MXPA99008037A - Pharmaceutical composition soluble in water in an ionic complex and the use of the mi - Google Patents

Abstract

A water soluble composition containing a complex of a pharmaceutically effective ionic polypeptide selected from the group consisting of hedgehog proteins, bone morphogenetic proteins, growth factors, erythropoietin, thrombopoietin, G-CSF, interleukins and interferons, characterized in that said composition contains , additionally, an amphiphilic compound, said polypeptide and said amphiphilic compound forming an ionic complex, so that the formation of the complex does not increase the solubility of said polypeptide, is suitable for increasing the activity of the polypeptide and / or for the controlled release of the polypeptide.

Description

PHARMACEUTICAL COMPOSITION SOLUBLE IN WATER IN AN IONIC COMPLEX AND THE USE OF THE SAME Field of the Invention The present invention relates to a composition of a polypeptide that is biologically effective through its interaction with an extracellular receptor of the cell membrane, said polypeptide being present in said composition in an ionic complex with an amphiphilic compound. The invention further relates to the use of said composition.

Background of the Invention The use of amphiphilic compounds as drug delivery systems is well known in the art (see U.S. Patent Nos. 5,650,393, 5,688,761, 5,665,328, 5,124,081, 5,109,038). The formation of micellar complexes between substances with surface activity and pharmaceutical agents is also known, for example in the improvement of the transdermal and transmembrane penetration of the active agent (To linson and REF .: 31114 Davis, J., Colloid Interf., Sci. 74 (1980) 349). It is also known that pharmaceutical agents usually have better transport properties through biological membranes in their non-ionized form with respect to their ionized state (Cools and Jansen, J. Pharm, Pharmacol, 35 (1983): 689-691). It is also known that peptides that are present in multiple ionization forms at physiological pH values not optimal for transport to the site of action (transport of the drug) since the charged molecules, and in particular the polypeptides, have a low solubility in lipids (Hirai et al., Int. J. Pharm. 7 (1991): 317-325). It is known from the works of O ada y cois., J. Pharm. Sci, 72 (1993): 75-78, that it is advantageous to bind a lipophilic counterion to the ionic part of the agent, thereby improving the interaction with the biological membrane in order to facilitate the transport of proteins through the intestinal membras . For example, Hazzenga and Berner describe an improved method for the transdermal transport of zwitterionic active agents in J. Controlled Relay 16 (1991): 77-88.

Other methods for improving the interaction of agents with biological membranes are described, for example, by Lee et al., Critical Rev. Therp. Drug Carrier Systems 8 (1991): 91-192, Morimoto et al., Arch. Int. Pharmcodyn. 302 (1989): 18-26 and Aungst, Int. J. Pharm. 33 (1986): 225-234. However, in all these methods the objective was to increase the hydrophobicity of the active agent in order to facilitate penetration through biological membranes such as the skin, and to bring said agent to the cell. Substances with surface activity are used for this purpose at a concentration that is above the critical micelle concentration (CMC, Womack et al., Biochim Biophys, Acta 733 (1983): 210). A disadvantage of these methods is that the high concentrations of substances with surface activity that are used have an important influence on the cell membrane and can damage it. It is known from WO 94/08599 that a homogeneous solution of an active agent can be prepared for the production of active agents attached to carrier vehicle by adding a suitable amount of an anionic detergent to form a precipitate, isolating the precipitate and dissolving it again in an organic solvent. This homogeneous solution containing a complex between the anionic detergent and the active agent can then be used to imbibe or disperse the active agent in a solid matrix. Additionally, WO 94/08599 mentions that a complex of the protein can be formed with an anionic detergent and that the active agent can be released therefrom for the controlled release of a protein. It is known that the activity of proteins can be improved by covalent coupling to hydrophobic compounds such as fatty acids or steroids. However, these methods are complicated and lead to the formation of non-homogeneous products due to the coupling chemical reaction (see for example Ekra i, HM et al., FEBS Letters 371 (1995): 283-286, Pepinski, RB and cois., J. Biol. Chem., 273 (1998): 14037-14045).

Description of the Invention The object of the invention is to provide pharmaceutically effective polypeptide compositions that improve the activity of the polypeptide contained therein. The object is achieved by a composition, preferably a pharmaceutical composition, containing a pharmaceutically effective polypeptide selected from the group consisting of hedgehog proteins, bone morphogenetic proteins, growth factors, erythropoietin, rhombopoietin, G-CSF, interleukins and interferons, characterized in that said composition additionally contains an amphiphilic compound, said polypeptide and said amphiphilic compound forming an ionic complex, so that complex formation does not increase the solubility of said polypeptide. The composition does not contain any organic solvent. The composition can be lyophilized for conservation reasons. In the invention, the polypeptide and the amphiphilic compound are soluble indi idually at the concentrations used in aqueous solutions, preferably buffered, being only the combination of the two substances which results in the formation of a complex by ionic interactions that hydrophobize the polypeptide and thus worsens, or at least does not improve, its solubility in water. It has surprisingly been found that in this way the activity of said polypeptides can be significantly improved. According to the invention, the amount and proportion of the amphiphilic compound and the polypeptide are preferably selected such that the aqueous composition containing the ionic complex is a clear solution. If complex formation between the polypeptide and the amphiphilic compound results in turbidity, then the solution is filtered to obtain a solution without turbidity, if the solution is to be used directly as a solution for administration to a patient. If the composition is to be immobilized in a carrier vehicle prior to administration to the patient, then it is not necessary to avoid turbidity.

