or myofibroblasts with sllbst~nti~lly no stimlll~ting effect on keratinocytes or skin fibroblasts. Reference above to a "wound" in~lu~5 refelence to a bone fracture.
The animal to be treated may be a hnm ~n, livestock anim al as hereinbefore ~l.ofin~
10 a laboratoly test animal as hereinbefore defin~-l, a companion animal (e.g dog or cat) or a captive wild ~nim~l Most pl~,relably, ho~ er, the ~nimal is a hnm~n The wound he.~l in~ protocol may ~n~l ~e a single aprli~tion of VBHF or a regime involving mllltiple applir~tion~ of VBHF on an hourly, twice daily, daily, mlll~iple 15 daily, weekly, multirle weekly or monthly basis, depen-ling on the wou~d to be treated, the extent of the wound and the healing capacity of the p~ti--nt The method may also Colll~l~SC further tre~tment wit_ one or more other agents such as antibiotics or other ~ntimi~robial agents, ~n~esthetics or other wound healing promotents. VBHF may be used as i~ol~tecl~ or may require cc~nce~ ation or 20 dilution. Dilutions contemrl~ted herein are in the order of 1:2 to about 1:10although dilutions of the order 1:2 to 1:5 are ~r~r~.l e~ If the VBHF is cc).~cenLI ated this may be done by any number of procedures inrln~ling lyophili~tio~ or filtration and extend to a concell~ ion factor of 2 to 10.
25 In ~nr-ther embo-liment, the VBHF is used in a non-liquid form such as following lyophili~tio~ Such a form may be particulary useful for the applic~tion to a solid matrix such as a bandage, dressing, gauze or sutures.
WO 94/09800 pcr/Au93/oos54 21~7~
g The present invention is further described byreÇele,lce to the following non-limiting Figures and/or F~Ample~c.
S In the Figures:
Figure 1 is a graphical represe-nt~tion showing t_e phases of wound repair.
Figure 2 is a gr~phic~l represent~tion of the effect of VBHF on tritiated - thymidine 10 incorporation into vascular smooth muscle cells.
Figure 3 is a graphical representation showing the effect of VBHF on tritiated -thymidine incol~olation into myofibroblasts.
lS Figure 4 is a graphical representation showing the effect of VBHF on tritiated thymidine incol~ration into osteoblasts.
FSgure S is a gr~phiç~l repr~.sent~tion of the effect of VBHF on tritiated - thymidine incolporation into skin fibroblasts.
Figure 6 is a gr~phic~l represçnt~tion showing the effect of VBHF on tritiated -thymidine h~col~uralion into keratinocytes.
Figure 7 is a grap_ical rc~lese.~ ;on of the effect of VBHF on tritiated - thymidine 25 i~lcol~lalion into vascular endothçlinm Figure 8 is a gr~rhi~l lc~lese~ t;on showing the effect of VBHF on proliferationof vascular smooth muscle ce~ls.
30 Figure 9 is a gr~phi~l repr~o~entation showing the effect of VBHF on proliferation of myofibroblasts.
WO 94/09800 PCr/AU93/00554 ~ 4~
Figure 10 is a gr~phiç~l repres~ont~tion showing the effect of VBHF on proliferation of skin fibroblasts.
Figure 11 is a graphical repr~sent~tion showing the effect of VBHF on proliferation 5 of keratinocytes.
Figure 12 is a graphical re~lesent~tion showing the effect of VBHF on proliferation of vascular endothelil-m 10 Figure 13 is a gr~phic~l represe-nt~tion showing the effect of VBHF on migration of vascular smooth muscle cells.
Figure 14 is a graphical repr~s~.nt~tion showing the effect of VBHF on migrationof myofibroblasts.
Figure 15 is a gr~rhic ~l repre~ent~tion showing the effect of VBHF on migrationof skin fibroblasts.
Figure 16 is a gr~rhic~l repr~-~çnt~tion showing the effect of VBHF on migration20 of ~eratil~ocytes.
Figure 17 is a photographic repr~se-nt~tion showing the effect of VBHF on morphology of vascular smooth muscle cells. In the presence of: A. DMEM to 0.5%
v/v PCS; B. DMEM to 0.5% v/v FCS and 1:2 diluted VBHF; C. DMEM to 0.5%
25 v/v FCS to 1:4 diluted VBHF.
Figure 18 is a photographic represe-nt~tion showing the effect of VBHF on morphology of myofibroblasts. In the presence of: A. DMEM to 0.5% v/v FCS; B.
DMEM to 0.5% v/v FCS and 1:2 diluted VBHF; C. DMEM to 0.5% v/v FCS to 1:4 30 diluted VBHF.
Wo 94/09800 PCr/AU93/00554 21~7~08 Figure 19 is a photographic represent~tion showing the effect of VBHF on morphology of skin fibroblasts. In the presence of: ~ DMEM to 0.5% v/v FCS;
B. DMEM to 0.5~o v/v FCS and 1:2 diluted VBHF; C. DMEM to 0.5% v/v FCS to 5 1:4 diluted VBHF.
Figure 20 is a photographic represent~tion showing the effect of VBHF on morphology of keratinocytes. In the presence of: A. DMEM to 0.5% v/v FCS; B.
DMEM to 0.5% v/v FCS and 1:2 diluted VBHF; C. DMEM to 0.5% v/v FCS to 1:4 10 cliluted VBHF.
EX,~MPLE 1 Isolation of vitreous 15 The vitreous is removed from 2 freshly sl~ htered bovine's eye in the following m~nner. Pr~rerably, all procedures outlined below are carried out in a l~min~r flow hood.
(i) the outside of the eye is rinsed in 70% v/v eth~nol (ii) a syringe fitted with a 19G needle is inserted into the cavity cont~iningthe aqueous humour and the aqueous humour is collectecl (iii) an in~ ion is made around the cornea with a scalpel and the cornea is removed.
(iv) using a pair of forceps, the lens is removed.
(v) the vitreous is then collected in a 10ml sterile syringe.
(vi) the syringe and its contents are then frozen at -70 degrees ~e WO 94/09800 PCr/AU93/00554 Preparation of vitreous based h~z-l;ng forrmll~*~n S The VBHF is used as icol~te~ according to the method of FY~mple 1 or additional conl~ollcnts added such as one or more pharma~e~ltie~lly acceptable carriers and/or dilll-ontc, emnlcifying com~onents such as Be~lometh~cone or Cetrimide or components s~lecte~l from saline, protein, antibiotic, carbohydrates. Alternatively, or in addition to, the vitreous is fractionated to remove ullw~lted com~llents or to 10 isolate and purify desired active components.
Once the VBH~ is ~le~aled, it is then stored or transported as is or in a frozen or lyophilised condition.
Wound Tl~l..- -,' (i) The wound is debrided and if infection is present it is removed by the appli~tion of a suitable antibiotic or ~ntimi~robial agent.
(ii) The wound is washed in 0.9% w/v sterile saline.
(iii) An aliquot of lml of the VBHF is applied from a syringe and dispersed evenly over the wound.
(iv) A piece of plastic backed gauze (cut to the same size of the wound) is moictçnecl with 0.9% w/v sterile saline and laid over the wound.
(v) Plastic wrap is placed over the gauze.
(vi) Tre~tment is repeated every 12hrs.
WO 94/09800 2 14 7 ~1~ 8 pcr/Al)93/ooss4 E~MPLE 4 tion for Skin G~
(i) Steps (i) to (v) of FY~mrle 3 are carried out until a suitable bed of gr~n~ tion tissue is present.
(ii) A skin graft can then be applied to the growing dermal tissue.
(iii) Treatment with VBHF can be contin--ed if required.
