HRP990036A2

HRP990036A2 - Pharmaceutical composition of hedgehog proteins and the use thereof

Info

Publication number: HRP990036A2
Authority: HR
Inventors: Apollon Papadimitriou
Original assignee: Roche Diagnostics Gmbh
Priority date: 1998-02-04
Filing date: 1999-02-03
Publication date: 1999-08-31
Also published as: TR199900197A2; AR014530A1; ZA99887B; BR9900523A; ID22356A; PE20000241A1; PL331201A1

Description

Izum se odnosi na farmaceutski sastav hedgehog proteina i njegovu upotrebu posebno za lokalno oslobađanje ovih proteina na kostima i hrskavici. The invention relates to a pharmaceutical composition of hedgehog proteins and its use, especially for the local release of these proteins on bones and cartilage.

Pod hedgehog (hh) proteinima podrazumijeva se porodica izlučenih signalnih proteina koji su odgovorni za tvorbu brojnih struktura u embriogenezi (J.C. Smith, Celi 76 (1994) 193-196, N. Perrimon, Celi 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). Tijekom biosinteze 20 kD N-terminalna domena i 25 kD C-terminalna domena dobiju se nakon odcjepljenje signalne sekvence i autokatalitičkog cijepanja. N-terminalna domena se modificira u svom prirodnom obliku s kolesterolom (J.A.Porter et al., Science 274 (1996) '255-259). U višim oblicima života porodica hh sastavljena je od najmanje tri člana tj . sunčanog, indijskog i pustinjskog hh (Shh, Ihh, Dhh; M. Fietz et al., Development (Suppl.) (1994) 43-51). Razlike u aktivnosti rekombinantno proizvedenih hedgehog proteina opažene su nakon produkcije u prokariotima i eukariotima (M. Hynes et al., Neuron 15 (1995) 35-44 i T. Nakamura et al., Biochem. Biophys. Res. Comm. 237 (1997).465-469. Hedgehog (hh) proteins are a family of secreted signaling proteins that 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 biosynthesis, the 20 kD N-terminal domain and the 25 kD C-terminal domain are obtained after cleavage of the signal sequence and autocatalytic cleavage. The N-terminal domain is modified in its native form with cholesterol (J.A. Porter et al., Science 274 (1996) '255-259). In higher forms of life, the hh family is composed of at least three members, i.e. sunny, Indian and desert hh (Shh, Ihh, Dhh; M. Fietz et al., Development (Suppl.) (1994) 43-51). Differences in the activity of recombinantly produced hedgehog proteins have been observed after production in prokaryotes and eukaryotes (M. Hynes et al., Neuron 15 (1995) 35-44 and T. Nakamura et al., Biochem. Biophys. Res. Comm. 237 (1997 ).465-469.

Hynes et al usporedili su aktivnost hh proteina u supernatantu transformiranih humanih embrionskih bubrežnih 293 stanica (eukariotski hh) s hh proteinom kojeg je proizvela E. coli i našli su četverostruko višu aktivnost hh iz supernatanta bubrežne stanične linije. Kao razlog za tu povišenu aktivnost razmatran je mogući dodatni dopunski faktor koji se umnaža samo u eukariotskim stanicama, post-translacijska modifikacija, različit N-kraj, budući da hh izoliran iz E. coli sadrži 50% hh koji nosi dvije dodatne N-terminalne amino kiseline (Gly-Ser) ili je skraćen za 5 -6 amino kiseline, ili više stanje agregacije (npr. vezanjem nikal agaroznih zrnaca). Hynes et al compared hh protein activity in the supernatant of transformed human embryonic kidney 293 cells (eukaryotic hh) with hh protein produced by E. coli and found a fourfold higher activity of hh from the supernatant of a renal cell line. As the reason for this increased activity, a possible additional complement factor that is reproduced only in eukaryotic cells, post-translational modification, different N-terminus, since hh isolated from E. coli contains 50% of hh carrying two additional N-terminal amino acids, was considered. acid (Gly-Ser) or shortened by 5-6 amino acids, or a higher state of aggregation (eg by binding nickel agarose beads).

Nakamura et al. usporedili su aktivnost shh u supernatantu transfomiranih fibroplasta iz pilećeg embrija s shh fuzijskim proteinom izoliranim iz E. coli koji još uvijek ima N-terminalni polihistidinski dio. Shh u supernatantu fibroplasta ima sedmerostruko višu aktivnost u usporedbi s očišćenim E. coli proteinom s obzirom na stimulaciju alkalijske fosfataze (AP) u C3H10T 1/2 stanicama. Kao razlog za povišenu aktivnost prisutnu samo u supernatantu eukariotskih stanica i uzrok jače indukcije AP-a, razmatrane su molekule kao što su koštani morfogenetički proteini (BMPs). Nakamura et al. compared shh activity in the supernatant of transformed chicken embryo fibroplasts with an shh fusion protein isolated from E. coli that still has the N-terminal polyhistidine portion. Shh in fibroplast supernatant has a sevenfold higher activity compared to purified E. coli protein with respect to alkaline phosphatase (AP) stimulation in C3H10T 1/2 cells. Molecules such as bone morphogenetic proteins (BMPs) were considered as the reason for the increased activity present only in the supernatant of eukaryotic cells and the cause of stronger AP induction.

Kinto et al., FEBS Letters, 404 (1997) 319-323 opisali su da fibroplasti koji izlučuju hh induciraju ektopijsku tvorbu kosti kod i.m. implatacije na koleganu. Zbog toga hedgehog proteini djeluju osteoinduktivno. Kinto et al., FEBS Letters, 404 (1997) 319-323 reported that hh-secreting fibroblasts induce ectopic bone formation in i.m. implations on the colleague. This is why hedgehog proteins have an osteoinductive effect.

Postupak za proizvodnju sistema koji isporučuje proteine s usporenim oslobađanjem upotrebom alginata poznat je iz WO 90/08551. Po tom postupku oblikuje se dvofazni sistem, pri čemu prva faza sadrži visoku koncentraciju proteina (zasićena otopina), a druga faza sadrži alginat. Međutim, takovo odvajanje faza je teško i komplicirano za provedbu pri proizvodnji farmaceutskih sastava u velikim količinama. A process for producing a system that delivers proteins with a sustained release using alginate is known from WO 90/08551. According to this procedure, a two-phase system is formed, where the first phase contains a high concentration of protein (saturated solution), and the second phase contains alginate. However, such phase separation is difficult and complicated to implement in the production of pharmaceutical compositions in large quantities.

Ritmičko oslobađanje dekstrana iz kompleksa kalcijevog alginata poznato je iz Kikuchi, A. et al., J. Controlled Release 47 (1997) 21-29. Međutim, povezivanje hedgehog proteina na takove komplekse Kikuchi nije opisao. Rhythmic release of dextran from calcium alginate complexes is known from Kikuchi, A. et al., J. Controlled Release 47 (1997) 21-29. However, Kikuchi did not describe the connection of hedgehog proteins to such complexes.

