DK171308B1 - Stable pharmaceutical composition containing granulocyte colony stimulating factor and method for preparing this - Google Patents
Stable pharmaceutical composition containing granulocyte colony stimulating factor and method for preparing this Download PDFInfo
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Description
DK 171308 B1DK 171308 B1
Opfindelsen angår et stabilt farmaceutisk præparat indeholdende en granulocytkolonistimulerende faktor og især et stabiliseret farmaceutisk præparat indeholdende en granulocytkolonistimulerende faktor, 5 der er beskyttet mod tab eller inaktivering af den aktive komponent (dvs. den granulocytkolonistimulerende faktor) pga. absorption på væggen af beholderen, hvori præparatet findes eller pga. association, polymerisation eller oxidation af denne komponent. Desuden angår 10 opfindelsen en fremgangsmåde til fremstilling af et sådant præparat.The invention relates to a stable pharmaceutical composition containing a granulocyte colony stimulating factor and in particular to a stabilized pharmaceutical composition containing a granulocyte colony stimulating factor which is protected against loss or inactivation of the active component (i.e. the granulocyte colony stimulating factor) due to absorption on the wall, are present or due to association, polymerization or oxidation of this component. In addition, the invention relates to a process for the preparation of such a composition.
Kemoterapi udføres som en metode til behandling af en mangfoldighed af infektionssygdomme, men det har fornylig vist sig, at kemoterapi giver nogle alvorlige 15 kliniske problemer, såsom udvikling af medikamentresistente organismer, ændring af de sygdomsfremkaldende organismer og store bivirkninger. Til undgåelse af disse problemer forbundet med kemoterapi, der involverer anvendelse af terapeutiske midler, såsom antibiotika og 20 baktericider, gøres der forsøg på at anvende et materiale, der aktiverer den profylaktiske evne hos værten for en infektionsfremkaldende organisme, og derved tilvejebringe en fuldstændig løsning på de ovennævnte problemer ved kemoterapi. Af værtens forskellige profylak-25 tiske egenskaber formodes leukocyternes fagocytiske baktericide virkning at have den største indflydelse under den indledende periode af en bakteriel infektion, og den antages derfor at være vigtig til forstærkning af værtens infektionsbeskyttende evner ved at 30 fremme væksten af neutrofiler og deres differentiering til det modne stadium. En granulocytkolonistimulerende faktor (G-CSF) er et af de meget nyttige materialer, der udviser sådanne virkninger, og den samme ansøger som for den foreliggende opfindelse indleverede tidli-35 gere en patentansøgning på et infektionsbeskyttende middel med anvendelse af G-CSF (Japansk Patentansøgning 2 DK 171308 B1 nr. 23777/1985).Chemotherapy is conducted as a method of treating a variety of infectious diseases, but it has recently been found that chemotherapy presents some serious clinical problems, such as developing drug-resistant organisms, altering the pathogenic organisms, and major side effects. In order to avoid these problems associated with chemotherapy involving the use of therapeutic agents such as antibiotics and bactericides, attempts are made to use a material that activates the prophylactic ability of the host of an infection-causing organism, thereby providing a complete solution to the aforementioned problems in chemotherapy. Of the host's various prophylactic properties, the phagocytic bactericidal activity of the leukocytes is thought to have the greatest influence during the initial period of a bacterial infection, and is therefore believed to be important in enhancing the host's infection-protective capabilities by promoting the growth of neutrophils and their differentiation. to the mature stage. A granulocyte colony stimulating factor (G-CSF) is one of the very useful materials exhibiting such effects, and the same applicant as for the present invention previously filed a patent application for an infectious agent using G-CSF (Japanese Patent Application 2 DK 171308 B1 No. 23777/1985).
Som nævnt i det foregående involverer kemoterapi/ som det idag praktiseres, forskellige uundgåelige problemer, og der har været gjort intensive forsøg på 5 at anvende et medikament, der kan aktivere de profylaktiske funktioner hos værten eller hos den person, som er inficeret.As mentioned above, chemotherapy / as it is practiced today involves various unavoidable problems and intensive attempts have been made to use a drug that can activate the prophylactic functions of the host or of the person infected.