EX~MPLE S
T~ of Isch~ Ulcer 15 A clinical trial was carried out in a hospital e~lvho~ ent. A patient was a male 72 years of age in good health with the exception of a Scm x 3cm ulcer on the lower left leg The ulcer had failed to respond to various proprietary dressings which had been applied over a period of six month~. In ~ litioll during this tre~tment period, skin grafting had also been carried out and as a result, the patient required frequent 20 hospit~ tioJl All of these l,c~ cnt regimes were lm~uccec~ful. The wound lacked epiderm~l and dermal tissue, secor~ ry inte-nti~n he~ling by ~le~ive granulation tissue growth was required.
The patient was started on a tre~tm~nt regime which involved an appliç~tion, every 25 12 hours, of the VBHF, as outlined in Fy~mrle 3. As a result of this tre~tment regime, the patient's ulcer healed completely in 14 days with no recurrence for the period of the test which was 18 months.
WO 94/09800 PCr/AU93/00554 Q~ - 14-EX~MPLE 6 Tre~ nt of Diabetic Ulcer S A ~ linic~l test was carried out in a hospital ellvi,o~lent. A patient was a male 76 years of age in good health with the exception of a chronic ulcer me~cl~ring 7cm x 4cm on lower right leg. The ulcer had been re~;u"ent over 25 years during which time he~ling had never been complete using various proprietary dressings and skin grafting techniques. Accelerated growth of gr~n~ tion tissue was required to 10 sll~ceccfully heal the wound.
The patient was commenced on a tre~tment regime using a topical appli--~tion of the VBHF as outlined in Fx~mple 2. As a result of this tre~tment regime, the patient's ulcer was 90% healed after 12 dayc and comrletely healed after 22 days, with no 15 recurrence of the ulcer for the time of the test which was 17 monthc.
Tr ~ of ro~L L~ c Ulcer 20 A rlinic~l trial was carried out initially in a hospit~l setting and later by the patient at home. The patient was a female 48 years of age with a post tr~llm~tic non-healing ulcer.
The ulcer measured Scm x Scm and was sit l~ted on the lower left leg.
No progress had been made in he~ling the ulcer using various dressing techniques25 and there was no evidence of new skin growth. In ~ lition~ over the S year history of the ulcer, repeated skin grafting reslllted in short term coverage of the wound, but each skin graft eventually degenerated. The wound lacked epicl~rmic tissue int ~ ing both epithei~l and dermal tissue and required gr~nlll~qtion tissue growth for sllcceccf wound healing.
Wo 94/09800 21~ 7 ~ ~ 8 pcr/Au93/oo554 .
The patient was commenced on a tre~tm~nt regime which involved the topical aprlic~tion of the VBHF as outlined in FY~mrle 3. As a result of this tre~tment, the ulcer was completely healed after 21 days.
S
E~MPLE 8 T~ of Diabetic Ulce~
A clinical trial was carried out in a hospit~l setting on a female 82 year old patient.
10 The patient had 2 ulcers: one on the front of the right foot (Scm x 2cm) and the other at the rear of the right leg (lOcm x 10cm). Both ulcers were the result ofperipheral vascular restriction resulting from diabetes. The ulcers had failed to respond to various proprietary dressings and hyperbaric treatment over S months.The ulcer at the rear had penetrated through the tendon and ~mputation of the limb 15 was likely.
The patient was commen~e~ on a tre~tm~-nt regime for the ulcer on the back of the right leg which involved the topical applic~tion of the VBHF (as o-lLlilled in FY~mrle 3). The ulcer on the front of the right foot was dressed with N-saline as a control.
20 After 14 days the ulcer on the back of the right leg had ~lelsivt; growth of gr~mll~tion tissue, whereas the ulcer on the front of the right foot showed no change.
Tre~tment of the ulcer on the rear of the right leg was then commçnt~e(l as outlined in ry~lnple 4. In both cases rapid growth of gr~n-ll~tinn tissue occurred, the ulcer at the front of the right foot was comrlet~ly healed after æ days whereas the ulcer 25 on the front of the right foot was comrletely healed after 38 days.
EXM~PLE 9 Differen~ ect of VBHF
1. Sources of Cells V~c~ll~r Smnoth Mllcrle ('o.11~
Vascular smooth muscle cells were obtained by enzymatic di~el~ion of 12 week oldrabbit aortae by s~n~l~rd procedures (Campbell and Campbell, 1993). Cells were grown in Dulbecco's Minim~l F.ccçnti~l Medium plus 0.5% v/v foetal calf serurn 10 (DMEM + 0.5% v/v FCS).
Myofibrobl ~cf.c Myofibroblasts were obtained by enzyme dispersion of Selye's gr~nll10m~ pouch in~ ecl in rats by subcllt~nrQus mjection of 1 ml of 1% v/v croton oil in corn oil 15 following a 20 ml bolus of air (Selye, 1953). Cells were grown in DMEM + 0.55'o v/v FCS.
~kin Fihrobl~ct.c Human skin fibroblasts were obtained from Dr Rebecca Mason of the U11iVt;1~ilY of 20 Sydney, New South Wales, Australia and were grown in DMEM + 0.5% v/v FCS.
Ker~tinncyt~.c - Humankeratinocytes (epi(le~nic)were obtainedfromDrRebeccaMason, Ulliv~l~ily of Sydney, New South Wales, Australia together with k~l ati~ocyte growth medium 25 (KGM). For ~~ ent.~, cells were grown in DMEM.
F.n~ fheli~
Endothelial cells were i~ol~terl from bovine aorta by the method of Horrigan et al (1988) grown in me~ lm RPMI + 0.5% v/v FCS.
WO 94/09800 pcr/Au93/oo5s4 214750~-2. VB~
VBHF was prepared according to FY~mrles 1 and 2 in plastic syringes on ice.
The VBHF was stored frozen until required, then serially diluted at the time of use 5 at 1:2, 1:4, 1:8 and 1:16 in either DMEM + 0.5% v/v FCS or RPMI + 0.5% v/v FCS (for endothelial cells). P~nicillin G was usèd to control co..~lnin~tion.
3. Incorporation of 3H-Thymidine V~c~ll~r.~mooth Mnccle ~ell~
10 Cells were seeded at S x 104 cells/well into a 24 well plant in DMEM + 10% v/v FCS. The cells were allowed to attach and flatten for 18 hours, then the cells thoroughly washed in DMEM + 0.5% v/v FCS. The wells were then divided into six groups, four wells per group and exposed to the following con-litions:
15 1. DMEM + 0.5% v/v FCS
2. DMEM + 0.5% v/vFCS + 1:2 VBHF
3. DMEM + 0.5% v/v FCS + 1:4 VBHF
4. DMEM + 0.5% v/v FCS + 1:8 VBHF
5. DMEM + 0.5% v/v FCS + 1:16 VBHF
The wells cont~inin~ DMEM + 0.5% v/v FCS acted as controls for those with added VBHF.
The cells were further incubated at 37C for 24 hours, at which time 0.5 mCi 3H-25 thymidine (~mçrsh~m) was added to each well. After 4 hours incubation, the cells were harvested onto 2.5 cm ~ass mi--lurll)r~, f~ters (Wh~tm~n Intern~tion~l, UK), washed and then lysed with 3 x 3 ml H20. The filters were air-dried, then placed in srintill~tion vials to which 7 ml Berkm~n Ready Safe scintill~nt was added. Samples were counted in a Be~m~n LS6000TA ,3-Counter ~g~inct a blank.
The quadr lplic~te wells in each e~elilllental con~ition were meaned and comr~red st~tictir~lly to the control (DMEM + 0.5% v/v FCS). A .~igm~ct~t st~tictic~l WO 94/09800 PCr/AU93/00554 program was lltili7ecl for a one way analysis of variance.
