US20050123610A1 - Composition of a polypeptide and an amphiphilic compound in an ionic complex and the use thereof - Google Patents

Composition of a polypeptide and an amphiphilic compound in an ionic complex and the use thereof Download PDF

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US20050123610A1
US20050123610A1 US11/032,492 US3249205A US2005123610A1 US 20050123610 A1 US20050123610 A1 US 20050123610A1 US 3249205 A US3249205 A US 3249205A US 2005123610 A1 US2005123610 A1 US 2005123610A1
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pharmaceutical composition
controlled
release pharmaceutical
polypeptide
amphiphilic compound
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Apollon Papadimitriou
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
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Definitions

  • amphiphilic compounds as a drug delivery system is well known in the state of the art (cf. US Patents U.S. Pat. No. 5,650,393; U.S. Pat. No. 5,688,761; U.S. Pat. No. 5,665,328; U.S. Pat. No. 5,124,081; U.S. Pat. No. 5,109,038).
  • Formation of complexes in the form of micelles between surface-active substances and pharmaceutical agents is also known for example for improving the transdermal and transmembrane penetration of the active agent (Tomlinson and Davis, J. Colloid. Interf., Sci. 74 (1980) 349).
  • a homogeneous solution of an active agent can be prepared for the production of carrier-bound active agents by adding an adequate amount of an anionic detergent to form a precipitate, isolating the precipitate and dissolving it again in an organic solvent.
  • This homogeneous solution which contains a complex between the anionic detergent and the active agent can then be used to embed or disperse the active agent in a solid matrix.
  • WO 94/08599 mentions that a complex of the protein with an anionic detergent can be formed and the active agent can be released from it for the controlled release of a protein.
  • This invention provides a composition
  • a composition comprising an aqueous buffered solution having a protein and an amphiphilic agent dissolved therein, wherein the protein is selected from the group consisting of hedgehog proteins, bone morphogenetic proteins, growth factors, erythropoietin, thrombopoietin, G-CSF, interleukins, and interferons; the protein is at a concentration of from 0.1 to 10 mg/ml in the solution; the amphiphilic compound is at a concentration of at least 0.001 to 0.1% weight per volume in the solution; the solution is buffered with a buffer to a pH of from 4 to 10 when measured at a temperature of from 4° C. to 30° C.; and the buffer is present in the solution in a concentration of from 10 to 500 mmol/liter in the solution.
  • the protein is selected from the group consisting of hedgehog proteins, bone morphogenetic proteins, growth factors, erythropoietin, thrombopoie
  • compositions in accordance with this invention improve the activity of the cell surface active protein contained therein. It is believed that the increase in biological activity results from the formation of an ionic complex between the protein and the amphiphilic compound. Accordingly, the invention further provides a method of increasing the activity of cell surface active proteins. In addition, the invention provides solid storage forms of such proteins and uses of the composition of this invention.
  • the compositions of this invention can be used in the same way as the proteins contained therein are used, for example in in vitro assays, diagnostic methods and as therapeutic substances.
  • FIG. 1 shows the dependency of the induction of alkaline phosphatase in a cell test by shh on increasing concentrations of deoxycholate.
  • FIG. 2 shows the dependency of aggregate formation on the concentration of deoxycholate.
  • composition preferably a pharmaceutical composition, containing a pharmaceutically effective 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, in addition, an amphiphilic compound, said polypeptide and said amphiphilic compound forming an ionic complex, whereby the forming of the complex does not enhance the solubility of said polypeptide.
  • the composition does not contain any organic solvent. For storage purposes the composition can be lyophilized.
  • the polypeptide and the amphiphilic compound are individually soluble at the concentrations used in aqueous, preferably buffered solutions, and it is only the combination of the two substances that results in the formation of a complex by means of ionic interactions which hydrophobizes the polypeptide and thus worsens or at least does not improve its water-solubility. It has surprisingly turned out that in this manner the activity of such polypeptides can be significantly improved.
  • the amount and ratio of the amphiphilic compound and the polypeptide are selected preferably in such a way that the aqueous composition containing the ionic complex is a clear solution. If the complex formation between the polypeptide and the amphiphilic compound results in turbidity, then the solution is filtered to give a turbidity-free solution, if the composition will be used directly as a solution for administration to a patient. If the composition will be immobilized on a carrier prior to administration to the patient, turbidity need not be avoided.
  • the pharmaceutically effective polypeptide is a polypeptide which can be present in an ionic form and which is recognized and bound by a cell surface receptor (extracellular receptor) to develop its biological activity.
  • a cell surface receptor extracellular receptor
  • Such polypeptides are growth factors (e.g. NGF, TGF-13, FGF, GDF, insulin-like growth factors), erythropoietin, thrombopoietin, G-CSF, interferons such as Interferon- ⁇ 2b, interleukins such as Interleukin-2 or Interleukin-12, bone morphogenetic proteins such as BMP-2, or hedgehog proteins such as sonic, indian or desert hedgehog proteins. Especially preferred are hedgehog proteins.
  • Polypeptides are preferably used which have an activity (therapeutic effect and/or protein activity in vitro) that is increased preferably 10-fold or more in the complex according to the invention compared to the uncomplexed form.
  • the ionic form of the polypeptide can be obtained by its being present in an environment which advantageously differs by at least an 0.5 pH unit from its pK value.
  • amphiphilic compound according to the invention is to be understood as an anionic, zwitterionic or cationic hydrophobic surfactant, a fatty acid, an alkyl sulfonate or a lipid.
  • any amphiphilic compound can be utilized.
  • Preferred anionic surfactants are anionic detergents such as steroidic tensides like deoxycholates, cholates, taurocholates, taurodeoxcholates, dehydrocholates (useful for cationic polypeptides); preferred zwitterionic surfactants are CHAPS (3 [(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate) and Zwittergent® (N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate); and preferred cationic detergents are cetyltrimethylammonium bromide or dodecylammonium chloride (useful for anionic polypeptides); preferred fatty acids are fatty acids such as palmitic acid (useful for cationic polypeptides).
  • Preferred alkyl sulfates are alkyl sulfonates such as decyl sulfonate (useful for cationic polypeptides); and preferred lipids are lipids such as phosphatidyl serine and phosphatidate (useful for cationic polypeptides).
  • the amphiphilic compound is added to the composition under conditions which hydrophobise the polypeptide and therefore reduce, or at least do not improve, the water-solubility of the polypeptide. It is important that according to the invention a water-soluble ionic complex between the polypeptide and amphiphilic compound is formed in this process.
  • the ratio of polypeptide to amphiphilic compound in the complex depends on the pH value used and the pK values of the two substances and on the concentration ratio.
  • a pH value is preferably used which differs by at least one half pH unit from the pK values of the polypeptide and of the auxiliary substance. The more amphiphilic compound is added, the more amphiphilic compound binds to the polypeptide and the more hydrophobic the complex becomes.
  • a non-ionic detergent such as of a polyoxamer like Tween® can, however, at least partly restore the water-solubility of the complex, or the composition can be filtered, if necessary.
  • the non-ionic detergent can also be present at concentrations which lead to micelle formation. It must be noted that the type and concentration of the amphiphilic compound are selected such that, especially with proteins as a polypeptide, the molecular structure of the polypeptide is retained in its natural active form and thus the activity of the polypeptide is not reduced.
  • amphiphilic compound usually a 10-fold molar excess of amphiphilic compound is sufficient for this purpose.
  • a protein amount of 5 ⁇ g protein per ml 0.001 to 0.05% (weight per volume) of amphiphilic compound are added.
  • the protein is a hedgehog protein in a concentration of about 1 mg/ml in phosphate buffer solution, pH 7.4, and the amphiphilic agent is cholate, deoxycholate, taurocholate or taurodeoxycholate
  • the optimal concentration of the amphiphilic agent is from 0.001 to 0.1% (w/v) in the solution.
  • the amphiphilic agent is phosphatidate, phosphatidylserine or sodium palmitate
  • the optimal concentration of the amphiphilic agent is from 0.001 to 0.05% (w/v) in the solution.
  • a denaturing surfactant such as sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • amphiphilic compounds like SDS can also form micelles at higher concentrations in addition to the desired complexes according to the invention which then in turn increase the solubility of the polypeptide.
  • Whether the amphiphilic compound causes an undesired denaturation of the polypeptide can be easily determined by methods familiar to a person skilled in the art. Such methods are for example activity determination or physicochemical methods for checking the structure such as IR, CD and fluorescence spectroscopy.
  • an aqueous water-soluble pharmaceutical composition in the sense of the invention is to be understood as a composition which essentially comprises no insoluble particles which contain the pharmaceutically effective polypeptide.
  • an aqueous pharmaceutical composition is to be understood according to the invention as a composition which does not have visible turbidity.
  • Such soluble compositions are possible when the ionic complex according to the invention is completely water-soluble at the concentrations used of polypeptide and surfactant or undissolved complex is removed by filtration.