Det er unødvendigt at sige, at G-CSP i sig selv udviser evne til at aktive værtens profylaktiske funk-10 tioner, og det har også vist sig, at G-CSF udviser større terapeutiske virkninger ved kliniske anvendelser, hvis det anvendes i kombination med et materiale, der aktiverer værtens profylaktiske evner.Needless to say, G-CSP itself exhibits the ability to activate the prophylactic functions of the host, and it has also been found that G-CSF exhibits greater therapeutic effects in clinical applications if used in combination with a material that activates the prophylactic capabilities of the host.
G-CSF anvendes i en meget lille mængde, og et 15 farmaceutisk præparat indeholdende 0,1-500 pg (fortrinsvis 5-50 pg) G-CSF administreres sædvanligvis med en doseringsrate på 1-7 gange pr. uge til en voksen person. G-CSF har imidlertid tendens til at blive adsorberet på væggen af beholderen, hvori det 20 findes, såsom en ampul til injektion eller en sprøjte. Hvis medikamentet anvendes som et injektionspræparat i form af en vandig opløsning, vil det derfor blive adsorberet på væggen af beholderen, hvori det findes, såsom en ampul eller sprøjte. Dette resulterer enten i at 25 G-CSF'et ikke udviser dets fulde aktivitet som farmaceutisk middel eller nødvendiggør inkorporering af G-CSF i en mængde, der er større end den nødvendige, for at tage hensyn til det mulige tab pga adsorbtion.G-CSF is used in a very small amount and a pharmaceutical composition containing 0.1-500 µg (preferably 5-50 µg) is usually administered at a dosage rate of 1-7 times per day. week for an adult. However, G-CSF tends to be adsorbed on the wall of the container in which it is found, such as a vial for injection or a syringe. Therefore, if the drug is used as an aqueous solution in the form of an aqueous solution, it will be adsorbed on the wall of the container in which it is contained, such as an ampoule or syringe. This either results in the G-CSF not exhibiting its full activity as a pharmaceutical agent or necessitating the incorporation of G-CSF in an amount greater than necessary to account for the potential loss due to adsorption.
Desuden er G-CSF labilt og stærkt følsomt over 30 for miljøfaktorer, såsom temperatur, fugtighed, oxygen og ultraviolette stråler. Ved indvirkning af sådanne faktorer undergår G-CSF fysiske eller kemiske ændringer, såsom association, polymerisation og oxidation, og den lider et stort tab af aktivitet. Disse fænomener 35 gør det vanskeligt at sikre fuldstændig opnåelse af en terapeutisk virkning ved administrering af en meget 3 DK 171308 B1 lille mængde G-CSF på en meget præcis måde.In addition, G-CSF is labile and highly sensitive to 30 environmental factors such as temperature, humidity, oxygen and ultraviolet rays. In effecting such factors, G-CSF undergoes physical or chemical changes, such as association, polymerization and oxidation, and suffers a great loss of activity. These phenomena 35 make it difficult to ensure complete attainment of a therapeutic effect by administering a very small amount of G-CSF in a very precise manner.
Derfor er det nødvendigt at udvikle et stabilt, farmaceutisk præparat af G-CSF, der er fuldstændigt beskyttet mod et fald i dets virksomme komponents 5 aktivitet. Dette er hovedformålet med den foreliggende opfindelse, ifølge hvilken der tilvejebringes et stabilt farmaceutisk præparat af G-CSF samt en fremgangsmåde til fremstilling af et sådant præparat.Therefore, it is necessary to develop a stable pharmaceutical composition of G-CSF that is completely protected against a decrease in the activity of its active component. This is the main object of the present invention, which provides a stable pharmaceutical composition of G-CSF as well as a process for the preparation of such a preparation.