It was found that VBHF at a dilution of 1:2 and 1:4 stim~ te~l the 3H-thymidine uptake into smooth musde cells by about 6.4 fold above controls in each of theseS e~eriments (Figure 2). Dilutions of 1:8 and 1:16 also stim~ tecl 3H-thymidine uptake but to a lesser extent and dose depen~le-ntly. Stimtll~tion of 3H-thymidine uptake was significant for all dilutions tested. VBHF at dilution 1:2 and 1:4 ctimlll~te~ 3H-thymidine uptake above that of the positive control (DMEM + 10%
v/v FCS) (Figure 2).
Myofihrobl~ctc Cells were seeded at 5 x 104 cells/well. The protocol was identical to that described above.
15 VBHF at 1:2 dilution si~nific~ntly stim~ ted 3H-thymidine uptake into myofibrobla~t~ ab~ve con~ol~ (1.7 fold) in ea~h of the ~ree ~~ ent~ (Fi~re ~).
There w~c no si~nifi~nt increase in 3H-thymidine incol~ration with any of the other dilutions (1:4,1:8 and 1:16).
20 ~kin fihrobl~ctc Cells were seeded at 5 x 104 cells/well. The protocol was j~lentic~l to that described above.
VBHF at 1:4 and 1:8 dillltionc ci~nific~ntly decreased the incc,.~ration of 3H-25 thymidine into skin fibroblasts while 1:2 and 1:16 had no effect (Figure 5).
Ker~tinncyt.oc The protocol was iclentit~l to that described above.
30 VBHF at 1:4, 1:8 and 1:15 dilution decreased inco~ aLion ~vhile 1:2 dilution had no effect (Figure 6).
Wo 94/09800 2117 ~ 0 8 PCr/AU93/00554 Fn~lr~th~
The protocol was as described above except that the control medium was RPMI +
0.5% v/v FCS.
5 VBHF at all ~ ltion~ inrll)re~ a ~i nific~nt decrease in inool~o,ation of 3H-thymidine into endothelial cells (Figure 7).
4. Cell Prolife~ation Experiments were carried out to determine the effect of VBHF at dilutions 1:2, 1:4, 10 1:8 and 1:16 on cell proliferation. Cells were plated out and left to attach and flatten for 18 hours. Quadrl-plic~te wells were then counted using a haemocytometer, in order to determine number of cells at e~el ;., .~nt~l time ~. The rem~inin~ wells were washed with DMEM + 0.5% v/v FCS (or RPMI + 0.5% v/v FCS for endothelial cells) then fed with their a~ropliate me~ m as follows:
1. DMEM + 0.5% v/v FCS
2. DMEM + 0.5% v/v FCS + 1:2 VBHF
3. DMEM + 0.5% v/v FCS + 1:4 VBHF
4. DMEM + 0.5% v/v FCS + 1:8 VBHF
5. DMEM + 0.5% v/v FCS + 1:16 VBHF
For endothelial cells, the protocol for 1 to 5 above was repeated using RPMI +
0.5% v/v FCS.
25 An ~c~ cnt for each cell type was ~elÇulll,ed with quadrllplic~te wells per con-lition Cells were counted at day 2 for vascular smooth muscle cells and myofibroblasts, day 3 for skin fibroblasts and endothelial cells and day 5 for kelalinocytes. This reflected the dirrerellt rates of proliferation of the cell types.
30 V~c~ll~r~Smooth Mllc~le (~toll~
The number of smooth muscle cells was si~nific~ntly increased in the presence of 1:2 and 1:4 diluted VBHF by 2.5 fold, with no effect on 1:8 and 1:16 (Figure 8).
2~
Mynfihrobl~et.e The number of myofibroblasts was si~nifi~ntly increased above control by VBHF
diluted at 1:2 only (Figure 9).
~;
5 ~kin Fihrobl~c~.c There was no si~nifi~ ~nt change in nllmber of skin fibroblasts with any dilution of VBHF (Figure 10).
K~.r~tinncyttoe 10 Similarly, there was no si~nific~nt change in number of keratinocytes with any lti~ln of VBHF (Figure 11).
Fn~loth~ l Celle VBHF diluted at 1:2, 1:4 and 1:8 was growth inhibitory for endothelial cells (Figure 15 12). The number of cells at day 0 was 4.08 + 0.51 x 104 cells/well, while at day 3 with 1:2 diluted VBHF there were 6.78 _ 0.45 x 104 cells per well, and in RPMI +0.5% v/v serum alone at day 3 there were 25.09 _ 3.19 x 104 cells/well. The growth inhibitory effect was dose depen~ent and cell number returned to the level of control RPMI + 0.5% v/v FCS at ~lillltion of VBHF of 1:16 (Figure 12).
5. Cell Migration All five cell types were ~u~pended in DMEM + 10% v/v FCS (RMPI + 15% v/v FCS for endothelial cells) at 1 x 105 cells/ml, then 200 ,ul ~ ed into a circular well insert placed in the centre of each well. The cells were allowed to attach and 25 flatten into a conflllent layer overnight, the insert re~l,uv~d, the cells thoroughly washed and the ~ .nt~l meclillm added.
For each cell type, quadrllrlic~te wells were used for each experimental con~litio;
The experimental conrl;tions were the same as that described above (1 to 5).
Once the insert was removed and the experiment~l mç~ m added the cells were allo~ved to migrate in a radial m~nn~r from the central circle of cells over 7 days.
WO 94/09800 21 4 7 ~) O ~ pcr/Aus3/oo554 Fresh medium and VBHF were added on day 3. On day 7, cells were fixed in 10%
v/v neutral buffered form~lin and stained with 1% v/v to~ ine blue. The area of culture substrate covered by cells was determmed by pomt cou~lLi"g morphometry and ~, essed as a percentage of the total well surface. Quadr lrli~ate wells for each 5 cell type were fixed and stained at ~,;.,.ent~l time 0.
V~c~ll~r~Smooth Mllc-~le Cellc In the presence of 1:2 and 1:4 dillltionc of VBHF vascular smooth muscle cell migration was signific~ntly increased above control wells C~r~t~ g DMEM + 05%
10 v/v FCS (Figure 13). The effect was dose depen-l~nt It should be noted that vascular smooth muscle cells do not grow as a mon~l~yer~and in sit~l~tions where cell growth is stim~ te~l, the cells can grow in mnltirle layers. This was especially evident in the presence of 1:2 and 1:4 diluted VBHF and 15 the ~o~itive control, where st~inin~ with toluidine blue was not only more ~le,~ive over the area of the culture substrate but was markedly more int.onse.
Mynfihrobl~ctc Only the dilution of 1:2 VBHF ci~nifir~ntly stim~ ted ~ vrlbroblast migration above 20 control (Figure 14).
~Skin Fihrobl~tc There was no signific~nt dirr~lel,ce in degree of skin fibroblast Out~owlh with any ~lilntion of VBHF coll~ d with control (Figure 15).
K~. ~I;..n~yt~
Simil~rly, there was no effect on keratinocyte migrationby any ~lihltion of VBHF(Figure 16).
30 F.n~ theli~l ('.~11~
VBHF at 1:2 and 1:4 dilutions a~ated to be toxic forendothelial cells, since thearea of culture substrate covered by cells after 7 days ~s less than that at day 0 WO 94/09800 ~,3~ PCr/AU93/00554 prior to ~o~ e to VBHF. The effect was dose dep~o-n-lent, with less toxic effectoccurring at 1;8 and 1:16 ~ ltion~.
The wells cont~inin~ DMEM + 0.5% v/v FCS acted as controls for those with added VBHF.
The cells were further incubated at 37C for 24 hours, at which time 0.5 mCi 3H-25 thymidine (~mçrsh~m) was added to each well. After 4 hours incubation, the cells were harvested onto 2.5 cm ~ass mi--lurll)r~, f~ters (Wh~tm~n Intern~tion~l, UK), washed and then lysed with 3 x 3 ml H20. The filters were air-dried, then placed in srintill~tion vials to which 7 ml Berkm~n Ready Safe scintill~nt was added. Samples were counted in a Be~m~n LS6000TA ,3-Counter ~g~inct a blank.
