AU613776B2 - Wound healing - Google Patents

Description

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PCT

OPI DATE 19/07/89 AOJP DATE 17/08/89 APPLN ID 37472 89 PCT NUMBER PCT/US88/04557 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (1 ter tion aI ubficaon iWber,:- WO 89/ 05656 A61K 37/36 29 June 1989 (29.06.89) (21) International Application Number: PCT/US88/04557 (22) International Filing Date: 20 December 1988 (20.12.88) (31) Priority Application, 136,399 (32) Priority Date: 2 e ember 1987 (22.12.87) (33) Priority Country 'i US SECTION 113 DIRE ION SEE FOLIO NAME DIRECTED IeS iden+ a Iod S o rec C c- 'I Qake\LC Si CGat-nI sKQssqCJ'L At v L t SI-.A 3 1 i t 'c Qtjtlvua. MdS0-c.Act-l-uSa U r (74) Agent: CLARK, Paul, Fish Richardson, One Financial Center, Suite 2500, Boston, MA 02111 (US), (81) Designated States: AT (European patent), AU, BE (European patent), BJ (OAPI patent), CF (OAPI patent), CG (OAPI patent), CH (European patent), CM (OA- PI patent), DE (European patent), DK, FR (European patent), GA (OAPI patent), GB (European patent), IT (European patent), JP, KR, LU (European patent), ML (OAPI patent), MR (OAPI patent), NL' patent), NO, SE (European patent), SN ent), SU, TD (OAPI patent), TG (OAPI pa- ,I;actNS Euoenptn) N 0 1 Qr-VQK Igto"3 national search report.

expiration of the time limit for amending the I to be republished in the event of the receipt nients, (54) Title: WOUND HEALING (57) Abstract Healing an external wound or regenerating bone of a mammal by administering to the mammal a composition containing purified platelet/derived growth factor and purified transforming growth factor alpha.

,r I .i i i i;i L I' L L C WO 69/05656 PCT/US88/04557 WOUND HEALING Background of the Invention This invention relates to healing wounds.

Growth factors are polypeptide hormones which stimulate a defined population of target cells.

Examples of growth factors include platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor beta (TGF-3), transforming growth factor alpha (TGF-a), epidermal growth factor (EGF), and fibroblast growth factor (FGF). PDGF is a cationic, heat-stable protein found in the granules of circulating platelets which is known to stimulate in vitro protein synthesis and collagen production by fibroblasts. It is also known to act as an vitro mitogen and chemotactic agent for fibroblasts, and smooth muscle cells, It has been proposed to use PDGF to promote in vivo wound healing. For example, Grotendorst (1984) J.

Trauma 24:549-52 describes adding PDGF to Hunt-Schilling wire mesh chambers impregnated with a collagen gel and implanted in the backs of rats; PDGF was found to increase the amount of new collagen synthesized.

However, Leitzel et al, (1985) J. Dermatol, Surg, Oncol.

11:617-22 were unable to accelerate normal wound healing in hamsters using PDGF alone or in combination with FGF and EGF, Michaeli, et al. (1984) In Soft and Hard Tissue Repair (Hunt, T.K, et al., Eds), Praeger Publishers, New York, pp. 380-394, report that application of a partially purified preparation of PDGF obtained from platelet-rich plasma stimulated angiogenesis when I j Ii WO 89/05656 PCT/US88/04557 2 implanted in rabbit corneas. Because PDGF is not an angiogenic growth factor the investigators suggested that an unknown factor in their partially purified PDGF preparation was responsible for the angiogenic effect, Schultz, G.S. et al. (1987) Science reported that local application of TGF-a to partial thickness skin burns in pigs accelarated epidermal regeneration, in comparison with untreated burns.