Robinson, C. J. et al. u Trends in Biotechnology 14 (1996) 451-452 opisali su intraventrikularnu implantaciju alginatnih mikro kuglica kao metodu za lokalnu aplikaciju NGF-a ili stanica koje izlučuju NGF. Međutim, aplikacija hedgehog proteina nije opisana. Robinson, C.J. et al. in Trends in Biotechnology 14 (1996) 451-452 described intraventricular implantation of alginate microspheres as a method for local application of NGF or NGF-secreting cells. However, the application of hedgehog protein has not been described.

Downs, E.C. et al u J. Celi. Physiol. 152 (1992) 422-429 opisali su primjenu kalcijevih alginatnih kuglica kao sistema isporuke faktora angiogeneze. Međutim, primjena tog postupka ili tog sistema isporuke za hedgehog proteine nije opisana. Downs, E.C. et al in J. Cela. Physiol. 152 (1992) 422-429 described the use of calcium alginate beads as an angiogenesis factor delivery system. However, the application of this method or delivery system for hedgehog proteins has not been described.

Crey, C. J. i J. Dowsett u Biotechnol. Bioeng. 31 (1988) 607-612 opisuju upotrebu kalcij/cink alginatnih kuglica kao sistema isporuke za inzulin. Međutim, iz toga nije poznata primjena ovog postupka za proizvodnju sistema isporuke hedgehog proteina. Crey, C.J. and J. Dowsett in Biotechnol. Bioeng. 31 (1988) 607-612 describe the use of calcium/zinc alginate beads as a delivery system for insulin. However, the application of this process for the production of hedgehog protein delivery systems is not known from this.

Od Yang et al., Development 124 (1997) 4393-4404 poznato je da se za farmaceutski učinkovito djelovanje in vivo visoke lokalne koncentracije hedgehog proteina na strani djelovanja u tijelu moraju održavati tijekom perioda od najmanje 16 sati. Sistem nosača kao kromatografski medij Affigel CM napunjen s hedgehog proteinom, kojeg su opisali Yang et al., Ni-agaroza koju su opisali Marti et al. u Nature 375 (1995) 322-325 ili Affigel Blue kojeg su upotrijebili Lopez-Martinez et al. u Curr. Biol 5 (1995) 791-796, ili čestice heparin-agaroze, koje su tamo upotrijebljene, nisu prikladne za farmaceutsku primjenu, jer su one imunogene i mogu uzrokovati upalne reakcije. It is known from Yang et al., Development 124 (1997) 4393-4404 that for a pharmaceutically effective action in vivo, high local concentrations of hedgehog protein on the side of action in the body must be maintained for a period of at least 16 hours. Carrier system as chromatographic medium Affigel CM loaded with hedgehog protein, described by Yang et al., Ni-agarose described by Marti et al. in Nature 375 (1995) 322-325 or Affigel Blue used by Lopez-Martinez et al. in Curr. Biol 5 (1995) 791-796, or the heparin-agarose particles used there are not suitable for pharmaceutical use, because they are immunogenic and can cause inflammatory reactions.

Izumitelji su pronašli da je biološki kompatibilan i biološki razgradijiv kolagenski nosač, kojeg su opisali Kinto et al. za stanice koje umnažaju hh, također neprikladan za optimalnu lokalnu farmaceutsku aplikaciju hedgehog proteina sve dok se ti hedgehog proteini vežu na nosač samo putem ionskih interakcija. Nađeno je da kolagenski nosači opterećeni s hedgehog proteinima pod fiziološkim uvjetima (pH pribl. 7 i u slabim kiselinama (sve do pH 4,5)) glavninu apliciranog hedgehog proteina oslobađaju s matrice za par minuta. Prema otkriću izumitelja, uzrok tog nedovoljnog vezanje je nedostatak dovoljnih ionskih interakcija između hedgehog proteina i nosača. U tom slučaju stavljanja hh pod kiselim uvjetima (ispod pH 4,5), velika količina stavljenog hedgehog proteina se denaturira i ireverzibilno se veže na nosač. The inventors have found that the biocompatible and biodegradable collagen carrier described by Kinto et al. for hh-amplifying cells, also unsuitable for optimal local pharmaceutical application of hedgehog proteins as long as these hedgehog proteins bind to the carrier only through ionic interactions. It was found that collagen carriers loaded with hedgehog proteins under physiological conditions (pH approx. 7 and in weak acids (up to pH 4.5)) release most of the applied hedgehog protein from the matrix in a few minutes. According to the discovery of the inventor, the cause of this insufficient binding is the lack of sufficient ionic interactions between the hedgehog protein and the carrier. In this case of inserting hh under acidic conditions (below pH 4.5), a large amount of inserted hedgehog protein is denatured and irreversibly binds to the carrier.

Stoga je predmet izuma osigurati farmaceutski sastav hedgehog proteina s biološki kompatibilnim nosačem pri čemu nosač veže hedgehog protein u njegovoj aktivnoj, složenoj strukturi i može ga usporeno osloboditi in vivo u njegovom aktivnom obliku. Takove formulacije su posebno prikladne za ispravljanje defekata kosti i hrskavice, ali one se također mogu upotrijebiti i za ispravljanje defekata neurona ili za sistemsko oslobađanje. Therefore, the object of the invention is to provide a pharmaceutical composition of hedgehog protein with a biologically compatible carrier, whereby the carrier binds the hedgehog protein in its active, complex structure and can slowly release it in vivo in its active form. Such formulations are particularly suitable for the correction of bone and cartilage defects, but they can also be used for the correction of neuronal defects or for systemic release.

Taj cilj postignut je s farmaceutskim sastavom hedgehog proteina koji je karakteriziran time da je hedgehog protein vezan na hidrofilnom nosaču koji je biološki kompatibilan, pri čemu nosač je polimer koji This goal was achieved with the pharmaceutical composition of the hedgehog protein, which is characterized by the fact that the hedgehog protein is attached to a hydrophilic carrier that is biologically compatible, wherein the carrier is a polymer that

- veže hedgehog protein kao nosač negativnog naboja što je posljedica ionskih interakcija, - binds the hedgehog protein as a negative charge carrier, which is a consequence of ionic interactions,

- ne denaturira hedgehog protein kad je on vezan na nosaču, - does not denature the hedgehog protein when it is attached to the carrier,

- pod neutralnim uvjetima nosač sadrži najmanje 0,1 do 1, ponajprije 0,1 do 2 ostatka negativnog naboja po monomeru, - under neutral conditions, the carrier contains at least 0.1 to 1, preferably 0.1 to 2 residues of negative charge per monomer,

- naboj je dobiven u obliku kiselinskih skupina kao npr. sulfatnih, kabroksilriih ili fosfatnih skupina, i - the charge is obtained in the form of acid groups such as sulfate, carboxyl or phosphate groups, and

- prosječna molekulsa masa nosača je najmanje 50.000 Da. - the average molecular weight of the carrier is at least 50,000 Da.