Opfinderne har udført intensive undersøgelser 10 med henblik på forøgelse af stabiliteten for G-CSF-holdige farmaceutiske præparater og fundet, at dette effektivt kan opnåes ved tilsætning af visse nærmere definerede farmaceutisk acceptable overfladeaktive midler, saccharider, proteiner eller forbindelser med høj 15 molekylevægt.The inventors have conducted intensive studies 10 to increase the stability of G-CSF-containing pharmaceutical compositions and found that this can be effectively achieved by the addition of certain more defined pharmaceutically acceptable surfactants, saccharides, proteins or high molecular weight compounds.
Opfindelsen angår således et stabilt farmaceutisk præparat indeholdende granulocytkolonistimulerende faktor, hvilket præparat er ejendommeligt ved, at den granulocytkolonistimulerende faktor er humant G-CSF med 20 de følgende fysisk-kemiske egenskaber: i) molekylvægt: ca. 19.000 ± 1.000 målt ved elektroforese gennem en natrium-dodecylsulfat - polyacrylamid-gel, 25 ii) isoelektrisk punkt: med mindst ét af de tre isoelektriske punkter, pi = 5,5 ± 0,1, pi = 5,8 ± 0,1 og pi 6,1 ± 0,1, iii) ultraviolet absorption: med en maksimal ab- 30 sorption ved 280 nm og en mi nimal absorption ved 250 nm, iv) aminosyresekvens for de 21 rester fra N-ter minus : H2N-Thr-Pro-Leu-Gly-Pro-Ala-Ser-Ser-Leu-Pro-Gln-Ser-35 Phe-Leu-Leu-Lys-Cys-Leu-Glu-The invention thus relates to a stable pharmaceutical composition containing granulocyte colony-stimulating factor, which is characterized in that the granulocyte colony-stimulating factor is human G-CSF having the following physicochemical properties: i) molecular weight: ca. 19,000 ± 1,000 measured by electrophoresis through a sodium dodecyl sulfate-polyacrylamide gel; ii) Isoelectric point: with at least one of the three isoelectric points, pi = 5.5 ± 0.1, pi = 5.8 ± 0.1 and pi 6.1 ± 0.1, iii) ultraviolet absorption: with a maximum absorption at 280 nm and a minimum absorption at 250 nm, iv) amino acid sequence for the 21 residues from N-minus: H2N-Thr -Pro-Leu-Gly-Pro-Ala-Ser-Ser-Leu-Pro-Gln-Ser-35 Phe-Leu-Leu-Lys-Cys-Leu-Glu-
Gln-Val-, 4 DK 171308 B1 og at præparatet foruden den granulocytkolonistimule-rende faktor, der er den virksomme bestanddel, indeholder mindst ét materiale valgt blandt farmaceutisk acceptable overfladeaktive midler valgt blandt ikke-5 ioniske, anioniske og naturlige overfladeaktive midler, hvor det ikke-ioniske overfladeaktive middel kan være en ester af sorbitan, en polyoxyethylensorbitan eller en polyethylenglycol med en alifatisk syre, en polyoxy-ethylenpolyoxypropylenalkylether eller en polyoxyethy-10 leret hærdet castorolie, det anioniske, overfladeaktive middel kan være et alkylsulfatsalt og det naturlige overfladeaktive middel, kan være lecithin, saccharider valgt blandt glycerol, xylitol, sorbitol, mannitol, glucuronsyre, neuraminsyre, ketoglycolsyre, hyaluronsy-15 re og salte deraf, heparin, chitin og derivater deraf, chitosan og derivater deraf, dextrin, dextran med en middelmolekylvægt på 5.000-150.000, og alginsyre og salte deraf, proteiner valgt blandt humant serumalbumin, humant serumglobulin, gelatine, syre- eller base-20 behandlet gelatine med en middelmolekylvægt på 7.000- 100.000, og collagen og forbindelser med høj molekylvægt valgt blandt hydroxypropylcellulose, hydroxyme-thylcellulose, natriumcarboxymethylcellulose, hydroxy-ethylcellulose, polyethylenglycol med en molekylvægt på 25 300-6.000, polyvinylalkohol