The quadr lplic~te wells in each e~elilllental con~ition were meaned and comr~red st~tictir~lly to the control (DMEM + 0.5% v/v FCS). A .~igm~ct~t st~tictic~l WO 94/09800 PCr/AU93/00554 program was lltili7ecl for a one way analysis of variance.
It was found that VBHF at a dilution of 1:2 and 1:4 stim~ te~l the 3H-thymidine uptake into smooth musde cells by about 6.4 fold above controls in each of theseS e~eriments (Figure 2). Dilutions of 1:8 and 1:16 also stim~ tecl 3H-thymidine uptake but to a lesser extent and dose depen~le-ntly. Stimtll~tion of 3H-thymidine uptake was significant for all dilutions tested. VBHF at dilution 1:2 and 1:4 ctimlll~te~ 3H-thymidine uptake above that of the positive control (DMEM + 10%
v/v FCS) (Figure 2).
Myofihrobl~ctc Cells were seeded at 5 x 104 cells/well. The protocol was identical to that described above.
15 VBHF at 1:2 dilution si~nific~ntly stim~ ted 3H-thymidine uptake into myofibrobla~t~ ab~ve con~ol~ (1.7 fold) in ea~h of the ~ree ~~ ent~ (Fi~re ~).
There w~c no si~nifi~nt increase in 3H-thymidine incol~ration with any of the other dilutions (1:4,1:8 and 1:16).
20 ~kin fihrobl~ctc Cells were seeded at 5 x 104 cells/well. The protocol was j~lentic~l to that described above.
VBHF at 1:4 and 1:8 dillltionc ci~nific~ntly decreased the incc,.~ration of 3H-25 thymidine into skin fibroblasts while 1:2 and 1:16 had no effect (Figure 5).
Ker~tinncyt.oc The protocol was iclentit~l to that described above.
30 VBHF at 1:4, 1:8 and 1:15 dilution decreased inco~ aLion ~vhile 1:2 dilution had no effect (Figure 6).
Wo 94/09800 2117 ~ 0 8 PCr/AU93/00554 Fn~lr~th~
The protocol was as described above except that the control medium was RPMI +
0.5% v/v FCS.
5 VBHF at all ~ ltion~ inrll)re~ a ~i nific~nt decrease in inool~o,ation of 3H-thymidine into endothelial cells (Figure 7).
4. Cell Prolife~ation Experiments were carried out to determine the effect of VBHF at dilutions 1:2, 1:4, 10 1:8 and 1:16 on cell proliferation. Cells were plated out and left to attach and flatten for 18 hours. Quadrl-plic~te wells were then counted using a haemocytometer, in order to determine number of cells at e~el ;., .~nt~l time ~. The rem~inin~ wells were washed with DMEM + 0.5% v/v FCS (or RPMI + 0.5% v/v FCS for endothelial cells) then fed with their a~ropliate me~ m as follows:
1. DMEM + 0.5% v/v FCS
2. DMEM + 0.5% v/v FCS + 1:2 VBHF
3. DMEM + 0.5% v/v FCS + 1:4 VBHF
4. DMEM + 0.5% v/v FCS + 1:8 VBHF
5. DMEM + 0.5% v/v FCS + 1:16 VBHF
For endothelial cells, the protocol for 1 to 5 above was repeated using RPMI +
0.5% v/v FCS.
25 An ~c~ cnt for each cell type was ~elÇulll,ed with quadrllplic~te wells per con-lition Cells were counted at day 2 for vascular smooth muscle cells and myofibroblasts, day 3 for skin fibroblasts and endothelial cells and day 5 for kelalinocytes. This reflected the dirrerellt rates of proliferation of the cell types.
30 V~c~ll~r~Smooth Mllc~le (~toll~
The number of smooth muscle cells was si~nific~ntly increased in the presence of 1:2 and 1:4 diluted VBHF by 2.5 fold, with no effect on 1:8 and 1:16 (Figure 8).
2~
Mynfihrobl~et.e The number of myofibroblasts was si~nifi~ntly increased above control by VBHF
diluted at 1:2 only (Figure 9).
~;
5 ~kin Fihrobl~c~.c There was no si~nifi~ ~nt change in nllmber of skin fibroblasts with any dilution of VBHF (Figure 10).
K~.r~tinncyttoe 10 Similarly, there was no si~nific~nt change in number of keratinocytes with any lti~ln of VBHF (Figure 11).
Fn~loth~ l Celle VBHF diluted at 1:2, 1:4 and 1:8 was growth inhibitory for endothelial cells (Figure 15 12). The number of cells at day 0 was 4.08 + 0.51 x 104 cells/well, while at day 3 with 1:2 diluted VBHF there were 6.78 _ 0.45 x 104 cells per well, and in RPMI +0.5% v/v serum alone at day 3 there were 25.09 _ 3.19 x 104 cells/well. The growth inhibitory effect was dose depen~ent and cell number returned to the level of control RPMI + 0.5% v/v FCS at ~lillltion of VBHF of 1:16 (Figure 12).
5. Cell Migration All five cell types were ~u~pended in DMEM + 10% v/v FCS (RMPI + 15% v/v FCS for endothelial cells) at 1 x 105 cells/ml, then 200 ,ul ~ ed into a circular well insert placed in the centre of each well. The cells were allowed to attach and 25 flatten into a conflllent layer overnight, the insert re~l,uv~d, the cells thoroughly washed and the ~ .nt~l meclillm added.
For each cell type, quadrllrlic~te wells were used for each experimental con~litio;
The experimental conrl;tions were the same as that described above (1 to 5).
Once the insert was removed and the experiment~l mç~ m added the cells were allo~ved to migrate in a radial m~nn~r from the central circle of cells over 7 days.
WO 94/09800 21 4 7 ~) O ~ pcr/Aus3/oo554 Fresh medium and VBHF were added on day 3. On day 7, cells were fixed in 10%
v/v neutral buffered form~lin and stained with 1% v/v to~ ine blue. The area of culture substrate covered by cells was determmed by pomt cou~lLi"g morphometry and ~, essed as a percentage of the total well surface. Quadr lrli~ate wells for each 5 cell type were fixed and stained at ~,;.,.ent~l time 0.
V~c~ll~r~Smooth Mllc-~le Cellc In the presence of 1:2 and 1:4 dillltionc of VBHF vascular smooth muscle cell migration was signific~ntly increased above control wells C~r~t~ g DMEM + 05%
10 v/v FCS (Figure 13). The effect was dose depen-l~nt It should be noted that vascular smooth muscle cells do not grow as a mon~l~yer~and in sit~l~tions where cell growth is stim~ te~l, the cells can grow in mnltirle layers. This was especially evident in the presence of 1:2 and 1:4 diluted VBHF and 15 the ~o~itive control, where st~inin~ with toluidine blue was not only more ~le,~ive over the area of the culture substrate but was markedly more int.onse.
Mynfihrobl~ctc Only the dilution of 1:2 VBHF ci~nifir~ntly stim~ ted ~ vrlbroblast migration above 20 control (Figure 14).
~Skin Fihrobl~tc There was no signific~nt dirr~lel,ce in degree of skin fibroblast Out~owlh with any ~lilntion of VBHF coll~ d with control (Figure 15).
K~. ~I;..n~yt~
Simil~rly, there was no effect on keratinocyte migrationby any ~lihltion of VBHF(Figure 16).
30 F.n~ theli~l ('.~11~
VBHF at 1:2 and 1:4 dilutions a~ated to be toxic forendothelial cells, since thearea of culture substrate covered by cells after 7 days ~s less than that at day 0 WO 94/09800 ~,3~ PCr/AU93/00554 prior to ~o~ e to VBHF. The effect was dose dep~o-n-lent, with less toxic effectoccurring at 1;8 and 1:16 ~ ltion~.
