MXPA99003976A - Conjugate of protein erizo with an increased activity, process for its production and its employment terapeut - Google Patents

Abstract

A hedgehog protein conjugate characterized in that it contains: a) a polypeptide composed of 10 to 30 amino acids that form transmembrane helicals and is positively charged, b) 1 to 4 aliphatic hydrocarbon residues, saturated or unsaturated with a chain length of 10 to 24 atoms of cy with a hydrophobic action, or c) a hydrophobic thiocompound, covalently linked to a hedgehog protein, which has several times greater activity and is suitable as a pharmaceutical agent.

Description

PRODUCTION AND ITS EMPLOYMENT TISRAP13JTICO Description of the invention: The invention relates to a conjugate of a hedgehog protein with increased activity, to a process for its production, and to its therapeutic use. The hedgehog proteins (hh) are known as a family of secreted signal proteins, responsible for the formation of numerous structures in embryogenesis (JC Smith, Cell 76 (1944) 193-196, N. Perrimon, Cell 80 (1995) 517- 520, C. Chiang et al., Nature 83 (1996) 407, MJ Bitgood et al., Curr. Biol. 6 (1966) 296, A. Vortkamp et al., Science 273 (1996), 613, CJ Lai and col., Development 121 (1995) 1 2349). During its biosynthesis a N-terminal domain of 20 kD and a C-terminal domain of 25 kD is obtained, after the excision of the signal sequence and autocatalytic cleavage. In the wild-type 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 the higher forms of life, the family of the hh consists of at least three members, namely the sonic hh, the Indian hh and the hh desert (shh, Ihh, Dhh, M. Fietz et al., Development (Suppl.) (1994) 43-51). Differences in the activity of the hedgehog proteins obtained by recombinant route have been observed, after obtaining them 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. , have compared the activity of the hh of the supernatant of 293 transformed human embryonic kidney cells (hh eukaryotic), with the hh obtained from REF .: 29963 E. coli and isolated from the cytoplasm, and have found an activity four times higher in the hh of the supernatants of the kidney cell line. It has been discussed whether the cause of this increased activity of hh was due to a potential additional accessory factor that is only expressed in eukaryotic cells, to a post-translational modification, to a different N-terminus, since the hh isolated from E coli contains 50% of a form of hh that contains two additional N-terminal amino acids (Gly-Ser) or is shortened by 5-6 amino acids, or to a higher state of aggregation (eg by binding to beads of nickel agarose). Nakamura et al. Compare the shh activity of the supernatant of the transformed chicken embryo fibroblasts with a shh fusion protein isolated from E. Coli which still has a part of N-terminal polyhistidine.

The shh of the supernatant of the fibroblasts has an activity seven times greater than the protein of purified E. coli, in what refers to the stimulation of the alkaline phosphatase (AP) in C3H10T cells. It has been discussed whether the greater activity was due to the synergism of the hh with molecules such as the orfogenetic proteins of the bone.

(BMPs), which are only present in the supernatant of eukaryotic cells and which in combination with the hh cause a greater induction of the AP. Kinto et al., FEBS Letters, 404 (1997) 319-323 discloses that fibroblasts secreting hh induce ectopic bone formation in an i.m. about collagen. However, this activity is not known for the isolated hh protein. The object of the invention is to produce hh proteins (polypeptides) having a considerably increased activity, compared to the known forms of hh. This goal is achieved by a hedgehog protein that has been obtained recombinantly and that has been artificially converted into lipofluol. This lipophilization is preferably achieved by a chemical modification. This hedgehog protein conjugate preferably contains an additional polypeptide that is covalently linked (preferably the C or / and N-terminus) and is composed of 10-30 amino acids, preferably hydrophobic and / or amino acids that form transmembrane helicals. The additional polypeptide contains particularly preferably 2-12 lysines and / or arginines, but no polyhistidine fragment that would be suitable for the purification of the conjugate in a Ni chelate column. It is also preferable to covalently link (preferably to the C-terminus and / or to the N-terminus), 1-4 saturated or unsaturated hydrocarbon radicals, with a chain length of 8-24 atoms or steroids with lipophilic (hydrophobic) activity. In addition, 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 by oxidation (preferably at the C-terminus and / or at the N-terminus, and at this end). case in the N-terminus of cysteine). The protein is hydrophobicized by these lipophilizing radicals which improves its interaction with the lipid membranes of eukaryotic cells, in particular mammalian cells. Accordingly, a lipophilized protein according to the invention is understood as a hydrophobized protein having an increased surface hydrophobicity, compared to an unmodified protein, which increases its affinity for apolar or amphiphilic molecules. The increase in the degree of lipophilicity of the protein can be measured by the degree of integration of a lipid layer as described for example by Haque, Z et al., J. Agrie. Food Chem. 30 (1982), 481. Methods for the hydrophobic modification (lipophilization) of protein are described for example by Haque, Z et al., J. Agrie. Food Chem. 31 (1983), 1225-1230; ebb, R.J. et al., Biochemistry 37 (1998) 673-679; Hancock, J.F., Cell 63 (1990) 133-139; A Practical guide to membrane protein purification ("A practical guide for the purification of membrane proteins"), Ed. G.v. Jagow, Hermann Schágger (1994), (chapter 16, pages 535-554). It has surprisingly been found that such freeze-dried hedgehog proteins (also called hedgehog conjugates (conjugated hh) hereafter] have a drastically increased activity of at least 10 times, in particular preferably 103-105 compared with the hedgehog proteins. unmodified (eg after cytoplasmic expression in E. coli) especially in pharmaceutical formulations and in vitro. Furthermore, it is particularly surprising that the hedgehog conjugates according to the invention can be used with particular advantage for a local therapy preferably in bones, cartilages, nerve cells, (in nerve lesions or neurodegenerative diseases) or in muscle tissue. It is known, by Yang et al., Development 124 (1997) 4393-4404 that the high local concentrations of hedgehog protein should be maintained for at least a period of 16 hours at the site of action in the body for a pharmaceutically in vivo activity. effective The support system described for this by Yang et al., Ie, the affigel CM medium of chromatography loaded with protein hedgehog, the Ni agarose described by Marti et al., In Nature 375 (1995) 322-325 or the blue 1 Affigel used by López-Martínez 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. The conjugates according to the invention serve as new active substances for the preparation of pharmaceutical dosage forms. Taken together, the coupling results in an improved pharmacokinetic profile of the hedgehog protein. Hydrophobic hydrocarbon radicals result in localization of the hedgehog protein in the membrane of the target cells which, in addition to facilitating integration within the cell interior, and above all, result in a substantially longer presence on the cell surface, which is optimal for the pharmacological effect. The conjugates according to the invention do not necessarily need to be additionally coupled to a support for a 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, from a support for a long period (several days). However, it is convenient to use a pharmaceutical composition for the local application of hedgehog conjugates according to the invention, which contains the conjugate according to the invention together with a support matrix. The support matrix serves essentially to facilitate local application, in particular providing a pharmaceutical composition with a minimum viscosity suitable for local application. The pharmaceutical composition is preferably buffered in the pH range between 4 and 9 and contains one or more non-ionic detergents such as polyoxysilobate or polyoxyethylene detergents (eg T een®20, T een®80, Triton®X- l00), octylglucoside or ionic detergents such as sodium deoxycholate, sodium cholate, sodium taurodeoxycholate. In a preferred embodiment, a hh protein is expressed which contains about 10-30 amino acids, mainly hydrophobic at the N-terminus and / or the C-terminus, since these are also incorporated into the membrane of the cells [ebb et al., Biochemistry 37 (1998) 673-679, Skolnick et al., Biol. Membranes (1996) 536-554; ed. : Merz and Roux]. Hydrophobic amino acids in the context of the invention, are understood amino acids that have a negative free energy in the transition from the aqueous phase to the hydrophobic / organic phase. In addition, the N-terminal and / or C-terminal sequences that are known to form transmembrane helioids, such as p. ex. the M28 peptide or interacting as a helical with the surface of the membranes such as p. ex. maginina 2 (Skolnick et al., 1996) are also suitable for increasing the activity of the hh proteins. In another preferred embodiment, the N-terminus and / or C-terminus of the hedgehog protein is modified by a polypeptide residue containing 2-12 lysines and / or arginines. In this case it is possible to dispense with the modification with the hydrocarbon residue. A saturated or unsaturated aliphatic hydrocarbon radical with a hydrophobic action and a chain length of 8-24, preferably 10-24, more preferably 12-18 C atoms, is preferably a saturated or monounsaturated fatty acid at polyunsaturated or an alkyl alcohol radical optionally interrupted by an oxygen or sulfur atom or a carbonyl group. In particular, the following saturated fatty acids are preferred: capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidonic acid and behenic acid. Preferred mono-unsaturated fatty acids are myristic acid, palmitoleic acid and oleic acid. Particularly preferred polyunsaturated fatty acids are linoleic acid, linolenic acid and arachidonic acid. These fatty acid residues are preferably coupled by an ester, acid amide, disulfide or thioes attached to reactive protein groups.