Iznenađujuće se je pokazalo da se hedgehog proteini mogu osloboditi reverzibilno iz nosača in vivo u aktivnom obliku i usporeno bez uzrokovanja homogenih i/ili upalnih reakcija in vivo ako su oni vezani na topivu ili netopivu polimernu matricu negativnog naboja. Surprisingly, it has been shown that hedgehog proteins can be released reversibly from the carrier in vivo in an active form and slowly without causing homogeneous and/or inflammatory reactions in vivo if they are attached to a soluble or insoluble polymer matrix of negative charge.

Upotrebljava se ponajprije hidrofilna noseća matrica, a posebno prednosno topiva ili netopiva organska hidrofilna noseća matrica. Noseća matrica sastoji se posebno prednosno od anionskog poli-saharida kao ponajprije hijaluronske kiseline (kao i njenih kemijski umreženih oblika), kondroitoin sulfata, polivinil sulfata, keratin sulfata, dekstran sulfata, pektina, karaginana i drugih hidrokoloida, sulfatiranog alginata, dermatan sulfata, alginata, ponajprije kalcijevog alginata ili kombinacije od najmanje dva takova anionska polisaharida ili kombinacije od tih polisaharida s nabojem i drugih polimera, kao što je posebno kolagen, u kojem maseni postotak polisaharida s nabojem iznosi 10-50%. Netopiva matrica u smislu izuma znači da se matrica uglavnom ne razgrađuje ili da se ne otapa vidljivo u puferiranoj otopini in vitro za 10 - 20 sati pri sobnoj temperaturi. S tim u svezi posebno prednosno je da nosač upotrijebljen prema izumu sadrži manje od 50%, ponajprije manje od 20%, a posebno prednosno da ne sadrži nikakvu količinu neutralnog polisaharida. Kao noseća matrica posebno prikladna je hijaluronska kiselina s molekulskom masom od najmanje 10 Daltona, posebno s molekulskom masom od 4 x 106 Daltona. Preferably, a hydrophilic carrier matrix is used, and especially preferably a soluble or insoluble organic hydrophilic carrier matrix. The carrier matrix consists especially preferably of an anionic polysaccharide, primarily hyaluronic acid (as well as its chemically cross-linked forms), chondroitin sulfate, polyvinyl sulfate, keratin sulfate, dextran sulfate, pectin, carrageenan and other hydrocolloids, sulfated alginate, dermatan sulfate, alginate , preferably calcium alginate or a combination of at least two such anionic polysaccharides or a combination of these charged polysaccharides and other polymers, such as especially collagen, in which the mass percentage of charged polysaccharides is 10-50%. An insoluble matrix in the sense of the invention means that the matrix is generally not degraded or does not visibly dissolve in a buffered solution in vitro for 10-20 hours at room temperature. In this regard, it is especially preferable that the carrier used according to the invention contains less than 50%, preferably less than 20%, and especially preferably that it does not contain any amount of neutral polysaccharide. Hyaluronic acid with a molecular weight of at least 10 Daltons, especially with a molecular weight of 4 x 106 Daltons, is particularly suitable as a carrier matrix.

U daljnjoj izvedbi pokazalo se je da su hidrofilni nosači na osnovi anorganskih netopivih sulfata, kao što je hidroksilapatit ili trikalcijev fosfat, također prikladni kao netopive noseće matrice prema izumu. In a further embodiment, it has been shown that hydrophilic carriers based on inorganic insoluble sulfates, such as hydroxylapatite or tricalcium phosphate, are also suitable as insoluble carrier matrices according to the invention.

Kao usporeno oslobađanje prema izumu podrazumijeva se oslobađanje farmakološki učinkovite doze hedgehog proteina tijekom definiranog perioda od najmanje 14 sati. Kao farmakološki učinak podrazumijeva se neurološki učinak na nervne stanice, kondrogenezu i/ili indukciju kondrogeneze, a ponajprije osteogenezu i/ili osteoindukciju kako su opisali Kinto et al., FEBS Letters, 404 (1997) 319-232 za koštanu indukciju, Miao et al. u J. Neurosci. 17 (1997) 5891-5899 za učinak na nervnim stanicama i Stott et al. u J. Celi. Sci. 110 (1997) 2691-2701 za indukciju stanica hrskavice. Slow release according to the invention is understood as the release of a pharmacologically effective dose of hedgehog protein during a defined period of at least 14 hours. A pharmacological effect is understood to mean a neurological effect on nerve cells, chondrogenesis and/or induction of chondrogenesis, and primarily osteogenesis and/or osteoinduction as described by Kinto et al., FEBS Letters, 404 (1997) 319-232 for bone induction, Miao et al. . in J. Neurosci. 17 (1997) 5891-5899 for effect on nerve cells and Stott et al. in J. Cela. Sci. 110 (1997) 2691-2701 for the induction of cartilage cells.

Enzimski razgradljiv nosač upotrebljava se ponajprije kao nosač koji se može razgraditi s enzimima (npr. s proteinazom) izlučenim iz stanica na kojima se vrši lokalna aplikacija in vivo. Međutim, vrijeme poluraspadanja nosača mora biti najmanje 12 sati, ali ono također može biti i nekoliko tjedana. Ako se nosač sastoji od polisaharida, taj se nosač razgrađuje ponajprije s glikozidazom i hidrolazom koje su prisutne u stanici i izlučene. Međutim, takav biološki razgradljiv nosač nije nužan u svakom slučaju. Ako se oslobađanje provodi zbog liječenja osteoporoze ili bolesti neurona, biološka razgradijivost nije potrebna. Međutim, takovi nosači imaju ponajprije skromnu topivost pod fiziološkim uvjetima i zbog toga se apsorbiraju u tijelu tijekom relativno dugog perioda (nekoliko tjedana do nekoliko mjeseci). An enzymatically degradable carrier is primarily used as a carrier that can be degraded with enzymes (eg with proteinase) secreted from cells on which local application is performed in vivo. However, the half-life of the carrier must be at least 12 hours, but it can also be several weeks. If the carrier consists of polysaccharides, this carrier is degraded primarily by glycosidase and hydrolase, which are present in the cell and secreted. However, such a biodegradable carrier is not necessary in every case. If the release is for the treatment of osteoporosis or neuron disease, biodegradability is not required. However, such carriers primarily have modest solubility under physiological conditions and are therefore absorbed in the body over a relatively long period (several weeks to several months).