  • the aqueous composition does not contain in addition organic solvents.
  • it might be necessary, for the manufacture of the compositions to solve such amphiphilic compounds like fatty acids in a small amount of organic solvent (up to 5%, preferably up to 1% of the volume of the composition).
  • An additional subject matter of the invention is a process for producing an aqueous pharmaceutical composition according to the invention which is characterized in that a pharmaceutically effective polypeptide and an amphiphilic compound which worsens or at least does not improve the water-solubility of the pharmaceutically effective polypeptide are combined in such a concentration ratio and at such a pH value that an ionic complex forms between the polypeptide and auxiliary substance by ionic interaction.
  • a further subject matter of the invention is the use of the pharmaceutical composition according to the invention for a systemic or local administration to the body of humans or mammals.
  • 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).
  • the activity of hedgehog proteins can be increased to a considerable extent by forming an ionic complex between a hedgehog protein and an amphiphilic compound.
  • an increase in the activity of the hedgehog protein (compared to a recombinant hedgehog protein produced in E. coli ) by 10-fold or more is obtained.
  • a preferred subject matter of the invention is a pharmaceutical composition containing a complex of a hedgehog protein and an amphiphilic compound formed by ionic interactions wherein the compound is present at a concentration which worsens or at least does not improve the solubility of the said hedgehog protein.
  • Hedgehog (hh) proteins are understood as a family of secreted signal proteins which are responsible for the formation of numerous structures in embryogenesis (J. C. Smith, Cell 76 (1994) 193-196, N. Perrimon, Cell 80 (1995) 517-520, C. Chiang et al., Nature 83 (1996) 407, M. J. Bitgood et al., Curr. Biol. 6 (1996) 296, A. Vortkamp et al., Science 273 (1996) 613, C. J. Lai et al., Development 121 (1995) 2349).
  • hh family is composed of at least three members namely sonic, indian and desert hh (Shh, Ihh, Dhh; M. Fietz et al., Development (Suppl.) (1994) 43-51).
  • Sonic, indian or desert hh are particularly preferably used (Fietz M. et al., Development (Suppl.) (1994) 43-51).
  • a hh protein having a sequence described in the EMBL data bank under the No. L38518 is preferably used.
  • Proteins of the hedgehog family have a pronounced homology in their amino acid sequence which is why it is also preferable to express those nucleic acids which code for hedgehog proteins that are 80% or more homologous to the above-mentioned sequence of sonic hedgehog protein.
  • Hedgehog proteins are preferably used as they are for example described in the International Application No. WO 99/28454 and in European Patent Application No. 99108032.6.
  • the human sonic hedgehog precursor protein is composed of the amino acids 1-462 of the sequence described in the EMBL databank under No. L38518.
  • the amino acids 1-23 represent the signal peptide
  • the amino acids 24-197 represent the mature signal domain
  • the amino acids 32-197 represent the signal domain shortened by eight amino acids
  • the amino acids 198-462 represent the autoprocessed C-terminal domain after autoproteolytic cleavage.
  • 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 cartilage cell induction.
  • Solutions of the hedgehog proteins at high concentrations are necessary to prepare carrier matrices that are coated or embedded with the complexes according to the invention in such a manner that they have an adequate pharmaceutical efficacy for a local application. It has turned out that carriers that can be used pharmaceutically should preferably contain a concentration of the hedgehog protein of 0.1-10 mg/ml carrier and more. Hedgehog proteins are intrinsically poorly soluble. It has, however, surprisingly turned out that the solubility of hedgehog proteins is drastically increased and the stability of hh proteins is improved at low concentrations ( ⁇ 1 mg/ml or less) in solutions which contain arginine or argininium ions. It is therefore preferable to add arginine or argininium ions to the aqueous solution and to the carrier bound complex.
  • Activity of the hedgehog protein within the sense of the invention is understood as the activity of alkaline phosphatase (stimulation of the expression of alkaline phosphatase) which the polypeptide can induce in mammalian cells (activity in the alkaline phosphatase test).
  • alkaline phosphatase stimulation of the expression of alkaline phosphatase
  • mammalian cells activity in the alkaline phosphatase test.
  • a mouse fibroblast cell line is cultured in a medium which contains foetal calf serum.
  • sterile filtered sample is added, the cells are lysed after ca. 5 days and alkaline phosphatase is determined in the cell lysate by means of the cleavage of a chromogenic substrate (pNP, p-nitrophenyl) (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 systemically or 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 transforming growth factor family (TGF- ⁇ , GDF). These other proteins can also, but do not have to be, present in the complexes according to the invention.
  • BMPs bone morphogenetic proteins
  • IGF-I or II insulin-like growth factors
  • TGF- ⁇ , GDF transforming growth factor family
  • a further subject matter of the invention is a process for the production of a preferably water-soluble pharmaceutical composition of a hedgehog protein by combining the said hedgehog protein with an amphiphilic compound under conditions which allow an ionic complex to form between the hedgehog protein and the amphiphilic compound.
  • An additional subject matter of the invention is the use of such a complex of a hedgehog protein 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 suitable additional pharmaceutical auxiliary substances, preferably in a buffered aqueous solution.
  • the hedgehog complex according to the invention is present in a mixture of a dissolved and precipitated form or only in a precipitated form which enables a delayed release of the hedgehog protein or a local application at the site of action in vivo. Release of the protein at the site of action is slower from this mixture than from a completely dissolved pharmaceutical formulation.
  • auxiliary substances such as sodium chloride, sugars or sugar alcohols (mannitol, sucrose, lactose, trehalose, preferably 20-100 mg/ml) or amino acids such as glycine or arginine, methionine, cysteine as well as antioxidants such as EDTA, citrate, thioglycerol, acetylcysteine, polyethylene glycol (1-10% by weight), anti-inflammatory agents, local anaesthetics, antibiotics and/or stabilizers.
  • auxiliary substances such as sodium chloride, sugars or sugar alcohols (mannitol, sucrose, lactose, trehalose, preferably 20-100 mg/ml) or amino acids such as glycine or arginine, methionine, cysteine as well as antioxidants such as EDTA, citrate, thioglycerol, acetylcysteine, polyethylene glycol (1-10% by weight), anti-inflammatory agents, local anaesthetics
  • a pharmaceutical composition of the hedgehog protein according to the invention containing suramin is preferred and this can be used advantageously.
  • the pharmaceutical composition can contain additional pharmaceutical auxiliary substances and is preferably lyophilized.
  • the pharmaceutical composition contains hedgehog protein at a concentration of 0.1-10 mg/ml, preferably 0.1-5 mg/ml. In a more specific embodiment, the concentration of the hedgehog protein is from 1-5 mg/ml.
  • the pharmaceutical composition additionally contains a pharmaceutically acceptable buffer which is biocompatible, preferably in a range between pH 4 and pH 10, particularly preferably in a range between pH 6 and 8.
  • concentration of the buffer is preferably 10-500 mmol/l, more preferably 10-100 mmol/l. It is expedient to select the salt concentrations such that they do not interfere with the complex formation due to high ionic strength.
  • the pharmaceutical composition contains the complex according to the invention embedded in a carrier which is biocompatible and can for example be used as an implant.
  • the carrier is preferably a polymer which
  • Such polymers are, for example, hyaluronic acid, collagen, alginate or organic polymers such as PLGA (copolymer of polylactic and glycolic acid) or derivatives thereof. If the complex is embedded in a carrier it is not necessary that the complex is completely soluble in solution like it is useful for the aqueous pharmaceutical composition described above. As the carrier bound complex is applied locally in the body, preferably as a complex of hedgehog polypeptide in bone or cartilage, it is slowly released in soluble form from the complex, thus developing its desired biological effect.
  • a further subject matter of the invention is the use of the pharmaceutical composition according to the invention which is immobilized on (bound reversibly to) a biocompatible carrier for local application on the human body or on animals.
  • biocompatible carriers are for example hyaluronic acid, collagen, alginate or organic polymers such as PLGA or derivatives thereof.
  • the complex according to the invention is preferably localized on a biocompatible carrier wherein the carrier can release the complex locally in vivo in an active form.
  • a biocompatible carrier wherein the carrier can release the complex locally in vivo in an active form.
  • Such formulations are especially suitable for the repair of bone and cartilage defects but can also be used to repair neuronal defects or for a systemic delivery.
  • the pharmaceutical composition according to the invention preferably contains a polymer which essentially acts as a structural substance which preferably also has an adhesion function for cells.
  • a structural substance is for example collagen.
  • the pharmaceutical composition according to the invention is used to reduce systemic side-effects outside the desired site of action when administered locally.
  • the local administration of a pharmaceutically effective polypeptide which is not completely immobilized or does not have an extremely short local half-life can lead to spreading of the polypeptide or at least a part of it beyond the desired site of action where it leads to undesired systemic actions. These undesired systemic effects can be considerably reduced or even avoided by the invention.