The pharmaceutically effective polypeptide is a polypeptide which can be present in an ionic form and which is recognized and binds to cell surface receptors (extracellular receptor) to develop its biological activity. Said polypeptides are growth factors (for example NGF, TGF-β, FGF, GDF, insulin-like growth factors), erythropoietin ina, thrombopoietin ina, G-CSF, interferons such as interferon a2b, interleukins such as interleukin 2 or interleukin 12, orfogenetic bone proteins such as' BMP-2, or hedgehog proteins such as hedgehog proteins soni cr iridi an or desert. Hedgehog proteins are especially preferred. Polypeptides having an activity (therapeutic effect and / or in vitro protein activity) which preferably increases 10 times or more in the complex according to the invention compared to the non-complexed form are preferably used. The ionic form of the polypeptide can be obtained if it is present in an environment that advantageously differs by at least 0.5 pH units from its pK value.

The amphiphilic compound according to the invention is understood as an anionic, zwitterionic or cationic hydrophobic surfactant, a fatty acid, an alkyl sulfonate or a lipid. Preferred anionic surfactants are anionic detergents such as steroidal surfactants such as deoxycholates, cholates, taurocholates, taurodeoxycholates, dehydrocholates (useful for cationic polypeptides); Preferred zwitterionic surfactants are CHAPS (3 [(3-colamidopropyl) imethylammonium] -1-propane sulfonate) and Z ittergent® (N-dodecyl-N, N-imethyl-3-a-n-l-propanesulfonate); and preferred cationic detergents are cetyltrimethalmmonium bromide or dodecylammonium chloride (useful for anionic polypeptides); Preferred fatty acids are fatty acids such as palmitic acid (useful for cationic polypeptides). Preferred alkyl sulphates are alkylsulfonates such as decylsulfonate (useful for cationic polypeptides); and preferred lipids are lipids such as phosphatidyl serine (useful for anionic polypeptides) and phosphatidate (useful for cationic polypeptides).

The amphiphilic compound is added to the composition under conditions that render the polypeptide hydrophobic and thereby reduce, or at least not improve, the water solubility of the polypeptide. It is important that according to the invention, a water-soluble ionic complex is formed between the polypeptide and the amphiphilic compound in this process. The ratio of polypeptide to amphiphilic compound in the complex will depend on the pH value used and on the pK values of the two substances, as well as on the concentration ratio. A pH value that differs by at least half a pH unit from the pK values of the polypeptide and the auxiliary substance is used. The more amphiphilic compound is added, the more amphiphilic compound binds to the polypeptide, and the more hydrophobic the complex becomes. This can lead to the precipitation of the complex, and consequently to the presence of a mixture of soluble and insoluble compound that is no longer completely soluble in water. However, the addition of a non-ionic detergent such as a polyoxamer such as Tween® can restore, at least partially, the water solubility of the complex, or the composition can be filtered if necessary. In this case, the non-ionic detergent can also be present in concentrations that lead to the formation of micelles. It should be noted that the type and concentration of amphiphilic compound is selected so that, especially in the case of proteins such as polypeptides, the molecular structure of the polypeptide is retained in its natural active form, so that activity is not reduced in consequence of the polypeptide. Normally, a 10-fold molar excess of an amphiphilic compound is sufficient for this purpose. Preferably, with an amount of protein of 5 μg of protein per ml, 0.001 to 0.05% (weight per volume) of amphiphilic compound are added. If a denaturing surfactant, such as sodium dodecyl sulfate (SDS) according to the invention is used, this compound can be used only at low concentrations. It is known that SDS denatures proteins at high concentrations, which improves the water solubility of these proteins, albeit in an inactive denatured form. Such amphiphilic compounds such as SDS can also form micelles at higher concentrations together with the desired complexes according to the invention, which can in turn increase the solubility of the polypeptide. A person skilled in the art will readily be able to determine by customary methods whether the amphiphilic compound causes undesired denaturation of the polypeptide. Such methods include for example the determination of the activity or physicochemical methods to control the structure, such as IR, CD and fluorescence spectroscopy. A water-soluble aqueous pharmaceutical composition in the sense of the invention should be interpreted as a composition that essentially does not comprise any insoluble particle containing the pharmaceutically effective polypeptide. In particular, an aqueous pharmaceutical composition should be interpreted according to the invention as a composition that does not possess visible turbidity. Said soluble compositions are possible when the ionic complex according to the invention is completely soluble in water at the used concentrations of polypeptide and surfactant or the undissolved complex is removed by filtration. According to the invention, the aqueous composition does not additionally contain organic solvents. Additionally, it may be necessary, for the manufacture of the compositions, to dissolve the amphiphilic compounds such as the fatty acids in a small amount of organic solvent (up to 5%, preferably up to 1% of the volume of the composition). A further object of the invention is a process for the production of an aqueous pharmaceutical composition according to the invention that is characterized in that a pharmaceutically effective polypeptide and an amphiphilic compound that worsens or at least does not improve the water solubility of the pharmaceutically effective polypeptide are combined in a concentration ratio and at a pH value such that an ionic complex is formed between the polypeptide and a auxiliary substance by ionic interaction. A further object of the invention is the use of the pharmaceutical composition according to the invention for a local or systemic administration in the body of humans or mammals.