The number of hydrophobic hydrocarbon chains per protein molecule can be adequately controlled by the reaction conditions (eg 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 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 approx. 4 chains per molecule. Although a greater number of hydrocarbon chains per molecule are suitable, the solubility in a pharmaceutical composition decreases therewith and can lead to alterations in the active three-dimensional structure of the protein. When copulating with long chain alkyl groups (from 14 to 24 carbon atoms, preferably from 16 to 24 carbon atoms), it is preferable to splice only 1-2 carbon chains and when copying short carbon chains, it is preferable, however, copulate 2-3 alkyl groups. In a preferred embodiment, the derivatization may also comprise the coupling of two hydrophobic hydrocarbon chains to an amino acid. This can be achieved for example by coupling a diglyceride of fatty acid to the amino acid. As the hedgehog proteins are very unstable, in a preferred version a hedgehog protein is used for coupling in which the SH group of the N-terminal cysteine is protected. A coupling product is then obtained SH, by reduction of protected SH groups immediately before or during the coupling procedure. It is preferable to protect the SH group from said cysteine by forming a homologous hedgehog disulfide or a mixed disulfide (eg with GSH or β-mercaptoethanol). Thiol protecting groups are known in the art, including but not limited to triphenylmethyl (trityl) and st-butyl, sp-nitrobenzyl and sp-methoxy-benzyl (see, eg, Greene and Wuts, Protective Groups in Organic Synthesis ("Protective groups in organic synthesis"), second edition, John iley &sons, New York (1991), and Atherton et al., The Peptides ("The peptides") Gross and Meienhofer, eds., Academic Press , New York (1983), vol 19, 1-38). Said thiol-protected or homo-dimerized hedgehog proteins are valuable intermediate products for obtaining H-modified hedgehog proteins and are also object of the present invention. The invention also relates to the use of hedgehog proteins in which the SH group of the N-terminal cysteine is protected or homodimerized, as stable intermediates for the production of modified hedgehog SH proteins. In this process, the protecting group is removed by cleavage or the homologous dimer is cleaved and reacted with the activated derivatization reagent. The following methods are preferred for the coupling of SH-protected hedgehog proteins: I. Reduction of the disulfide or cleavage of the protective group in the coupling mixture, preferably when coupling takes place by imidazolides or CoA derivatives. II. Reduction of disulphides, isolation of unprotected monomers in an acid medium and immediate coupling in a neutral range using an activated disulfide (eg pyridyl SS) as a coupling reagent. It has been found that a double acylation in the N-terminal cysteine takes place mainly in the coupling by imidazolide or CoA derivatives (approximately 60-70%) in which case the coupled hydrophobic compound is present in case I, bound as an acid amide and in case II, it is attached as a thioester. Selective cleavage of the bound hydrophobic compound as a thioester allows the synthesis of a monohydrophobized hedgehog protein. Said selective cleavage is carried out with a reducing agent such as DTE or hydroxylamine. Coupling by an activated disulfide such as, for example, the pyridyl-SS derivatives, leads to a mono-alkylation. The process described according to the invention allows numerous hydrophobic substances such as steroids, carbon chains (eg fatty acids) containing thiol groups, to be copulen to hedgehog proteins. Accordingly, another object of the invention is a process for the production of a SH-modified hedgehog protein, or of an N-terminal fragment thereof, preferably a fragment essentially containing the N-terminal domain which is characterized in that the The thiol group of the N-terminal cysteine of the hedgehog protein is protected, the protein modified in this way is isolated and, after cleavage of the protective group, the hedgehog protein is derivatized to the N-terminal cysteine, preferably via a linkage. covalent of a hydrophobic compound such as a fatty acid or a steroid of the SH group. This coupling is preferably reversible and can be reversed by the physiological reduction conditions present in the cell of a mammal and resulting in vivo, the formation of a hydrophobized hedgehog protein. Another subject matter of the invention is a method for the coupling of hydrophobic compounds to hedgehog proteins or fragments thereof by the SH group of the N-terminal cysteine which is characterized in that the thiol group of the N-terminal cysteine of the hedgehog protein is protected, said protected SH group is reduced and said hedgehog protein is reacted with an activated derivative of the hydrophobic compound to form a thiol bond between the hedgehog protein and the hydrophobic compound. In a preferred embodiment, an additional molecule of the hydrophobic compound can be coupled to the hedgehog protein via an amide bond.

The imidazolide and the CoA derivatives of hydrophobic compounds are, for example, suitable as activated hydrophobic compounds. Such activated compounds are preferably used in method I (see above). The pyridyl disulfide derivatives of the hydrophobic compounds are also suitable as hydrophobic activated compounds. These activated compounds are preferably used in method II (see above). Monounsaturated or polyunsaturated fatty acids, natural or unnatural, and in particular, saturated fatty acids with a chain length of 4-18, preferably 8-18 carbon atoms or steroids, are preferably used to acylate hedgehog proteins by the imidazolide process, whereby conjugates are obtained in which the hydrocarbon radicals have a chain length of 8-24 C atoms. In order to splice the hydrophobic compounds with hedgehog proteins by the pyridyl disulfide process, it is preferable to use natural or non-natural saturated and monounsaturated or polyunsaturated mercaptoalkanes, and in particular, saturated carbon chains containing a thiol group with a chain length of 8-24 carbon atoms, mercapto-steroids and in particular thiocholesterol.

In the process according to the invention it is preferable to use the hedgehog protein at a concentration of 0.01-10 mg / ml, particularly preferably 3 mg / ml. The salt concentration is preferably 0-2 moles / liter. Sodium chloride is preferably used. The molar ratio of the coupling reagent to the protein is convenient to be 1: 2 to 20: 1. Preferred buffers are: Hepes buffer, phosphate buffer and MES buffer. The coupling reactions are preferably carried out in a pH range between 4.5 and 8.5, preferably at pH 6.5-7.5. The duration of the reaction depends on the conditions used and it is advisable that it be from 5 minutes to 5 days for the preparation of the dipalmoylated shh. The reaction is preferably carried out in a temperature range between 0-40 ° C. The proportion of secondary products is low at low temperatures, preferably at temperatures below 10 ° C, p. ex. 4 ° C. The addition of detergents is particularly advantageous for the process according to the invention. These reagents can be, for example, ionic or non-ionic detergents. Amphoteric detergents are preferred. Preferred concentrations are about 0.5-3% (v / v). The hydrocarbon chain or the hydrocarbon chains or the steroids are conveniently coupled to the reactive groups of the protein, for example to free hydroxyl, mercapto, carboxyl or amino groups via an amino bond, an ester, disulfide or thioester linkage. These processes are already known to those skilled in the art and are described for example in ong, SS, Chemistry of Protein Conjugation and Cross Linking ("Chemistry of conjugation and protein cross-linking), CRC Press, Boca Raton, FL., USA. , 1993. For example, fatty acids can be coupled to thioesters with coenzyme A (eg palmitoyl coenzyme A) by a succinimide ester or N-maleimide coupling (eg the N-hydroxy succinimide ester of palmitic acid). ) by a fatty acid anhydride, fatty acid imidazolide or acid chloride The coupling methods for palmitoyl-CoA, stearoyl-CoA or myristoyl-CoA are described, for example, by Ross et al., J. Neurosci. (1988) 35-44, Bizzorero et al., J. Biol. Chem. 262 (1987) 2138-2145 or for tubulin, by Ozols, J. et al., Molec. Biol. Of the Cell 8 (1997) 637-645 A derivatization with fatty acid anhydrides has been described for example for ovalbumin to (Segawa, A., et al., Int. Archs Allergy appl. Immun. 66 (1981) 189-199) or for the peptides (Yadav, S.P. et al., Biochem.