6. Cell Morphology S In order to observe any change in cell shape, size or orient~tion in~ ecl by VBHF, quadrllplic~te wells of each cell type were exposed to:
1. DMEM + 0.5% v/v FCS
2. DMEM + 0.5% v/v FCS + 1:2 diluted VBHF
10 3. DMEM + 0.5% v/v FCS + 1:4 diluted VBHF
For Endothelial cells, 1 to 3 were repeated with RPMI + 0.5% v/v FCS.
V~ r.~;mooth Mn~le C~
15 In the presence of both 1:2 and 1:4 diluted VBHF, the number of smooth musclecells was clearly greater than in control (DMEM + 0.5% FCS), but there was little change in cell shape, size or orient~tion (Figure 17), except that the cells were confluent.
20 Myofihrobl~tc In conL~ myofibroblasts changed shape from flat, broad cells in control (Figure 18A), to an elongated ribbon shape characteristic of motile cells in the presence of 1:2 diluted VBHF (Figure 18B). This was also the case, although to a lesser extent, with 1:4 dilution VBHF (Figure 18C).
Skin Fibrobl~ct~
Skin fibroblast morphology did not alter appreciably in the presence of 1:2 or 1:4 diluted VBHF or 0.5% FCS (Figure 19).
30 K~r~tinocyt~c When the keratinocytes were first passaged, the cells were flat, epidermal-like and growing in a monolayer. Iwo lictin~t cell populations were evident, with a sm~ller 2147~
WO 94/09800 PCr/AUs3/00s54 .
pop~ tion of larger cells spread uniformly throughout the monol~yer. All proliferation and migration studies were carried out with the cells in this phenotype, and 1:2 and 1:4 ~lih~tion.~ of VBHF had no effect on morphology. The results areshown in Figure 20.
S
E~XAMPLE 10 Prolif~dLi.~ Effect of VBHF on Os~
Cultures of human foetal osteoblast-like cells were treated with VBHF and 10 compared to the effects produced by vitamin D 1,25(0H)2D3 and TGF-,B, both ofwhich modulate the rate of cell division of osteoblast-like cells in vitr~
Parameters tested included comparison of cell numbers and [3H]- thyrnidine inco~ ation into the cells after 48 hours treatment. The known and unknown 15 ~reatments were tested against two control tre~tmçnt~. The first was a culture which had received no treatment and the second set of controls in~ lded cells cultivated in medium that had been ~ lted to 75% w/v of its initial concenlrdtion using Dulbecco's PBS.
20 Con~posilions were received on ice and stored in a 0-4C refrigcratol-.
Two flasks of the primary cell culture FBC200893 (from the trabecular ends of long bones) were L~y~h~ised and the cells were washed and r~ le~l at a co..~e-~ ion of 6 x 104 cells/ml of BGJ meclil-m (GIBCO), suppl~mçntecl with 25 phosphoascorbate and 10% v/v FCS. The cells were plated in one ml aliquots in 24 well culture plates overnight.
The me~ m was aspired the following day and repl~l~e-l and 1 ml/well 2% v/v FCS
supplemented BGJ medium. The methods by which the v~ious factors were 30 delivered to the cultures are ou~lh~cd in Table 1. Tre~tm~.nt~ rwlned in quadruplicate, were r~n-lomly allocated to the col~lmn~ of the 24 well plates.
WO 94/09800 PCI'/AU93/005~4 Tre~trnentc used in the study and the cc"n~silion of those tre~qtmentc T~ c Used in Study Cc,.ll~,osilion PBS control PBS:BGJ 1:3 TGF- ,3 0.5~1 of 1 ~g/ml TGF- ,3 VBHF VBHF:BGJ 1:3 Nil Control 2% FCS in BGJ*
10-81,25(0H)~D3 1~1 10-5M 1,25D dissolved in the EtOH
* All treatment cultures used this form~ tion as the basis of the tre~trnent medium.
The cultures with various tre~tmçntc were incubated for 48 hours after which cultures were used to me~Cllre cell density or [3H]-thymidine ill~,~ration.
Variations in the cell cultures as a result of tre~tmçnt were tested using ANOVAfrom the MicroSoft Excel package. .signi~lc~n~e of dir~lc~ces between tre~tmentCwas performed using Tukey's method of mllltiple co~ isc,~ at p~0.05.
Tre~trn~o-nt of the cells with TGF-~ at a concenLralion of 0.5 ng/ml affected the morphology of the celL. Cultures appeared more conflll-ont than those treated with either PBS or Nil treatment. Cultures treated with TGF- ,3 appeared to be con,~osed of a higher pclce,ltage of cells with cuboidal morphology while PBS control cultures were ~lomin~ted by cells e~ics~ g a fibroblastic morphology.
Cultures treated with VBHF exhibited similar morphology and culture surface coverage as cultures treated with TGF- ,~. Cultures ~sed to these two tre~tmentcappeared to be fully confl~l~nt (totally covering the culture surface).
WO 94/09800 . 214 7 5 ~ 8 PCr/AUs3/00554 Cell Coumts T1P~ Cel1 NU~
(c~s/well) PBS control 105000 0.5 ng/ml TGF- ,~ 72800 Nil Control 87600 10-8M 1,25-(OH)2D3 88800 Using ANOVA at the 95% conficle.nce interval, there is a st~ti~tic~lly signifie~nt dirre~ ce in cell number between cultures treated with VBHF and TGF- ~ and PBS
treated cultures.
[3H]~ 3inP I"c~ .ion T,~ cpm/well 0.5 ng/ml TGF- ,B 52907 Nil Control 90929 10 8M 1,25(0H)2D3 69245 The results of 3H-th~nidine incol~lation are shown in Figure 4.
WO 94/09800 ~ ) PCI/AU93/00554 Analysing these data using ANOVA shows there is no signifiç~nt dirr~cl~ce in therate of thymidine uptake between cultures m~ .çd in untreated 2% FCS
suppl~mente~l BGJ medium and cultures in which the me~ m has been diluted to 75% of its original co~ce~ ation with Dulbecco's PBS. There is a cignific~nt dirrclcllce in the rate of thymi~line uptake betwe~en cultures treated with PBS and those treated with VBHF ~p ~ 0.05).
Dilution of the culture merlitlm to 75% of its original concç.~l~ aLion using PBS
appears to have had no signific~nt effect on the cell numbers or the rate of cell division of the human foetal bone derived cells ~.~p~rcd to the untreated control cultures. This was, therefore, concicl~red to be more a~o~liate as a control for the purpose of testing the effect of the supplied c~,lllpo~;l;on, as the use of these com~osi~ions entailed liilnting the culture medillm to 75% of its normal concellLl ation.
TGF- ~ has been shown to have a number of effects on bone cells, de~el~ g on thesystem in which the factor is used. In the present study, TGF-,B decreased ceIl proliferation and there is a cwlcs~ll~ling decrease in thymi&e uptake. This inhibition of cell division by TGF-,3 tre~tm~nt was probably related to the celldensity used in this study. At low cell d~ncities~ osteobl~ct-like cell cultures tend to be domin~ted by cells which are at an early stage of dirrcl~ tio~ and col~ ingly, a stage of rapid cell division. In such cases the pre-lomin~nt effect of TGF- ,~ is to cause the cells to advance their stage of dirrclc " i~ti-~n simnlt~n-~ously decreasing the rate of cell division. In contl a~l, when bone cell cultures are denser, the cells tend to dirr~le..~;~te This leads to a decrease in the rate of cell division.