In a preferred embodiment, a hedgehog protein is used in the pharmaceutical composition as the pharmaceutically effective polypeptide. It is known that the activity of hedgehog proteins can be improved by covalent hydrophobic modification (European Patent Application No. 99108032.6). In accordance with the invention it has surprisingly been found that the activity of the hedgehog proteins can be greatly increased by the formation of an ionic complex between a hedgehog protein and an amphiphilic compound. In a preferred embodiment, an increase in the activity of the hedgehog protein (compared to a recombinant hedgehog protein produced in E. Coli) of 10 times or more is obtained. Accordingly, a preferred object of the invention is a pharmaceutical composition containing a complex of a hedgehog protein and an amphiphilic compound formed by ionic interactions in which the compound is present at a concentration that worsens, or at least does not improve, the solubility of said hedgehog protein.

Hedgehog proteins (hh) are known as a family of secreted signaling proteins, which are responsible for the formation of numerous structures during embryogenesis (JC Smith, Cell 76 (1994) 193-196, N. Perrimon, Cell 80 ( 1995) 517-520, C. Chiang et al., Nature 83 (1996) 407, MJ 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 N-terminal domain of 20 kDa and a C-terminal domain of 25 kDa are obtained after excision of the signal sequence and autocatalytic cleavage. In its natural form the N-terminal domain is modified with cholesterol or palmitoyl (JA Porter et al., Science 274 (1996) 255-259, Pepinski et al., J. Biol. Chem. 273 (1998) 14037-14045) . In the higher forms the family hh is composed of at least three members called hh sonx c, i ndi an and deser t (Shh, Ihh, Dhh, M. Fietz et al., Development (Suppl.) (1994) 43-51 ). Differences in the activity of hedgehog proteins that were produced recombinantly after production in prokaryotes and eukaryotes were observed (M. Hynes et al., Neuron 15 (1995) 35-44 and T. Nakamura et al., Biochem. Biophys. Res. Com. 237 (1997) 465-469). They are preferably used hh soni c, i n di a n or desert (Fietz M., et al., Development (suppl.) (1994): 43-51). A hh protein having a sequence described in the EMBL data bank with accession number L38518 is preferably used. The proteins of the family of hedgehog proteins have a pronounced homology in their amino acid sequence, which is why it is preferable to express those nucleic acids that encode hedgehog proteins that are 80% or more homologous to the aforementioned sequences of the hedgehog protein soni c. The hedgehog proteins are preferably used as described for example in International Application No. WO 99/28454 and in European Patent Application No. 99108032.6. The human hedgehog protein precursor protein is composed of amino acids 1-462 of the sequence described in the EMBL databank under 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 shortened signal domain at eight amino acids and amino acids 198-462 represent the self-processed C-terminal domain after autoproteolytic cleavage. The pharmacological effect of the hedgehog protein is preferably understood as a neurological effect on nerve cells, preferably osteogenesis and / or osteoinduction, and especially preferably chondrogenesis and / or chondroinduction, as described in Kinto et al., FEBS Letters, 404 ( 1997): 319-323 for bone induction, by Miao et al., In J. Neurosci. 17 (1997): 5891-5899 for the effect on nerve cells, and by Stott et al., in J. Cell. Sci. 110 (1997): 2691-2701 for the induction of cartilage cells. Protein solutions of hedgehog at high concentrations are required to produce carrier matrices that are coated or imbibed with hedgehog proteins so that they exhibit adequate pharmaceutical efficacy when applied locally. It has been found that pharmaceutically suitable carriers, coated with hedgehog protein, should preferably contain a concentration of hedgehog protein between 0.1-10 mg / ml of vehicle and more. Hedgehog proteins inherently are poorly soluble. However, it has surprisingly been found that the solubility of the hedgehog proteins increases considerably and that the stability of the hedgehog proteins is improved at low concentrations (<1 mg / ml or less) in solutions containing arginine or argininium ions. It is therefore preferable to add arginine or arginine ions to the aqueous solution and to the complex bound to the vehicle. The activity of the hedgehog protein is understood within the meaning of the invention as the activity of alkaline phosphatase (stimulation of alkaline phosphatase expression) that the polypeptide can induce in mammalian cells (activity in the alkaline phosphatase test). In this method a mouse fibroblast cell line is cultured in a medium containing fetal calf serum. Subsequently, the sterile filtered sample is added, the cells are lysed after about 5 days and the 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)). The pharmaceutical composition according to the invention contains a pharmacologically effective dose of the hh protein and can be administered locally or systemically, preferably locally. It is preferable to use the proteins according to the invention in combination with other proteins of the hedgehog family or bone growth factors such as bone morphogenetic proteins (BMPs).