Biophys. Res. Comm. 205 (1994) 1688-1695). There are also numerous examples for an acylation with succinimide esters of fatty acid p. ex. for casein (Haque, Z. et al., J. Agrie. Food Chem. 31 (1983) 1225-1230), (Haque, Z. et al., Agrie. Biol. Chem. 46 (1982) 597-599). An N-terminal coupling to the cysteine can also be effected by an aldehyde group on the fusion partner (eg palmitoyl-Cys-CHO) (Liu et al., Proc. Nati, Acad. Sci USA 91 (1994) 6584 -6588). N-terminal coupling to serine can be achieved by conversion to an aldehyde group, reaction with a hydrazide (eg palmitoyl-Cys hydrazide) and stabilization of the hydrazone formed (eg by reduction with NaBH 3 CN) (Gaertner et al. col., Bioconjugate Chem. 3 (1992) 262-268). The hydrophobic chain of a hydrocarbon is bonded depending on the coupling chemistry, for example, in the form of an ether, thioether, ester, thioester, disulfide or amide to the secondary groups of reactive amino acids serine, threonine, glutamic acid, acid Aspartic, cysteine, arginine or lysine. Methods for specific coupling to particular amino acids is described by ong, S.S. in Chemistry of Protein Conjugatipn and Cross Linking ("Chemistry of Conjugation and Protein Reticulation"), CRC Press Inc., Boca Raton, FL, USA (1993) and Lundblad in Techniques in Protein Modification ("Techniques in Protein Modification" ") (nineteen ninety five) . In a preferred embodiment of the invention, the hydrophobic compounds are solubilized in an organic solvent or mixture of an organic solvent and water, preferably containing more than 10% (v / v) organic solvent. These organic solvents are preferably dioxane, tetrahydrofuran or isopropanol. For the coupling of the hydrophobic compound and the hedgehog protein, these solutions of hydrophobic compounds are combined with a solution of the hedgehog protein, preferably in its protected form, containing a detergent, such that the mixture contains 10% or less of the solvent organic. It has been found that hedgehog protein solutions containing more than 10% of an organic solvent lead to precipitation and / or denaturation of the hedgehog protein. In another preferred embodiment, the thiocholesterol is coupled to the thiol group in particular of the N-terminal cysteine, by means of a disulfide bridge formed by oxidation in the presence of solubilizing detergents such as, in particular, sodium deoxycholate, sodium cholate, taurodeoxycholate of sodium, octyl glucoside or Triton® X-100. In contrast to the N-terminal fragment of hh, which is naturally modified at the C-terminus by cholesterol, a hh form containing a thiocholesterol at the N-terminus is produced in this process. This form has a greater activity similar to the form natural, but can be obtained much more easily and in large quantities. Due to the cytoplasmic instability of the disulfide bridges, this hh form has no immunogenic potential, or has only a slight immunogenic potential. In order to increase the solubility of the lipophilically modified hh proteins, it is preferable to further carry out the derivatization and / or subsequent purification or pharmaceutical formulation in the presence of soluble anionic polysaccharides, such as suramin and heparin. Activity within the context of the invention, is understood as the activity of the alkaline phosphatase that can induce the polypeptide in mammalian cells (activity in the alkaline phosphatase assay). In this method, a mouse fibroblast cell line is cultured in a medium containing fetal bovine serum. Then the sterile filtered sample is added, the cells are lysed after ca. 5 days, and alkaline phosphatase is determined in the cell lysate by cleaving a chromogenic substrate (pNP, p-nitrophenol) (J. Asahina, Exp. Cell. Res. 222 (1996) 38-47 and T. Nakamura (1997)). A hedgehog protein according to the invention is understood as a secreted signal protein (N-terminal signaling domain of 19 kD) which is responsible for the formation of numerous structures in embryogenesis. From pre •. . . . ferencia, the hh sonic, Indian or desertic are particularly used (Fietz M. et al., Development (Suppl.) (1994) 43-51). Preferably, an hh protein with a sequence as described by the EMBL data bank is used with the number L38518. The proteins of the hedgehog family have a marked homology in their amino acid sequence, so it is also preferable to express the nucleic acids that encode the hedgehog proteins, which are 80% or more homologous with the sequence mentioned above. sonic hedgehog protein (shh). The homology of the protein can be determined with the help of Gap or BestFit computer programs (University of Wisconsin; Needleman and unsch, J. Mol. Biol. 48 (1970) 443-153 453; Smith and aterman, Adv. Appl. Math. 2 (1981) 482-489). The human hedgehog precursor protein is composed of amino acids 1-462 of the sequence described in the EMBL data bank with No. L38518. Amino acids 1-23 represent the signal peptide, amino acids 24-197 represent the mature signal domain, amino acids 32-197 represent the signal domain shortened by eight amino acids, and amino acids 198-462 represent the self-processed C-terminal domain, after autoproteolytic cleavage. According to the N-terminus or the C-terminus of the hh protein where it has Instead of copulation, it is understood according to the invention as the first amino acids (N-terminus) or the last amino acids (C-terminus) of the N-terminal signaling domains 24-197. It is preferable to copulate to one or several amino acids of the first or last 10 amino acids. It is particularly preferable to copulate the first or second amino acid of the N-terminus or the N-terminal domain (AA 24 or 25) or the last or next to the last amino acid of the C-terminus of the N-terminal domain (AA 196 or 197). In the hedgehog conjugates according to the invention, the lipophilic groups or hydrocarbon chain (s) are preferably coupled to the N-terminal domain of an hh protein and the coupling product is in particular a thioester or an amide of the N-terminal cysteine at position 24 with lauric, myristic, palmitic, palmitoleic, stearic or oleic acid or a steroid or is an hh protein, to which is attached a thiocholesterol or a mercaptoalkane / -alkene, by means of a bridge disulfide. The production of an unmodified hh protein is preferably carried out recombinantly using methods familiar to a person skilled in the art, preferably in a prokaryotic expression system (eg E. coli). The hedgehog protein is preferably produced recombinantly as a fusion protein in a soluble form, isolated from the supernatant of the cell culture or, after lysis of the host cells, the fusion part (preferably N-terminal) (eg polyHis, streptavidin, etc.) is cleaved by a specific sequence protease such as an enterokinase, and its free thiol group of the N-terminal cysteine is protected by reaction with a thiol-protective reagent or by dimerization of the protein hedgehog by a disulfide bridge in said cysteine.

