CA2123803A1 - Bone regeneration - Google Patents

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

. WO93/10810 PCT/US92~t0214 2 1 2 3 ~

BONE REGENERATION

Backaround of the Invent~
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 ~omposition which will stimula~e and enhance bone regeneration in a ma~mal, 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 po~ypeptide hormones which stimulate a defined population of target cells. As multifunctional hormone-like molecules, they may stimulate or inhibit cell prolif~ration as well as affect cell function, depending on the state of dif~rentia~ion of the target cell and the combination of other signal peptides present. ~xamples of growth-factors include PDGF, IGF~s, transforming growth factor beta 5TGF-~), trans~orming growth factor alpha (TGF;~), epidermal gro~th factor ~EGF) and acidic or basic f~broblast grow*h factor (aFGF or bFGF).
IGF-l alone has also been studied fvr 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, cta Orth~p. Scand.
1989, 60: 607-l0). Continuous systemic administration of somatomedin C (IGF~I) also failed to promote the repair WO93/10810 ~ ? ~ PCT/US92/10214 of bone wounds resulting from a femoral osteotomy in rats (Kirkeby and Ekeland, Acta OrthoP._Scand.) l990; 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. rhe actîon appeared to be the result of an increased number of osteoblasts and decreased number o~ osteoclasts (Spencer, et al, Bone l99l; 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 appaxently conflicting data onthe effect of IGF-I on bvne cells. Pfeilschifter e~ al 2S (Endocrinolooy 1990; 127: 69-75) reported only a modest effect of IGF-I alone on bone matrix apposition in cultured fetal rat calvarial. Signi~icant effects on bone matrix formation were seen when IGF-I was combined with PDGF-BB, TGF-B, or both PD~F-BB and TGF-B. In contrast, McCarthy et al (Endocrinoloqy 19~9; 124: 301-7) reported that IGF-I and IGF-II stimulate significant DNA
and collogen synthesis in bone cultures. Hock et al (Endocrinoloqv 1988; 122:254-60) found that IGF-I
stimulates primarily pre-osteoblast replication in vitro and that collagen and bone matrix synthesis is stimulated WO93/10810 21~ 3 8 0 ~ PCT/US92/10214 independently of cell replication. Canalis et al (J.
Cell. Physiol. 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 fet~l rat bone (J. Cell Biol.) (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 ~ractures (Joyce et al 1991; in Clinical and Experimental Approaches to Dermal and Epidermal Repair: Normal and Chronic wounds, A. Bar~ul et al ed. pp 391 416). Both aFGF and bFGF have been isolated from bone matrix (Hauschka et al 1986). bFGF and I&F-I have been u~ed 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 WO 93/10~10 21 2 ~ 8 0 3 PCT/US92/10214 generally requires higher concentrations (Canalis J.
Clin. Invest. 1987; 7g: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 EndocrinoloaY 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; 250:61~-21).
Both aFGF and bFGF increased DN~ synthesis in cells cultured from parietal bones while bFGF was a more potent stimulator of alpha 1 Type 1 procollagen mRNA (McCarthy et al ~ndocrinolooy 1989; 12S:2118-26).
In vitro, acldic and basic FGF have been shown to be mitogenic and chemotactic for cells derived fr~m the periodontal ligament and bind to pretreated dentin slab~.
(Terranova et al J. Periodontol.. 1989;60: 293-301;
Terranova et al J. Periodontol. 1987; 58:247-257;
Terranova In The 8iological Mechanisms o~ Tooth Extraction and Root Resorption, Davidovitch 2. ed. 1989;
pp. 23-34).

Summary of the Invention ~5 The present in~ention provides novel methods for stimulating and enhancing bone and periodontal regeneration. The methods of the invention employ IGF's, preferably IGF-I, and a~idic 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 .~W093/10810 212 3 ~ O ~ PCT/US92/tO214 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, poly~ers or liquids (e.g., saline supplemented wîth albumin or methyl cellulose), purified a:idic 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 lOO:l and l:250. .
Nore preferably the purified FGF and IGF-I are combined in a weight ratio of between 50:l and l:lOO. Most .
preferably the FGF and IGF-I are combined in a weight ration of between lO:l and l:50. :

~etailed escri~tion .
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 212~803 compositions of the invention is pure as judged by amino-terminal 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 preferr~d, 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 WO93/10810 ~1 2 ~ ~ ~ 3 PCT/US92/10214 and l:250 more preferably from 50:l to l:l00, and most preferably from l0:l to l: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.l-l000~g, more preferably l-l00ag, of biologically active growth ~actors/cm2 of the area of injured or depleted bone.
The above description illustrates preferred embodiments of the in~ention. Other variations and modifications are within the scope of the invention and the following claims.
What is claimed is:

Claims (8)

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.