  • the method is suitable for polypeptides which have a 10-fold or more increased activity in the ionic complex as compared to the uncomplexed form, wherein the complex has a lower solubility in a buffered aqueous solution than the uncomplexed polypeptide.
  • Such polypeptides are preferably hedgehog proteins, cytokines and growth factors such as NGF.
  • an ionic complex of the polypeptide and the amphiphilic compound is applied preferably locally in this method in such an amount 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 such that when the complex dissociates, which for example occurs when it is diluted 10- to 20-fold under physiological conditions e.g. in blood, the activity of the polypeptide is then only 20% or less of the therapeutic dose.
  • the pharmaceutically effective polypeptide exhibits its full therapeutic effect such as bone growth locally at the desired site of action when the polypeptide is a bone growth factor such as a cytostatic or apoptosis-inducing effect when the polypeptide is a tumoricidal agent.
  • the complex diffuses from the site of action, the complex is diluted under the physiological conditions prevailing outside the site of action which leads to dissociation. This results in a decrease of 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 subject matter 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 such an amount that the complexed polypeptide exhibits an activity which corresponds to its therapeutic dose whereby the same amount of polypeptide in an uncomplexed form would exhibit an activity of 20% or less of the therapeutic dose.
  • a further subject matter 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 of an amphiphilic compound formed by ionic interaction is used as an essential component, in which the compound is present at a concentration which worsens the water-solubility of the pharmaceutically effective polypeptide and the complex is administered in an amount in which the complexed polypeptide exhibits an activity which corresponds to its therapeutic dose whereas the same amount of polypeptide in an uncomplexed form would exhibit an activity of 20% or less of the therapeutic dose.
  • this invention provides a method for increasing the activity of a cell surface active protein, comprising: a) dissolving the protein in an aqueous solution; and b) adjusting the aqueous solution with an amphiphilic compound and a buffer so that the aqueous solution having the protein dissolved therein contains the protein at a concentration of from 0.1 to 10 mg/ml; contains the amphiphilic compound at a concentration of at least 0.001 to 0.1% weight per volume; is buffered with the buffer to a pH of from 4 to 10 when measured at a temperature of from 4° C. to 30° C.; and contains the buffer in a concentration of from 10 to 500 mmol/liter.
  • cell surface active proteins include hedgehog proteins, bone morphogenetic proteins, growth factors, erythropoietin, thrombopoietin, G-CSF, interleukins, and interferons.
  • 5000 cells of the murine mesenchymal plutipotent line C3H10T1/2 (ATCC CCL-226) are sown in each well of a 96-well microtitre plate.
  • the cells are in DMEM, 2 mM glutamine, 100 IU penicillin/ml, 100 ⁇ g streptomycin/ml and 10% foetal calf serum.
  • the medium is replaced by medium which contains human shh (0, 5 or 50 ⁇ g/ml) in different formulations (0, 0.00016, 0.00052, 0.0013, 0.0019 or 0.01% sodium deoxycholate), or the various hedgehog formulations are added directly.
  • the test is stopped after 5 days.
  • the supernatants are decanted and the cells are washed once with PBS.
  • the cells are lysed in 50 ⁇ l 0.1% Triton® X-100 and frozen at ⁇ 20° C. After thawing, 25 ⁇ l aliquots are used for protein determination and to determine the activity of alkaline phosphatase.
  • reaction buffer (Sigma 221) is added to the mixture.
  • a substrate capsule (Sigma 104-40) is dissolved in 10 ml H 2 O, then 100 ⁇ l is added by pipette to the test mixture.
  • the absorbance is measured at 405 nm.
  • alkaline phosphatase converts p-nitrophenylphosphate to p-nitrophenyl (pNP).
  • the measured absorbances are converted into nmol pNP by means of standard curves.
  • FIG. 1 The activities of various hedgehog formulations in nmol pNP/min/mg protein are plotted in FIG. 1 . This shows that, at the same protein concentrations, the activities of the examined hedgehog formulations increased considerably with increasing deoxycholate concentrations.
  • Recombinant human sonic hedgehog protein (dimer, 0.8 mg/ml in 50 mM Tris-Cl, pH 7.4 or in 0.1% Tween 80, 50 mM Tris-Cl, pH 7.4) is admixed with increasing concentrations of sodium deoxycholate.
  • the absorbance at 360 nm is measured as an indicator for turbidity (formation of water-insoluble aggregates composed of ionic protein-detergent complexes). It is clear from FIG. 2 that the transition to water-insoluble aggregates occurs above ca. 0.04% Na deoxycholate. The formation of water-insoluble aggregates can be largely prevented in the presence of 0.1% Tween 80.
  • the stated absorbances are not corrected for dilution.
  • NGF bioactivity was determined by morphometric analysis of dorsal root ganglion (DRG) neurons developing in vitro. Briefly, lumbar DRG's were dissected from E7-E8 embryonic chickens, freed from surrounding connective tissue and dissociated by triturgation through a fire polished pasteur pipette following digestion with 0.1% trypsin for 20 min at 37° C. Contaminating cells, such as fibroblasts were removed by preplating the entire cell preparation onto plastic tissue culture dishes for 2 h. Under these conditions neurons do not attach to the substrate, while fibroblasts and other non-neuronal cells adhere to the tissue culture plastic.
  • DRG dorsal root ganglion
  • “Clean” neurons were harvested by collecting the supernatant and plated onto poly-ornithine/laminin coated plastic dishes (48 wells) at a density of 10,000 cells/well in HAM's F14 medium containing 5% FBS. A dose-response curve for NGF was titrated from approximately 1 pg/ml to 15 ng/ml. Neurotrophic activity was quantified by counting viable differentiated neurons that developed neurites larger than twice the diameter of the perikaryon following 48 h of incubation with different NGF formulations. Data were plotted as mean number of double determinations of differentiated neurons vs. concentration of NGF test formulation and halfmaximal stimulatory activities of NGF (EC50) in several different formulations were determined (Table 1). TABLE 1 Halfmaximal stimulatory activity of NGF formulations (EC50) Formulation EC50 (pg/ml) NGF (no additive) 75 NGF (0.006% sodium deoxycholate) 17 NGF (0.02% sodium deoxycholate) 10
  • aqueous solutions of 5 mg/ml or 1 mg/ml Hshh (human sonic hedgehog protein) in 50 mmol/l Tris buffer, pH 7.4, are dialysed against formulation solution without deoxycholate for 24 h at 4° C.
  • sodium deoxycholate is added from a stock solution while stirring to obtain an aqueous pharmaceutical composition of 1 mg/ml or 5 mg/ml Hshh in formulation solution.
  • the solution is sterile filtered and stored at 4° C. 0.05 to 2 ml of the solution is used for the injection into man or animals.
  • the solution is sterile filtered and stored at 4° C. 0.05 to 2 ml of the solution is used for the injection into man or animals.
  • Formulation solution NaCl: 150 mmol/l Sodium phosphate buffer: 10 mmol/l Phosphatidate: 0.01% (w/v) pH: 7.4 NaCl: 100 mmol/l Sodium phosphate buffer: 10 mmol/l Phosphatidylserine: 0.01% (w/v) pH: 7.4 NaCl: 150 mmol/l Potassium phosphate buffer: 20 mmol/l Sodium palmitate: 0.01% (w/v) pH: 7.4 NaCl: 150 mmol/l Sodium phosphate buffer: 10 mmol/l Sodium cholate 0.05% (w/v) pH: 7.4 NaCl: 100 mmol/l Sodium phosphate buffer: 10 mmol/l Sodium taurodeoxycholate 0.05% (w/v) pH: 7.4 Na
  • aqueous solution of 0.4 mg/ml BMP-2 are dialysed against 500 mmol/l arginine in 10 mmol/l potassium phosphate buffer, pH 6.0, for 24 h at 4° C. After dialysis, 0.01 g (palmitate) or 0.05 g (deoxycholate or taurodeoxycholate) is added from a stock solution while stirring to obtain an aqueous pharmaceutical composition of 0.4 mg/ml BMP in formulation solution. The solution is sterile filtered and stored at 4° C. 0.05 to 2 ml of the solution is used for the injection into man or animals.
  • Formulation solution Arginine 500 mmol/l Potassium phosphate buffer: 10 mmol/l Sodium deoxycholate: 0.05% (w/v) pH: 6.0 Arginine 500 mmol/l Potassium phosphate buffer: 10 mmol/l Sodium palmitate: 0.01% (w/v) pH: 6.0 Arginine 500 mmol/l Potassium phosphate buffer: 10 mmol/l Sodium taurodeoxycholate: 0.05% (w/v) pH: 6.0
  • aqueous solution of 1 or 2 million IU Interleukin-2 in 50 mmol/l Tris buffer, pH 7.4 are dialysed against formulation solution without the amphiphilic compound for 24 h at 4° C.
  • 0.05 g deoxycholate, 0.01 g phosphatidyl serine or 0.01 g of sodium palmitate is added from stock solutions while stirring to obtain an aqueous pharmaceutical composition of 1 or 2 million IU Interleukin-2 in formulation solution.
  • the solution is sterile filtered and stored at 4° C. 0.05 to 2 ml of the solution is used for the injection into man or animals.
  • Formulation solution NaCl: 150 mmol/l Sodium phosphate buffer: 10 mmol/l Deoxycholate: 0.05% (w/v) pH: 7.4 NaCl: 150 mmol/l Sodium phosphate buffer: 10 mmol/l Phosphatidylserine 0.01% (w/v) pH: 7.4 NaCl: 150 mmol/l Potassium phosphate buffer: 20 mmol/l Sodium palmitate: 0.01% (w/v) pH: 7.4
  • Formulation solution NaCl: 150 mmol/l Sodium phosphate buffer: 10 mmol/l Deoxycholate: 0.05% (w/v) pH: 7.4 NaCl: 100 mmol/l Sodium phosphate buffer: 10 mmol/l Phosphatidylserine: 0.01% (w/v) pH: 7.4 NaCl: 150 mmol/l Potassium phosphate buffer: 20 mmol/l Sodium taurodeoxycholate: 0.05% (w/v) pH: 7.4
  • Human NGF 1 mg/ml Sodium acetate buffer: 100 mmol/l Deoxycholate: 0.05% (w/v) pH: 6.0
  • Human NGF 2 mg/ml Sodium acetate buffer: 100 mmol/l Phosphatidate: 0.01% (w/v) pH: 6.0
  • Human NGF 1 mg/ml Sodium acetate buffer: 100 mmol/l Sodium taurodeoxycholate: 0.05% (w/v) pH: 6.0
  • Example 4.1 An aliquot of formulation solution of Example 4.1 is stirred with 1% (w/v) aqueous sodium alginate stock solution (Pronova Biopolymer, Norway) in such a way that a gelatinous alginate protein mixture is formed. This gel is directly used as an injectable matrix in an amount of 0.05 to 2 ml.
  • Example 6 100 ⁇ l of one of the formulations solutions of Example 6 is added dropwise onto collagen sponges (Helistat, Integra Life Science, USA) with a size of 10 ⁇ 10 ⁇ 3 mm. The loaded carriers are then frozen ( ⁇ 70° C.) and lyophilized. The sponge is used locally for the healing of bone fractures.
  • collagen sponges Helistat, Integra Life Science, USA