The pharmaceutical composition according to the invention contains an effective dose of the conjugate of hh and can preferably be administered locally. It is preferable to use the conjugates according to the invention in combination with other proteins of the hedgehog family or bone growth factors such as bone orfogenetic proteins (BMPs) (Wozney et al., Cell.Mol. Biol. Of Bone, Bone Morphogenetic Proteins an their Gene Expression ("Morphogenetic proteins of bone and their gene expression") (1993) Academic Press Inc., 131-167) or parathyroid hormones (Karablis et al., Genes and Development ("Genes and development") , 8 (1994) 277-289) or insulin-like growth factors (IGF-I or II) or growth-transforming factors (TGF-3). In another preferred embodiment, a pharmaceutical composition of the hedgehog protein conjugate according to the invention, containing suramin, which may be used advantageously, is preferred. In a preferred embodiment the pharmaceutical composition contains the hedgehog protein conjugate at a concentration of 0.01-10 mg / ml, in particular 0.01 to 1 mg / ml. In a preferred embodiment, the pharmaceutical composition additionally contains a pharmaceutically acceptable buffer which is biocompatible, preferably in a pH range between 4 and 10, in particular preferably in a pH range between 6 and 9, in particular at a pH of approx. of 7. The pH value of the pharmaceutical composition is convenient to be greater than pH 4, in order to prevent the denaturing of the folded structure and the separation of the zinc from the complex in the hedgehog protein. The concentration of the buffer is preferably 1-500 mmol / liter, preferably 10-100 mmol / liter. For this reason, in a suitable version, 20 mmoles / liter of potassium phosphate buffer pH 7.2 are used as a buffer. In addition, it is preferable for the preparation of the pharmaceutical composition, add auxiliary substances such as a sugar (mannitol, sucrose, lactose, glucose, sucrose, trehalose, preferably 20-100 mg / ml) or an amino acid such as glycine or arginine as well as antioxidants such as EDTA, citrate, polyethylene glycol (1 to 10% by weight) of ascorbic acid, tocopherol, detergents, preferably non-ionic detergents (preferably 0.005-1% by weight) such as polysorbate or polyoxyethylene type detergents (eg Tween®20, Tween®80) or polyoxyethylenes or ionic detergents such as sodium cholate, sodium deoxycholate or sodium taurodeoxycholate, anti-inflammatory agents, local anesthetics, antibiotics and / or stabilizers such as lipids, fatty acids and glycerin. The conjugate according to the invention can be used advantageously to induce or stimulate chondrocytes and osteocytes in an osteoinductive pharmaceutical composition or also to induce muscle cells and nerve cells. Osteoinductive pharmaceutical compositions are for example known from US Pat. No. 5,364,839, WO 97/35607 and WO 95/16035. The activity of the hedgehog protein conjugates according to the invention can be evaluated in vivo according to Glansbeek, H.L., et al., Laboratory Investigation 78 (1998) 133-142; US Patent No. 5,270,300; Toriumi, D.M., et al., Arch. Otolaryngol. Head Neck Surg. 117 (1991) 1101-1112; Cook, S.D., et al., J. Bone and Joint Surgery 76-A (1994) 827-837; and Riley, E.H., et al., Clin. Orthopaed. and Related Research 324 (1996) 39-46. When the conjugate according to the invention is administered locally, it is preferable to use it in combination with an appropriate matrix as support and / or with a sequestering agent. Said matrix is suitable for a slow release of the protein in vivo in an active form, in particular in the vicinity of the bones or cartilaginous tissue. The sequestering agent is a substance that facilitates administration for example by injection and / or prevents or at least delays the migration of the protein according to the invention from the administration site. The pharmaceutical composition according to the invention preferably contains a polymer (structural substance), which has an adhesion function for the cells. Said structural substance is, for example, collagen. A biocompatible, degradable material, for example based on collagen or other polymers based on polylactic acid, polyglycolic acid or copolymers of lactic acid and glycolic acid, are particularly suitable as matrix material. Said polymer matrices are described, for example, in WO 93/00050. Sequestering agents are for example cellulose and cellulose-like materials, for example alkyl cellulose, carboxymethyl cellulose, hyaluronic acid, sodium alginate, polyethylene glycol and polyvinyl alcohol, of which hyaluronic acid is particularly preferred especially in a pharmaceutical composition including without support matrix. The following examples, publications, sequence protocol and figures clarify the invention in more detail, the protective purpose of which results from the patent claims. The described methods are to be understood as examples which still describe the object of the invention even after the modifications. Description of the figures: Fig. 1: Shows the activity of the recom- binant human shh after derivatization with palmy- cloyl-CoA. Fig. 2: Shows the activity of recombinant human shh after derivatization with thiocholesterol. Example 1 a) cloning of the human hedgehog protein with annexation of a His-6 anchor as well as an enterokinase cleavage site; expression in E. coli. The following procedure can be used to amplify the mature N-terminal part of the human hedgehog protein (aa 24-Cys to 197 Gly) from any plasmid that is desired, or the corresponding cDNA containing the sonic hedgehog protein: A fragment of the shh gene that extends from an internal RsrII cleavage site to the coding sequence of amino acid 198, was amplified with the help of two primers (344 and 345), and at the same time several codons could be annexed to the C-terminus. interruption, as well as a Pstl cleavage site. SEQ ID NO: l Primer 344: 5 '- ca gaattc ttg cggaccg ggc agg gg 26-mer EcoRI RsrII SEQ ID NO: 2 Primer 345: _ 5' - ga ctgcag tta a tea tta gee tec cga ttt ggc cgc 36-mer PstI stop stop stop A fragment of DNA amplified in this way could be rescinded with RsrII and PstI, and were needed in the following steps (fragment 344/345). A linker was constructed by annealing two more primers (346 and 347), with the aid of which 6 histidine residues and an enterokinase cleavage site (EK) were incorporated at the N-terminus: SEQ ID NO: 3 primer 346 : 5'- aatte atg cat cat falls falls falls gat gac gac gac aaa tg cg | His 6 Ek EcoRI pendant SEQ ID NO.4 primer 347: 5 '-gtc cgc att tgt cgt cgt cat cgt ggt ggt ggt gat gat gca tg RsrII hanging adapter after the reassociation of 346 and 347: aattc atg cat cat falls falls falls gat gac gac gac aaa tgc gg tac gta gta gtg gtg gtg gtg cta ctg ctg ctg ttt acg c ctg For expression, the PCR fragment 344/345 was cloned together with the adapter 346/347 in a vector cleaved with EcoRl / Pstl. This could be done directly or after the intermediate cloning of fragment 344/345 in another vector. The vector cleaved with EcoRI / PstI should contain a promoter suitable for expression in E. coli at the EcoRI end, preferably T5, tac, lac, etc. Shh expression plasmids were transfected into a suitable strain of E. coli for expression. The expression system also contained nucleic acids encoding the arginine-tRNANAGA / AGG contained in prokaryotic cells (Brinkmann et al., Gene 85 (1989) 109-114). b) Fermentation Fermentation of 10 liters of the E. coli expression clone for the hedgehog protein: Pre-cultures were prepared from standard cultures (plate smears or ampoules stored at -20 ° C), which were incubated at 30-37 ° C. C with agitation. The volume of inoculation in the next highest dimension was in each case, from 1 to 10% by volume. Ampicillin (50-100 mg / liter) was added to the preculture and the main culture to screen against the loss of plasmid. The nutrients that can be used are enzymatically digested protein and / or yeast extract as source of N and C, as well as glycerin and / or glucose as additional source of C. The medium is buffered to pH 7 and metal salts can be added to Physiologically tolerated concentrations to stabilize the fermentation process. The fermentation temperature is 25-37 ° C. The growth is determined by measuring the optical density at 528 nm. The expression is induced by IPTG. After a fermentation period of approx. hours, the biomass is separated by centrifugation with a stabilized OD. Example 2 a) Preparation of the dimeric recombinant human shh 55 g of the biomass prepared in Example Ib was smooth by a high pressure press, centrifuged and the supernatant was applied to 50 ml of Chelating Sepharose (Pharmacia Biotech) which had previously been loaded with Zn. The shh fusion protein was eluted by a gradient of 0 to 200 mM imidazole in 50 mM Hepes; 250 mM NaCl; pH 7.4. Shh fractions were identified by SDS-PAGE analysis, pooled and diluted with a volume of 50 mM Hepes; pH 7.4. The precipitates formed during the dilution were centrifuged and the supernatant dialysed at 4CC against 50 mM Hepes; pH 7.4. Enterokinase (1: 500, w / w, Boehringer Mannheim GmbH) and 5-ME (up to 10 mM) were added to 500 mg of the shh fusion protein obtained in this manner and incubated for 16 hours at 35 ° C in a Water bath. Solid DTT was then added at a concentration of 10 mM. The sample was applied to 166 ml of SP-Sepharose (Pharmacia Biotech) and eluted with a gradient of 0-800 mM NaCl in 20 mM HEPES, pH 7.4. After the analysis of the peak fractions by means of SDS-PAGE, the main fractions were pooled, e? aliquots and stored at -80 ° C until further use. These main fractions contain shh under non-reducing conditions such as a dimer which is cross-linked by a reducible disulfide bridge and has an apparent molecular weight of approx. 38 kDa. b) Modification by incubation under reducing conditions Shh dimer (c = 1 mg / ml) was reduced with 10 mM of DTE (30 minutes, 25 aC) and dialyzed overnight against PBS containing 0.5 mM of DTT. The mass spectrum of the dialysate showed after desalination by RP-HPLC, that most of the monomerized shh had among others (ie, in addition to other modifications that led to a spreading of the peak in the mass spectrum, and that they are specified later), adducts of 32 ± 4 Da (a double oxidation of the N-terminal cistern) and 47 ± 4 Da. The forms of shh modified in this way could not be dimerized again by reoxidation. This showed that the SH group of the N-terminal cysteine had been modified in a stable manner. Consequently, this SH group is no longer available for subsequent reactions p. ex. to form a thioester and the yield of the derivatization with hydrophobic compounds is therefore strongly reduced. Therefore, in order to acylate the N-terminal cysteine in vitro, the periods in which the reduced shh is present in solution should be kept as short as possible or, in case of an appropriate coupling chemistry, the reaction should carried out in the presence of Tris (2-carboxyethyl) phosphine hydrochloride (TCEP.HC1) since it only attacks disulfide compounds but not thioesters (activated). c) Kinetics of the reoxidation of the shh to form the dimer, with respect to the value of the -pH of the solvent 0.5 ml of shh dimer (c = 2.7 mg / ml) was monomerized by reducing the disulfide intermolecular bridge, adding 2 mM of TCPE (Tris (2-carboxyethyl) phosphine hydrochloride) for 15 minutes at 25 ° C. The sample was then re-buffered by a PD-10 column (Pharmacia) in PBS pH 7.0 or PBS pH 5.0 and the spontaneous dimerization (at 25 ° C) was analyzed with respect to the incubation period, then to eliminate the reducing agent by RP-HPLC. The result is summarized in table 2: Table 2 It can be seen that the monomeric shh dimerizes spontaneously. This dimerization is very fast at pH 7.0 but at pH 5.0 takes place with a slower kinetics. Example 3 a) Preparation of coupling reagents for selective acylation of the SH groups on cysteines (I) Imidazolide process The starting materials (imidazolides) are prepared by general procedures A, B or C analogously to the literature (Leksyszyn, J. et al., Synthesis (1978) 478-479; Staab HA, Angew. Chem. 74 (1962) 407-423; Fahrenholtz, K.E. et al., J. Med. Chem. 17 (1974) 337-342). la) General procedure 10 mmol of the corresponding carboxylic acid and 10 mmol of 1,1 '-carbonyldiimidazoles are dissolved in absolute tetrahydrofuran and stirred for 30 minutes - 24 hours at room temperature. The mixture was evaporated in vacuo and used without further purification. Ib) General procedure B 10 mmoles of the corresponding acid chloride and 20 mmoles of imidazole are heated for 1-24 hours under reflux in absolute toluene in the absence of moisture. The imidazole hydrochloride that forms is filtered and the filtrate evaporated in vacuo. The residue is purified by chromatography on silica gel using ethyl acetate / heptane, or by recrystallization. Ic) General procedure C 10 mmoles of the corresponding carboxylic acid are stirred for 1-48 hours at room temperature with 10 mmoles of dicyclohexylcarbodiimide and 10 mmoles of imidazole in dichloromethane. After cooling to 0 ° C the formed urea is removed by filtration and the filtrate is extracted with H20, dried over sodium sulfate, evaporated in vacuo and recrystallized.