Da bi se dobile noseće matrice prevučene s hedgehog proteinima na takav način da one kod lokalne aplikacije pokazuju adekvatnu farmaceutsku učinkovitost, potrebne su otopine hedgehog proteina visoke koncentracije. Pokazalo se je da nosači prevučeni s hedgehog proteinom, a koji se mogu upotrijebiti u farmaciji, moraju sadržavati ponajprije hedgehog protein koncentracijom od l do 5 mg/ml, ponajprije 3 mg/ml nosača, ili višom. Posebno su prednosni nosači koji sadrže hedgehog proteine koncentracijom od 10 mg/ml ili višom. Hedgehog proteini su strukturno slabo topivi. Međutim, iznenađujuće je nađeno da se topivost hedgehog proteina jako povisuje u otopinama koje sadrže arginin ili argininijeve ione (ponajprije argininijev sulfat). Stoga daljnji predmet izuma su vodene otopine hedgehog proteina koncentracije od 3 mg/ml i više koje sadrže arginin i argininijeve ione i koje su puferirane. Daljnji predmet izuma je postupak za proizvodnju noseće matrice prevučene s hedgehog proteinom, koji je karakteriziran time da se noseća matrica inkubira s otopinom hedgehog proteina koncentracije 3 mg/ml, koja otopina sadrži arginin ili argininijeve ione i na taj način prevučena noseća matrica se izolira. In order to obtain carrier matrices coated with hedgehog proteins in such a way that they show adequate pharmaceutical efficacy during local application, hedgehog protein solutions of high concentration are required. It has been shown that carriers coated with hedgehog protein, which can be used in pharmacy, must preferably contain hedgehog protein in a concentration of 1 to 5 mg/ml, preferably 3 mg/ml carrier, or higher. Carriers containing hedgehog proteins at a concentration of 10 mg/ml or higher are particularly preferred. Hedgehog proteins are structurally poorly soluble. However, it was surprisingly found that the solubility of hedgehog protein is greatly increased in solutions containing arginine or arginine ions (primarily arginine sulfate). Therefore, a further subject of the invention are hedgehog protein aqueous solutions with a concentration of 3 mg/ml and more, which contain arginine and arginine ions and which are buffered. A further subject of the invention is a process for the production of a carrier matrix coated with hedgehog protein, which is characterized by the fact that the carrier matrix is incubated with a hedgehog protein solution with a concentration of 3 mg/ml, which solution contains arginine or arginine ions, and in this way the coated carrier matrix is isolated.

Takove otopine prikladne su za proizvodnju matrica koje sadrže hedgehog proteine u farmaceutski učinkovitim koncentracijama i one su prikladne za farmaceutsku primjenu. Stoga je daljnji predmet izuma noseća matrica koja po ml noseće matrice sadrži 3 mg hedgehog proteina ili više, ponajprije 10 mg ili više, te arginin ili argininijeve ione. Koncentracija arginina je ponajprije između 10 i 500 mmolova/l, ponajprije u pH području između 6 i 8. Such solutions are suitable for the production of matrices containing hedgehog proteins in pharmaceutically effective concentrations and they are suitable for pharmaceutical use. Therefore, a further object of the invention is a carrier matrix which per ml of the carrier matrix contains 3 mg of hedgehog protein or more, preferably 10 mg or more, and arginine or arginine ions. The concentration of arginine is preferably between 10 and 500 mmol/l, preferably in the pH range between 6 and 8.

Kao aktivnost u smislu izuma podrazumijeva se aktivnost alkalijske fosfataze (stimulacija ekspresije alkalijske fosfataze) koja može inducirati polipeptid u stanicama sisavca (aktivnost u pokusu s alkalijskom fosfatazom). U tu svrhu mišja mezenhimalna stanična linija uzgaja se u mediju koji sadrži fetalni goveđi serum. Zatim se doda sterilno filtrirani uzorak, stanice liziraju nakon pribl. 5 dana i u stanici lizata utvrdi se alkalijsku fosfatazu cijepanjem kromogenog supstrata (pNP, p-nitro-fenol) (J. Asahina, Exp. Celi. Res. 222 (1996) 38-47 i T. Nakamura (1997)). Activity in the sense of the invention means alkaline phosphatase activity (stimulation of alkaline phosphatase expression) which can induce a polypeptide in mammalian cells (activity in the alkaline phosphatase experiment). For this purpose, a mouse mesenchymal cell line is cultured in a medium containing fetal bovine serum. A sterile filtered sample is then added, the cells are lysed after approx. 5 days and alkaline phosphatase was detected in the cell lysate by cleaving the chromogenic substrate (pNP, p-nitro-phenol) (J. Asahina, Exp. Cell. Res. 222 (1996) 38-47 and T. Nakamura (1997)).

Prema izumu kao hedgehog protein podrazumijeva se izlučeni signalni protein koji je odgovoran za tvorbu brojnih struktura u embriogenezi. Posebno prednosno se upotrebljavaju sunčani, indijski ili pustinjski hh (Fietz M. et al., Development (Suppl.) (1994) 43-51). Upotrebljava se ponajprije hh protein sa sekvencom kako je opisana u banci podataka EMBL pod brojem L38518. Proteini hedgehog porodice pokazuju izrazitu homologiju u njihovim sekvencama amino kiselina, što je razlog da oni ponajprije umnažaju one nukleinske kiseline koje kodiraju za hedgehog proteine koji su 80% ili više homologni s gore spomenutom sekvencom sunčanog hedgehog proteina. According to the invention, hedgehog protein is a secreted signal protein responsible for the formation of numerous structures in embryogenesis. Sunny, Indian or desert hh are particularly preferably used (Fietz M. et al., Development (Suppl.) (1994) 43-51). The hh protein with the sequence as described in the EMBL database under the number L38518 is primarily used. Proteins of the hedgehog family show marked homology in their amino acid sequences, which is the reason that they primarily amplify those nucleic acids that code for hedgehog proteins that are 80% or more homologous to the above-mentioned sequence of the sunny hedgehog protein.

Humani sunčani hedgehog prekurzorski protein sastavljen je od sekvence amino kiselina 1-462 opisane u banci podataka EMBL pod brojem L38518. Amino kiseline 1-23 predstavljaju signalni peptid, amino kiseline 24-197 predstavljaju zrelu signalnu domenu, amino kiseline 32-197 predstavljaju signalnu domenu skraćenu za 8 amino kiselina i amino kiseline 198-462 predstavljaju samo-preradivačku domenu nakon autoproteolitskog cijepanja. The human sunny hedgehog precursor protein consists of the amino acid sequence 1-462 described in the EMBL data bank under number L38518. Amino acids 1-23 represent the signal peptide, amino acids 24-197 represent the mature signal domain, amino acids 32-197 represent the signal domain shortened by 8 amino acids and amino acids 198-462 represent the self-processing domain after autoproteolytic cleavage.