When such cultures are treated with TGF- ,B they have a ten~e-ncy to increase the rate of cell division. This accounl~ for the dirLrcllt effects of TGF- ,B on the rate of cell division under dirr~ ienl c~ itionc Wo 94/09800 214 7 ~ ~ 8 PCr~AUs3/00554 The state of tlifferentiation in the present study can be ~ccecsed qualitatively by observation of the cell morphology. Dirrele,~ ted osteoblasts in culture tend toexhibit a more cuboidal morphology while ceLc that are less well dirrerf "ti~tedappear more like cultured fibroblastic cells. TGF-~ and VBHF appeared to affect the morphology of the cells in culture ac a greater percel-tage of the cells in the culture were cuboidal in shape. More qualitative methods of ~Cceccing state of dirr~"..li~tion can be made by me~cllring biochemical markers of dirr~rP~-ti~tion such as pro~ tion of ~lk~line phosph~t~ce Cells treated with VBHF were more cuboidal and larger than cells of control cultures. Further, cultures treatedwith this co..,l.osil;o~ were c~mpletelyconfl-lent at the end of 48 hours. Tre~tment of the bone cell cultures with VBHF appeared to si~nific~ntly re~lllred cell number compared with control cultures over the 48 hour period of incubation. There was, howGvt:" a stimlll~tion of thyrnidine incorporation at the end of the 48 hour period.
The a~ enL discrepancy bet~,veen cell numbers at the end of 48 hours (a me~cllreof proliferation during the period of treatment) and thymidine incorporation measured over 4 hours at the end of the 48 hour tre~tm~ont~ may reflect the activity of the cells at two dirf~,rel,t time periods. It is likely that VBHF has a dirreL.~"~ ;~tin~
effect. The dirr~ tion of the cells in these cultures may be followed by an increase in the proli~laliv~ activity as me~llred by thymidine i.~ alion. This Lclea_e in proliferation may not be me~cllrable in terms of cell numbers for some period of time beyond the initial 48 hour period.
WO 94/09800 ~4~ ~,Q~ PCI/AU93/OOS54 REFE~RENOES:
Knighton, D., Doucette, M., Fiegel, V., Ciresi, K., Butler, E. and Austin, L.
(1988) The Use of Platelet Derived Wound Healing Formula in Human ~linic~l Trial, in p.p. 319-329.
Carter, D., Balin, A., Gottlieb, M., Eisinger, A., Lin, A., Pratt, L., Sherbany,A. and Caldwell, D. (1988) ~lini~l Experience with Crude Pr~ ~aLations of Growth Factors in Healing of Chronic Wounds in Human Subjects, in p.p.
303-317.
Van Brunt, J. and Kl~nc~r, A. (1988) Growth Factor Speed Wound Healing.
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Buckley, A., Dandson, J., Kamerath, C., Wolt, T. and Woodward, S. (1985) S~ ;,.c~1 Release of Fpi(lerm~1 Growth Factor Accelerates Wound Repair.
Proc Natl Acad Sci USA, 92, p.p. 7340-7344.
Balæs, E. and Denlinger, J. (1984) The Vitreus in Davvson, H. The eye (3rd Ed) Vol. la Vegetalive Physiology and Biol~l.f~ -y ~r~tlemic Press, NY., p.p. 533-580.
Clark, R~F. (1988). Overview and general con~i~lerations of wound repair.
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Gabbiani, G., Hirshel, BJ., Ryan, G.B., Sta~ov, P.R. and Majno, G. (1972).
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Wo 94/09800 ~ 1 4 7 5 ~ ~ Pcr/Aug3/oo554 Horrig-an, S., Campbell, J.H. and Campbell, G.R. (1988). Effect of endothelium on ,~-VLDL metabolism by cultured smooth muscle cells of ~rr~lillg phenotype. Atherosclerosis. 71: 57-69.
Kurkinen, M., Vaheri, A., Roberts, P.J. and Stçnm~n, S. (1980). Sequential appearance of fibronectin and collagen in experim~-nt~l gr~mll~tion tissue.
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Leibovich, S.J. and Ross, R. (1975). The role of the macrophage in wound repair: A study with hydrocollisolle and anti-macrophage serum. Am. J.
Pathol. 78: 71-100.
Roberts, A B., Sporn, M.B., Assovan, RK., Smith, J.M., Roche, M.S., Heine, U.F., Liottay, L., Falanga, V., Kehrl, J.H. and Fancie, A S. (1986).
T~ ro,~ g growth factor beta: rapid inr~ ion of fibrosis and angiogenesis in vivo and stimnl~ti~n of collage form~tion Proc. Natl. Acad. Sci. USA 83:
4167-4171.
Selye, H. (1953~. On the merh~ni~m through which hydroco- l.sone affects the le~ re of tissues to injury. An ~ ent~l study with the gr~nlllom pouch terhniqlle J. Am. Med. Assoc. 152: 1207-1213.
1. DMEM + 0.5% v/v FCS
2. DMEM + 0.5% v/v FCS + 1:2 diluted VBHF
10 3. DMEM + 0.5% v/v FCS + 1:4 diluted VBHF
For Endothelial cells, 1 to 3 were repeated with RPMI + 0.5% v/v FCS.
V~ r.~;mooth Mn~le C~
15 In the presence of both 1:2 and 1:4 diluted VBHF, the number of smooth musclecells was clearly greater than in control (DMEM + 0.5% FCS), but there was little change in cell shape, size or orient~tion (Figure 17), except that the cells were confluent.
20 Myofihrobl~tc In conL~ myofibroblasts changed shape from flat, broad cells in control (Figure 18A), to an elongated ribbon shape characteristic of motile cells in the presence of 1:2 diluted VBHF (Figure 18B). This was also the case, although to a lesser extent, with 1:4 dilution VBHF (Figure 18C).
Skin Fibrobl~ct~
Skin fibroblast morphology did not alter appreciably in the presence of 1:2 or 1:4 diluted VBHF or 0.5% FCS (Figure 19).
30 K~r~tinocyt~c When the keratinocytes were first passaged, the cells were flat, epidermal-like and growing in a monolayer. Iwo lictin~t cell populations were evident, with a sm~ller 2147~
WO 94/09800 PCr/AUs3/00s54 .
pop~ tion of larger cells spread uniformly throughout the monol~yer. All proliferation and migration studies were carried out with the cells in this phenotype, and 1:2 and 1:4 ~lih~tion.~ of VBHF had no effect on morphology. The results areshown in Figure 20.
S
E~XAMPLE 10 Prolif~dLi.~ Effect of VBHF on Os~
Cultures of human foetal osteoblast-like cells were treated with VBHF and 10 compared to the effects produced by vitamin D 1,25(0H)2D3 and TGF-,B, both ofwhich modulate the rate of cell division of osteoblast-like cells in vitr~
Parameters tested included comparison of cell numbers and [3H]- thyrnidine inco~ ation into the cells after 48 hours treatment. The known and unknown 15 ~reatments were tested against two control tre~tmçnt~. The first was a culture which had received no treatment and the second set of controls in~ lded cells cultivated in medium that had been ~ lted to 75% w/v of its initial concenlrdtion using Dulbecco's PBS.
20 Con~posilions were received on ice and stored in a 0-4C refrigcratol-.
Two flasks of the primary cell culture FBC200893 (from the trabecular ends of long bones) were L~y~h~ised and the cells were washed and r~ le~l at a co..~e-~ ion of 6 x 104 cells/ml of BGJ meclil-m (GIBCO), suppl~mçntecl with 25 phosphoascorbate and 10% v/v FCS. The cells were plated in one ml aliquots in 24 well culture plates overnight.
The me~ m was aspired the following day and repl~l~e-l and 1 ml/well 2% v/v FCS
supplemented BGJ medium. The methods by which the v~ious factors were 30 delivered to the cultures are ou~lh~cd in Table 1. Tre~tm~.nt~ rwlned in quadruplicate, were r~n-lomly allocated to the col~lmn~ of the 24 well plates.