Abstract

A water-soluble composition containing a complex of an ionic pharmaceutically effective interleukin, characterized in that said composition contains, in addition, an amphiphilic compound, said interleukin and said amphiphilic compound forming an ionic complex, whereby the forming of the complex does not enhance the solubility of said interleukin, is suitable for increasing the activity of the interleukin and/or for the delayed release of the interleukin.

Description

    PRIORITY TO RELATED APPLICATIONS
  • This application is a continuation of U.S. application Ser. No. 09/953,721, filed Sep. 17, 2001, pending, which is a division of U.S. application Ser. No. 09/385,404, filed Aug. 30, 1999, now abandoned, which claims the benefit of European Application No. 98116494.0, filed Sep. 1, 1998; all of which are hereby incorporated herein by reference in their entirety.
    • BACKGROUND OF THE INVENTION
  • The use of amphiphilic compounds as a drug delivery system is well known in the state of the art (cf. US Patents U.S. Pat. No. 5,650,393; U.S. Pat. No. 5,688,761; U.S. Pat. No. 5,665,328; U.S. Pat. No. 5,124,081; U.S. Pat. No. 5,109,038). Formation of complexes in the form of micelles between surface-active substances and pharmaceutical agents is also known for example for improving the transdermal and transmembrane penetration of the active agent (Tomlinson and 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 than in the ionized state (Cools and Jansen, J. Pharm. Pharmacol. 35 (1983) 689-691). It is also known that peptides which are present in a multiple ionized form at physiological pH values, are also not optimal for transport to the site of action (drug delivery) since charged molecules and in particular polypeptides have a low solubility in lipids (Hirai et al., Int. J. Pharm. 7 (1991) 317-325). It is known from Okada et al., J. Pharm. Sci. 72 (1993) 75-78 that it is advantageous to bind a lipophilic counterion to the ionic part of the agent and thus improve the interaction with the biological membrane in order to facilitate transport of proteins through intestinal membranes. For example Hazzenga and Berner describe an improved method for the transdermal transport of zwitterionic active agents in J. Controlled Release 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. Pharmacodyn. 302 (1989) 18-26 and Aungst, Int. J. Pharm. 33 (1986) 225-234. However, in all these methods the aim was to increase the hydrophobicity of the active agent in order to facilitate its penetration through biological membranes such as skin and deliver said agent into the cell. The surface-active substances are used for this at a concentration which was above the critical micelle concentration (CMC, Womack et al., Biochim. Biophys. Acta 733 (1983) 210). A disadvantage of such methods is that the high concentrations of the surface-active substances that are used have a massive influence on the cell membrane and may damage it.
  • It is known from WO 94/08599 that a homogeneous solution of an active agent can be prepared for the production of carrier-bound active agents by adding an adequate amount of an anionic detergent to form a precipitate, isolating the precipitate and dissolving it again in an organic solvent. This homogeneous solution which contains a complex between the anionic detergent and the active agent can then be used to embed or disperse the active agent in a solid matrix. In addition WO 94/08599 mentions that a complex of the protein with an anionic detergent can be formed and the active agent can be released from it 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, such methods are complicated and lead to inhomogeneous products due to the chemical reaction of the coupling (cf. e.g. Ekrami, H. M. et al., FEBS Letters 371 (1995) 283-286, Pepinski, R. B. et al., J. Biol. Chem. 273 (1998) 14037-14045).
    • SUMMARY OF THE INVENTION
  • This invention provides a composition comprising an aqueous buffered solution having a protein and an amphiphilic agent dissolved therein, wherein the protein is selected from the group consisting of hedgehog proteins, bone morphogenetic proteins, growth factors, erythropoietin, thrombopoietin, G-CSF, interleukins, and interferons; the protein is at a concentration of from 0.1 to 10 mg/ml in the solution; the amphiphilic compound is at a concentration of at least 0.001 to 0.1% weight per volume in the solution; the solution is buffered with a buffer to a pH of from 4 to 10 when measured at a temperature of from 4° C. to 30° C.; and the buffer is present in the solution in a concentration of from 10 to 500 mmol/liter in the solution.
  • The compositions in accordance with this invention improve the activity of the cell surface active protein contained therein. It is believed that the increase in biological activity results from the formation of an ionic complex between the protein and the amphiphilic compound. Accordingly, the invention further provides a method of increasing the activity of cell surface active proteins. In addition, the invention provides solid storage forms of such proteins and uses of the composition of this invention. The compositions of this invention can be used in the same way as the proteins contained therein are used, for example in in vitro assays, diagnostic methods and as therapeutic substances.
    • BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 shows the dependency of the induction of alkaline phosphatase in a cell test by shh on increasing concentrations of deoxycholate.
  • FIG. 2 shows the dependency of aggregate formation on the concentration of deoxycholate.
    • DETAILED DESCRIPTION OF THE INVENTION
  • This invention provides 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, thrombopoietin, G-CSF, interleukins and interferons, characterized in that said composition contains, in addition, an amphiphilic compound, said polypeptide and said amphiphilic compound forming an ionic complex, whereby the forming of the complex does not enhance the solubility of said polypeptide. The composition does not contain any organic solvent. For storage purposes the composition can be lyophilized.
  • In the invention, the polypeptide and the amphiphilic compound are individually soluble at the concentrations used in aqueous, preferably buffered solutions, and it is only the combination of the two substances that results in the formation of a complex by means of ionic interactions which hydrophobizes the polypeptide and thus worsens or at least does not improve its water-solubility. It has surprisingly turned out that in this manner the activity of such polypeptides can be significantly improved.
  • According to the invention the amount and ratio of the amphiphilic compound and the polypeptide are selected preferably in such a way that the aqueous composition containing the ionic complex is a clear solution. If the complex formation between the polypeptide and the amphiphilic compound results in turbidity, then the solution is filtered to give a turbidity-free solution, if the composition will be used directly as a solution for administration to a patient. If the composition will be immobilized on a carrier prior to administration to the patient, turbidity need not be avoided.
  • The pharmaceutically effective polypeptide is a polypeptide which can be present in an ionic form and which is recognized and bound by a cell surface receptor (extracellular receptor) to develop its biological activity. Such polypeptides are growth factors (e.g. NGF, TGF-13, FGF, GDF, insulin-like growth factors), erythropoietin, thrombopoietin, G-CSF, interferons such as Interferon-α2b, interleukins such as Interleukin-2 or Interleukin-12, bone morphogenetic proteins such as BMP-2, or hedgehog proteins such as sonic, indian or desert hedgehog proteins. Especially preferred are hedgehog proteins. Polypeptides are preferably used which have an activity (therapeutic effect and/or protein activity in vitro) that is increased preferably 10-fold or more in the complex according to the invention compared to the uncomplexed form. The ionic form of the polypeptide can be obtained by its being present in an environment which advantageously differs by at least an 0.5 pH unit from its pK value.
  • The amphiphilic compound according to the invention is to be understood as an anionic, zwitterionic or cationic hydrophobic surfactant, a fatty acid, an alkyl sulfonate or a lipid. In accordance with this invention any amphiphilic compound can be utilized. Preferred anionic surfactants are anionic detergents such as steroidic tensides like deoxycholates, cholates, taurocholates, taurodeoxcholates, dehydrocholates (useful for cationic polypeptides); preferred zwitterionic surfactants are CHAPS (3 [(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate) and Zwittergent® (N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate); and preferred cationic detergents are cetyltrimethylammonium bromide or dodecylammonium chloride (useful for anionic polypeptides); preferred fatty acids are fatty acids such as palmitic acid (useful for cationic polypeptides). Preferred alkyl sulfates are alkyl sulfonates such as decyl sulfonate (useful for cationic polypeptides); and preferred lipids are lipids such as phosphatidyl serine and phosphatidate (useful for cationic polypeptides).
  • The amphiphilic compound is added to the composition under conditions which hydrophobise the polypeptide and therefore reduce, or at least do not improve, the water-solubility of the polypeptide. It is important that according to the invention a water-soluble ionic complex between the polypeptide and amphiphilic compound is formed in this process. The ratio of polypeptide to amphiphilic compound in the complex depends on the pH value used and the pK values of the two substances and on the concentration ratio. A pH value is preferably used which differs by at least one half pH unit from the pK values of the polypeptide and of the auxiliary substance. 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 precipitation of the complex and hence the presence of a mixture of soluble and insoluble complex which is no longer completely water-soluble. The addition of a non-ionic detergent such as of a polyoxamer like Tween® can, however, at least partly restore 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 at concentrations which lead to micelle formation. It must be noted that the type and concentration of the amphiphilic compound are selected such that, especially with proteins as a polypeptide, the molecular structure of the polypeptide is retained in its natural active form and thus the activity of the polypeptide is not reduced. Usually a 10-fold molar excess of amphiphilic compound is sufficient for this purpose. Preferably, with a protein amount of 5 μg protein per ml, 0.001 to 0.05% (weight per volume) of amphiphilic compound are added. When the protein is a hedgehog protein in a concentration of about 1 mg/ml in phosphate buffer solution, pH 7.4, and the amphiphilic agent is cholate, deoxycholate, taurocholate or taurodeoxycholate, the optimal concentration of the amphiphilic agent is from 0.001 to 0.1% (w/v) in the solution. When the amphiphilic agent is phosphatidate, phosphatidylserine or sodium palmitate, the optimal concentration of the amphiphilic agent is from 0.001 to 0.05% (w/v) in the solution.
  • If a denaturing surfactant such as sodium dodecyl sulfate (SDS) is for example used according to the invention, this compound can only be used at low concentrations. It is known that SDS denatures proteins at high concentrations which improves the water-solubility of these proteins but in a denatured inactive form. Such amphiphilic compounds like SDS can also form micelles at higher concentrations in addition to the desired complexes according to the invention which then in turn increase the solubility of the polypeptide. Whether the amphiphilic compound causes an undesired denaturation of the polypeptide can be easily determined by methods familiar to a person skilled in the art. Such methods are for example activity determination or physicochemical methods for checking the structure such as IR, CD and fluorescence spectroscopy.
  • An aqueous water-soluble pharmaceutical composition in the sense of the invention is to be understood as a composition which essentially comprises no insoluble particles which contain the pharmaceutically effective polypeptide. In particular an aqueous pharmaceutical composition is to be understood according to the invention as a composition which does not have visible turbidity. Such soluble compositions are possible when the ionic complex according to the invention is completely water-soluble at the concentrations used of polypeptide and surfactant or undissolved complex is removed by filtration. According to the invention, the aqueous composition does not contain in addition organic solvents. Moreover, it might be necessary, for the manufacture of the compositions, to solve such amphiphilic compounds like fatty acids in a small amount of organic solvent (up to 5%, preferably up to 1% of the volume of the composition).