Farmaceutski sastav prema izumu sadrži ponajprije dodatni polimer koji djeluje uglavnom kao potporna strukturna tvar i koji prednosno također ima adhezijsku funkciju za stanice, ali se ne veže na hedgehog proteine na osnovi ionskih interakcija. Prednosno ta tvar je biološki razgradijivi protein ili proizvod hidrolitičke razgradnje koji se može upotrijebiti, na primjer, u obliku intaktnih proteinskih vlakana kao solubilizirani protein ili kao djelomično hidrolizirani protein. Takova potporna strukturna tvar je ponajprije kolagen, želatina, elastin, ili fibrin. Potporna strukturna tvar prisutna je ponajprije manjom količinom od opisanog hidrofilnog biološki razgradijivog nosača prema izumu. Udio potporne strukturne tvari je stoga ponajprije 30% ili manje, ponajprije 10% ili manje. Međutim, potporna strukturna tvar može također biti prisutna u suvišku u odnosu prema hidrofilnom nosaču. U toj svezi potrebno je samo osigurati da količina hidrofilnog nosača u farmaceutskom sastavu prema izumu bude dovoljno visoka da osigura da je terapeutski učinkovita količina hedgehog proteina vezana na hidrofilni nosač. Zbog toga je prednosno upotrijebiti najmanje peterostruki višak hidrofilnog nosača u odnosu prema hedgehog proteinu. K tome, vezanje hedgehog proteina na nosač za pripravljanje farmaceutskog sastava treba izvršiti pri pH vrijednosti 4,5 ili višoj. Kako je gore istaknuto, nađeno je da se hedgehog proteini pri pH vrijednosti ispod 4,5 denaturiraju i ireverzibilno vežu na proteinski nosač sličan kolagenu. Zbog toga se vezanje hedgehog proteina mora izvršiti u neutralnom području pH vrijednosti. The pharmaceutical composition according to the invention primarily contains an additional polymer which acts mainly as a supporting structural substance and which preferably also has an adhesive function for cells, but does not bind to hedgehog proteins on the basis of ionic interactions. Preferably, the substance is a biodegradable protein or a hydrolytic degradation product that can be used, for example, in the form of intact protein fibers as a solubilized protein or as a partially hydrolyzed protein. Such a supporting structural substance is primarily collagen, gelatin, elastin, or fibrin. The supporting structural substance is preferably present in a smaller amount than the described hydrophilic biodegradable carrier according to the invention. The proportion of the supporting structural substance is therefore preferably 30% or less, preferably 10% or less. However, the supporting structuring agent may also be present in excess relative to the hydrophilic carrier. In this connection, it is only necessary to ensure that the amount of hydrophilic carrier in the pharmaceutical composition according to the invention is high enough to ensure that the therapeutically effective amount of hedgehog protein is bound to the hydrophilic carrier. For this reason, it is preferable to use at least a fivefold excess of the hydrophilic carrier in relation to the hedgehog protein. In addition, the binding of the hedgehog protein to the carrier for the preparation of the pharmaceutical composition should be carried out at a pH value of 4.5 or higher. As pointed out above, hedgehog proteins have been found to denature at pH values below 4.5 and bind irreversibly to a collagen-like protein carrier. Therefore, the binding of the hedgehog protein must be carried out in a neutral pH range.

Nosači koji neovisno o hidrofilnom nosaču sadrže i druge potporne strukturne spojeve jesu, na primjer, kompleksi protein/polisaharida. Prednosni kompleksi opisani su u U.S. patentnu 4,614,794. Carriers that independently of the hydrophilic carrier also contain other supporting structural compounds are, for example, protein/polysaccharide complexes. Advantageous complexes are described in U.S. Pat. patent 4,614,794.

Farmaceutski sastav proizveden je inkubacijom hedgehog proteina s hidrofilnim nosačem pri pH 4,5 ili višoj pH vrijednosti, ponajprije pri pH u granicama neutralnog područja (pH 6 do 8), čime se vrši vezanje hedgehog proteina na nosač. Inkubacija se provodi ponajprije u puferiranoj otopini. Ako se kao nosač upotrebljava matrica koja dodatno sadrži biološki razgradljiv protein kao što je kolagen, inkubacija pri pH 4,5 ili višoj pH vrijednosti osigurat će sprečavanje irevezibilnog vezanja hedgehog proteina (koji se denaturira pri nižim pH vrijednostima) na biološki razgradljiv protein. The pharmaceutical composition is produced by incubating the hedgehog protein with a hydrophilic carrier at pH 4.5 or a higher pH value, preferably at a pH within the neutral range (pH 6 to 8), which binds the hedgehog protein to the carrier. Incubation is preferably carried out in a buffered solution. If a matrix that additionally contains a biodegradable protein such as collagen is used as a carrier, incubation at pH 4.5 or higher will ensure that the hedgehog protein (which denatures at lower pH values) is not irreversibly bound to the biodegradable protein.

Nađeno je da pod tim uvjetima ne dolazi do vezanja ili dolazi samo do zanemarivog vezanja, hedgehog proteina na biološki razgradijiv protein sve dok hedgehog protein ne sadrži hidrofobnu modifikaciju i stoga biološki razgradljiv protein značajno djeluje kao potporna strukturna tvar. It was found that under these conditions there is no or only negligible binding of the hedgehog protein to the biodegradable protein as long as the hedgehog protein does not contain a hydrophobic modification and therefore the biodegradable protein significantly acts as a supporting structural substance.

Hidrofobno modificirani (lipofilizirani) hedgehog proteini su hedgehog proteini koji u odnosu prema nemodificiranim hh proteinima (npr. proizvedenim rekombinantno u prokariotima) pokazuju povišenu površinsku hidrofobnost. Stupanj lipofilizacije proteina izmjeren je integracijom u lipidni sloj prema Haque, Z. et al., J. Agric. Food Chem. 30 (1982) 481. Takovi lipofilizirani hh proteini vežu se prema izumu na hidrofilni nosač na isti način kao i ne-lipofilizirani hh protein, ali oni se hidrofobnim interakcijama dodatno vežu na biološki razgradljiv protein. Hydrophobically modified (lipophilized) hedgehog proteins are hedgehog proteins that show increased surface hydrophobicity compared to unmodified hh proteins (eg produced recombinantly in prokaryotes). The degree of protein lipophilization was measured by integration into the lipid bilayer according to Haque, Z. et al., J. Agric. Food Chem. 30 (1982) 481. Such lipophilized hh proteins bind according to the invention to a hydrophilic carrier in the same way as a non-lipophilized hh protein, but they are additionally bound to a biodegradable protein by hydrophobic interactions.