The following fatty acid imidazolide derivatives were obtained: 1- imidazol-1-yl-hexan-l-one (colorless oil, 66% yield) 1-imidazol-1-yl-o-n-1-one (colorless crystals, 59% yield) 1-imidazol-1-yl decan-1-one (colorless crystals, 79% yield) 1-imidazol-1-yl-dodecan-1-one (colorless crystals, 86% yield) 1-imidazol-1-yl-tetradecan-1-one (crystals colorless, yield 52%) 1- imidazol-l-yl-hexadecan-l-one (colorless crystals, 70% yield) 17- (4-imidazsl-l-yl-l-methyl-4-oxo-butyl) -10, 13 -dimethyl-dodecahydro-cyclopenta.a] -phenanthrene-3, 7, 12-trione) (colorless crystals; yield 56 %) 4- (10, 13-dimethyl-hexadecahydro-cyclopenta [a] phenanthrene-17-yl) -l-imidazol-1-yl-pentan-1-one (colorless crystals, yield 71%) (II) Pyridyl disulfide process The starting materials (pyridyl disulfides) are prepared by the general procedure A according to the literature (Lee, S. et al., Bull. Chem. Soc. Jpn. 64 (1991) 2019- 2021). 10 mmoles of the corresponding mercaptan and 10 mmoles of 2, 2'-dithiodipyridine are dissolved in 30 ml of absolute dichloromethane and stirred for 6-24 hours at room temperature. The solvent is removed in vacuo and diethyl ether is added to the residue and filtered. The filtrate is evaporated in vacuo and purified by chromatography on silica gel using ethyl acetate / heptane: The following compounds were obtained: 2-decildisulfanyl pyridine (colorless oil, 76% yield) 2-hexadecyldisulfanyl-pyridine (colorless crystals; 64%) 2 -. { 17 - (1, 5-dimethyl-hexyl) -10, 13-dimethyl-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 - 1etradecahydro - 1H- Cyclopenta [a] -phenanthrene-3-yl-disulfanyl] -pyridine (colorless crystals, 56% yield) b) Preparation of recombinant human ih-toilated shh, using palmitoyl imidazolide The recombinant dimeric human shh was used as an educt. modify, according to example 2. The protein was present in the PBS buffer (10 mM sodium phosphate buffer, 150 mM NaCl pH 7.4) at a concentration of 3 mg / ml. Detergent, preferably 200 μl of a 10% solution of Zwittergent 3-14, was added to 2 ml of this sample. Then, tri (2-carboxyethyl) phosphine was added (TCEP) at a concentration of 2 mM. After 15 minutes of incubation at room temperature, 50 μl of a 60 mM solution of palmitoyl imidazolide (l-imidazol-1-yl-hexadecan-1-one) in dioxane was added. The solution was incubated for 1-3 days, preferably at 4 ° C, with shaking. Under these conditions, the dipalmitoylated shh is formed as the main product (70-75%), which is identified as in Example 6 by means of RP-HPLC and mass spectrometry. The shh modified in this way presents a biological activity much higher than the shh unmodified in the activity assay described in example 7. The maximum half activity was reached with 70 ng / ml. The type of detergent used has an important influence on the yield of the dipalmitoylated shh and the level of biological activity (table 1). Table 1: Influence of the detergent used on the proportion of dipalmitoylated shh in the reaction mixture (carried out at room temperature). Method for quantification: as in example 6. c) Preparation of the alkylated recombinant human shh using 2-hexadecyldisulfanylpyridine The unmodified dimeric recombinant human shh was used as starting material, according to example 2a. The protein was present in the PBS buffer (10 mM sodium phosphate buffer, 150 mM NaCl pH 7.4) at a concentration of 3 mg / ml. After the addition of 2 mM of TCEP it was incubated during minutes for monomer formation. Then, the TCEP was removed by means of a PD-10 column equilibrated in 10 mM dp of potassium phosphate buffer, 150 mM NaCl, pH 5.0. Aliquots of 200 μl of the shh monomer prepared in this way were used to effect the reaction with 2-hexadecyl-disulfanyl-pyridine by two different methods. A) 20 μl of 10% solution of Zwittergent 3-15 14 was added; 20 μl of 0.5M HEPES pH 7.0 and an excess of one to twenty • Molar times of the coupling agent (dissolved in dioxane, 60 mM), to the shh monomer. B) 2 μl of 10% sodium deoxycholate, 20 μl of 1M potassium phosphate buffer, pH 7.6 and an excess of to 20 molar times of the coupling agent (dissolved in methanol, 60 mM), to the shh monomer. The reaction was completed after 15 minutes, in both variants. A tenfold molar excess of the coupling agent gave as a result in variant A), a proportion of 43% and in variant B), a 47% proportion of mono-alkylated shh, in the reaction mixture. In variant A, the biological activity was greatly increased compared to shh without modifying and the maximum half activity was 200 ng / ml shh. A simple molar excess of 2-hexadecyldisulfanylpyridine gave only slightly lower yields. Variant B also led to an increase in activity compared to the unmodified shh, but to a lesser extent: with the same amount of protein, only 30% of the biological activity was reached. Therefore, variant A is preferred. d) Purification of the alkylated derivatives of the shh The product of the coupling was diluted 1: 4 in 50 mM of HEPES, 0.1% Tween®80, pH 7.4 and then purified by SP-Sepharose HP (Pharmacia). Balancing buffer: 50 mM HEPES, 0.1% Tween®80, pH 7.4; Elution buffer: 50 mM HEPES, 0.2% Tween®80, pH 7.4 containing 0.8 M NaCl; gradient elution (2 x 6 column volume) and discontinuous elution. In the case of dipalmitoylated shh, it was possible to separate the excess palmitoylimidazolide and partially remove the shh without modification. In the case of the modification with 2-hexadecyldisulfanylpyridine, there was considerable enrichment of the monopalmitylated shh of 76%. The modified shh can for example be further purified by means of the heparin Sepharose. Example 4 a_ Preparation of palmi-toilated recombinant human shh using palmitol-CoA 2 ml of the purified dimeric shh sample, with a shh concentration of 0.35 mg / ml, was added and mixed with DTE at a final concentration of 20 mM, incubated for 2 hours at 37 ° C and then analyzed against a) 50 mM Tris / HCl, 1 mM DTE, 0.1 mM ZnCl2, 0.5% Triton®X -100, pH 8, 5 b) 100 mM MOPS, 1 mM DTE, 0.1% Triton®X-100, 0.1 mg / ml suramin, pH 7.4 c) 100 mM MOPS, 1 mM DTE, 0.1% Triton®X-100, pH 7.4. Then, different volumes of a solution of palmitoyl-CoA (10 mg / ml in 100 mM MOPS, 1 mM DTT, 0.2% Triton®X-100, pH 7.6) were added, to aliquots of 0.5 ml of the samples, which resulted in palmitoyl-CoA concentrations in the mixtures, of: 1) 0 μM 2) 50 μM 3) 500 μM after which they were incubated for 1 hour at 37 ° C . Before filtration and dilution 1/200 for cell assay, samples were added and mixed with BSA (1 mg / ml final concentration) and suramin (0.1 mg / ml final concentration). With the above samples, the activity test as described in example 7 for the shh, showed that the samples of shh incubated in buffers b) and c) containing 500 .muM of palmitoyl-CoA had a significantly increased biological activity (see Fig. 1) compared with samples without palmitoyl-CoA. Palmitoylated shh in this manner can be purified by methods that have been described for membrane proteins p. ex. in "A practical Guide to Membrane Protein Purification" ("Practical Guide for the Purification of Membrane Proteins") (1994); ed. : Jagow & Sclágger; Academic Press) or in the European patent application No. 98 102 095.1. b) Dependence of acylation in the concentration of shh and molar ratio between shh and palmitol-CoA (Pal-CoA) The dimeric shh (in PBS) was diluted with PBS a = 150 μM or 37.5 μM and Tween®80 (final concentration of 0.05%), DTE (3 mM final concentration) and TCEP (1 mM concentration) were added. final) . Next, Pal-CoA (20 mM in water) was added in the indicated amounts, and the mixture was incubated overnight at 25 ° C. The samples were analyzed by RP-HPLC or different dilutions in the assay with the cells after dilution in PBS, 1 mg / ml BSA, 0.05% Tween®80 pH 7.3 and sterile filtration. The results are summarized in table 3: Table 3 * EC50 is the concentration of shh that has to be used in the assay with the cells to achieve a maximum half induction of the alkaline phosphatase. Due to the differences in the number of passes of the cells and thus to the alterations in the cellular physiology, this value can vary slightly between the tests carried out at different times. A moderate excess of the coupling reagent (< 50 times) leads mainly to the formation of a shh derivative that is acylated twice in the N-terminal cysteine. With an increasing ratio of Pal-CoA to shh, the shh that is pal-mitilated more than twice is formed in a growing extension. A palmitylation with more than two fatty acid residues also increases the activity. With a shh concentration of 37.5 μM, an excess of approx. 10 times of Pal-CoA is optimal with respect to activity. At a concentration of shh of 150 M a 3-fold excess of Pal-CoA already achieves a high yield of twice-palmylated shh and high activity. c) Dependence of the acylation with respect to the type and concentration of detergent The dimeric shh was adjusted with PBS pH 7.4 to a concentration of 0.75 mg / ml, and mixed in the amounts indicated with the respective detergents as well as in each case with 500 μM of Pal-CoA and 3 mM of DTE or 3 mM of TCEP (final concentration). The coupling mixtures were incubated overnight at 25 ° C and then analyzed by RP-HPLC or used in different solutions in the cell assay after dilution in PBS, lmg / ml BSA, 0.05% of Tween®80 pH 7.3 and sterile filtration.

