Drug – bio-affecting and body treating compositions – Designated organic nonactive ingredient containing other... – Solid synthetic organic polymer
Reexamination Certificate
1999-08-02
2001-03-27
Page, Thurman K. (Department: 1615)
Drug, bio-affecting and body treating compositions
Designated organic nonactive ingredient containing other...
Solid synthetic organic polymer
C424S422000, C424S461000, C424S484000, C424S486000
Reexamination Certificate
active
06207718
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention concerns a composition, preferably a pharmaceutical composition, of hedgehog proteins and its use.
Hedgehog (hh) proteins are understood as a family of secreted signal proteins which are responsible for the formation of numerous structures in embryogenesis (J. C. Smith, Cell 76 (1994) 193-196, N. Perrimon, Cell 80 (1995) 517-520, C. Chiang et al., Nature 83 (1996) 407, M. J. Bitgood et al., Curr. Biol. 6 (1996) 298-304, A. Vortkamp et al., Science 273 (1996) 613, C. J. Lai et al., Development 121 (1995) 2349). During its biosynthesis a 20 kDa N-terminal domain and a 25 kDa C-terminal domain are obtained after cleavage of the signal sequence and autocatalytic cleavage. In its natural form the N-terminal domain is modified with cholesterol or palmitoyl (J. A. Porter et al., Science 274 (1996) 255-259, Pepinski et al., J. Biol.Chem. 273 (1998) 14037-14045). In higher life-forms the hh family is composed of at least three members namely sonic, indian and desert hh (shh, ihh, dhh; M. Fietz et al., Development (Suppl.) (1994) 43-51). Differences in the activity of hedgehog proteins that were produced recombinantly were observed after production in prokaryotes and eukaryotes (M. Hynes et al., Neuron 15 (1995) 35-44 and T. Nakamura et al., Biochem. Biophys. Res. Comm. 237 (1997) 465-469).
Hynes et al. compare the activity of hh in the supernatant of transformed human embryonic kidney 293 cells (eukaryotic hh) with hh produced from
E. coli
and find a four-fold higher activity of hh from the supernatants of the kidney cell line. The reason for this increased activity has been discussed to be a potential additional accessory factor which is only expressed in eukaryotic cells, a post-translational modification, a different N-terminus since the hh isolated from E. coli contains 50% of a hh form which carries two additional N-terminal amino acids (Gly-Ser) or is shortened by 5-6 amino acids, or a higher state of aggregation (e.g. by binding to nickel agarose beads).
Nakamura et al. compare the activity of shh in the supernatant of transformed chicken embryo fibroblasts with an shh fusion protein isolated from
E. coli
which still has an N-terminal polyhistidine part. The shh in the supernatant of the fibroblasts has a seven-fold higher activity than the purified
E. coli
protein with regard to stimulation of alkaline phosphatase (AP) in C3H10T ½ cells. The increased activity has been postulated to be due to molecules such as for example bone morphogenetic proteins (BMPs) which are only present in the supernatant of eukaryotic cells and cause the stronger induction of AP.
Pepinski et al. (J. Biol. Chem. 273 (1998) 14037-14045) have identified a shh form which is modified with palmitic acid. This shh mutant is 30-fold more potent than the unmodified form in the C3H10T ½ assay.
Kinto et al., FEBS Letters, 404 (1997) 319-323 described that fibroblasts which secrete hh induce ectopic bone formation in an i.m. implantation on collagen. Thus hedgehog proteins have an osteoinductive activity. Hedgehog proteins can also stimulate the formation of cartilage cells (Stott et al., 1997).
It is known from Yang et al., Development 124 (1997) 4393-4404 that high local hedgehog concentrations must prevail over a period of at least 16 h at the site of action in the body for a pharmaceutically effective in vivo activity. The carrier system described by Yang et al. i.e. the hedgehog-loaded chromatography medium Affigel CM, the Ni agarose described by Marti et al., in Nature 375 (1995) 322-325 or the Affigel blue used by Lopez-Martinez et al., in Curr.Biol. 5 (1995) 791-796 or the heparin agarose particles that they used are less suitable for a pharmaceutical application since they are immunogenic and can cause inflammatory reactions.