Za proizvonju farmaceutskog sastava dodatno je prednosno dodati pomoćne tvari kao šećere (manitol, saharozu, laktozu, glukozu, trehalozu, ponajprije 20-100 mg/ml) ili amino kiseline kao glicin ili arginin, kao i antioksidante kao EDTA, citrat, polietilen glikol (1-10 mas. %) , površinski aktivne tvari, ponajprije ne-ionske površinski aktivne tvari (ponajprije 0,005-1 mas. %) kao što su polisorbati (Tween® 20 ili Tween® 80) ili polioksietileni, anti-upalne sastojke, lokalne anestetike, antibiotike i/ili stabilizatore kao lipide, masne kiseline i glicerol. For the production of the pharmaceutical composition, it is also preferable to add auxiliary substances such as sugars (mannitol, sucrose, lactose, glucose, trehalose, preferably 20-100 mg/ml) or amino acids such as glycine or arginine, as well as antioxidants such as EDTA, citrate, polyethylene glycol ( 1-10 wt. %), surfactants, preferably non-ionic surfactants (preferably 0.005-1 wt. %), such as polysorbates (Tween® 20 or Tween® 80) or polyoxyethylenes, anti-inflammatory ingredients, local anesthetics, antibiotics and/or stabilizers such as lipids, fatty acids and glycerol.

U drugoj prednosnoj izvedbi, prednost se daje farmaceutskom sastavu hedgehog proteina prema izumu zajedno sa sumarinom i on se može upotrijebiti kao prednostan. In another preferred embodiment, the pharmaceutical composition of the hedgehog protein according to the invention together with sumarin is preferred and can be used preferentially.

Farmaceutski sastavi mogu sadržavati dodatne farmaceutske pomoćne tvari. Pharmaceutical compositions may contain additional pharmaceutical excipients.

U prednosnoj izvedbi farmaceutski sastav sadrži hedgehog protein koncentracijom od 0,1-100 mg/ml. In a preferred embodiment, the pharmaceutical composition contains hedgehog protein at a concentration of 0.1-100 mg/ml.

U prednosnoj izvedbi farmaceutski sastav dodatno sadrži farmaceutski prihvatljiv pufer, koji je biološki kompatibilan ponajprije u području od pH 4 do pH 10, posebno prednosno u području između pH 6 i pH 8, a naročito pri pH vrijednosti pribl. 7. Da se spriječi denaturiranje i otkidanje cinkovog kompleksa u hedgehog proteinu, pH vrijednost farmaceutskog sastava mora biti primjereno viša od pH 4. Koncentracija pufera je ponajprije 1-500 mmolova/l, prednosno 5-150 mmolova/l, a naročito prednosno 10-100 mmolova/l. U prikladnoj izvedbi upotrijebljen je pufer od 20 mmolova/l kalijevog fosfata, pH 7,2 ili je kao pufer upotrijebljeno 100 mmolova/l arginin klorida pH 7,2. In a preferred embodiment, the pharmaceutical composition additionally contains a pharmaceutically acceptable buffer, which is biologically compatible preferably in the range from pH 4 to pH 10, especially preferably in the range between pH 6 and pH 8, and especially at a pH value of approx. 7. In order to prevent the denaturation and breaking off of the zinc complex in the hedgehog protein, the pH value of the pharmaceutical composition must be appropriately higher than pH 4. The concentration of the buffer is preferably 1-500 mmol/l, preferably 5-150 mmol/l, and especially preferably 10- 100 mmol/l. In a suitable embodiment, a buffer of 20 mmol/l potassium phosphate, pH 7.2 was used, or 100 mmol/l arginine chloride, pH 7.2, was used as a buffer.

Slijedeći primjeri, literatura i slike dalje rasvjeljavaju izum, čija svrha zaštite proizlazi iz patentnih zahtjeva. The following examples, literature and pictures further elucidate the invention, the purpose of protection of which is derived from the patent claims.

Podrazumijeva se da opisane metode kao i primjeri također opisuju glavni predmet izuma čak i nakon modifikacija. It is understood that the described methods and examples also describe the main subject of the invention even after modifications.

Opis slika Description of images

Slika 1: In vitro oslobađanje shh iz kolagenske matrice. Figure 1: In vitro release of shh from the collagen matrix.

Slika 2: In vitro oslobađanje shh iz kapsule kalcijevog Figure 2: In vitro release of shh from the calcium capsule

alginata. alginate.

Slika 3: In vitro oslobađanje shh iz gela hijaluronske kiseline. Figure 3: In vitro release of shh from hyaluronic acid gel.

Slika 4: In vitro oslobađanje shh iz alginat/kolagenske matrice. Figure 4: In vitro release of shh from alginate/collagen matrix.

PRIMJERI EXAMPLES

Primjer 1 Example 1

Pripravljanje alginatnog gela koji sadrži hh protein Preparation of alginate gel containing hh protein

Alikvot otopine hh proteina (1 mg/ml otopine shh u 50 mg/ml saharoze, 50 mM kalijevog fosfata, pH 7,2) miješa se s osnovnom otopinom Na-alginata (Pronova, Biopolymer, NO) (u vodi, više od 0,1%) na takav način da se dobije želatinoznu mješavinu alginatnog proteina. Taj gel se može upotrijebiti izravno kao matrica koja se može ubrizgati ili se dalje prerađuje u kapsule kaićijevog alginata ili se pohranjuje kao liofilizat. An aliquot of the hh protein solution (1 mg/ml shh solution in 50 mg/ml sucrose, 50 mM potassium phosphate, pH 7.2) is mixed with Na-alginate stock solution (Pronova, Biopolymer, NO) (in water, more than 0 .1%) in such a way as to obtain a gelatinous mixture of alginate protein. This gel can be used directly as an injectable matrix or further processed into kaichi alginate capsules or stored as a lyophilisate.

Primjer 2 Example 2

Pripravljanje kolagenske mješavine koja sadrži hh protein (usporedbeni primjer) Preparation of collagen mixture containing hh protein (comparative example)

100 μl hh otopine (1 mg/ml hh) u 100 μl hh solution (1 mg/ml hh) in

a) 20 mM kalijevog fosfata, pH 7,4 ili a) 20 mM potassium phosphate, pH 7.4 or

b) u 50 mM natrijevog acetata, pH 4,5, ili b) in 50 mM sodium acetate, pH 4.5, or

c) u 0,1%-tnoj trifluoroctenoj kiselini, pH 2 c) in 0.1% trifluoroacetic acid, pH 2

doda se kap po kap na kolagenske spužve (Helisat, Integra Life Science, USA) veličine 10 x 10 x 3 mm. Zatim se tako opterećeni nosači smrznu (-70°C), liofiliziraju i analiziraju. U tu svrhu te se opterećene spužve inkubiraju pri 37°C u prikladnom volumenu pufera (10 mmolova/l kalijevog fosfata, 150 mmolova/l NaCl, pH 7,2). Količina oslobođenog hh određuje se pomoću RP-HPLC. is added drop by drop to collagen sponges (Helisat, Integra Life Science, USA) size 10 x 10 x 3 mm. The loaded carriers are then frozen (-70°C), lyophilized and analyzed. For this purpose, these loaded sponges are incubated at 37°C in a suitable volume of buffer (10 mmol/l potassium phosphate, 150 mmol/l NaCl, pH 7.2). The amount of released hh is determined by RP-HPLC.

