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Active hedgehog protein conjugateRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai, Cyclopeptides, 25 Or More Peptide Repeating Units In Known Peptide Chain StructureActive hedgehog protein conjugate description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070191274, Active hedgehog protein conjugate. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The invention concerns a hedgehog protein conjugate with increased activity, a process for its production and its therapeutic use. BACKGROUND OF THE INVENTION [0002] 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) 296, A. Vortkamp et al., Science 273 (1996) 613, C. J. Lai et al., Development 121 (1995) 2349). During its biosynthesis a 20 kD N-terminal domain and a 25 kD C-terminal domain are obtained after cleavage of the signal sequence and autocatalytic cleavage. In the naturally occurring protein the N-terminal domain is modified with cholesterol at its C-terminus after cleavage of the C-terminal domain (J. A. Porter et al., Science 274 (1996) 255-259). 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). [0003] 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 of hh 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). [0004] 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 1/2 cells. The increased activity has been postulated to be due to synergism of hh with molecules such as bone morphogenetic proteins (BMPs) which are only present in the supernatant of eukaryotic cells and in combination with hh cause the stronger induction of AP. [0005] Kinto et al., FEBS Letters, 404 (1997) 319-323 describe that fibroblasts which secrete hh induce ectopic bone formation in an i.m. implantation on collagen. However, such an activity is not known for an isolated hh protein. SUMMARY OF THE INVENTION [0006] This invention provides a hedgehog conjugate, comprising from 1 to 4 moieties covalently bound to a hedgehog protein; wherein each moiety is: a) a polypeptide composed of 10 to 30 hydrophobic amino acids and/or of amino acids which form transmembrane helices and are positively charged, b) an aliphatic, saturated or unsaturated hydrocarbon residues with a chain length of 8 to 24 C atoms and with a hydrophobic action, or c) a hydrophobic thio group. [0007] The object of the invention is to provide hh protein (polypeptide) conjugates which have a considerably improved activity compared to the known forms. The conjugates of this invention have the same utility as the corresponding unconjugated hedgehog protein. DESCRIPTION OF THE FIGURES [0008] FIG. 1: shows the activity of recombinant human shh after derivatization with palmitoyl-CoA. [0009] FIG. 2: shows the activity of recombinant human shh after derivitization with thiocholesterol. DETAILED DESCRIPTION OF THE INVENTION [0010] This invention provides a hedgehog protein produced recombinantly that has artificially been made lipophilic. Such a lipophilization is preferably achieved by chemical modification. Such a hedgehog conjugate preferably contains an additional polypeptide that is covalently bound (preferably at the C-terminus or/and N-terminus) and is composed of 10-30 preferably hydrophobic amino acids and/or those amino acids which form transmembrane helices. The additional polypeptide particularly preferably contains 2-12 lysines and/or arginines but no polyhistidine part that would be suitable for purifying the conjugate on a Ni chelate column. It is also preferable to covalently bind (preferably at the C-terminus and/or N-terminus) 1-4 aliphatic, saturated or unsaturated hydrocarbon residues with a chain length of 8-24 C atoms or steroids with a lipophilic (hydrophobic) action. Furthermore it is preferred to covalently couple hydrophobic thio compounds, such as in particular thiocholesterol and thioalkanes, thioalkenes, to hh proteins via a disulfide bridge formed oxidatively (preferably at the C-terminus and/or N-terminus and in this case on the N-terminal cysteine). [0011] The protein is hydrophobized by such lipophilizing residues which improves its interaction with lipid membranes of eukaryotic cells, in particular of mammalian cells. [0012] Consequently a lipophilized protein according to the invention is understood as a hydrophobized protein which has an increased surface hydrophobicity compared to an unmodified protein which increases its affinity for apolar molecules or arnphiphiles. The increase in the degree of lipophilicity of the protein can be measured by the degree of integration in a lipid layer as described for example by Haque, Z. et al., J. Agric. Food Chem. 30 (1982), 481. Methods for the hydrophobic (lipophilizing) modification of proteins are for example described by Haque, Z. et al., J. Agric. Food Chem. 31 (1983) 1225-1230; Webb, R. J. et al., Biochemistry 37 (1998) 673-679; Hancock, J. F., Cell 63 (1990) 133-139; A Practical guide to membrane protein purification, Ed. G. v. Jagow, Hermann Schagger (1994), (chapter 16, pages 535-554). [0013] It has surprisingly turned out that such lipophilized hedgehog proteins (also denoted hedgehog conjugates (hh conjugates) in the following) exhibit a drastically increased activity of preferably at least 10-fold, particularly preferably of 10.sup.3-10.sup.5-fold compared to non-modified hedgehog proteins (e.g. after cytoplasmic expression in E. coli) especially in a pharmaceutical formulation and in vitro. In addition it is particularly surprising that such hedgehog conjugates according to the invention can be used particularly advantageously for a local therapy preferably