WO1993010810A1

WO1993010810A1 - Bone regeneration

Info

Publication number: WO1993010810A1
Application number: PCT/US1992/010214
Authority: WO
Inventors: Samuel E. Lynch; Harry N. Antoniades
Original assignee: Institute Of Molecular Biology, Inc.; President And Fellows Of Harvard College
Priority date: 1991-11-27
Filing date: 1992-11-24
Publication date: 1993-06-10
Also published as: CA2123803A1; JPH07504160A

Abstract

Stimulation and enhancement of bone and periodontal regeneration using an insulin-like growth factor (IGF) and basic or acidic fibroblast growth factor (FGF).

Description

BONE REGENERATION

Background of the Invention The present invention relates to bone and periodontal regeneration.

Many physical conditions and diseases exist which cause bone loss in mammals, e.g., traumatic injuries and periodontal disease, and thus it is often desired in the medical and dental fields to provide a composition which will stimulate and enhance bone regeneration in a mammal, e.g. a human patient.

One composition which is known for this use is a combination of platelet derived growth factor (PDGF) and insulin-like growth factor I (IGF-l), described in U.S. Patent No. 4,861,757. Growth factors are polypeptide hormones which stimulate a defined population of target cells. As multifunctional hormone-like molecules, they may stimulate or inhibit cell proliferation as well as affect cell function, depending on the state of differentiation of the target cell and the combination of other signal peptides present. Examples of growth factors include PDGF, IGF's, transforming growth factor beta (TGF-/3) , transforming growth factor alpha (TGF-α) , epidermal growth factor (EGF) and acidic or basic fibroblast growth factor (aFGF or bFGF) .

IGF-l alone has also been studied for its effects on bone growth. In vivo, the continuous local application of IGF-I inside a titanium chamber implanted into the adult rabbit tibia did not significantly alter bone formation (Aspenberg, et al, Acta Orthop. Scand. 1989, 60: 607-10). Continuous systemic administration of somatomedin C (IGF-I) also failed to promote the repair of bone wounds resulting from a femoral osteotomy in rats ( irkeby and Ekeland, Acta Orthop. Scand.) 1990; 61: 335- 38) . A preliminary study in a small number of animals suggested that continuous infusion of IGF-I into the arterial supply of one hind limb for 14 days resulted in increased cortical bone formation in that limb in older but not young rats. The action appeared to be the result of an increased number of osteoblasts and decreased number of osteoclasts (Spencer, et al, Bone 1991; 12:21- 26) . The local application of IGF-l to the growth plate of young hypophysectomized rats resulted in a small but significant effect on unilateral longitudinal bone growth (Isgaard, et al, A.M. J. Phvsiol. 1986; 250-E367-372) . A single application of IGF-I in combination with platelet- derived growth factor-BB (PDGF-BB) has been reported to simulate striking bone formation around the teeth of dogs with natural periodontits (Lynch et al. J. Clin. Periodontol 1989; 16:545-598; J. Periodontol, 62: 458- 67) . IGF-I and PDGF have also been isolated from bone matrix (Hauschka et al J. Biol. Chem. 1986; 261:12665-74 and Canalis et al. Cal. Tiss. Internatl. 1988; 43:346- 51).