Primjer 3 Example 3

3.1 Pripravljanje kapsula Ca-alginata 3.1 Preparation of Ca-alginate capsules

Alginatni gel opisan u primjeru 1, koji sadrži hh protein doda se kap po kap k otopini CaCl2 (pribl. 1,5%) uz neprekidno miješanje. Spontano nastaju kompleksi Ca-alginata koji sadrže protein. Veličina nastalih kapsula ovisi o veličini kapi i može se po želji mijenjati. Nakon 5 do 10 minuta inkubacije u otopini CaCl2, kapsule se odfiltriraju i isperu u puferu (20 mmolova/l kalijevog fosfata, pH 7,2). Te se kapsule mogu upotrijebiti izravno kao implantati ili se dalje prerađuju liofilizacijom. The alginate gel described in example 1, which contains hh protein, is added drop by drop to the CaCl2 solution (approx. 1.5%) with continuous mixing. Ca-alginate complexes that contain protein are formed spontaneously. The size of the resulting capsules depends on the size of the drop and can be changed as desired. After 5 to 10 minutes of incubation in CaCl2 solution, the capsules are filtered and washed in buffer (20 mmol/l potassium phosphate, pH 7.2). These capsules can be used directly as implants or further processed by lyophilization.

3.2 Liofilizacija 3.2 Lyophilization

Kapsule C-alginata se smrznu pri -70°C i zatim se liofiliziraju. Liofilizacija omogućuje postojano skladištenje kapsula i dodatno olakšava implantaciju, jer je s kapsulama lakše rukovati u suhom stanju. C-alginate capsules are frozen at -70°C and then lyophilized. Lyophilization allows stable storage of capsules and additionally facilitates implantation, because it is easier to handle capsules in a dry state.

Primjer 4 Example 4

Oslobađanje shh in vitro Release of shh in vitro

Analiza kinetike oslobađanja in vitro pokazuje da je shh usporeno oslobođen iz kapsula Ca-alginata tijekom perioda od najmanje 70 sati (slika 2) , dok je shh iz kolagenske matrice oslobođen za nekoliko do najviše pribl. 20 minuta (slika 1). Liofilizirane alginatne kuglice koje sadrže shh inkubirane su pri 37°C u 10 mM kalijevom fosfatu, 150 mM NaCl, pH 7,2 (Rotatherm). Uzorci su uzeti i pomoću SDS poliakrilamidne elektroforeze (slika 2) analizirani su što se tiče njihovog sadržaja shh nakon 5 minuta, 1 sata, 5 sati, 10 sati, 1 dana, 34 sata, 2 dana, 3 dana i 6 dana. Ukupni sadržaj shh oslobođenog iz alginatnih kapsula grafički je prikazan kao funkcija vremena. Analysis of the release kinetics in vitro shows that shh is slowly released from Ca-alginate capsules over a period of at least 70 hours (Figure 2), while shh is released from the collagen matrix in a few to at most approx. 20 minutes (picture 1). Lyophilized alginate beads containing shh were incubated at 37°C in 10 mM potassium phosphate, 150 mM NaCl, pH 7.2 (Rotatherm). Samples were taken and analyzed by SDS polyacrylamide electrophoresis (Figure 2) for their shh content after 5 minutes, 1 hour, 5 hours, 10 hours, 1 day, 34 hours, 2 days, 3 days and 6 days. The total content of shh released from alginate capsules is shown graphically as a function of time.

Ispitivanje kinetike oslobađanja shh iz 2,5%-tnog gela hijaluronske kiseline, upotrebom tipova hijaluronske kiseline niske molekulske mase (LMW; molekulska masa pribl. 1,3 x 106 Da) ili visoke molekulske mase (HMW; molekulska masa pribl. 4 x 106 Da) prikazano je na slici 3. U tu svrhu gelovi hijaluronske kiseline, opterećeni sa shh, stavljeni su u cjevčice za dijalizu (odvajanje veličine 300.000 Da) i cjevčice su inkubirane u PBS-u pri 37°C. Nakon utvrđenog vremena uzeti su uzorci medija za oslobađanje i analizirani u pogledu njihovog sadržaja shh pomoću HPLC reverznih faza. Vidi se da je udio stavijenog hedgehog proteina oslobođen usporeno. Drugi dio proteina ostaje vezan na hijaluronsku kiselinu i može se osloboditi in vivo razgradnjom hijaluronske kiseline. Examining the kinetics of shh release from a 2.5% hyaluronic acid gel, using low molecular weight (LMW; molecular weight approx. 1.3 x 106 Da) or high molecular weight (HMW; molecular weight approx. 4 x 106) types of hyaluronic acid. Da) is shown in Figure 3. For this purpose, hyaluronic acid gels, loaded with shh, were placed in dialysis tubes (size separation 300,000 Da) and the tubes were incubated in PBS at 37°C. After the determined time, samples of the release media were taken and analyzed for their shh content by reverse phase HPLC. It can be seen that the part of the inserted hedgehog protein is released slowly. Another part of the protein remains bound to hyaluronic acid and can be released in vivo by degradation of hyaluronic acid.

Ispitivanje kinetike oslobađanja shh iz kolagen-alginatne matrice (Fibracol™, vidi U.S. patent 4,614,794) prikazano je na slici 4. U tu svrhu Fibracol spužva (1x1 x 0,3 cm) opterećena je s 0,2 mg shh u PBS-u. Opterećene spužve su smrznute (-70°C), liofilizirane i inkubirane u odgovarajućem volumenu PBS-a pri 37°C. Nakon utvrđenog vremena uzeti su uzorci medija za oslobađanje i analizirani pomoću HPLC reverznih faza u pogledu njihovog sadržaja shh. Vidi se je da samo pribl. 10 do 20% stavljenog hedgehog proteina oslobođeno u medij, a da je glavni dio ostao vezan na kolagen-alginatnu matricu. Taj udio se može osloboditi in vivo razgradnjom matrice. An examination of the kinetics of shh release from a collagen-alginate matrix (Fibracol™, see U.S. Patent 4,614,794) is shown in Figure 4. For this purpose, a Fibracol sponge (1 x 1 x 0.3 cm) was loaded with 0.2 mg of shh in PBS. Loaded sponges were frozen (-70°C), lyophilized and incubated in the appropriate volume of PBS at 37°C. After the determined time, samples of the release media were taken and analyzed by reverse phase HPLC for their shh content. It can be seen that only approx. 10 to 20% of the inserted hedgehog protein was released into the medium, while the main part remained attached to the collagen-alginate matrix. This fraction can be released in vivo by degradation of the matrix.