Summary of the Invention In general, the invention features healing an external wound in a mammal, a human patient, by applying to the wound an effective amount of a composition that includes a combination of purified PDGF and purified TGF-a. Preferably, the TGF-a is human TGF-a but can also be of another mammalian species, rat, The TGF-a can be isolated from natural sources or, more preferably, produced by recombinant cells or solid phase peptide syothesis. The composition of the invention aids in healing the wound, at least in part, by promoting the growth of epithelial and connective tissue and the synthesis of total protein and collagen. Wound healing using the composition of the invention is more effective than that achieved in the absence of treatment without applying exogenous agents) or by treatment with purified PDGF alone, or purified TGF-a alone.

In preferred embodiment of the invention, the composition is prepared by combining, in a pharmaceutically acceptable carrier substance, e.g., commercially available inert gels or liquids saline supplemented with albumin or methyl cellulose), purified PDGF and TGF-a (both of which are commercially available). Most preferably purified PDGF and TGF-a are combined in a weight-to-weight ratio of between 1:4 i WO 89/05656 PCT/US88/04557 3 and 2511, preferably between 1:2 and 10:1, and more preferably 1:1 or 2:1, The purified PDGF may be obtained from human platelets or by recombinant DNA technology. Thus, by the term "PDGF" we mean both platelet-derived and recombinant materials of mammalian, preferably primate, origin; most preferably, the primate is a human, but can also be a chimpanzee or other primate. Recombinant PDGF can be recombinant heterodimer, made by inserting into cultured prokaryotic or eukaryotic cells DNA sequences encoding both subunits, and then allowing the translated subunits to be processed by the cells to form heterodimer, or DNA encoding just one of the subunits (preferably the beta or chain) can be inserted into cells, which then are cultured to produce homodimeric PDGF (PDGF-1 or PDGF-2 homodimer).

The term "purified" as used herein refers to PDGF or TGF.-a which, prior to mixing with the other, is 95% or greater, by weight, PDGF or TGF-a, is substantially free of other proteins, lipids, and carbohydrates with which it is naturally associated.

A purified protein preparation will generally yield a single major band on a polyacrylamide gel for each PDGF or TGF-a component. Most preferably, the purified PDGF or TGF-a used in the composition of the invention is pure as judged by amino-terminal amino acid sequence analysis, The composition of the invention provides a fast, effective method for healing external wounds of mammals, bed sores, lacerations and burns. The composition enhances connective tissue formation compared to natural healing no exogenous agents added) or pure PDGF or TGF-a alone. Unlike pure PDGF alone, the composition promotes a significant increase

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WO 89/05656 PCT/US88/04557 4 in both new connective tissue and epithelial tissue.

The epithelial layer obtained is thicker than that created by natural healing or by TFG-a alone, and also contains more epithelial projections connecting it to the new connective tissue; it is thus more firmly bound and protective.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

Description of the Preferred Embodiments We now describe preferred embodiments of the invention.

External wounds, bed sores and burns, are treated, according to the invention, with PDGF/TGF-a mixtures prepared by combining pure PDGF and TGF-a.

Chemically synthesized human and rat TGF-a are commercially available from Peninsula Laboratories (Belmont, CA). Purified recombinant PDGF and purified PDGF derived from human platelets are commercially available from PDGF, Inc. (Boston, MA), Collaborative Research (Waltham, MA), and Amgen Corp. (Thousand Oaks, CA). Purified PDGF can also be prepared as follows.

Five hundred to 1000 units cf washed human platelet pellets are suspended in 1M NaC1 (2ml per platelet unit) and heated at 100 0 C for 15 minutes. The supernatant is then separated by centrifugation and the precipitate extracted twice with the 1M NaCl.

The extracts are combined and dialyzed against 0.08M NaCl-0.01M sodium phosphate buffer (pH 7.4) and mixed overnight at 4 0 C with CM-Sephadex equilibrated with the buffer. The mixture is then poured into a column (5 x 100 cm), washed extensively with 0.08M NaCl-0.O1M sodium phosphate buffer (pH 7.4), and eluted with 1M NaCl while 10 ml fractions are collected, WO 89/05656 PCT/US88/04557 5 Active fractions are pooled and dialyzed against 0.3M NaCl-0.O1M sodium phosphate buffer (pH centrifuged, and passed at 4°C through a 2.5 x cm column of Blue Sepharose (Pharmacia) equilibrated with 0.3M NaCl-0.01M sodium phosphate buffer (pH 7.4).