SUMMARY OF THE INVENTION
The object of the present invention is to provide a stable, preferably aqueous (preferably pharmaceutical) composition of a hedgehog protein.
The object is achieved by a, preferably pharmaceutical, composition of a hedgehog protein which contains a hedgehog protein in a pharmaceutically effective amount and, an additive being present in an effective amount to stabilize said hedgehog protein. The additive is preferably selected from the group consisting of ionic salts, cyclodextrin, non-ionic detergent, anionic saccharide and mixtures thereof. Preferred ionic salts are those containing an ion selected from the group consisting of zinc, sulfur, magesium, calcium, arginine, argininium and mixtures thereof.
In accordance with the present invention, compositions are provided which stabilize hedgehog proteins and allow the activity of the hedgehog protein to be maintained over a long period for example at room temperature. Thus, the compositions of the present invention allow for longer storage of pharmaceutically effective hedgehog protein compositions prior to administration to a patient. The compositions described herein are also particularly suitable for producing carrier matrices containing a pharmaceutically effective amount of hedgehog protein. The carrier matrices produced from the compositions of the present invention can be administered to a human patient to provide delayed release of pharmaceutically effective hedgehog protein in the human body.
DETAILED DESCRIPTION OF THE INVENTION
It has surprisingly turned out that one or several additives selected from the group ionic salts, cyclodextrin, non-ionic detergents and anionic saccharides such as chondroitin sulfate or heparin are able to stabilize hedgehog proteins (as a pharmaceutical composition or in another form), preferably in an aqueous solution. As a result the activity of the hedgehog protein can be maintained over a long period for example at room temperature and the compositions of hedgehog proteins can be stored in suitable containers, such as, vials, for a long period at room temperature prior to administration to a human patient in unit dosage form. The additives according to the invention are also suitable for stabilizing hedgehog lyophilisates (preferably as a bulk or pharmaceutical composition) and also stabilize the hedgehog proteins during the production of hedgehog preparations such as implants, microparticles, gels etc. and at increased temperatures (e.g. 37° C.).
In accordance with the present invention a composition is provided comprising an aqueous solution containing a hedgehog protein and an additive being present in an effective amount to stabilize said hedgehog protein, wherein said additive is selected from the group consisting of ionic salts, cyclodextrin, non-ionic detergent, anionic saccharide and mixtures thereof, said ionic salt containing an ion selected from the group consisting of zinc, sulfur, magesium, calcium, arginine, argininium and mixtures thereof.
In the aqueous solution of the hedgehog protein according to the invention the additive is in a molar excess relative to the hedgehog proteins. This excess is preferably 1-1000-fold and particularly preferably 1-100-fold. The aqueous solution according to the invention is especially suitable for producing combinations of hedgehog proteins with carrier substances.
Hence a further subject matter of the invention is an aqueous solution of a hedgehog protein which is characterized in that it contains a molar excess of the additive according to the invention relative to the hedgehog protein. The aqueous solution is preferably buffered and/or lyophilized. The composition of the present invention is adapted for formulating unit dosage forms for administration to a patient. Parenteral administration of the composition to a patient is preferred. The amount of the aqueous solution administered to a human patient in unit dosage form is from about 0.05 ml to about 2.0 ml, preferably, from about 0.5 ml to about 2.0 ml.
The amount of additives according to the invention is per se uncritical and can be varied over a wide range. Suitable amounts depend on the pharmaceutical compatibility of the additive and the extent of the stabilizing action at a pharmaceutically accept
Foley Hoag & Eliot LLP
Ontogeny, Inc.
Page Thurman K.
Vincent Matthew P.
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