(Wozney et al., Cell .Mol. Biol. Of Bone, Bone Morphogenetic Proteins and their Gene Expression (1993) Academic Press Inc., 131-167) or parathyroid hormones (Karablis et al., Genes and Development 8 (1994) 277-289) or insulin-like growth factors (IGF-I) or II) or the family of transforming growth factors (TGF-β, GDF). These other proteins may be present, although their presence is not essential, in the complexes according to the invention. Therefore, a further object of the invention is a process for the production of a preferably water-soluble pharmaceutical composition of a hedgehog protein by combining said hedgehog protein with an amphiphilic compound under conditions that allow the formation of a complex ion between the hedgehog protein and the amphiphilic compound. A further object of the invention is the use of said hedgehog protein complex according to the invention to produce a pharmaceutical composition in which the complex is used as an essential component of the composition and is optionally combined with additional pharmaceutically acceptable auxiliary substances, preferably in an aqueous buffered solution. In a further preferred embodiment, the hedgehog complex according to the invention is present in a mixture of a dissolved and precipitated form or only in a precipitated form that allows a controlled release of the hedgehog protein or a local application at the site of action in vivo. The release of the protein at the site of action is slower from this mixture than from a completely dissolved pharmaceutical formulation. Furthermore, it is preferable for the production of the composition to add auxiliary substances such as sugars (mannitol, sucrose, lactose, glucose, sucrose, trehalose, preferably between 20-100 mg / ml) or amino acids such as glycine or arginine, methionine, cysteine, as well as antioxidants as EDTA, citrate, thioglycerol, acetylcysteine, polyethylene glycol (1-10% by weight), anti-inflammatory agents, local anesthetics, antibiotics and / or stabilizers. In a further preferred embodiment a hedgehog-containing protein composition according to the invention containing suramin is preferred and can be used vent aj amenté. The composition may contain additional pharmaceutical auxiliaries and is preferably lyophilized. In a preferred embodiment the pharmaceutical composition contains hedgehog protein at a concentration between 0.1-10 mg / ml, preferably between 0.1 and 5 mg / ml. In a preferred embodiment the pharmaceutical composition additionally contains a pharmaceutically acceptable buffer which is biocompatible, preferably in the range between pH 4 and pH 10, particularly preferably in the range between pH 6 and pH 8. The concentration of the buffer is preferably between 10 - 500 mmol / 1, more preferably between 10 - 100 mmol / 1. It is essential to select the salt concentrations so that they do not interfere with the formation of the complex due to the high ionic strength. In another embodiment of the invention, the pharmaceutical composition contains the complex according to the invention embedded in a vehicle which is biocompatible and which can for example be used as an implant. The carrier is preferably a polymer that: - does not denature the hedgehog protein when it is embedded in the vehicle, - has an average molecular weight of at least 10000 Da. Such polymers are for example hyaluronic acid, collagen, alginate or organic polymers such as PLGA (polylactic and glycolic acid copolymer) or derivatives thereof. If the complex is embedded in a vehicle, it is not necessary for the complex to be completely soluble in solution, as was useful for the aqueous pharmaceutical composition described above. Since the complex bound to the vehicle is applied locally to the body, preferably in the form of a hedgehog polypeptide complex in bone or cartilage, it is released slowly in soluble form from the complex thus developing its desired biological effect. A further object of the invention is the use of a pharmaceutical composition according to the invention which is immobilized on (or which is reversibly linked to) a biocompatible vehicle for local application to the body of humans or animals. Such biocompatible vehicles are for example hyaluronic acid, collagen, alginate or organic polymers such as PLGA or derivatives thereof. The complex according to the invention is preferably located in a biocompatible vehicle in which the vehicle can release the complex locally in vivo in an active form. Said formulations are especially suitable for the repair of bone or cartilage defects, but they can also be used for the repair of neuronal defects or for a systematic administration.