The column is then washed with the buffer and partially purified PDGF eluted with a 1:1 solution of 1M NaC1 and ethylene glycol, The partially purified PDGF fractions are diluted with 1M NaCi, dialyzed against 1M acetic acid, and lyophilized. The lyophilized samples are dissolved in 0.8M NaCl-0.01M sodium phosphate buffer (pH 7.4) and passed through a 1.2 x 40 cm column of CM-Sephadex C-50 equilibrated with the buffer. PDGF is then eluted with a NaCl gradient (0.08 to IM).

The active fractions are combined, dialyzed against.1M acetic acid, lyophilized, and dissolved in a small volume of lM acetic acid. 0.5 ml portions are applied to a 1.2 x 100 cm column of Biogel P-150 (100 to 200 mesh) equilibrated with IM acetic acid. The PDGF is then eluted with 1M acetic acid while 2 ml fractions are collected.

Each active fraction containing 100 to 200 mg of protein is lyophilized, dissolved in 100 ml of 0.4% trifluoroacetic acid, and subjected to reverse phase high performance liquid chromatography on a phenyl Bondapak column (Waters). Elution with a linear acetonitrile gradient (0 to 60%) yields pure PDGF, PDGF made by recombinant DNA technology can be prepared as follows: Platelet-derived growth factor (PDGF) derived from human platelets contains two polypeptide sequences (PDGF-1 and PDGF-2 polypeptides; Antoniades, H.N. and Hunkapiller, M. (1983) Science 220:963-965). PDGF-1 is u_

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1 WO 89/05656 PCT/US88/04557 6encoded by a gene localized in chromosome 7 (Betshol:z, C. et al., Nature 320:695-699), and PDGF-2 is encoded by the sis oncogene (Doolittle, R. et al, (1983) Science 221:275-277) localized in chromosome 22 (Dalla-Favera, R, (1982) Science 218:686-688). The sis gene encodes the transforming protein of the Simian Sarcoma Virus (SSV) which is closely related to PDGF-2 polypeptide.

The human cellular c-sis also encodes the PDGF-2 chain (Rao, C.D. et al. (1986) Proc. Natl. Acad. Sci. USA 83:2392-2396). Because the two polypeptide chains of PDGF are coded by two different genes localized in separate chromosomes, the possibility exists that human PDGF consists of a disulfide-linked heterodimer of PDGF-1 and PDGF-2, or a mixture of the two homodimers (homodimer of PDGF-1 and homodimer of PDGF-2), or a mixture of the heterodimer and the two homodimers.

Mammalian cells in culture infected with the Simian Sarcoma Virus, which contains the gene encoding the PDGF-2 chain, were shown to synthesize the PDGF-2 polypeptide and-to process it into a disulfide-linked homodimer (Robbins, K. et al. (1983) Nature 305:605-608). In addition, PDGF-2 homodimer reacts with antisera raised against human PDGF, Furthermore, the functional properties of the secreted PDGF-2 homodimer are similar to those of platelet-derived PDGF in that it stimulates DNA synthesis in cultured fibroblasts, it induces phosphorylation at the tyrosine residue of a 185 kd cell membrane protein, and it is capable of competing with human 125 1)-PDGF for binding to specific cell surface PDGF receptors (Owen, A. et al. (1984) Science 225:54-56), Similar properties were shown for the sis/PDGF-2 gene product derived from cultured normal human cells (for example, human arterial endothel.al cells), or from human malignant cells expressing the II

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WO 89/05656 PC/US8804557 7 sis/PDGF-2 gene (Antoniades, H. et al. (1985) Cancer Cells 3:145-151), The recombinant PDGF-2 homodimer (referred to as recombinant PDGF herein) is obtained by the introduction of cDNA clones of c-sis/PDGF-2 gene into mouse cells using an expression vector. The c-sis/PDGF-2 clone used for the expression was obtained from normal human cultured endothelial cells (Collins, et al. (1985) Nature 216:748-750).