In vitro, there are apparently conflicting data on the effect of IGF-I on bone cells. Pfeilschifter et al (Endocrinology 1990; 127: 69-75) reported only a modest effect of IGF-I alone on bone matrix apposition in cultured fetal rat calvarial. Significant effects on bone matrix formation were seen when IGF-I was combined ith PDGF-BB, TGF-B, or both PDGF-BB and TGF-B. In contrast, McCarthy et al (Endocrinology 1989; 124: 301-7) reported that IGF-I and IGF-II stimulate significant DNA and collogen synthesis in bone cultures. Hock et al (Endocrinology 1988; 122:254-60) found that IGF-I stimulates primarily pre-osteoblast replication in vitro and that collagen and bone matrix synthesis is stimulated independently of cell replication. Canalis et al (J. Cell. Phvsiol. 1989; 140:530-537) reported that PDGF-BB opposed the stimulatory effect of IGF-I on collagen synthesis, IGF-I prevented the PDGF effect on collagen degradation and that PDGF-BB and IGF-I had additive effects on calvarial DNA synthisis. Piche and Graves (Bone 1989; 10: 131-8) also reported that in vitro IGF-I did not stimulate significant ³H-thymidine incorporation into bone derived cells nor did it enhance the activity of PDGF in this regard. IGF-I in combination with PDGF, EGF and TGF-B resulted in uptake by the bone cells nearly equal to that achieved by 10% fetal bovine serum. Receptors for IGF-I and II have been demonstrated in osteoblast-enriched cultures from fetal rat bone (J. Cell Biol.l (abstract) 1988; 107:62a). The role of IGF-I in bone metabolism has been most recently reviewed by Canalis et al (J. Endocrinol. Invest 1989; 12: 577-84). Another growth factor which has been studied for its effect on bone growth is fiberblast growth factor (FGF) . In vivo, both aFGF and bFGF and their respective mRNA's have been detected at the site of bone fractures (Joyce et al 1991; in Clinical and Experimental Approaches to Dermal and Epidermal Repair: Normal and Chronic wounds, A. Barbul et al ed. pp 391-416) . Both aFGF and bFGF have been isolated from bone matrix

(Hauschka et al 1986) . bFGF and IGF-I have been used in combination to promote the healing of skin wounds (Lynch et al, J. Clin. Invest. 84:640-646 1989).

In vitro, bFGF did not significantly alter ³Hy- thymidine incorporation in bone fracture calluses (Joyce et al 1991) . bFGF has been reported to enhance mitogenesis in fetal calvarial bone cultures but did not simulate differentiated function of osteoblasts directly (Canalis et al J. Clin. Invest. 1988; 81:1572). aFGF has the same reported biological effects on bone as bFGF but generally requires higher concentrations (Canalis J____ Clin. Invest. 1987; 79:52-58). Both aFGF and bFGF tend to decrease matrix synthesis in the fetal rat calvarial model (Canalis et al 1989) . Cultured bovine bone cells synthesize both bFGF and aFGF and store it in their extracellular matrix (Globus et al Endocrinology 1989; 124:1539) . bFGF has also been reported to enhance the capacity of bone marrow cells to form bone-like nodules in vitro (Noff et al F.E.B.S. Letters 1989; 250:619-21). Both aFGF and bFGF increased DNA synthesis in cells cultured from parietal bones while bFGF was a more potent stimulator of alpha 1 Type 1 procollagen mRNA (McCarthy et al Endocrinology 1989; 125:2118-26). In vitro, acidic and basic FGF have been shown to be mitogenic and chemotactic for cells derived from the periodontal ligament and bind to pretreated dentin slabs. (Terranova et al J. Periodontol.. 1989;60: 293-301; Terranova et al J. Periodontol. 1987; 58:247-257; Terranova In The Biological Mechanisms of Tooth

Extraction and Root Resorption, Davidovitch Z. ed. 1989; pp. 23-34) .

Summary of the Invention The present invention provides novel methods for stimulating and enhancing bone and periodontal regeneration. The methods of the invention employ IGF's, preferably IGF-I, and acidic or basic (a or b respectively) FGF. The invention aids in regeneration, at least in part, by promoting the growth of bone, cementum, and ligament by stimulating protein and collagen synthesis. Bone regeneration using the invention is more effective than that achieved in the absence of treatment (i.e. without applying exogenous agents) or by treatment with similar levels of purified IGF-I or purified FGF's alone.

In the method of regenerating bone of a mammal e.g., a human patient, according to the invention there is administered to the patient, preferably by application to the area of injured or depleted bone, an effective amount of a composition that includes purified acidic or basic FGF and purified IGF-I. Alternatively, the two factors can be applied sequentially, close enough in time to effect synergistic bone regeneration.