  • An additional subject matter of the invention is a process for producing an aqueous pharmaceutical composition according to the invention which is characterized in that a pharmaceutically effective polypeptide and an amphiphilic compound which worsens or at least does not improve the water-solubility of the pharmaceutically effective polypeptide are combined in such a concentration ratio and at such a pH value that an ionic complex forms between the polypeptide and auxiliary substance by ionic interaction.
  • A further subject matter of the invention is the use of the pharmaceutical composition according to the invention for a systemic or local administration to 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).
  • According to the invention it has surprisingly turned out that the activity of hedgehog proteins can be increased to a considerable extent by forming 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) by 10-fold or more is obtained.
  • Consequently a preferred subject matter of the invention is a pharmaceutical composition containing a complex of a hedgehog protein and an amphiphilic compound formed by ionic interactions wherein the compound is present at a concentration which worsens or at least does not improve the solubility of the said hedgehog protein.
  • Hedgehog (hh) proteins are understood as a family of secreted signal proteins which are responsible for the formation of numerous structures in embryogenesis (J. C. Smith, Cell 76 (1994) 193-196, N. Perrimon, Cell 80 (1995) 517-520, C. Chiang et al., Nature 83 (1996) 407, M. J. Bitgood et al., Curr. Biol. 6 (1996) 296, A. Vortkamp et al., Science 273 (1996) 613, C. J. Lai et al., Development 121 (1995) 2349). During its biosynthesis a 20 kD N-terminal domain and a 25 kD C-terminal domain are obtained after cleavage of the signal sequence and autocatalytic cleavage. In its natural form the N-terminal domain is modified with cholesterol and palmitoyl (J. A. Porter et al., Science 274 (1996) 255-259 and Pepinski et al., J. Biol. Chem. 273 (1998) 14037-14045). In higher life-forms the hh family is composed of at least three members namely sonic, indian and desert hh (Shh, Ihh, Dhh; M. Fietz et al., Development (Suppl.) (1994) 43-51). Differences in the activity of hedgehog proteins that were produced recombinantly were observed after production in prokaryotes and eukaryotes (M. Hynes et al., Neuron 15 (1995) 35-44 and T. Nakamura et al., Biochem. Biophys. Res. Comm. 237 (1997) 465-469).
  • Sonic, indian or desert hh are particularly preferably used (Fietz M. et al., Development (Suppl.) (1994) 43-51). A hh protein having a sequence described in the EMBL data bank under the No. L38518 is preferably used. Proteins of the hedgehog family have a pronounced homology in their amino acid sequence which is why it is also preferable to express those nucleic acids which code for hedgehog proteins that are 80% or more homologous to the above-mentioned sequence of sonic hedgehog protein. Hedgehog proteins are preferably used as they are for example described in the International Application No. WO 99/28454 and in European Patent Application No. 99108032.6.
  • The human sonic hedgehog precursor protein is composed of the amino acids 1-462 of the sequence described in the EMBL databank under No. L38518. The amino acids 1-23 represent the signal peptide, the amino acids 24-197 represent the mature signal domain, the amino acids 32-197 represent the signal domain shortened by eight amino acids and the amino acids 198-462 represent the autoprocessed C-terminal domain after autoproteolytic cleavage.
  • Pharmaceutical 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 cartilage cell induction.
  • Solutions of the hedgehog proteins at high concentrations are necessary to prepare carrier matrices that are coated or embedded with the complexes according to the invention in such a manner that they have an adequate pharmaceutical efficacy for a local application. It has turned out that carriers that can be used pharmaceutically should preferably contain a concentration of the hedgehog protein of 0.1-10 mg/ml carrier and more. Hedgehog proteins are intrinsically poorly soluble. It has, however, surprisingly turned out that the solubility of hedgehog proteins is drastically increased and the stability of hh proteins is improved at low concentrations (<1 mg/ml or less) in solutions which contain arginine or argininium ions. It is therefore preferable to add arginine or argininium ions to the aqueous solution and to the carrier bound complex.
  • Activity of the hedgehog protein within the sense of the invention is understood as the activity of alkaline phosphatase (stimulation of the expression of alkaline phosphatase) which 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 which contains foetal calf serum. Subsequently sterile filtered sample is added, the cells are lysed after ca. 5 days and alkaline phosphatase is determined in the cell lysate by means of the cleavage of a chromogenic substrate (pNP, p-nitrophenyl) (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 systemically or 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 transforming growth factor family (TGF-β, GDF). These other proteins can also, but do not have to be, present in the complexes according to the invention.
  • Hence a further subject matter of the invention is a process for the production of a preferably water-soluble pharmaceutical composition of a hedgehog protein by combining the said hedgehog protein with an amphiphilic compound under conditions which allow an ionic complex to form between the hedgehog protein and the amphiphilic compound.
  • An additional subject matter of the invention is the use of such a complex of a hedgehog protein 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 suitable additional pharmaceutical auxiliary substances, preferably in a buffered aqueous 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 which enables a delayed release of the hedgehog protein or a local application at the site of action in vivo. 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 pharmaceutical composition to add auxiliary substances such as sodium chloride, sugars or sugar alcohols (mannitol, sucrose, lactose, trehalose, preferably 20-100 mg/ml) or amino acids such as glycine or arginine, methionine, cysteine as well as antioxidants such as EDTA, citrate, thioglycerol, acetylcysteine, polyethylene glycol (1-10% by weight), anti-inflammatory agents, local anaesthetics, antibiotics and/or stabilizers.
  • In a further preferred embodiment a pharmaceutical composition of the hedgehog protein according to the invention containing suramin is preferred and this can be used advantageously.
  • The pharmaceutical composition can contain additional pharmaceutical auxiliary substances and is preferably lyophilized.
  • In a preferred embodiment the pharmaceutical composition contains hedgehog protein at a concentration of 0.1-10 mg/ml, preferably 0.1-5 mg/ml. In a more specific embodiment, the concentration of the hedgehog protein is from 1-5 mg/ml.
  • In a preferred embodiment the pharmaceutical composition additionally contains a pharmaceutically acceptable buffer which is biocompatible, preferably in a range between pH 4 and pH 10, particularly preferably in a range between pH 6 and 8. The concentration of the buffer is preferably 10-500 mmol/l, more preferably 10-100 mmol/l. It is expedient to select the salt concentrations such that they do not interfere with the complex formation due to high ionic strength.
  • In another embodiment of the invention the pharmaceutical composition contains the complex according to the invention embedded in a carrier which is biocompatible and can for example be used as an implant. The carrier is preferably a polymer which
      • does not denature the hedgehog protein when it is embedded in the carrier,
      • has an average molecular weight of at least 10,000 Da.
  • Such polymers are, for example, hyaluronic acid, collagen, alginate or organic polymers such as PLGA (copolymer of polylactic and glycolic acid) or derivatives thereof. If the complex is embedded in a carrier it is not necessary that the complex is completely soluble in solution like it is useful for the aqueous pharmaceutical composition described above. As the carrier bound complex is applied locally in the body, preferably as a complex of hedgehog polypeptide in bone or cartilage, it is slowly released in soluble form from the complex, thus developing its desired biological effect.
  • A further subject matter of the invention is the use of the pharmaceutical composition according to the invention which is immobilized on (bound reversibly to) a biocompatible carrier for local application on the human body or on animals. Such biocompatible carriers are for example hyaluronic acid, collagen, alginate or organic polymers such as PLGA or derivatives thereof.
  • The complex according to the invention is preferably localized on a biocompatible carrier wherein the carrier can release the complex locally in vivo in an active form. Such formulations are especially suitable for the repair of bone and cartilage defects but can also be used to repair neuronal defects or for a systemic delivery.
  • The pharmaceutical composition according to the invention preferably contains a polymer which essentially acts as a structural substance which preferably also has an adhesion function for cells. Such a 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. The local administration of a pharmaceutically effective polypeptide which is not completely immobilized or does not have an extremely short local half-life can lead to spreading of the polypeptide or at least a part of it beyond the desired site of action where it leads to undesired systemic actions. These undesired systemic effects can be considerably reduced or even avoided by the invention. The method is suitable for polypeptides which have a 10-fold or more increased activity in the ionic complex as compared to the uncomplexed form, wherein the complex has a lower solubility in a buffered aqueous solution than the uncomplexed polypeptide. Such 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 is applied preferably locally in this method in such an amount 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 such that when the complex dissociates, which for example occurs when it is diluted 10- to 20-fold under physiological conditions e.g. in blood, the activity of the polypeptide is then only 20% or less of the therapeutic dose. Hence in such a local application of the complex according to the invention the pharmaceutically effective polypeptide exhibits its full therapeutic effect such as bone growth locally at the desired site of action when the polypeptide is a bone growth factor such as a cytostatic or apoptosis-inducing effect when the polypeptide is a tumoricidal agent. When the complex diffuses from the site of action, the complex is diluted under the physiological conditions prevailing outside the site of action which leads to dissociation. This results in a decrease of 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 subject matter 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 such an amount that the complexed polypeptide exhibits an activity which corresponds to its therapeutic dose whereby the same amount of polypeptide in an uncomplexed form would exhibit an activity of 20% or less of the therapeutic dose.
  • A further subject matter 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 of an amphiphilic compound formed by ionic interaction is used as an essential component, in which the compound is present at a concentration which worsens the water-solubility of the pharmaceutically effective polypeptide and the complex is administered in an amount in which the complexed polypeptide exhibits an activity which corresponds to its therapeutic dose whereas the same amount of polypeptide in an uncomplexed form would exhibit an activity of 20% or less of the therapeutic dose.
  • In accordance with the composition described herein this invention provides a method for increasing the activity of a cell surface active protein, comprising: a) dissolving the protein in an aqueous solution; and b) adjusting the aqueous solution with an amphiphilic compound and a buffer so that the aqueous solution having the protein dissolved therein contains the protein at a concentration of from 0.1 to 10 mg/ml; contains the amphiphilic compound at a concentration of at least 0.001 to 0.1% weight per volume; is buffered with the buffer to a pH of from 4 to 10 when measured at a temperature of from 4° C. to 30° C.; and contains the buffer in a concentration of from 10 to 500 mmol/liter. Examples of such cell surface active proteins include hedgehog proteins, bone morphogenetic proteins, growth factors, erythropoietin, thrombopoietin, G-CSF, interleukins, and interferons.
  • The contents of European Patent Application No. 98116494.0, filed Sep. 1, 1998, are incorporated herein by reference.
  • The following examples, publications and the figures further elucidate the invention, the protective scope of which results from the patent claims. The described methods are to be understood as examples which still describe the subject matter of the invention even after modifications.