Claims

1. Farmaceutski sastav hedgehog proteina, naznačen time, da je hedgehog protein vezan na hidrofilnom nosaču koji je biološki kompatibilan, pri čemu nosač je polimer koji - - veže hedgehog protein kao nosač negativnog naboja što je posljedica ionskih interakcija, - ne denaturira hedgehog protein kad je on vezan na nosaču, - pod neutralnim uvjetima sadrži najmanje 0,1 do 2 ostatka negativnog naboja po monomeru, - sadrži naboj u obliku kiselinskih skupina, - ima prosječnu molekulsku masu od najmanje 50.000 Da i - ne sadrži agarozu.1. Pharmaceutical composition of hedgehog protein, characterized by the fact that the hedgehog protein is attached to a hydrophilic carrier that is biologically compatible, wherein the carrier is a polymer that - - binds the hedgehog protein as a negative charge carrier, which is a consequence of ionic interactions, - does not denature the hedgehog protein when it is attached to the carrier, - under neutral conditions, it contains at least 0.1 to 2 residues of negative charge per monomer, - contains a charge in the form of acid groups, - has an average molecular weight of at least 50,000 Da and - does not contain agarose.

2. Farmaceutski sastav prema zahtjevu 1, naznačen time, da se nosač sastoji od organske hidrofilne noseće matrice ili anorganskog netopivog fosfata.2. Pharmaceutical composition according to claim 1, characterized in that the carrier consists of an organic hydrophilic carrier matrix or an inorganic insoluble phosphate.

3. Farmaceutski sastav prema zahtjevima 1 ili 2, naznačen time, da se nosač sastoji od anionskog polisaharida ili anorganskog netopivog fosfata.3. Pharmaceutical composition according to claims 1 or 2, characterized in that the carrier consists of an anionic polysaccharide or an inorganic insoluble phosphate.

4. Farmaceutski sastav prema zahtjevima 1-3, naznačen time, da je hidrofilni nosač hijaluronska kiselina.4. Pharmaceutical composition according to claims 1-3, characterized in that the hydrophilic carrier is hyaluronic acid.

5. Farmaceutski sastav prema zahtjevima 1-4, naznačen time, da sadrži hedgehog protein koncentracijom od 0,1-100 mg/ml.5. Pharmaceutical composition according to claims 1-4, characterized in that it contains hedgehog protein in a concentration of 0.1-100 mg/ml.

6. Farmaceutski sastav prema zahtjevima 1-5, naznačen time, da je sastav puferiran u području između pH 4 i pH 10.6. Pharmaceutical composition according to claims 1-5, characterized in that the composition is buffered in the range between pH 4 and pH 10.

7. Farmaceutski sastav prema zahtjevima 1-6, naznačen time, da sastav sadrži arginin ili argininijeve ione.7. Pharmaceutical composition according to claims 1-6, characterized in that the composition contains arginine or arginine ions.

8. Postupak za proizvodnju farmaceutskog sastava koji sadrži hedgehog protein vezan na hidrofilnom nosaču koji je biološki kompatibilan, pri čemu je nosač polimer koji - veže hedgehog protein kao nosač negativnog naboja što je posljedica ionskih interakcija, - ne denaturira hedgehog protein kad je vezan na nosaču, - pod neutralnim uvjetima sadrži najmanje 0,1 do 2 ostatka negativnog naboja po monomeru, - sadrži naboj u obliku kiselinskih skupina, - ima prosječnu molekulsku masu od najmanje 50.000 Da, i - ne sadrži agarozu, naznačen time, da se hedgehog protein u farmaceutski učinkovitoj količini upotrebljava kao bitna komponenta tog sredstva.8. Process for the production of a pharmaceutical composition containing hedgehog protein attached to a hydrophilic carrier that is biologically compatible, wherein the carrier is a polymer that - binds the hedgehog protein as a negative charge carrier, which is a consequence of ionic interactions, - does not denature the hedgehog protein when it is attached to the carrier, - under neutral conditions, it contains at least 0.1 to 2 residues of negative charge per monomer, - contains a charge in the form of acid groups, - has an average molecular weight of at least 50,000 Da, and - does not contain agarose, characterized by the fact that the hedgehog protein is used in a pharmaceutically effective amount as an essential component of this agent.

9. Postupak prema zahtjevu 8, naznačen time, da se hedgehog protein upotrebljava koncentracijom od 0,1-100 mg/ml.9. The method according to claim 8, characterized in that the hedgehog protein is used at a concentration of 0.1-100 mg/ml.

10. Postupak za usporeno oslobađanje hedgehog proteina u ljudskom tijelu, naznačen time, da se hedgehog protein aplicira lokalno u ljudskom tijelu u farmaceutskom sastavu prema zahtjevima 1-6.10. Method for slow release of hedgehog protein in the human body, characterized in that the hedgehog protein is applied locally in the human body in a pharmaceutical composition according to claims 1-6.

11. Postupak prema zahtjevu 10, naznačen time, da se hedgehog protein aplicira pri koncentraciji od 0,1-100 mg/ml.11. The method according to claim 10, characterized in that the hedgehog protein is applied at a concentration of 0.1-100 mg/ml.

12. Postupak prema zahtjevima 8-11, naznačen time, da je hidrofilni nosač hijaluronska kiselina.12. The method according to claims 8-11, characterized in that the hydrophilic carrier is hyaluronic acid.

13. Postupak prema zahtjevima 8-12, naznačen time, da se vezanje hedgehog proteina na hidrofilni nosač vrši inkubacijom pri pH 4,5 ili višoj pH vrijednosti.13. The method according to claims 8-12, characterized in that the binding of the hedgehog protein to the hydrophilic support is carried out by incubation at pH 4.5 or a higher pH value.

14. Netopiva noseća matrica, naznačena time, da sadrži najmanje 3 mg hedgehog proteina i najmanje 10 mmolova/l arginina ili argininijevih iona po ml noseće matrice.14. Insoluble carrier matrix, characterized in that it contains at least 3 mg of hedgehog protein and at least 10 mmol/l of arginine or arginine ions per ml of the carrier matrix.

15. Postupak za proizvodnju netopive noseće matrice koja sadrži hedgehog protein, naznačen time, da se noseća matrica inkubira s otopinom koja sadrži hedgehog protein koncentracijom od 3 mg/ml ili više i arginin ili argininijeve ione koncentracijom od 10 mmolova/l ili više i na taj način prevučena noseća matrica se izolira.15. Method for the production of an insoluble carrier matrix containing hedgehog protein, characterized in that the carrier matrix is incubated with a solution containing hedgehog protein at a concentration of 3 mg/ml or more and arginine or arginine ions at a concentration of 10 mmol/l or more and at in this way, the coated carrier matrix is isolated.

16. Postupak prema zahtjevu 15, naznačen time, da se vezanje hedgehog proteina na hidrofilni nosač vrši inkubacijom pri pH 4,5 ili višoj pH vrijednosti.16. The method according to claim 15, characterized in that the binding of the hedgehog protein to the hydrophilic carrier is carried out by incubation at pH 4.5 or a higher pH value.