Drug – bio-affecting and body treating compositions – Extract – body fluid – or cellular material of undetermined... – Derived from mollusk
Reexamination Certificate
1998-10-16
2001-06-26
Lankford, Jr., Leon B. (Department: 1651)
Drug, bio-affecting and body treating compositions
Extract, body fluid, or cellular material of undetermined...
Derived from mollusk
C424S115000, C424S520000
Reexamination Certificate
active
06251438
ABSTRACT:
The present invention relates to a method of preparing biologically active substances from nacre and to the uses of the purified products which can be obtained in this way.
These products are, in particular, useful in bone and, in particular, maxillofacial surgery, or for the treatment of degenerative pathologies of the cartilage, as well as in dermatology.
Nacre has been proposed as a material for bone substitution and/or regeneration, in particular in orthopedic or maxillofacial surgery, and also for the manufacture of dental implants.
It has furthermore been shown (Lopez et al., Tissue and Cell, 24, 667-679, 1992) that nacre exerted, at a distance, osteoinducing and osteogenic effects on bone cells in vitro.
It was therefore desirable to be able to isolate the biologically active substances present in nacre.
It is known that nacre, or aragonite chonchylifer, is a biogenic mineralized formation; it consists of an organic matrix of fibrous and non-fibrous substances representing about 1.7% of the total mass (Taylor et al., Bulletin of the British Museum (Natural History) Zoology, Suppl. 3. 125 pp. +29 plates, 1969) and calcium carbonate crystallized in the form of aragonite. The structure of nacre therefore exhibits similarities with that of bone, which comprises an organic matrix and a mineral phase, consisting of calcium phosphate in the form of hydroxyapatite. In contrast, about 50% of the organic matrix of nacre is soluble.
Authors have described the extraction of proteins which are active on the formation of bone, BMPs (or Bone Morphogenetic Proteins) from the insoluble fraction of the matrix of bone (Urist et al., Proc. Natl. Acad. Sci. USA 76: 1828-1832, 1979). This method involves an acidic demineralization, which entails the risk of denaturing the products of interest.
Although nacre has for a long time been suggested as a biomaterial, proteins similar to BMPs, for example, have never been demonstrated.
Applicant has now found a method making it possible to separate substances having a set of particularly beneficial biological properties, in particular because it does not include a denaturing step.
This is why the present invention relates to a method for preparing biologically active substances from nacre, characterized in that:
a) nacre is reduced to a powder whose particle size is less than about 200 &mgr;m (that is to say in general from 50 to 150 &mgr;m),
b) this is brought into intimate contact with an aqueous solvent, optionally supplemented by salts,
c) at the end of the contact period, the insoluble fraction is separated so as to recover the aqueous fraction,
d) the solvent of the aqueous fraction is concentrated so as to recover the soluble or water-transportable fraction of the nacre, which consists of biologically active substances essentially free of mineral constituents.
This method is one which is simple to employ, and is preferably carried out at a temperature of between about 4° C. and 30° C.; good results are obtained at room temperature.
The nacre used may be obtained from shells of molluscs, in particular oysters such as
Pinctada maxima.
Raw nacre is used, which is free of other calcite-rich shell elements; the starting material is preferably white nacre, otherwise a step of removing the pigments must be provided.
This is a readily available raw material, the use of which does not impact negatively on natural populations; indeed, most of these oysters or other nacrous molluscs are farmed.
Furthermore, an additional advantage is given by the fact that the raw material can be obtained from oyster shells which have produced pearls; indeed, a pearl oyster is removed from the productive pool after having produced at most 3 pearls in succession, even though it has a thick layer of nacre of excellent quality (grade A). The present invention therefore provides an extra opportunity for using nacre downstream of pearl farming.
The nacre can be used without prior decontamination. The purified biological fractions extracted from nacre originating from bivalves filtering on a large scale (800 l water/day) are free of pathogenic agents which may be active in man, which constitutes an essential part of the conditions for an implantable biomaterial, for which harmlessness in the short and long term is currently a major preoccupation. This preoccupation is justified by the concern to avoid the serious pathologies which may occur through the use of lyophilized bone, for example in bone surgery.
In one of the embodiments of the method, the nacre is reduced to a particle size of between 50 and 100 &mgr;m.
In another embodiment, the nacre is reduced to a powder with particle size between 15 and 50 &mgr;m, which makes it possible to improve efficiency by approaching the size of a crystalline unit.
In all cases, crushing will firstly be carried out, followed by grinding, since a one-off size reduction procedure is not suited to the consistency of the starting material.
The solvent in step b) may be selected from pure, double-distilled or apyrogenic water, or water supplemented by salts selected, in particular, from NaCl or guanidine hydrochloride.
The invention has in fact made it possible to demonstrate that the substances responsible for the biological activity of nacre are hydrophilic or transportable in water, in particular water-soluble, and can be extracted as early as the first fractionation step, on condition that finely reduced nacre is treated, which limits its production cost.
The product which can be obtained directly by the method defined above comprises a mixture of proteins which have biological activities stimulating cell proliferation, in particular of osteoblasts, chondrocytes, keratinocytes and fibroblasts. These proteins may or may not be fibrous. The invention relates not only to the product obtained in this way, but also to the various substances purified by fractionation from the complete soluble product.
It comprises in particular proteins which may be likened to ancestral proteins associated with the BMP/TGF &bgr; (transforming growth factor) family.
More particularly, the invention relates to one or more non-fibrous osteoinducing proteins which can be obtained from nacre and have the following characteristics:
they are soluble in water or are transportable in water,
they are non-cytotoxic,
they increase the alkaline phosphatase activity and
the type I collagen synthesis by the osteoblasts.
It also relates to nuclootide sequences coding for a protein of this type. It should therefore be understood that proteins of this type, obtained from another source or by in vivo synthesis, by isolated tissues or organs or by cells in culture are also included in the invention, as are products obtained by chemical synthesis or genetic engineering.
The biologically active product may be concentrated in step d) by dialysis and/or lyophilization.
In one variant of the method, after step d), precipitation with ethanol is carried out and the residue which will constitute a biologically active sub-fraction is recovered, the products which remain in solution constituting another biologically active sub-fraction.
Step b) may, in particular, be carried out by suspending the nacre powder in the aqueous solvent under mechanical agitation; one possibility is to carry out the procedure at 4° C. for 24 hours, but the times and temperatures may be adapted by the person skilled in the art, for example according to the starting particle size.
In another embodiment, step b) is carried out by passing the solvent under pressure through the nacre powder which is rendered imobile. This immobilization is, for example, brought about using a column of the HPLC type, optionally as a mixture with filler substances allowing better solvent diffusion and avoiding compacting of the nacre powder.
The various products obtained according to the invention are useful as an implant or medicament, in particular intended to increase the regeneration of tissues, in particular osseous, cartilaginous or cutaneous tissues.
There are many fields of application, including:
Various tissues:
skel
Berland Sophie
Giraud Michel
Gutierrez Gilles
Lopez Evelyne
Milet Christian
Centre National de la Recherche Scientifique "CNRS"
Foley & Lardner
Lankford , Jr. Leon B.
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