    • EXAMPLE 1
  • Analysis of the Activity of Different Hedgehog Formulations in a Cell Test: Induction of Alkaline Phosphatase
  • 5000 cells of the murine mesenchymal plutipotent line C3H10T1/2 (ATCC CCL-226) are sown in each well of a 96-well microtitre plate. The cells are in DMEM, 2 mM glutamine, 100 IU penicillin/ml, 100 μg streptomycin/ml and 10% foetal calf serum. On the next day the medium is replaced by medium which contains 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 formulations are added directly. The test is stopped after 5 days. For this the supernatants are decanted and the cells are washed once with PBS. The cells are lysed in 50 μl 0.1% Triton® X-100 and frozen at −20° C. After thawing, 25 μl aliquots are used for protein determination and to determine the activity of alkaline phosphatase.
  • Protein Determination According to the Instructions of the Manufacturer Pierce.
  • 75 μl redistilled H2O is added to the mixture, then 100 μl BCA protein reagent is added (Pierce Micro BCA, No. 23225). The absorbance is measured at 550 nm after 60 min.
  • Determination of the Alkaline Phosphatase Activity According to the Instructions of the Manufacturer Sigma:
  • 100 μl reaction buffer (Sigma 221) is added to the mixture. A substrate capsule (Sigma 104-40) is dissolved in 10 ml H2O, then 100 μl is added by pipette to the test mixture. The absorbance is measured at 405 nm. During the reaction alkaline phosphatase converts p-nitrophenylphosphate to p-nitrophenyl (pNP). The measured absorbances are converted into nmol pNP by means of standard curves.
  • The activities of various hedgehog formulations in nmol pNP/min/mg protein are plotted in FIG. 1. This shows that, at the same protein concentrations, the activities of the examined hedgehog formulations increased considerably with increasing deoxycholate concentrations.
    • EXAMPLE 2
  • Hydrophobic Ion Pair Titration of Hshh (Dimer)
  • Recombinant human sonic hedgehog protein (dimer, 0.8 mg/ml in 50 mM Tris-Cl, pH 7.4 or in 0.1% Tween 80, 50 mM Tris-Cl, pH 7.4) is admixed with increasing concentrations of sodium deoxycholate. The absorbance at 360 nm is measured as an indicator for turbidity (formation of water-insoluble aggregates composed of ionic protein-detergent complexes). It is clear from FIG. 2 that the transition to water-insoluble aggregates occurs above ca. 0.04% Na deoxycholate. The formation of water-insoluble aggregates can be largely prevented in the presence of 0.1% Tween 80. The stated absorbances are not corrected for dilution.
    • EXAMPLE 3
  • Analysis of NGF Formulations in a Bioactivity Assay: Dorsal Root Ganglion Neuron Development Assay
  • NGF bioactivity was determined by morphometric analysis of dorsal root ganglion (DRG) neurons developing in vitro. Briefly, lumbar DRG's were dissected from E7-E8 embryonic chickens, freed from surrounding connective tissue and dissociated by triturgation through a fire polished pasteur pipette following digestion with 0.1% trypsin for 20 min at 37° C. Contaminating cells, such as fibroblasts were removed by preplating the entire cell preparation onto plastic tissue culture dishes for 2 h. Under these conditions neurons do not attach to the substrate, while fibroblasts and other non-neuronal cells adhere to the tissue culture plastic. “Clean” neurons were harvested by collecting the supernatant and plated onto poly-ornithine/laminin coated plastic dishes (48 wells) at a density of 10,000 cells/well in HAM's F14 medium containing 5% FBS. A dose-response curve for NGF was titrated from approximately 1 pg/ml to 15 ng/ml. Neurotrophic activity was quantified by counting viable differentiated neurons that developed neurites larger than twice the diameter of the perikaryon following 48 h of incubation with different NGF formulations. Data were plotted as mean number of double determinations of differentiated neurons vs. concentration of NGF test formulation and halfmaximal stimulatory activities of NGF (EC50) in several different formulations were determined (Table 1).
    TABLE 1
    Halfmaximal stimulatory activity of NGF formulations (EC50)
    Formulation EC50 (pg/ml)
    NGF (no additive) 75
    NGF (0.006% sodium deoxycholate) 17
    NGF (0.02% sodium deoxycholate) 10
  • These data clearly show that the specific activity of NGF is increased in formulations containing an amphiphilic additive (here: sodium deoxycholate).
    • EXAMPLE 4
  • Pharmaceutical Compositions with Deoxycholate
  • For the production of the pharmaceutical composition 100 ml of an aqueous solution of 5 mg/ml or 1 mg/ml Hshh (human sonic hedgehog protein) in 50 mmol/l Tris buffer, pH 7.4, are dialysed against formulation solution without deoxycholate for 24 h at 4° C. After dialysis, sodium deoxycholate is added from a stock solution while stirring to obtain an aqueous pharmaceutical composition of 1 mg/ml or 5 mg/ml Hshh in formulation solution. The solution is sterile filtered and stored at 4° C. 0.05 to 2 ml of the solution is used for the injection into man or animals.
  • 4.1 Formulation of Ionically Hydrophobized Hedgehog Protein in Aqueous Phosphate Buffered Saline (Low 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 Ionically Hydrophobized Hedgehog Protein in Aqueous Phosphate Buffered Saline (High 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 Ionically Hydrophobized Hedgehog Protein in Low Ionic Strength Phosphate Buffer (Low Sodium Deoxycholate)
    Formulation solution:
    NaCl: 30 mmol/l
    Potassium phosphate buffer: 20 mmol/l
    Sodium deoxycholate: 0.05% (w/v)
    pH: 6.5
  • 4.4 Formulation of Ionically Hydrophobized Hedgehog Protein in Low Ionic Strength Phosphate Buffer (High Sodium Deoxycholate)
    Formulation solution:
    NaCl: 30 mmol/l
    Potassium phosphate buffer: 20 mmol/l
    Sodium deoxycholate: 0.1% (w/v)
    pH: 6.5
    • EXAMPLE 5
  • Pharmaceutical Compositions of Ionically Hydrophobized Hedgehog Protein in Phosphate Buffered Saline with Lipids, Fatty Acids or Steroids
  • For the production of the pharmaceutical composition 100 ml of an aqueous solution of 1 mg/ml or 2 mg/ml Hshh in 50 mmol/l Tris buffer, pH 7.4, are dialysed against formulation solution without lipid, fatty acid or cholate for 24 h at 4° C. After 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 of taurocholate is added from stock solutions while stirring to obtain an aqueous pharmaceutical composition of 1 mg/ml or 2 mg/ml Hshh in formulation solution. The solution is sterile filtered and stored at 4° C. 0.05 to 2 ml of the solution is used for the injection into man or animals.
    Formulation solution:
    NaCl: 150 mmol/l
    Sodium phosphate buffer:  10 mmol/l
    Phosphatidate: 0.01% (w/v)
    pH: 7.4
    NaCl: 100 mmol/l
    Sodium phosphate buffer:  10 mmol/l
    Phosphatidylserine: 0.01% (w/v)
    pH: 7.4
    NaCl: 150 mmol/l
    Potassium phosphate buffer:  20 mmol/l
    Sodium palmitate: 0.01% (w/v)
    pH: 7.4
    NaCl: 150 mmol/l
    Sodium phosphate buffer:  10 mmol/l
    Sodium cholate 0.05% (w/v)
    pH: 7.4
    NaCl: 100 mmol/l
    Sodium phosphate buffer:  10 mmol/l
    Sodium taurodeoxycholate 0.05% (w/v)
    pH: 7.4
    NaCl: 150 mM
    Potassium phosphate buffer:  20 mM
    Sodium taurocholate 0.05% (w/v)
    pH: 7.4
    • EXAMPLE 6
  • Pharmaceutical Compositions of Ionically Hydrophobized Bone Morphogenetic Protein (BMP-2) in Arginine Buffers
  • For the production of the pharmaceutical composition 100 ml of an aqueous solution of 0.4 mg/ml BMP-2 are dialysed against 500 mmol/l arginine in 10 mmol/l potassium phosphate buffer, pH 6.0, for 24 h at 4° C. After dialysis, 0.01 g (palmitate) or 0.05 g (deoxycholate or taurodeoxycholate) is added from a stock solution while stirring to obtain an aqueous pharmaceutical composition of 0.4 mg/ml BMP in formulation solution. The solution is sterile filtered and stored at 4° C. 0.05 to 2 ml of the solution is used for the injection into man or animals.
    Formulation solution:
    Arginine 500 mmol/l
    Potassium phosphate buffer:  10 mmol/l
    Sodium deoxycholate: 0.05% (w/v)
    pH: 6.0
    Arginine 500 mmol/l
    Potassium phosphate buffer:  10 mmol/l
    Sodium palmitate: 0.01% (w/v)
    pH: 6.0
    Arginine 500 mmol/l
    Potassium phosphate buffer:  10 mmol/l
    Sodium taurodeoxycholate: 0.05% (w/v)
    pH: 6.0
    • EXAMPLE 7
  • Pharmaceutical Compositions of Ionically Hydrophobized Interleukin-2 in Phosphate Buffered Saline
  • For the production of the pharmaceutical composition 100 ml of an aqueous solution of 1 or 2 million IU Interleukin-2 in 50 mmol/l Tris buffer, pH 7.4, are dialysed against formulation solution without the amphiphilic compound for 24 h at 4° C. After dialysis, 0.05 g deoxycholate, 0.01 g phosphatidyl serine or 0.01 g of sodium palmitate is added from stock solutions while stirring to obtain an aqueous pharmaceutical composition of 1 or 2 million IU Interleukin-2 in formulation solution. The solution is sterile filtered and stored at 4° C. 0.05 to 2 ml of the solution is used for the injection into man or animals.
    Formulation solution:
    NaCl: 150 mmol/l
    Sodium phosphate buffer:  10 mmol/l
    Deoxycholate: 0.05% (w/v)
    pH: 7.4
    NaCl: 150 mmol/l
    Sodium phosphate buffer:  10 mmol/l
    Phosphatidylserine 0.01% (w/v)
    pH: 7.4
    NaCl: 150 mmol/l
    Potassium phosphate buffer:  20 mmol/l
    Sodium palmitate: 0.01% (w/v)
    pH: 7.4
    • EXAMPLE 8
  • Pharmaceutical Compositions of Ionically Hydrophobized Interferon-Alpha 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-α2b in 50 mmol/l Tris buffer, pH 7.4, are dialysed against formulation solution without the amphiphilic substance for 24 h at 4° C. After dialysis, 0.05 g deoxycholate, 0.01 g phosphatidyl serine or 0.05 g taurodeoxycholate is added from a stock solution to obtain an aqueous pharmaceutical composition of 4 or 40 million IU Interferon-α2b in formulation solution. The solution is sterile filtered and stored at 4° C. 0.05 to 2 ml of the solution is used for the injection into man or animals.
    Formulation solution:
    NaCl: 150 mmol/l
    Sodium phosphate buffer:  10 mmol/l
    Deoxycholate: 0.05% (w/v)
    pH: 7.4
    NaCl: 100 mmol/l
    Sodium phosphate buffer:  10 mmol/l
    Phosphatidylserine: 0.01% (w/v)
    pH: 7.4
    NaCl: 150 mmol/l
    Potassium phosphate buffer:  20 mmol/l
    Sodium taurodeoxycholate: 0.05% (w/v)
    pH: 7.4
    • EXAMPLE 9
  • Pharmaceutical Compositions of Ionically Hydrophobized Human NGF in Acetate Buffers
  • For the production of the pharmaceutical composition to 100 ml of an aqueous solution of 1 or 2 mg/ml human NGF in 100 mmol/l sodium acetate buffer, pH 6.0, are added 0.05 g deoxycholate, 0.01 g phosphatidate or 0.05 g taurodeoxycholate from a stock solution while stirring to obtain aqueous pharmaceutical compositions. The solution is sterile filtered and stored at 4° C. 0.05 to 2 ml of the solution is used for the injection into man or animals.
    Compositions:
    Human NGF:  1 mg/ml
    Sodium acetate buffer: 100 mmol/l
    Deoxycholate: 0.05% (w/v)
    pH: 6.0
    Human NGF:  2 mg/ml
    Sodium acetate buffer: 100 mmol/l
    Phosphatidate: 0.01% (w/v)
    pH: 6.0
    Human NGF:  1 mg/ml
    Sodium acetate buffer: 100 mmol/l
    Sodium taurodeoxycholate: 0.05% (w/v)
    pH: 6.0
    • EXAMPLE 10
  • Production of an Alginate Gel Containing Hedgehog Proteins
  • An aliquot of formulation solution of Example 4.1 is stirred with 1% (w/v) aqueous sodium alginate stock solution (Pronova Biopolymer, Norway) in such a way that a gelatinous alginate protein mixture is formed. This gel is directly used as an injectable matrix in an amount of 0.05 to 2 ml.
    • EXAMPLE 11
  • Production of a Collagen Mixture Containing BMP-2
  • 100 μl of one of the formulations solutions of Example 6 is added dropwise onto collagen sponges (Helistat, Integra Life Science, USA) with a size of 10×10×3 mm. The loaded carriers are then frozen (−70° C.) and lyophilized. The sponge is used locally for the healing of bone fractures.
  • Bibliography
    • Asahina, J., Exp. Cell. Res. 222 (1996) 38-47
    • Aungst, Int. J. Pharm. 33 (1986) 225-234
    • Bitgood, M. J. et al., 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. et al. FEBS Letters 371 (1995) 283-286
    • European Patent Application No. 99108032.6
    • Fietz, M. et al., Development (Suppl) (1994) 43-51
    • Hazzenga and Berner, J. Controlled Release 16 (1991) 77-88
    • Hirai et al., Int. J. Pharm. 7 (1991) 317-325
    • 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, 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. Comm. 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. et al., 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
    • U.S. Pat. No. 5,650,393
    • U.S. Pat. No. 5,109,038
    • U.S. Pat. No. 5,124,081
    • U.S. Pat. No. 5,665,328
    • U.S. Pat. No. 5,688,761
    • Vortkamp, A. et al., Science 273 (1996) 613
    • WO 99/28454
    • Womack et al., Biochim. Biophys. Acta 733 (1983) 210
    • Wozney et al., Cell. Mol. Biol. of Bone, Bone Morphogenetic Proteins and their Gene Expression, (1993) Academic Press Inc. 131-167