The pharmaceutical composition according to the invention preferably contains a polymer which acts essentially as a structural substance which also preferably has an adhesion function for the cells. Said structural substance is for example collagen. In a further preferred embodiment, the pharmaceutical composition according to the invention is used to reduce systemic side effects outside the desired site of action when administered locally. Local administration of a pharmaceutically effective polypeptide that is not completely immobilized or that does not have an extremely short local half-life can lead to dispersion of the polypeptide or at least part of it beyond the desired site of action, which leads to actions unwanted systemic These undesired systemic effects can be considerably reduced or even avoided by the invention. The method is suitable for polypeptides having an activity increased 10-fold or more in the ionic complex compared to the non-complexed form, so that the complex has a lower solubility in a buffered aqueous solution than the non-complexed polypeptide. Said polypeptides are preferably hedgehog proteins, cytokines and growth factors such as NGF. According to the invention, an ionic complex of the polypeptide and the amphiphilic compound are preferably applied locally in this method in an amount such that the polypeptide exhibits an activity in the complex which corresponds to its therapeutic dose (effective dose) in vivo. The amount of complex must be selected so that the complex dissociates, which for example occurs when it is diluted 10 to 20 times under physiological conditions, for example in the blood, the activity of the polypeptide is then only 20% or less. the therapeutic dose. Thus, in said local application of the complex according to the invention the pharmaceutically effective polypeptide shows a complete therapeutic effect such as local bone growth at the desired site of action when the polypeptide is a bone growth factor such as a cytostatic or an apoptosis-inducing effect when the polypeptide is a tumoricidal agent. When the complex diffuses from the site of action, the complex is diluted in the physiological conditions that prevail outside the site of action, which leads to dissociation. This results in a decrease in the concentration of the complexed polypeptide and an increase in the concentration of non-complexed polypeptide. Since the activity of the non-complexed polypeptide is considerably less than that of the complexed polypeptide, its therapeutic effect is also reduced outside the site of action. A further object of the invention is the use of a pharmaceutical composition according to the invention for local application in humans characterized in that the complex is administered in an amount such that the complexed polypeptide exhibits an activity that corresponds to its dose therapeutic, while the same amount of polypeptide in a non-complexed form would exhibit an activity of 20% or less of the therapeutic dose. A further object of the invention is a process for the production of a pharmaceutical composition for local administration in humans characterized in that a complex of a pharmaceutically effective polypeptide and an amphiphilic compound formed by ionic interaction is used as an essential component, in that the compound is present at such a concentration as to worsen the water solubility of the pharmaceutically effective polypeptide, and the complex is administered in an amount such that the complexed polypeptide exhibits an activity that corresponds to its therapeutic dose, while the same amount of polypeptide in a non-complexed form would exhibit an activity of 20% or less of the therapeutic dose. The following examples, publications and figures will contribute to a better understanding of the invention, the protective scope of which is derived from the patent claims. The described methods should be understood as examples that continue to describe the object of the invention even after modifications.

Brief description of the Figures Figure 1: The dependence of the alkaline phosphatase induction in a cell assay by shh at increasing concentrations of deoxycholate is shown. Figure 2: shows the dependence of aggregate formation on deoxycholate concentration.

Example 1 Analysis of the activity of different hedgehog protein formulations in a cell assay: Induction of alkaline phosphatase. 5000 cells of mouse mesenchymal pluripotential line C3H10T1 / 2 (ATCC CCL-266) are shown in each well of a 96-well microtiter plate. The cells are in DMEM, 2 mM glutamine, 100 U / ml penicillin, 100 μg / ml estroptimicin and 10% fetal bovine serum. The following day the medium is replaced by means containing human shh (0, 5 or 50 μg / ml) in different formulations (0, 0.00016, 0.00052, 0.0013, 0.0019 or 0.01% sodium deoxycholate), or the various hedgehog protein formulations are added directly. The test is stopped after 5 days have elapsed. The supernatants are decanted and the cells are washed once with PBS. The cells are lysed in 50 μl of Triton "X-100 0.1% and frozen at -20 ° C. After thawing, 25 μl aliquots are used for the determination of proteins and to determine the activity of alkaline phosphatase Determination of proteins according to the instructions of the manufacturer Pierce: 75 μl of redistilled H20 is added to the mixture, and then 100 μl of BCA protein reagent (Pierce Micro BCA, No. 23225) is added. 550 nm after 60 min Determination of the alkaline phosphatase activity in accordance with the manufacturer's instructions Sigma: 100 μl of reaction buffer (Sigma 221) is added to the mixture A substrate capsule (Sigma 104-40) is dissolved in 10 ml of H 0 and then add 100 μl with pipette to the test mixture Absorbance is measured at 405 nm During the reaction, alkaline phosphatase converts p-nitrophenyl phosphate to p-nitrophenol (pNP). becomes n in nmol of pNP using standard curves. The activities of various hemolymph protein formulations in nmol / pNP / min / mg protein are depicted in Figure 1. It is shown that, at the same protein concentrations, the activities of the hedgehog protein formulations examined increased considerably with increasing concentrations of deoxycholate. Example 2 Titration of hydrophobic ion pair of hshh (dimer) Recombinant human sonic hedgehog protein (dimer, 0.8 mg / ml in 50 M Tris-Cl, pH 7.4, or 0.1% Tween 80, 50 mM Tris-Cl, pH 7.4) is mixed with increasing concentrations of sodium deoxycholate. The absorbance at 360 nm is measured as an indicator of turbidity (formation of water-insoluble aggregates composed of protein-detergent ion complexes). It is obvious from Figure 2 that the transition to insoluble aggregates in water occurs approximately above 0.04% sodium deoxycholate. The formation of insoluble aggregates in water can be avoided to a large extent in the presence of 0.1% Tween 80. The mentioned absorbances are not corrected for dilution. Example -3 Analysis of the NGF formulations in a bioactivity assay: development test of the dorsal root ganglion neuron. The NGF activity was determined by the morphometric assay of development of dorsal ganglionic root (DRG) neurons in vitro. Briefly, lumbar DRGs were dissected from E7-E8 chicken embryos, stripped of adjacent connective tissue and dissociated by trituration through a fire-molded pasteur pipette, after digestion with 0.1% trypsin for 20 minutes. minutes at 37 ° C. Polluting cells, such as fibroblasts, were removed by preplating the entire cell preparation in plastic tissue culture plates for 2 hours. Under these conditions, neurons do not bind to the substrate, while fibroblasts and other non-neuronal cells adhere to the tissue culture plastic. The "clean" neurons were harvested by recovering the supernatant and plating it on plastic plates coated with polyornithine / laminin (48 wells) at a density of 10,000 cells per well in HAM's F14 medium containing 5% FBS. A dose response curve for NGF was titrated from about 1 pg / ml to 15 ng / ml. Neurotrophic activity was quantified by counting viable differentiated neurons that developed neurites greater than twice the diameter of the pericarion after 48 hours of incubation with different formulations of NGF. The data were represented as the mean number of double determinations of differentiated neurons with respect to the concentration of the NGF test formulation, with half of the maximal stimulatory activities (EC50) of NGF being determined in several different formulations (Table 1). i Table 1 MAXIMUM STIMULATING ACTIVITY OF GF FORMULATIONS (CE50) 1 i t- r \ "** - ™ 11". 2_ ^ "» Ó T ^ E, 50 (pg / r.I1 1 v- * i: s? R. additive '! -7 1 : NGF ídesoxicolatc scd ec C, 00D ° =. ' -" 1 INGF ¡¡Esox Colanc Sodium C, 02 ° o) '10 | These data clearly show that the specific activity of NGF is increased in ormulations containing an amphiphilic additive (in this case sodium deoxycholate).