WO 94/09800 PCI'/AU93/005~4 Tre~trnentc used in the study and the cc"n~silion of those tre~qtmentc T~ c Used in Study Cc,.ll~,osilion PBS control PBS:BGJ 1:3 TGF- ,3 0.5~1 of 1 ~g/ml TGF- ,3 VBHF VBHF:BGJ 1:3 Nil Control 2% FCS in BGJ*
10-81,25(0H)~D3 1~1 10-5M 1,25D dissolved in the EtOH
* All treatment cultures used this form~ tion as the basis of the tre~trnent medium.
The cultures with various tre~tmçntc were incubated for 48 hours after which cultures were used to me~Cllre cell density or [3H]-thymidine ill~,~ration.
Variations in the cell cultures as a result of tre~tmçnt were tested using ANOVAfrom the MicroSoft Excel package. .signi~lc~n~e of dir~lc~ces between tre~tmentCwas performed using Tukey's method of mllltiple co~ isc,~ at p~0.05.
Tre~trn~o-nt of the cells with TGF-~ at a concenLralion of 0.5 ng/ml affected the morphology of the celL. Cultures appeared more conflll-ont than those treated with either PBS or Nil treatment. Cultures treated with TGF- ,3 appeared to be con,~osed of a higher pclce,ltage of cells with cuboidal morphology while PBS control cultures were ~lomin~ted by cells e~ics~ g a fibroblastic morphology.
Cultures treated with VBHF exhibited similar morphology and culture surface coverage as cultures treated with TGF- ,~. Cultures ~sed to these two tre~tmentcappeared to be fully confl~l~nt (totally covering the culture surface).
WO 94/09800 . 214 7 5 ~ 8 PCr/AUs3/00554 Cell Coumts T1P~ Cel1 NU~
(c~s/well) PBS control 105000 0.5 ng/ml TGF- ,~ 72800 Nil Control 87600 10-8M 1,25-(OH)2D3 88800 Using ANOVA at the 95% conficle.nce interval, there is a st~ti~tic~lly signifie~nt dirre~ ce in cell number between cultures treated with VBHF and TGF- ~ and PBS
treated cultures.
[3H]~ 3inP I"c~ .ion T,~ cpm/well 0.5 ng/ml TGF- ,B 52907 Nil Control 90929 10 8M 1,25(0H)2D3 69245 The results of 3H-th~nidine incol~lation are shown in Figure 4.
WO 94/09800 ~ ) PCI/AU93/00554 Analysing these data using ANOVA shows there is no signifiç~nt dirr~cl~ce in therate of thymidine uptake between cultures m~ .çd in untreated 2% FCS
suppl~mente~l BGJ medium and cultures in which the me~ m has been diluted to 75% of its original co~ce~ ation with Dulbecco's PBS. There is a cignific~nt dirrclcllce in the rate of thymi~line uptake betwe~en cultures treated with PBS and those treated with VBHF ~p ~ 0.05).
Dilution of the culture merlitlm to 75% of its original concç.~l~ aLion using PBS
appears to have had no signific~nt effect on the cell numbers or the rate of cell division of the human foetal bone derived cells ~.~p~rcd to the untreated control cultures. This was, therefore, concicl~red to be more a~o~liate as a control for the purpose of testing the effect of the supplied c~,lllpo~;l;on, as the use of these com~osi~ions entailed liilnting the culture medillm to 75% of its normal concellLl ation.
TGF- ~ has been shown to have a number of effects on bone cells, de~el~ g on thesystem in which the factor is used. In the present study, TGF-,B decreased ceIl proliferation and there is a cwlcs~ll~ling decrease in thymi&e uptake. This inhibition of cell division by TGF-,3 tre~tm~nt was probably related to the celldensity used in this study. At low cell d~ncities~ osteobl~ct-like cell cultures tend to be domin~ted by cells which are at an early stage of dirrcl~ tio~ and col~ ingly, a stage of rapid cell division. In such cases the pre-lomin~nt effect of TGF- ,~ is to cause the cells to advance their stage of dirrclc " i~ti-~n simnlt~n-~ously decreasing the rate of cell division. In contl a~l, when bone cell cultures are denser, the cells tend to dirr~le..~;~te This leads to a decrease in the rate of cell division.
When such cultures are treated with TGF- ,B they have a ten~e-ncy to increase the rate of cell division. This accounl~ for the dirLrcllt effects of TGF- ,B on the rate of cell division under dirr~ ienl c~ itionc Wo 94/09800 214 7 ~ ~ 8 PCr~AUs3/00554 The state of tlifferentiation in the present study can be ~ccecsed qualitatively by observation of the cell morphology. Dirrele,~ ted osteoblasts in culture tend toexhibit a more cuboidal morphology while ceLc that are less well dirrerf "ti~tedappear more like cultured fibroblastic cells. TGF-~ and VBHF appeared to affect the morphology of the cells in culture ac a greater percel-tage of the cells in the culture were cuboidal in shape. More qualitative methods of ~Cceccing state of dirr~"..li~tion can be made by me~cllring biochemical markers of dirr~rP~-ti~tion such as pro~ tion of ~lk~line phosph~t~ce Cells treated with VBHF were more cuboidal and larger than cells of control cultures. Further, cultures treatedwith this co..,l.osil;o~ were c~mpletelyconfl-lent at the end of 48 hours. Tre~tment of the bone cell cultures with VBHF appeared to si~nific~ntly re~lllred cell number compared with control cultures over the 48 hour period of incubation. There was, howGvt:" a stimlll~tion of thyrnidine incorporation at the end of the 48 hour period.
The a~ enL discrepancy bet~,veen cell numbers at the end of 48 hours (a me~cllreof proliferation during the period of treatment) and thymidine incorporation measured over 4 hours at the end of the 48 hour tre~tm~ont~ may reflect the activity of the cells at two dirf~,rel,t time periods. It is likely that VBHF has a dirreL.~"~ ;~tin~
effect. The dirr~ tion of the cells in these cultures may be followed by an increase in the proli~laliv~ activity as me~llred by thymidine i.~ alion. This Lclea_e in proliferation may not be me~cllrable in terms of cell numbers for some period of time beyond the initial 48 hour period.
WO 94/09800 ~4~ ~,Q~ PCI/AU93/OOS54 REFE~RENOES:
Knighton, D., Doucette, M., Fiegel, V., Ciresi, K., Butler, E. and Austin, L.
(1988) The Use of Platelet Derived Wound Healing Formula in Human ~linic~l Trial, in p.p. 319-329.
Carter, D., Balin, A., Gottlieb, M., Eisinger, A., Lin, A., Pratt, L., Sherbany,A. and Caldwell, D. (1988) ~lini~l Experience with Crude Pr~ ~aLations of Growth Factors in Healing of Chronic Wounds in Human Subjects, in p.p.
303-317.
Van Brunt, J. and Kl~nc~r, A. (1988) Growth Factor Speed Wound Healing.
Biote~hnology, 6. p.p. 25-30.
Buckley, A., Dandson, J., Kamerath, C., Wolt, T. and Woodward, S. (1985) S~ ;,.c~1 Release of Fpi(lerm~1 Growth Factor Accelerates Wound Repair.
Proc Natl Acad Sci USA, 92, p.p. 7340-7344.
Balæs, E. and Denlinger, J. (1984) The Vitreus in Davvson, H. The eye (3rd Ed) Vol. la Vegetalive Physiology and Biol~l.f~ -y ~r~tlemic Press, NY., p.p. 533-580.
Clark, R~F. (1988). Overview and general con~i~lerations of wound repair.
In "The Molecular and ~ r Biology of Wound repair", editors Clark, R~F. and Henson, P.M. Plenum Press, New York, p.p. 3-33.