Claims (27)

1. A controlled-release pharmaceutical composition comprising an ionic complex precipitate comprising a polypeptide and an amphiphilic compound, wherein the ionic complex is precipitated in an environment which differs by at least 0.5 pH units from both the polypeptide's pK value and the amphiphilic compound's pK value.
2. The controlled-release pharmaceutical composition of claim 1 wherein the complex is embedded in a carrier.
3. The controlled-release pharmaceutical composition of claim 1 wherein the complex is embedded in a biocompatible carrier.
4. The controlled-release pharmaceutical composition of claim 1 wherein the polypeptide is a hedgehog protein.
5. The controlled-release pharmaceutical composition of claim 1 wherein the polypeptide is a bone morphogenetic protein.
6. The controlled-release pharmaceutical composition of claim 1 wherein the polypeptide is a growth factor.
7. The controlled-release pharmaceutical composition of claim 1 wherein the polypeptide is erythropoietin.
8. The controlled-release pharmaceutical composition of claim 1 wherein the polypeptide is thrombopoietin.
9. The controlled-release pharmaceutical composition of claim 1 wherein the polypeptide is G-CSF.
10. The controlled-release pharmaceutical composition of claim 1 wherein the polypeptide is an interleukin.
11. The controlled-release pharmaceutical composition of claim 1 wherein the polypeptide is an interferon.
12. The controlled-release pharmaceutical composition of claim 1 wherein the amphiphilic compound is deoxycholate.
13. The controlled-release pharmaceutical composition of claim 1 wherein the amphiphilic compound is cholate.
14. The controlled-release pharmaceutical composition of claim 1 wherein the amphiphilic compound is taurcholate.
15. The controlled-release pharmaceutical composition of claim 1 wherein the amphiphilic compound is taurodeoxcholate.
16. The controlled-release pharmaceutical composition of claim 1 wherein the amphiphilic compound is dehydrocholate.
17. The controlled-release pharmaceutical composition of claim 1 wherein the amphiphilic compound is 3[(3-cholamidopropyl)dimethylammonio]-1 propane sulfonate.
18. The controlled-release pharmaceutical composition of claim 1 wherein the amphiphilic compound is N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate.
19. The controlled-release pharmaceutical composition of claim 1 wherein the amphiphilic compound is palmitic acid.
20. The controlled-release pharmaceutical composition of claim 1 wherein the amphiphilic compound is alkyl sulfonate.
21. The controlled-release pharmaceutical composition of claim 1 wherein the amphiphilic compound is decyl sulfonate.
22. The controlled-release pharmaceutical composition of claim 1 wherein the amphiphilic compound is phosphatidyl serine.
23. The controlled-release pharmaceutical composition of claim 1 wherein the amphiphilic compound is phosphatidate.
24. A method of treating a disease or condition comprising locally applying the composition of claim 1 to the body of a patient in need of treatment.
25. The method of claim 24 wherein the disease or condition is a defect in bone.
26. The method of claim 24 wherein the disease or condition is a defect in cartilage.
27. The controlled-release pharmaceutical composition of claim 1 wherein the activity of the polypeptide is more active in said ionic complex than when said polypeptide is not in said ionic complex.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070042041A1 (en) * 2005-08-17 2007-02-22 Board Of Trustees Of The University Of Arkansas Drug-surfactant complexes for sustained release
US20090275066A1 (en) * 2006-11-13 2009-11-05 Universite Paris 7 - Denis Diderot Immobilization of membrane porteins onto supports via an amphiphile

Families Citing this family (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050112087A1 (en) * 2003-04-29 2005-05-26 Musso Gary F. Pharmaceutical formulations for sustained drug delivery
US20060193825A1 (en) * 2003-04-29 2006-08-31 Praecis Phamaceuticals, Inc. Pharmaceutical formulations for sustained drug delivery
MXPA05013398A (en) * 2003-06-10 2006-03-09 Lg Life Sciences Ltd Stable, aqueous solution of human erythropoietin, not containing serum albumin.
DK1758590T3 (en) * 2004-05-19 2011-11-21 Los Angeles Biomed Res Inst Use of a detergent for non-surgical removal of fat
US20060127468A1 (en) 2004-05-19 2006-06-15 Kolodney Michael S Methods and related compositions for reduction of fat and skin tightening
US7754230B2 (en) * 2004-05-19 2010-07-13 The Regents Of The University Of California Methods and related compositions for reduction of fat
US20090214625A1 (en) * 2005-07-15 2009-08-27 Mizuo Nakayama Drug delivery patch
JP2007037868A (en) * 2005-08-05 2007-02-15 Transcutaneous Technologies Inc Transdermal administration device and its controlling method
CN101262905A (en) * 2005-09-16 2008-09-10 Tti优而美株式会社 Catheter type iontophoresis apparatus
AU2006299520A1 (en) * 2005-09-30 2007-04-12 Tti Ellebeau, Inc. Iontophoresis device to deliver multiple active agents to biological interfaces
US20080033338A1 (en) * 2005-12-28 2008-02-07 Smith Gregory A Electroosmotic pump apparatus and method to deliver active agents to biological interfaces
WO2007079193A2 (en) * 2005-12-30 2007-07-12 Tti Ellebeau, Inc. Iontophoretic systems, devices, and methods of delivery of active agents to biological interface
WO2007123707A1 (en) * 2006-03-30 2007-11-01 Tti Ellebeau, Inc. Controlled release membrane and methods of use
RU2009103771A (en) * 2006-07-05 2010-08-10 ТиТиАй ЭЛЛЕБО, ИНК. (JP) DELIVERY DEVICE, INCLUDING SELF-ASSEMBLY BRANCHED POLYMERS, AND METHOD OF APPLICATION
US20090306337A1 (en) 2006-07-31 2009-12-10 Novo Nordisk A/S Pegylated, Extended Insulins
KR101729986B1 (en) 2006-09-22 2017-04-25 노보 노르디스크 에이/에스 Protease resistant insulin analogues
US20080193514A1 (en) * 2006-11-02 2008-08-14 Transcu Ltd. Compostions and methods for iontophoresis delivery of active ingredients through hair follicles
EP2123259A1 (en) * 2007-01-16 2009-11-25 Hokkaido University Liposome preparation for iontophoresis having antioxidant component encapsulated therein
WO2008132224A2 (en) 2007-04-30 2008-11-06 Novo Nordisk A/S Method for drying a protein composition, a dried protein composition and a pharmaceutical composition comprising the dried protein
JP5675347B2 (en) * 2007-06-01 2015-02-25 ノボ・ノルデイスク・エー／エス Stable non-aqueous pharmaceutical composition
JP2010187707A (en) * 2007-06-12 2010-09-02 Hokkaido Univ Liposome preparation for iontophoresis comprising insulin encapsulated therein
ES2618073T3 (en) 2008-03-14 2017-06-20 Novo Nordisk A/S Insulin analogs stabilized against proteases
US8691759B2 (en) 2008-03-18 2014-04-08 Novo Nordisk A/S Protease stabilized, acylated insulin analogues
CA2736495A1 (en) * 2008-09-10 2010-03-18 Gregory A. Smith Apparatus and method to dispense hpc-based viscous liquids into porous substrates, e.g., continuous web-based process
WO2012134540A2 (en) * 2010-10-22 2012-10-04 Vanderbilt University Injectable synthetic pur composite
US8101593B2 (en) 2009-03-03 2012-01-24 Kythera Biopharmaceuticals, Inc. Formulations of deoxycholic acid and salts thereof
CN107412754A (en) * 2009-12-22 2017-12-01 塞尔德克斯医疗公司 Vaccine combination
US20120237492A1 (en) 2011-02-18 2012-09-20 Kythera Biopharmaceuticals, Inc. Treatment of submental fat
US8653058B2 (en) 2011-04-05 2014-02-18 Kythera Biopharmaceuticals, Inc. Compositions comprising deoxycholic acid and salts thereof suitable for use in treating fat deposits
JP6224069B2 (en) 2012-03-23 2017-11-01 アミクローブ，インコーポレイテッド Compositions and uses of antibacterial agents with histocompatibility properties
CN104364260B (en) 2012-04-11 2017-02-22 诺和诺德股份有限公司 insulin formulations
AU2014333979B2 (en) 2013-10-07 2018-02-15 Novo Nordisk A/S Novel derivative of an insulin analogue
EP3372247B1 (en) * 2015-11-04 2022-01-05 Postech Academy-Industry Foundation Composition for skin permeation comprising cationic molecular transporter and protein
ES2930149T3 (en) 2016-12-16 2022-12-07 Novo Nordisk As Pharmaceutical compositions containing insulin
RU2019133628A (en) 2017-04-06 2021-05-06 Амикроб, Инк. COMPOSITIONS AND APPLICATIONS OF ANTIMICROBIAL AGENTS FOR LOCAL APPLICATION WITH IMPROVED PERFORMANCE AND SAFETY
US11198831B2 (en) 2019-01-31 2021-12-14 Kvi Llc Lubricant for a device
AR118536A1 (en) * 2019-04-01 2021-10-20 Genentech Inc COMPOSITIONS AND METHODS TO STABILIZE FORMULATIONS CONTAINING PROTEIN