The results are summarized in table 4: Table 4 pal. = palmitylated * was reduced with 3 mM of TCEP instead of 3 mM of DTE It was found that it is possible to obtain high proportions of palmitylation using 0.05% Tween®80 or 0.5% octylglycoside; also the palmitilation in 0.05% of Zwittergent led to a high increase in activity. A simultaneous reduction with TCEP led to similar results to reduction with DTE. d) Acylation of the reduced monomeric shh, with respect to the pH value of the coupling mixture The dimeric shh (c = 0.7 mg / ml) was reduced with 10 mM DTE and dialyzed overnight against PBS, 0.05% Tween®80, 0.5 mM DTE. The dialysate was adjusted to a final concentration c = 0.25 mg / ml and at the respective pH value with PBS, 0.05% Tween®80, 0.5 mM DTE and 125 μM Pal-CoA were added. The mixtures were incubated for 2 hours at 37 ° C and then analyzed at a dilution of 1: 500 in the assay with the cells. The activity of the samples is summarized in Table 5 as a stimulation of AP activity with respect to the basal activity of unstimulated C3H10T cells (= 100%) .- Table 5 Thus, the maximum activation takes place from a neutral to slightly alkaline pH. e) Copulation of the acyl-CoA derivatives containing acyl groups having carbon chains of different length The dimeric shh was adjusted to a concentration of 0.75 mg / ml in PBS of 0.05% Tween®80 pH 7, 4, 3 mM of DTE and 0.5 mM of the respective acyl-CoA derivative were added and incubated overnight at 25 ° C. The samples were analyzed by RP-HPLC or used in the assay with the cells at various dilutions after dilution with PBS, 1 mg / ml BSA, 0.05% Tween®80 pH 7.3 and sterile filtration. The change in retention in the RP-HPLC as well as the yields of the diacetylated shh and the activity in the assay with the cells are summarized in Table 6.