EXAMPLE 4 Pharmaceutical Compositions With Deo Icolate For the production of the pharmaceutical composition, 100 ml of an aqueous solution of 5 mg / ml or 1 mg / ml of Hshh (human sonic hedgehog protein) in 50 mmol / l of Tris buffer, pH 7.4, are dialyzed against the formulation solution without deoxycholate for 24 h at 4 ° C. After the dialysis, sodium deoxycholate is added from a stock solution with stirring to obtain an aqueous pharmaceutical composition of 1 mg / ml or 5 mg / ml of Hshh in formulation solution. The solution is sterilized by filtration and stored at 4 ° C. 0.05 to 2 ml of the solution are used for injection in humans or mammals. 4.1 Formulation of hedgehog protein hydrophobized ionically in phosphate buffered saline (low concentration of sodium deoxycholate) Formulation solution: NaCl: 150 mmol / l Sodium phosphate buffer 10 mmol / l Sodium deoxycholate: 0.05% (w / v) pH. 7.4 4. 2 Formulation of hedgehog protein hydrophobized ionically in phosphate buffered saline (high concentration of sodium deoxycholate) Formulation solution NaCl: 150 mmol / l Sodium phosphate buffer 10 mmol / l Sodium deoxycholate: 0.1% (w / v) PH 7, 4 4. 3 Formulation of hedgehog protein hydrophobized ionically in phosphate buffer of low ionic strength (low concentration of sodium deoxycholate) Formulation solution NaCl: 30 mmol / l Sodium phosphate buffer 20 mmol / l Sodium deoxycholate: 0.05% (? / V) pH. 6, 5 4. 4 Formulation of hedgehog protein hydrophobic ionically in phosphate buffer of low ionic strength (high concentration of sodium deoxycholate) Formulation solution mmol / l NaCl Sodium phosphate buffer 20 mmol / l Sodium deoxycholate: 0.1% (w / v) pH. 6.5 Example 5 Pharmaceutical compositions of hedgehog protein hydrophobized ionically in phosphate buffered saline with lipids, fatty acids or steroids. For the production of the pharmaceutical composition, 100 ml of aqueous solution of 1 mg / ml or 2 mg / ml of Hshh are dialysed in 50 mmol / l of Tris buffer, pH 7.4, against a formulation solution without lipids, fatty acids or cholate for 24 hours at 4 ° C. After the dialysis, 0.01 g of phosphatidate, 0.01 g of phosphatidyl serine, 0.01 g of palmitate, 0.05 g of cholate, 0.05 g of taurodeoxycholate or 0.05 g are added from stock solutions. of taurocholate with stirring to obtain an aqueous pharmaceutical composition of 1 mg / ml or 2 mg / ml of Hshh in formulation solution. The solution is sterilized by filtration and stored at 4 ° C. 0.05 to 2 ml of the solution are used for injection in humans or animals.