Wound Healing To determine the effectiveness of PDGF/TGF-a mixtures in promoting wound healing, the following experiments were performed, Young white Yorkshire pigs (Parson's Farm, Hadley, MA) weighing between 10 and 15 kg were fasted for at least 6 hours prior to surgery and then anesthetized. Under aseptic conditions, the back and thoracic areas were clipped, shaved, and washed with mild soap and water. The area to be wounded was then disinfected with 70% alcohol, Wounds measuring 1 cm x 2 cm were induced at a depth of 0.5 mm using a modified Castroviejo electrokeratome (Storz, St. Louis, MO, as modified by Brownells, Inc.). The wounds resulted in complete removal of the epithelium, as well as a portion of the underlying dermis (comparable to a second degree burn injury). Individual wounds were separated by at least mm of unwounded skin. Wounds receiving identical treatment were organized as a group and separated from other groups by at least 3 cm, Wounds receiving no growth factor treatment were separated from wounds receiving such treatment by at least 10 cm, The wounds were treated directly with a single application of the following growth factors suspended in i

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i i j r, WO 89/05656 PCT/US88/04557 8 biocompatible gel: 1) 500 ng pure human PDGF (purified by high performance liquid chromatography) or recombinant PDGF alone; 2) 500 ng pure recombinant PDGF in combination with each of the following: a) 500 ng human TGF-a; b) 500 ng rat TGF-a; 3) 500 ng human or rat TGF-a alone.

Following wounding, biopsy specimens were taken on days 3 through 10. Biopsy specimens for histologic evaluation were taken as wedges approximately 3 mm deep and placed in 10% formalin, Specimens for biochemical analysis and autoradiography were obtained using an electrokeratome. The final dimensions of the specimens were 1.5 mm x 10 mm x 1.5 mm, Three specimens per wound were collected for biochemical analysis, while two specimens per wound collected for autoradiography.

Following collection, the specimens were stored in cold Eagle's Modified Essential Medium (EMEM) media supplemented with.10% fetal calf serum, The biopsy specimens were analyzed as follows.

Histologic Evaluation Histologic specimens were prepared using standard paraffin impregnating and embedding techniques. Four micron sections were made and stained using filtered Harris hemotoxylin and alcoholic eosin; they were then observed under a microscope. All specimens were scored blindly by two investigators at equally distributed points throughout the sections. The widths of the epithelial and connective tissue layers were scored using a grid placed within the ocular of.the microscope; the measurement was then converted into millimeters using a micrometer viewed under the same conditions.

i WO 89/05656 PCT/US88/04557 9 DNA and Protein Determination DNA determination was performed using a modification of the method of Labarca et al. (1980) Anal. Biochem. 120:344-52. A 50 il aliquot of tissue extract in concentrated ammonium hydroxide was added to 400 pl of a buffer solution containing 1 M sodium phosphate and 2M sodium chloride (pH the pH of the resulting solution was adjusted to 7.4 using HC1.

Afterwards, the final solution volume was brought to 500 pl, while maintaining the pH at 7.4. The solution was then added to 2.5 ml of a buffered solution (0.05 M sodium phosphate, 2M sodium chloride, pH 7.4) of Hoesht dye (1.14 mg/ml). Fluorescence was induced at an excitation wavelength of 352 nm and emission measured at 454 nm. Calf thymus DNA prepared by identical treatment was used to develop standard curves.

Protein content of the tissue extract in concentrated ammonium hydroxide was measured by the Bradford method (Bradford (1976) Anal. Biochem.

72:248-54), with bovine serum albumin as a standard, Results The results from histologic evaluation indicated that wounds treated with the combination ofpurified human PDGF or recombinant PDGF and chemically synthesized human or rat TGF-a had thicker connective tissue and epithelial layers, and more extensive epithelial projections connecting these layers, than wounds receiving no treatment, human or rat TGF-a alone, or pure PDGF alone.

The PDGF/TGF-a treated wounds had greater DNA, protein and collagen contents.