Table 6 The table shows that acyl groups with different length chains can be efficiently transferred by using the method that uses Pal-CoA derivatives. The concentration of detergent as well as the type of detergent used requires a fine optimization as a function of the chain length of the acyl residue transferred. A chain of decreasing length of the acyl residue or an increasing number of carbon unsaturated bonds leads to a decrease in the specific activity of the adh derivative of the shh. Example 5 a) Derivatization of the recombinant human shh, with thiocholesterol In order to modify the shh with a thiocholesterol locked to the amino terminal cysteine by a disuifuro bridge, the reduced monomeric shh is diluted to a concentration of 0.66 mg / ml in 0.5 mM DTT pH 7.0, in a ratio of 1: 3 in the following reaction buffer: 100 mM ethanolamine 100 mM 50 μM NaCl CuCl2 pH 9.5 0.8% (p. / v) of sodium deoxycholate or 0.4% (w / v) of sodium cholate or 1.3% (w / v) of n-octyl glycoside or 0.3% of Triton®X-100. The reaction started by addition (final concentration): 5% (v / v) acetone or 100 μM thiocholesterol (from a 10 mM solution in acetone) or 500 μM thiocholesterol (from a 10 mM solution in acetone) and the mixture was stirred for 30 minutes at room temperature. Already before the sterile filtration, the samples were diluted with nine volumes of 20 mM sodium phosphate, 0.9% NaCl, 0.05% Tween®80, 1 mg / ml, 0.1 mg / ml. ml of suramin, pH 7.2, and analyzed in the assay with the cells at an additional 1/20 dilution. As shown in Figure 2, there is an increase in activity that depends on the concentration of thiocholesterol, which is more efficiently generated or stabilized in samples containing anionic detergents. b) Derivatization of the recombinant human shh with thiocholesterol pyridyl disulfide In order to modify the shh with a thiocholesterol locked to an amino terminal cysteine via a disulfide bridge, the shh dimer that was covalently crosslinked between the N-terminal cysteines via a linkage Disulfide was reduced by 2 mM TCEP for 15 minutes at room temperature and then re-buffered in PBS buffer pH 5 to remove the reducing agent via a PD10 column (Pharmacia). For the coupling, the concentration of the shh ac = 1 mg / ml was adjusted with PBS pH 5. The pH was adjusted to a pH value from neutral to slightly alkaline (eg pH 7.6) by adding a concentrated solution of buffer (eg 40 volume percent 0.4 M Na phosphate pH 9.2) and immediately the detergent was added first and then the coupling reagent (5 mM thiocholesterol pyridyl disulfide) dissolved in an organic solvent (eg methanol, 40 ° C). The reaction mixture was incubated for several hours at room temperature and then analyzed by HPLC and mass spectrometry or different dilutions were used in the assay with the cells after dilution in PBS, 1 mg / ml BSA, 0.05 % Tween®80 pH 7.3 and sterile filtration. The results of some examples of coupling mixtures are summarized in table 7.

Table 7 As can be seen from table 7, a thiolsterol derivative copolymerized with the disulfide of the shh can be obtained under suitable conditions with a yield greater than 50%, which has at least 10% of the specific activity of the dipalmylated shh in the assay with the cells. Example 6 Characterization of a preparation of a shh coupling with thiocholesterol pyridyl disulfide, by RP-HPLC and mass spectrometry 100 μl of a preparation of coupling dialyzed against PBS, 0.05% of Tween 80 containing 0.7 mg / ml of shh, 1% sodium cholate, 250 μM thiocholesterol pyridyl disulfide, pH 7.6, was applied to a 1 x 150 mm butyl column (Vydac ™ 214TP5115) which had been equilibrated in 18% acetonitrile, 0, 1% trifluoroacetic acid (TFA). It was eluted at 25 ° C in a gradient of 18-90% acetonitrile in 0.1% TFA. The eluate was divided and analyzed on the one hand on-line by detecting the absorbance at 220 nm and on the other hand on-line using an electrospray mass spectrometer (API100, Sciex; adjustment parameters: starting mass (m / z) 900 units of atomic mass (amu), mass of stop (m / z) 1500 amu, stop 0.3 amu, dwell time 0.5 ms, orifice voltage 30 V). The shh. reduced monomer and the reoxidized shh with masses of 19560 Da and 39120 Da eluted at 18 min (42-44% acetonitrile), the shh crosslinked with disulfide containing thiocholesterol with a mass of 19962.7 Da eluted at 24.5 min ( 48.5% acetonitrile). Example 7 Induction of alkaline phosphatase in the cell assay (determination of alkaline phosphatase activity) 5000 cells were seeded from a mouse mesenchymal pluripotent line C3H10T ^ (ATCC CCl-226), in each well of a plaque. 96 well microtitre. The cells were in 100 μl of DMEM, 2 mM of glutamine, 100 IU / ml of penicillin, 100 μg / ml of streptomycin and 10% of fetal bovine serum, FCS. The next day, the active substances to be examined were added, at the appropriate concentration, in a volume of 100 μl after dilution in the culture medium. The trial was stopped after 5 days. For this purpose, the supernatant was discarded and the cells were washed once with PBS. Cells were used in 50 μl of 0.1% Triton®X-100 and frozen at -20 ° C. After thawing, 25 μl was used for the determination of the protein and 25 μl was used to determine the activity of the alkaline phosphatase. Determination of the protein according to the instructions of the manufacturer Pierce: 75 μl of redistilled H20 is added to the mixture, then 100 μl of BCA 1 protein reagent (Pierce Micro BCA No. 23225) is added. After 60 minutes the optical density (OD) is measured at 550 nm. Alkaline phosphatase activity according to the manufacturer's instructions Sigma: 100 μl of reaction buffer (Sigma 221) is added to the preparation. A substrate capsule (Sigma 104-40) is dissolved in 10 ml of redistilled H20 and then 100 μl is added to the test mixture by pipette. OD at 405 nm is measured after yellow staining. In the reaction, alkaline phosphatase converts p-nitrophenyl phosphate to p-nitrophenol. Each of the ODs was converted to nmoles or μg by standard curves. The evaluation was made according to the formula: n moles of PNP per minute (measured) per mg of protein (cell) List of references A Practical guide to membrane protein purification, Ed. G. v. Jagow, Hermann Schágger (1994), Chapter 16, pp. 535-554 Asahina, J., Exp. Cell. Res. 222 (1996) 38-47 Atherton et al., The Peptides, Gross and Meienhofer, eds., Academic Press, New York (1983), Vol. 19, 1-38 Bitgood, M.J. et al., Curr. Biol. 6 (1996) 296 Bizzozero et al., J. Biol. Chem. 262 (1987) 2138-2145 Brinkmann et al., Gene 85 (1989) 109-114 Chiang, C. et al, Nature 83 (1996) 407 Cook, SD, et al, J. Bone and Joint Surgery 76-A (1994) 827-837 European Patent Application No. 98 102 095.1 Fahrenholtz, KE et al., J. Med. Chem. 17 (1974) 337-342 Fietz, M. et al., Development (Suppl.) (1994) 43-51 Gaertner et al., Bioconjugate Chem. 3 (1992) 262- 268 Glansbeek, HL, et al., Laboratory Investigation 78 (1998) 133-142 Greene and Wuts, Protective Groups in Organic Synthesis, second edition, John Wiley 8c Sons, New York (1991) Hancock, JF, Cell 63 (1990) 133 - 139 Haque, Z. et al., Agrie. Biol. Chem. 46 (1982) 597-599 Haque, Z. et al., J. Agrie. Food Chem. 30 (1982) 481 Haque, Z. et al, J. Agrie. Food Chem. 31 (1983) 1225-1230 Hynes, M. et al., Neuron 15 (1995) 35-44 Karablis et al., Genes and Development 8 (1994) 277-289 Kinto et al., FEBS Letters, 404 (1997) 319-323 Lai, CJ et al., Development 121 (1995) 2349 Lee, S. et al., Bull. Chem.Soc. Jpn. 64 (1991) 2019-2021 Leksyszyn, J. et al., Synthesis (1978) 478-479 Liu et al., Proc. Nati Acad. Sci. USA 91 (1994) 6584-6588 Lopez-Martinez et al. in Curr. Biol. 5 (1995) 791-796 Lundblad, Techniques in Protein Modification, CRC Press, Boca Raton, FL, USA (1995) Marti et al., Nature 375 (1995) 322-325 Nakamura, T. et al., Biochem. Biophys. Res. Comm. 237 (1997) 465-469 Needleman and Wunsch, J. Mol. Biol. 48 (1970) 443-453 Ozols, J. et al., Molec. Biol. Of the Cell 8 (1997) 637-645 Perrimon, N., Cell 80 (1995) 517-520 Porter, J.A. et al., Science 274 (1996) 255-259 Riley, E.H., et al .; Clin. Orthopaed. and Related Research 324 (1996) 39-46 Ross et al., J. Neurosci. Res. 21 (1988) 35-44 Segawa, A. et al., Int. Archs Allergy appl. Immun. 66 (1981) 189-199 Skolnick et al., Biol. Membranes (1996) 536-554; ed .: Merz and Roux Smith, J.C .. Cell 76 (1994) 193-196 Smith and Waterman, Adv. Appl. Math. 2 (1981) 482-489 Staab H.A., Angew. Chem. 74 (1962) 407-423 Toriumi, D.M., et al, Arch. Otolaryngol. Head Neck Surg. 117 (1991) 1101-1112 US-Patent No. 5,270,300 US-Patent 5,364,839 Vortkamp, A. et al, Science 273 (1996) 613 Webb, RJ. et al, Biochemistry 37 (1998) 673-679 WO 93/00050 WO 95/16035 WO 97/35607 Wong, S.S., Chemistry of Protein Conjugation and Cross Linking, CRC Press, Boca Raton, USA, 1993 Wozney et al, Cell. Mol. Biol. Of Bone, Bone Morphogenetic Proteins and their Gene Expression (1993), Academic Press Inc., 131-166 Yadav, S.P. et al, Biochem. Biophys. Res. Comm. 205 (1994) 1688-1695 Yang et al., Development 124 (1997) 4393-4404 LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: ROCHE DIAGNOSTICS GMBH (B) STREET: Sandhofer Str. 116 (C) CITY: Mannheim (E) COUNTRY: Germany (F) ZIP CODE (ZIP) ): d-68305 (G) TELEPHONE: 08856 / 60-3446 (H) TELEFAX: 08856 / 60-3451 (ii) TITLE OF THE INVENTION: Active conjugate of hedgehog protein, procedure for its obtaining and use (iii) NUMBER OF SEQUENCES: 4 (iv) LEGIBLE FORM BY THE COMPUTER: (A) MIDDLE TYPE: Soft disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAM: Patentln Relay # 1.0 , version # 1.30B (? PO) (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) CHAIN: single-stranded (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Primer 344" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 1: CAGAATTCTT GCGGACCGGG CAGGGG 26 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) CHAIN: single-chain (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Primer 345" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: GACTGCAGTT AATCATTAGC CTCCCGATTT GGCCGC 36 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) CHAIN: single-stranded (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "Primer 346" (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: AATTCATGCA TCATCACCAC CACCACGATG ACGACGACAA ATGCG 45 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) CHAIN: single-stranded (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION : / desc = "Primer 347" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: GTCCGCATTT GTCGTCGTCA TCGTGGTGGT GGTGATGATG CATG 44 It is noted that in relation to this date, the best method known to the applicant to carry out the practice said invention is that which is clear from the present description of the invention.