Formulation solution: NaCl: 150 mmol / 1 Sodium phosphate buffer 10 mmol / l Phosphatidate: 0.01% (w / v) pH. 7.4 Formulation solution NaCl: 100 mmol / l Buffer sodium phosphate 10 mmol / l Phosphatidylserine 0.01% (w / v) PH 7.4 Formulation solution NaCl: 150 mmol / l Sodium phosphate buffer 20 mmol / l Sodium palmitate: 0.01% (w / v) pH. 7.4 Formulation solution NaCl: 150 mmol / 1 Sodium phosphate buffer 10 mmol / l Sodium colate: 0.05% (? / V) pH. 7.4 Formulation solution: 100 mmol / l NaCl Sodium phosphate buffer: 10 mmol / l 0.05% sodium taurodeoxycholate (w / v) pH. 7.4 Formulation solution 150 mmol / l NaCl Sodium phosphate buffer 20 mmol / l Sodium taurocholate: 0.05% (w / v) pH 7.4 Use 6 Pharmaceutical compositions of bone morphogenetic protein (BMP-2) hydrophobic ionically in arginine buffers. For the production of the pharmaceutical composition, 100 ml of aqueous solution of 0 are dialyzed, 4 mg / ml of BMP-2 is dialyzed against arginine 500 mmol / l in 10 mmol / 1 potassium phosphate buffer, pH 6.0, for 24 hours at 4 ° C. After dialysis, 0.01 g of palmitate or 0.05 g of deoxycholate or taurodeoxycholate are added with stirring to obtain an aqueous pharmaceutical composition of 0.4 mg / ml of BMP in formulation solution. The solution is sterilized by filtration and stored at 4 ° C. 0.05 to 2 ml of the solution are used for injection in humans or animals.

Formulation solution Arginine: 500 mmol / l Sodium phosphate buffer 10 mmol / l Sodium deoxycholate: 0.05% (w / v) pH. 6, 0 Formulation solution: Arginine: 500 mmol / l Potassium phosphate buffer. 10 mmol / l sodium deoxycholate: 0.01% (w / v) PH. 6, 0 Formulation solution Arginine: 500 mmol / l Potassium phosphate buffer: 10 mmol / l Sodium taurodeoxycholate 0.05% (w / v) pH. 6, 0 Example 7 Pharmaceutical compositions of interleukin 2 ion hydrophobized in phosphate buffered saline. For the production of the pharmaceutical composition, 100 ml of an aqueous solution of 1 or 2 million Ul of interleukin-2 in 50 mmol / 1 Tris buffer, pH 7.4, are dialysed against a formulation solution without the amphiphilic compound for 24 hours at 4 ° C. After the dialysis, 0.05 g of deoxycholate, 0.01 g of sodium palmitate or 0.01 g of phosphatidyl serine are added with stirring to obtain an aqueous pharmaceutical composition of 1 6 2 million Ul of interleukin. 2 in formulation solution The solution is sterilized by filtration and stored at 4 ° C. 0.05 to 2 ml of the solution are used for injection in humans or animals.

Formulation solution NaCl: 150 mmol / l Sodium phosphate buffer 10 mmol / l Deoxycholate 0.05% (w / v) pH. 7.4 Formulation solution 150 mmol / l NaCl Sodium phosphate buffer 10 mmol / l Phosphatidyl serine: 0.01% (w / v) pH. 7.4 Formulation solution NaCl: 150 mmol / 1 Potassium phosphate buffer 20 mmol / l Sodium palmitate: 0.01% (w / v) pH: 7.4 Example 8 Pharmaceutical compositions of interferon alpha hydrophobized ionically in phosphate buffered saline. For the production of the pharmaceutical composition, 100 ml of an aqueous solution of 4 or 40 million IU of interferon-a2b in Tris buffer 50 mmol / 1, pH 7.4, are dialyzed against a formulation solution without the amphiphilic compound for 24 hours at 4 ° C. After the dialysis, 0.05 g of deoxycholate, 0.05 g of taurodeoxycholate or 0.01 g of phosphatidyl serine are added with stock solutions with stirring to obtain an aqueous pharmaceutical composition of 4 or 40 million IU of interferon- a2b in formulation solution The solution is sterilized by filtration and stored at 4 ° C. 0.05 to 2 ml of the solution are used for injection in humans or animals.

Formulation solution 150 mmol / l NaCl Sodium phosphate buffer 10 mmol / l Deoxy cholate: 0.05% (w / v) pH. 7.4 Formulation solution NaCl: 100 mmol / l Sodium phosphate buffer 10 mmol / 1 Fosfat idilserin: 0.01% (? / V) PH 7.4 Formulation solution NaCl: 150 mmol / l Potassium phosphate buffer: 20 mmol / 1 Taurodeoxycholate sodium 0.05% (w / v) pH. 7.4 Example 9 Pharmaceutical compositions of human NGF hydrophobized ionically in acetate buffers. For the production of the pharmaceutical composition, 100 ml of aqueous solution of 1 or 2 mg / ml of human NGF in 100 mmol / 1 of sodium acetate buffer, pH 6.0, are dialysed, and are added with stirring from stock solutions 0, 05 g of deoxycholate, 0.05 g of taurodeoxycholate or 0.01 g of phosphatidate to obtain an aqueous pharmaceutical composition. The solution is sterilized by filtration and stored at 4 ° C. 0.05 to 2 ml of the solution are used for injection in humans or animals.