Gabbiani, G., Hirshel, BJ., Ryan, G.B., Sta~ov, P.R. and Majno, G. (1972).
C r~n~ t ~n tlss~le as a contractlle organ. A study o~ structure and ~ln~ t~on J. E~p. Med. 135: 719-734.
Wo 94/09800 ~ 1 4 7 5 ~ ~ Pcr/Aug3/oo554 Horrig-an, S., Campbell, J.H. and Campbell, G.R. (1988). Effect of endothelium on ,~-VLDL metabolism by cultured smooth muscle cells of ~rr~lillg phenotype. Atherosclerosis. 71: 57-69.
Kurkinen, M., Vaheri, A., Roberts, P.J. and Stçnm~n, S. (1980). Sequential appearance of fibronectin and collagen in experim~-nt~l gr~mll~tion tissue.
Lab. Invest. 43: 47-51.
Leibovich, S.J. and Ross, R. (1975). The role of the macrophage in wound repair: A study with hydrocollisolle and anti-macrophage serum. Am. J.
Pathol. 78: 71-100.
Roberts, A B., Sporn, M.B., Assovan, RK., Smith, J.M., Roche, M.S., Heine, U.F., Liottay, L., Falanga, V., Kehrl, J.H. and Fancie, A S. (1986).
T~ ro,~ g growth factor beta: rapid inr~ ion of fibrosis and angiogenesis in vivo and stimnl~ti~n of collage form~tion Proc. Natl. Acad. Sci. USA 83:
4167-4171.
Selye, H. (1953~. On the merh~ni~m through which hydroco- l.sone affects the le~ re of tissues to injury. An ~ ent~l study with the gr~nlllom pouch terhniqlle J. Am. Med. Assoc. 152: 1207-1213.
Claims (31)
1. A topical composition comprising vitreous or an extract thereof from mammalian ocular tissue wherein said vitreous or its extract preferentially accelerates growth of granulation tissue compared to skin tissue.
2. A topical composition according to claim 1 further comprising one or more pharmaceutically acceptable carriers and/or diluents.
3. A topical composition according to claim 1 or 2 further comprising one or more wound healing promoting agents and/or antimicrobial agents.
4. A topical composition according to claim 1, wherein the ocular tissue is froma livestock animal.
5. A topical composition according to claim 4 wherein the livestock animal is selected from the list consisting of bovine, ovine, equine, porcine species and goat.
6. A topical composition according to claim 5 wherein the livestock animal is a bovine species.
7. A topical composition according to claim 1 immobilized or otherwise impregnated onto a solid support.
8. A topical composition according to claim 7 wherein the solid support is a bandage, dressing, gauze or sutures.
9. A topical composition comprising vitreous or an extract thereof from mammalian ocular tissue wherein said vitreous or its extract preferentially stimulates in vitro growth of vascular smooth muscle cells and/or myofibroblasts while substantially not stimulating growth of skin fibroblasts and/or keratinocytes.
10. A topical composition according to claim 9 further comprising one or more pharmaceutically acceptable carriers and/or diluents.
11. A topical composition according to claim 9 or 10 further comprising one or more wound healing promoting agents and/or antimicrobial agents.
12. A topical composition according to claim 9 wherein the ocular tissue is froma livestock animal.
13. A topical composition according to claim 12 wherein the livestock animal is selected from the list consisting of bovine, ovine, equine, porcine species and goat.
14. A topical composition according to claim 13 wherein the livestock animal is a bovine species.
15. A topical composition according to claim 9 immobilized or otherwise impregnated onto a solid support.
16. A topical composition according to claim 15 wherein the solid support is a bandage, dressing, gauze or sutures.
17. A topical composition according to claim 1 or 9 in lyophilized form and which is optionally reconstituted in aqueous medium prior to use.
18. A method for enhancing or accelerating healing of a wound in an animal, saidmethod comprising contacting said wound with an effective amount of the composition according to claim 1 or 9 for a time and under conditions sufficient to promote healing of the wound.
19. A method according to claim 18 wherein the animal to be treated is a human, livestock animal or companion animal.
20. A method according to claim 20 wherein the animal to be treated is a human.
21. A method according to claim 18 wherein the wound is healed by secondary intention.
22. A method according to claim 21 wherein the wound is an ulcer.
23. A method according to claim 18 wherein the wound is damage to dermal tissue.
24. A method according to claim 19 wherein the wound is a bone fracture.
25. A method according to claim 18 wherein the wound to be healed is a skin graft.
26. A method according to claim 18 wherein the wound is a post-operative skin lesion lacking epidermis or dermis tissue.
27. A method according to claim 18 further comprising the topical application ofanother wound healing promoting agent and/or an antimicrobial agent.
28. A method according to claim 18 wherein the ocular tissue is from a livestock animal.
29. A method according to claim 28 wherein the livestock animal is selected fromthe list consisting of bovine, ovine, equine, porcine species and goat.
30. A method according to claim 29 wherein the livestock animal is a bovine species.
31. A method according to claim 18 wherein the composition is topically applied on a bandage, dressing, gauze or sutures.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPL555292 | 1992-10-28 | ||
| AUPL5552 | 1992-10-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2147508A1 true CA2147508A1 (en) | 1994-05-11 |
Family
ID=3776504
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002147508A Abandoned CA2147508A1 (en) | 1992-10-28 | 1993-10-27 | A method of promoting wound healing and compositions useful for same |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0669828A4 (en) |
| JP (1) | JPH08502496A (en) |
| AU (1) | AU5365894A (en) |
| CA (1) | CA2147508A1 (en) |
| WO (1) | WO1994009800A1 (en) |
| ZA (1) | ZA938054B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2835565B1 (en) | 2002-02-05 | 2004-10-22 | Saint Gobain Ct Recherches | METHOD FOR MANAGING MEANS FOR CLEANING A PARTICLE FILTER |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1140076A (en) * | 1977-04-07 | 1983-01-25 | Franco Donadelli | Solution of tissue-regenerating extracts treated electrolytically and with alternating electric field |
| FR2461002A1 (en) * | 1979-07-13 | 1981-01-30 | Inst Nat Sante Rech Med | METHOD FOR STIMULATING THE GROWTH OF HUMAN EPIDERMIC CELLS AND PRODUCTS USING THE SAME |
| US4477435A (en) * | 1981-05-19 | 1984-10-16 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Method for regenerating corneal epithelium |
| US4534967A (en) * | 1982-08-03 | 1985-08-13 | Boston Biomedical Research Institute | Cell proliferation inhibitor and method of preparation |
| US4770877A (en) * | 1982-08-03 | 1988-09-13 | Boston Biomedical Research Institute | Isolation of a high molecular weight aortic endothelial cell growth inhibitor |
| US5234914A (en) * | 1991-06-11 | 1993-08-10 | Patent Biopharmaceutics, Inc. | Methods of treating hemorrhoids and anorecial disease |
-
1993
- 1993-10-27 AU AU53658/94A patent/AU5365894A/en not_active Abandoned
- 1993-10-27 EP EP93923972A patent/EP0669828A4/en not_active Withdrawn
- 1993-10-27 CA CA002147508A patent/CA2147508A1/en not_active Abandoned
- 1993-10-27 JP JP6510465A patent/JPH08502496A/en active Pending
- 1993-10-27 WO PCT/AU1993/000554 patent/WO1994009800A1/en not_active Application Discontinuation
- 1993-10-28 ZA ZA938054A patent/ZA938054B/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| ZA938054B (en) | 1994-06-07 |
| EP0669828A1 (en) | 1995-09-06 |
| AU5365894A (en) | 1994-05-24 |
| JPH08502496A (en) | 1996-03-19 |
| EP0669828A4 (en) | 1996-07-31 |
| WO1994009800A1 (en) | 1994-05-11 |
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