Claims (13)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. Conjugate of hedgehog pro tein, ear clH- i adr) perqué arrejere: a) A polypeptide composed of 10 to 30 hydrophobic amino acids and / or amino acids that forms a trans-membranic helicoid, and is positively charged, b) 1 to 4 residues of aliphatic hydrocarbon, saturated or unsaturated, with a chain length of 8 to 24 C atoms and with hydrophobic activity, or c) A hydrophobic thiocompound covalently bound to a hedgehog protein.
2. 2 . Hedgehog protein conjugate as claimed in claim 1, characterized in that the polypeptide contains from 2 to 12 lysines and / or arginines.
3. 3 . Hedgehog protein conjugate as claimed in claim 1, characterized in that the hydrocarbon residue is a fatty acid or an alkyl alcohol residue, which is bound as an ester, thioester, acid amide or disulfide. Four . Hedgehog protein conjugate as claimed in claim 3, characterized in that the fatty acid is lauric acid, myristic acid, palmitic acid, tearic acid, arachidic acid, behenic acid, palmitoleic acid, oleic acid, linoleic acid , linolenic acid or arachidonic acid. 5 . Hedgehog protein conjugate as claimed in claim 1, characterized in that the hydrophobic thiocompound is thiocholesterol. 6 Hedgehog protein conjugate as claimed in claims 1 to 5, characterized in that the hydrocarbon residue, the polypeptide or the thiocompound is attached to the C-terminus and / or N-terminus of the hedgehog protein. 7 Process for obtaining a conjugate of hedgehog protein as claimed in claims 3 or 4 and 6, characterized by the fact that the hydrocarbon content is covalently coupled to a hydroxyl, mercapto, carboxyl or free amino group. the protein hedgehog. 8 Process for obtaining a hedgehog protein conjugate as claimed in claims 1 to 6, csracrjerizado because it is carried out rrediaits na c pilarrirh coval nbe of a repicar-o hydrocarbon or a thiocompound hydrophobic, a protein hedgehog, wherein the covalent coupling of the hydrocarbon residue, the hydrophobic polypeptide or thiocompound and / or the isolation is carried out in the presence of suramin, heparin, anionic polysaccharides or a detergent. 9. A method for obtaining a hedgehog protein conjugate as claimed in claims 1 to 6, characterized in that the battery is heated to a battery with a lens of a fatty acid resin or a piggyback h-d-S -acid to a hedgehog, where palmitoyl-CoA or palmitoylimidazolide is used as a coupling reagent of fatty acid and th.ocholesterol or carbon chains containing a thiol group are used as the hydrophobic thiocompound. 10 Copp-S-jciai f_ap.Bautica ca_3c ± erizaób because cent-Lene a hedgehog protein carjugab as claimed in claims 1 to 6, in a pharmaceutically effective amount as well as also pharmaceutical auxiliaries, detergents, stabilizers, matrix materials , suramin, heparin, anionic polysaccharides and / or sequestering agents. 11. Process for obtaining a pharmaceutical composition, characterized by percuta or carjugao de pxrtEána hedgehog chorus has been claimed in claims 1 to 6, is employed as the main component of this composition. 12. Hedgehog protein, CE- ^ r? Briza? B perqué the thiol group of the N-terminal cysteine is coupled to the thiol-protective group, or said hedgehog protein is a homodimer in which the N-terminal cysteines are linked by a disulfide bridge. 13. The use of a hedgehog protein as claimed in claim 12, in obtaining a protein conjugate which contains: a) A polypeptide composed of 10 to 30 hydrophobic amino acids and / or amino acids that form transmembrane helicals and It is positively charged. b) 1 to 4 aliphatic hydrocarbon residues, saturated or unsaturated with a chain length of 8 to 24 C atoms and with a hydrophobic activity, or c) A hydrophobic thiocompound covalently bound to a hedgehog protein.