Dosage To determine the appropriate dosage of purified PDGF, the above-described experiments were repeated i WO 89/05656 PCT/US88/04557 10 except that the wounds were treated with 2.5 ng, 5.0 ng, and 10 ng PDGF equivalents of purified PDGF per square millimeter of wound dispersed in 30pl of biocompatible gel. The results showed that optimum effects were produced when the PDGF content was 5.0 ng/mm 2 or higher.

To determine the appropriate dosage of pure PDGF plus TGF-a, combinations in which the weight to weight ratio of PDGF to TGF-a ranged from 1:10 to 25:1 were evaluated as described above, Optimum results were achieved with a ratio of between 1:1 and 2:1,

IS

Claims (10)

1. A method for healing an external wound of a mammal comprising applying to said wound a wound-healing amount of a composition comprising purified platelet-derived growth factor and purified transforming growth factor alpha.

2. The method of claim 1 wherein the weight to weight ratio of said platelet-derived growth factor to said transforming growth factor alpha in said composition is between 1:4 and 25:1.

3. The method of claim 2 wherein said ratio is between 1:2 and 10:1.

4. The method of claim 3 wherein said ratio is about 1:1 or 2:1.

5. A wound healing composition comprising purified platelet-derived growth factor and purified transforming growth factor alpha, in a weight to weight ratio of 1:4 to 25:1.

6. The composition of claim 5 wherein said ratio is between 1:2 and 10:1.

7. The composition of claim 6 wherein said ratio is about 1:1 or 2:1.

8. A method for preparing a composition for healing wounds, comprising mixing purified platelet-derived growth factor and purified transforming growth factor alpha in a weight to weight ratio of between 1:4 and 25:1. I i i vii Ao"- INTERNATIONAL SEARCH REPORT International Anplication No. PCT/TIRR RI04557 1. CLASSIFICATION OF SUBJECT MATTER ft seyorat l itssilicltiofl symbols iapply, Inicat~3e all) 6 Accororni to tIntenalonatl Patent Classification IPC) or to 00oth National Classiticatron ano IPC IPC(4: A61K 37/36 U.S. CL.: 514/2, 3, 21; 530/399 It FIELDS SEARCHED Minimamn Documentiin Searched Classification System Classification Symbols U.S. 514/2, 3, 21; 520'/399 Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fietds Searcheda Chemical Abstracts, "Growth Factors", "Growth Hormones" 111, DOCUMENTS CONSIDERED TO BE RELEVANT2 Category Citation ot Document, 11 witti indicartionl, where appropriate, of the relevant nassagesr. 7 7Relevat i Claim No. '3 Y J. TRAUMk, 24, No.

9, 549-552 (1984), 1-8 Grotendorst YJ. Clin. Invest. 76, 2323-2329 (1985) 1-8 Grotendorst k US, A, 4,742,003 (DERYNCK) 03 May 1988 1-8 X US, H, Serial Number 468,590 1-8 (SPORN)

10 June 1983 Special categories of cited documents: 10 tater document pubished alter the internationat riting date ocuentdefiingthegenist tat ottheart hic isPal or priority date and not in conflict with the application but ""dcnside gto eeatp rtiuarleae h r hc sntcited to understand the principle or theory undertying the consderd t be f prtiularrelvane rvention E" earlier document but published on or alter the international X' document of Particular retevance: the claimed invention fiing date cannot be considered novel or cannot be considered to L"document which may throw doubts on priority claimls) or involve an inventive step which is cited to establish the publor-ation date of another document oft pariular relevance; the claimed invention citation or other special reason (as specified) cannot be Considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one Or more other such docu- other means ments, such combination being obvious to a person skilled P71 document published Prior to the international riling date but in the art. later than the priority date claimed document member ot the same patent family IV. CERTIFICATION Dale at the Actual Completion ot the Inernatianal 'earch Date at Mailing at this International Search Report 31 March 1989 2 7APR 1989 International Searching Authority SignatureIatAuthor zd~flri) 1 ISA/US f; ~_A Formt PCT1SA/2 10 (Seond shom) Iov.t 1 "7)