on bones, on cartilage, on nerve cells (in nerve lesions or neurodegenerative diseases) or in muscle tissue. [0014] 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 for this 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. [0015] The conjugates according to the invention serve as new active substances for the production of pharmaceutical forms of administration. Overall the coupling results in an improved pharmacokinetic profile of the hedgehog protein. The hydrophobic hydrocarbon residue results in a localization of the hedgehog protein on the membrane of the target cells which, in addition to facilitating integration into the cell interior, above all results in a substantially more prolonged presence on the cell surface which is optimal for the pharmacological effect. [0016] The conjugates according to the invention do not necessarily need to be additionally coupled to a carrier for slow release. The hedgehog conjugates according to the invention are also highly active at the site of action in the body without a delayed release occurring from a carrier over a long period (several days). Nevertheless it is expedient to use a pharmaceutical composition for the local application of the hedgehog conjugates according to the invention which contains the conjugate according to the invention together with a carrier matrix. The carrier matrix essentially serves to facilitate the local application in particular by providing such a pharmaceutical composition with a suitable minimum viscosity for the local application. The pharmaceutical composition is preferably buffered in the pH range between pH 4 and 9 and contains one or several non-ionic detergents such as polyoxysorbate or polyoxyethylene type detergents (e.g. Tween.RTM.20, Tween.RTM.80, Triton.RTM.X-100), octylglucoside or ionic detergents such as sodium deoxycholate, sodium cholate, sodium taurodeoxycholate. [0017] In a preferred embodiment a hh protein is expressed which contains an additional 10-30 -mainly hydrophobic amino acids at the N-terminus and/or C-terminus since these are also incorporated into the membrane of cells [Webb et al., Biochemistry 37 (1998) 673-679, Skolnick et al., Biol. Membranes (1996) 536-554; ed.: Merz and Roux]. Hydrophobic amino acids within the sense of the invention are understood as amino acids which have a negative free energy in the transition from the aqueous phase into a hydrophobic/organic phase. Furthermore N-terminal and/or C-terminal sequences which are known to form transmembrane helices such as e.g. the M28 peptide or which interact as a helix with the surface of membranes such as e.g. maginin 2 (Skolnick et al., 1996) are also suitable for increasing the activity of hh proteins. [0018] In a further preferred embodiment the N-terminus and/or C-terminus of the hedgehog protein is modified by a polypeptide residue which contains 2-12 lysines and/or arginines. In this case it is possible to omit the modification with the hydrocarbon residue. [0019] An aliphatic, saturated or unsaturated hydrocarbon residue with a hydrophobic action and a chain length of 8-24, preferably 10-24, most preferably. 12-18 C atoms, is preferably a saturated or monounsaturated to polyunsaturated fatty acid or alkyl alcohol residue optionally interrupted by an oxygen or sulphur atom or a carbonyl group. Particularly preferred saturated fatty acids are: capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid and behenic acid. Preferred monounsaturated fatty acids are myristic acid, palmitoleic acid and oleic acid. Particularly preferred polyunsaturated fatty acids are linoleic acid, linoleic acid and arachidonic acid. Such fatty acid residues are preferably coupled via an ester, acid amide, disulfide or thioester bond to reactive groups of the protein. [0020] The number of hydrophobic hydrocarbon chains per protein molecule can be suitably controlled by the reaction conditions (e.g. dilution) or by the selection of the amino acid to be modified. For example shh contains three cysteines of which the N-terminal cysteine is particularly reactive. In this case the reaction procedure can lead to the N-terminal cysteine being modified with either one or more hydrophobic hydrocarbon chains. It is also possible to statistically modify two or almost all three cysteines. Although when modifying other amino acids it is preferable to modify defined amino acids, it is also possible to use derivatized hedgehog proteins for the pharmaceutical composition in which there is a statistical distribution of hydrocarbon chain modifications of ca. 1 to ca. 4 chains per molecule. Although a higher number of hydrocarbon chains per molecule is suitable, the solubility in a pharmaceutical composition is decreased by this and it can lead to disturbances in the active three dimensional protein structure. When coupling with long chain alkyl groups (C.sub.14-C.sub.24, preferably C.sub.16-C.sub.24) it is preferable to only link 1-2 carbon chains and when short-chain carbon chains are coupled, it is, however, preferable to couple 2-3 alkyl groups. In a preferred embodiment the derivatization can also comprise the coupling of two hydrophobic hydrocarbon chains to one amino acid. This can for example be achieved by coupling a fatty acid diglyceride to the amino acid. Continue reading about Active hedgehog protein conjugate... Full patent description for Active hedgehog protein conjugate Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Active hedgehog protein conjugate patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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