In a preferred embodiment of the invention, the composition is prepared by combining, in a pharmaceutically acceptable carrier substance, e.g., commercially available inert gels, polymers or liquids (e.g., saline supplemented with albumin or methyl cellulose) , purified acidic or basic FGF and an IGF, e.g. IGF-I (which are commercially available) . Preferably purified acidic or basic FGF and purified IGF-I are combined in a weight ratio of between 100:1 and 1:250. More preferably the purified FGF and IGF-I are combined in a weight ratio of between 50:1 and 1:100. Most preferably the FGF and IGF-I are combined in a weight ration of between 10:1 and 1:50.

Detailed Description The term ^••purified" as used herein refers to acidic or basic FGF or IGF which, prior to mixing with the other growth factor, is 90% or greater, by weight, FGF or IGF, i.e., 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 subunit of IGF or acidic or basic FGF. Most preferably, the purified a or b FGF or IGF used in the compositions of the invention is pure as judged by amino- ter inal amino acid sequence analysis.

The purified a or b FGF and IGF may be obtained by purifying them from natural sources e.g. brain or plasma, respectively, by recombinant DNA technology, or by chemical synthesis. Thus, by the terms -^■IGF" and "FGF", we mean naturally derived, recombinant, and synthesized materials of mammalian, preferably primate, origin; most preferably, the primate is a human, but can also be a chimpanzee or other primate. A method of making recombinant a and b FGF and analogues thereof is dislcosed in EP 88311099.1.

IGF's are commercially available from Amgen Corporation' (Thousand Oaks, California) and Kabi (Sweden) . a and b FGF are commercially available from R & D Systems (Minneapolis, MN) and AmGen Corporation.

The terms a or b FGF and IGF include active fragments and analogs thereof which mediate biological activity through their respective receptors. Analogs which are presently unknown may be made and tested for this purpose. Testing of these analogs for efficacy is routine, and may be easily accomplished by conventional methods, e.g. radioreceptor assays. Suitable analogs are disclosed in EP 88311099.1. While IGF-I is preferred, IGF-II or IGF-III may also be used in the invention.

The compositions of the invention are formed by combining an IGF with a or b FGF using known mixing methods or by attaching these proteins to polymers. In a preferred embodiment of the invention, the composition is prepared by combining the two growth factors in a pharmaceutically acceptable carrier substance, e.g., commercially available inert gels, polymers or liquids (e.g., saline polymers supplemented with albumin or methyl cellulose) . Preferably the purified growth factors are combined in a weight ratio of between 100:1 and 1:250 more preferably from 50:1 to 1:100, and most preferably from 10:1 to 1:50 aFGF or bFGF to IGF.

According to the invention, regenerating bone of a mammal, e.g. a human patient, is accomplished by administering to the patient, preferably by local administration to the area of injured or depleted bone, an effective amount of a composition of the invention. Systemic administration can also be used. A preferred dosage of the composition is about 0.1-1000μg, more preferably l-100μg, of biologically active growth factors/cm² of the area of injured or depleted bone.

The above description illustrates preferred embodiments of the invention. Other variations and modifications are within the scope of the invention and the following claims.

What is claimed is:

Claims

1. Use of an insulin-like growth factor and acidic or basic fibroblast growth factor in the manufacture of a medicament for regenerating bone of a mammal.

2. The use of claim 1 wherein each said growth factor is purified.

3. The use of claim 1 wherein the weight ratio of acidic or basic fibroblast growth factor to insulin-like growth factor is from 100:1 to 1:250.

4. The use of claim 3 wherein said weight ratio is from 50:1 to 1:100.

5. The use of claim 3 wherein said weight ratio is from 10:1 to 1:50.

6. The use of claim 1 wherein said IGF is IGF-I.

7. The use of claim 1 wherein said factors are admixed with a pharmaceutically acceptable carrier substance.

8. A bone regenerating composition comprising an insulin-like growth factor admixed with acidic or basic fibroblast growth factor.