Formulation solution human NGF: 1 mg / ml sodium acetate buffer: 100 mmol / l Desoxycholate: 0.05% (w / v, pH 6. 0 formulation solution Human NGF: 2 mg / ml Buffer sodium acetate 100 mmol / l Phosphatidate: 0.01% (w / v: pH 6.0) Formulation solution: Human NGF: 1 mg / ml Sodium acetate buffer: 100 mmol / l 0.05% sodium taurodeoxycholate (? / V: pH 6.0) Example 10 Production of an alginate gel containing hedgehog proteins. An aliquot of the formulation solution of Example 4.1 is stirred with a 1% (w / v) aqueous sodium alginate stock solution (Pronova Biopolymer, Norway) so that a mixture of gelatinous alginate protein is formed. This gel is used directly as an injectable matrix in an amount of 0.05 to 2 ml.

Example 11 Production of a mixture of collagen containing BMP-2. 100 μl of the formulation solutions of Example 6 are added dropwise onto collagen sponges (Helistat, Integra Life Science, USA) with a size of 10 x 10 x 3 mm. The loaded vehicles are frozen (-70 ° C) and lyophilized. The sponge is used locally for the healing of bone fractures.

LIST OF REFERENCES Ashanina, J. Exp. Cell. Res, 222 (1996) 38-47 Aungst, Int. J. Pharm. 33 (1986) 225-234 Bitgood, M.J. and cois., Curr. Biol. 6 (1996) 296 Chiang, C. et al., Nature 83 (1996) 407 Cools and Jansen, J. Pharm. Pharmacol., 35 (1983) 689-691 Ekrami, J.M. and you FEBS Letters 371 (1995) 283-286 European Patent Application No. 99108032.6 Fietz, M. et al. Development (Supl 1994) 43-51 Hazzenga and Berner, J. Controlled Reeléase 16 (1991 77-78 Hirai et al., Int. J. Pharm. 7 (1991) 317-325 Hynes, M. et al., Neuron 15 (1995) 34-44 Karablis et al., Genes and Development 8 (1994) 277-289 Kinto et al., FEBS Letters, 404 (1997) 319-323 Lai C.J. et al., Development 121 (1995) 2349 Lee et al., Critical Rev. Therap. Drug Carrier Systems 8 (1991) 91-192 Miao et al., J. Neurosci. 17 (1997) 5891-5899 Morimoto et al., Arch. Int Pharmacodyn. 302 (1989) 18-26 Nakamura T et al., Biochem. Biophys. Res. Co m., 237 (1997) 465-469 Okada et al., J. Pharm. Sci. 72 (1993) 75-78 Pepinski, R.B. et al., J. Biol. Chem. 273 (1989) 14037-14045 Perrimon, N. Cell 80 (1995) 517-520 Porter, J.A. and co., Science 274 (1996) 255-259 Smith, J.C. Cell 76 (1994) 193-196 Stott et al., J. Cell. Sci. 110 (1997) 2691-2701 Tomlinson and Davis, J. Colloid. Interf. Sci. 74 (1980) 349 US-P 5,650,393 US-P 5,109,038 US-P 5,124,081 US-P 5,665,328 US-P 5,688,761 Vortkamp, A. and cois. Science 273 (1996) 613 WO 99/28454 Womack et al., Biochim. Biophys. Act 733 (1983) 210 Wozney et al., Cell. Mol. Biol. Of Bone, Bone Morphogenetic Proteins and their Gene Expression (1993) Academic Press Inc., 131-167 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (11)

R E I V I N D I C A C I O N S

1. An aqueous composition containing a pharmaceutically effective polypeptide selected from the group consisting of hedgehog proteins, morphogenetic bone proteins, growth factors, erythropoietin, thrombopoietin, G-CSF, interleukins and interferons, characterized in that said composition additionally contains a compound amphiphilic, said polypeptide and said amphiphilic compound forming an ionic complex, so that complex formation does not increase the solubility of said polypeptide.

2. The composition as claimed in claim 1 in lyophilized form.

3. The composition as claimed in claim 2, characterized in that the complex is immobilized in a biocompatible vehicle.

4. The composition as claimed in claim 3, characterized in that the complex is present in a mixture of a dissolved and precipitated form.

5. The composition as claimed in claims 1, characterized in that said composition has a pH value in aqueous solution that differs by at least half a pH unit from the pK values of said polypeptide and said surfactant.

6. The composition as claimed in claims 1 to 5, characterized in that said polypeptide is a hedgehog protein. The composition as claimed in claims 1 to 6, characterized in that the amphiphilic compound is deoxycholate. 8. The use of a composition as claimed in claims 1 to 7 for the controlled release or local application of the hedgehog protein in the human body. 9. The process for the production of a pharmaceutical composition for local application in humans, characterized in that a pharmaceutically effective polypeptide complex selected from the group consisting of hedgehog proteins, bone morphogenetic proteins, growth factors, is used as an essential component, erythropoietin, thrombopoietin, G-CSF, interleukins and interferons, and of an amphiphilic compound formed by ionic interaction, in which the auxiliary compound is present at a concentration that does not increase the water solubility of said polypeptide. 10. Process as claimed in claim 9, characterized in that the complex is immobilized on a biocompatible vehicle. 11. A method for increasing the activity of a polypeptide that is recognized and binds to a cell surface receptor, characterized by the formation of an ionic complex between said polypeptide and an amphiphilic compound.