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4604377A (en) * 1984-03-28 1986-08-05 Cetus Corporation Pharmaceutical compositions of microbially produced interleukin-2
US5109038A (en) * 1987-05-11 1992-04-28 Rhone-Poulenc Chimie Polymer particles which carry, implanted on their surface, amphiphilic molecules carrying ion-forming or reactive groups, a process for the preparation of these particles and their use in biological applications
US5124081A (en) * 1985-06-25 1992-06-23 L'oreal Amphiphilic lipid compounds, process for their preparation and their applications especially in cosmetics and dermopharmacy
US5650393A (en) * 1992-08-06 1997-07-22 Alliance Pharmaceutical Corp. Amphiphilic compounds derived from amino acids or peptides, their methods of synthesis and their application as drug delivery systems
US5653987A (en) * 1995-05-16 1997-08-05 Modi; Pankaj Liquid formulations for proteinic pharmaceuticals
US5665328A (en) * 1988-05-02 1997-09-09 Phanos Technologies, Inc. Compounds, compositions and methods for binding bio-affecting substances to surface membranes of bio-particles
US5688761A (en) * 1991-04-19 1997-11-18 Lds Technologies, Inc. Convertible microemulsion formulations
US5853748A (en) * 1994-08-31 1998-12-29 Cortecs (Uk) Limited Pharmaceutical compositions
US5952008A (en) * 1993-06-24 1999-09-14 Ab Astra Processes for preparing compositions for inhalation
US6468978B1 (en) * 1998-04-30 2002-10-22 Curis, Inc. Active hedgehog protein conjugate
US6630148B1 (en) * 1993-12-30 2003-10-07 Imperial Cancer Research Technology Ltd. Compositions comprising hedgehog proteins

Family Cites Families (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61500790A (en) 1984-03-28 1986-04-24 シタス　コ−ポレイシヨン Composition containing microbially produced interleukin-2
EP0179583A1 (en) * 1984-10-04 1986-04-30 Merck & Co. Inc. A system for enhancing the water dissolution rate and solubility of poorly soluble drugs
JPS62207226A (en) 1986-03-07 1987-09-11 Sumitomo Pharmaceut Co Ltd Pharmaceutical for nasotracheal administration
AU5171293A (en) * 1992-10-14 1994-05-09 Regents Of The University Of Colorado, The Ion-pairing of drugs for improved efficacy and delivery
JP3290745B2 (en) 1993-03-31 2002-06-10 藤平 正道 Method for forming monolayer pattern
US5789543A (en) * 1993-12-30 1998-08-04 President And Fellows Of Harvard College Vertebrate embryonic pattern-inducing proteins and uses related thereto
EP0741783B1 (en) * 1993-12-30 2009-03-18 President And Fellows Of Harvard College Vertebrate embryonic pattern-inducing hedgehog-like proteins
FR2714621B1 (en) 1994-01-06 1996-02-23 Centre Nat Rech Scient Process for the preparation of liposomes without using an organic solvent.
US5750123A (en) * 1996-06-28 1998-05-12 Chesebrough-Pond's Co., Division Of Conopco, Inc. Vitamin C delivery system
WO1998030576A1 (en) * 1996-10-07 1998-07-16 The Johns Hopkins University School Of Medicine Novel hedgehog-derived polypeptides
EP1016672A4 (en) 1996-10-15 2001-04-11 Seikagaku Kogyo Co Ltd Polypeptide transition metal salts and method of enhancing anti-hiv activity of polypeptide
WO1998020298A1 (en) * 1996-11-08 1998-05-14 Northern Magnetics, Inc. Sensing and controlling the location of elements of a linear motor
US5759811A (en) * 1996-11-13 1998-06-02 The Regents Of The University Of California Mutant human hedgehog gene
JPH10194987A (en) * 1997-01-14 1998-07-28 Youtai Iwamoto Composition for ossification and chondrogenesis
JPH114695A (en) * 1997-04-25 1999-01-12 Hayashibara Biochem Lab Inc Hedgehog protein
US6639051B2 (en) * 1997-10-20 2003-10-28 Curis, Inc. Regulation of epithelial tissue by hedgehog-like polypeptides, and formulations and uses related thereto
PT917879E (en) 1997-11-22 2002-12-31 Roche Diagnostics Gmbh IMPROVED PROCESS FOR PROTEIN STABILIZATION
EP0919618A1 (en) 1997-11-28 1999-06-02 Boehringer Mannheim Gmbh Active Hedgehog protein mutants, methods of production and application
JP4289788B2 (en) 1997-12-03 2009-07-01 バイオジェン・アイデック・エムエイ・インコーポレイテッド Hydrophobically modified protein composition and production method
JP3648051B2 (en) * 1998-02-02 2005-05-18 富士通株式会社 Related information retrieval apparatus and program recording medium
EP0947201B1 (en) * 1998-02-04 2006-06-28 Curis, Inc. Pharmaceutical composition of hedgehog proteins and use thereof
CA2260423C (en) 1998-02-04 2006-06-20 Kurt Lang Pharmaceutical composition of hedgehog proteins and use thereof
US20050021440A1 (en) * 2003-04-04 2005-01-27 Scott Dresden Integrated dynamic pricing and procurement support for e-commerce advertising channels
US20070203906A1 (en) * 2003-09-22 2007-08-30 Cone Julian M Enhanced Search Engine
US20050114319A1 (en) * 2003-11-21 2005-05-26 Microsoft Corporation System and method for checking a content site for efficacy
US10515374B2 (en) * 2005-03-10 2019-12-24 Adobe Inc. Keyword generation method and apparatus

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4604377A (en) * 1984-03-28 1986-08-05 Cetus Corporation Pharmaceutical compositions of microbially produced interleukin-2
US5124081A (en) * 1985-06-25 1992-06-23 L'oreal Amphiphilic lipid compounds, process for their preparation and their applications especially in cosmetics and dermopharmacy
US5109038A (en) * 1987-05-11 1992-04-28 Rhone-Poulenc Chimie Polymer particles which carry, implanted on their surface, amphiphilic molecules carrying ion-forming or reactive groups, a process for the preparation of these particles and their use in biological applications
US5665328A (en) * 1988-05-02 1997-09-09 Phanos Technologies, Inc. Compounds, compositions and methods for binding bio-affecting substances to surface membranes of bio-particles
US5688761A (en) * 1991-04-19 1997-11-18 Lds Technologies, Inc. Convertible microemulsion formulations
US5650393A (en) * 1992-08-06 1997-07-22 Alliance Pharmaceutical Corp. Amphiphilic compounds derived from amino acids or peptides, their methods of synthesis and their application as drug delivery systems
US5952008A (en) * 1993-06-24 1999-09-14 Ab Astra Processes for preparing compositions for inhalation
US6630148B1 (en) * 1993-12-30 2003-10-07 Imperial Cancer Research Technology Ltd. Compositions comprising hedgehog proteins
US5853748A (en) * 1994-08-31 1998-12-29 Cortecs (Uk) Limited Pharmaceutical compositions
US5653987A (en) * 1995-05-16 1997-08-05 Modi; Pankaj Liquid formulations for proteinic pharmaceuticals
US6468978B1 (en) * 1998-04-30 2002-10-22 Curis, Inc. Active hedgehog protein conjugate

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070042041A1 (en) * 2005-08-17 2007-02-22 Board Of Trustees Of The University Of Arkansas Drug-surfactant complexes for sustained release
US20100105637A1 (en) * 2005-08-17 2010-04-29 Kim Cherng-Ju Drug-surfactant complexes for sustained release
US20090275066A1 (en) * 2006-11-13 2009-11-05 Universite Paris 7 - Denis Diderot Immobilization of membrane porteins onto supports via an amphiphile
US8207263B2 (en) * 2006-11-13 2012-06-26 Centre National De La Recherche Scientifique (Cnrs) Immobilization of membrane proteins onto supports via an amphiphile
US8674044B2 (en) 2006-11-13 2014-03-18 Centre National De La Recherche Scientifique (Cnrs) Immobilization of membrane proteins onto supports via an amphiphile

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