med en molekylvægt på 20.000-100.000 og polyvinylpyrrolidon med en molekylvægt på20.000-100.000.And that in addition to the granulocyte colony-stimulating factor which is the active ingredient, the composition contains at least one material selected from pharmaceutically acceptable surfactants selected from nonionic, anionic and natural surfactants wherein nonionic surfactant may be an ester of sorbitan, a polyoxyethylene sorbitan or a polyethylene glycol with an aliphatic acid, a polyoxyethylene polyoxypropylene alkyl ether or a polyoxyethylated cured castor oil, the anionic surfactant, the natural sulfate salt and the alkyl sulfate salt. may be lecithin, saccharides selected from glycerol, xylitol, sorbitol, mannitol, glucuronic acid, neuraminic acid, ketoglycolic acid, hyaluronic acid and its salts, heparin, chitin and derivatives thereof, chitosan and derivatives thereof, dextrin, dextran with an average weight of 5. 150,000, and alginic acid and salts thereof, proteins selected from human seru malbumin, human serum globulin, gelatin, acid or base-treated gelatin with an average molecular weight of 7,000-10000, and high molecular weight collagen and compounds selected from hydroxypropylcellulose, hydroxymethylcellulose, sodium carboxymethylcellulose, hydroxyethylcellulose, hydroxyethylcellulose, hydroxyethylcellulose, hydroxyethylcellulose 300-6,000, polyvinyl alcohol having a molecular weight of 20,000-100,000 and polyvinylpyrrolidone having a molecular weight of 20,000-100,000.
I en særlig udformning angår opfindelsen et stabilt farmaceutisk præparat indeholdende granulocytkolo-30 nistimulerende faktor, hvilket præparat er ejendommeligt ved, at den granulocytkolonistimulerende faktor indeholder enten et polypeptid, der har human granulo-cytstimulerende faktoraktivitet og er repræsenteret ved hele eller en del af aminosyresekvensen vist nedenfor, 35 eller et glycoprotein med både dette polypeptid og en sukkerkædedel: 5 DK 171308 B1 (Ket)nThr Pro Leu Gly Fro Ala Ser Ser Leu ProIn a particular embodiment, the invention relates to a stable pharmaceutical composition containing granulocyte colony stimulating factor, characterized in that the granulocyte colony stimulating factor contains either a polypeptide having human granulocyte stimulating factor activity and is represented by all or a portion of the amino acid sequence shown. below, or a glycoprotein with both this polypeptide and a sugar chain moiety: 5 DK 171308 B1 (Ket) nThr Pro Leu Gly Fro Ala Ser Ser Leu Pro
Gin Ser Phe Leu Leu Lys Cys Leu Glu Gin ValGin Ser Phe Leu Leu Lys Cys Leu Glu Gin Val
Arg Lys Ile Gin Gly Asp Gly Ala Ala Leu Gin 5Arg Lys Ile Gin Gly Asp Gly Ala Ala Leu Gin 5
Glu Lys Leu (Val Ser Glu)nCys Ala Thr Tyr LysGlu Lys Leu (Val Ser Glu) nCys Ala Thr Tyr Lys
Leu Cys His Pro Glu Glu Leu Val Leu Leu GlyLeu Cys His Pro Glu Glu Leu Val Leu Leu Gly
Bis Ser Leu Gly Ile Pro Trp Ala Pro Leu SerBis Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser
Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly 10Ser Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly 10
Cys Leu Ser Gin Leu Bis Ser Gly Leu Phe LeuCys Leu Ser Gin Leu To Ser Gly Leu Phe Leu
Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly IleTyr Gin Gly Leu Leu Gin Ala Leu Glu Gly Ile
Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr LeuSer Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu
Gin Leu Asp Val Ala As? Phe Ala Thr Thr Ile 15 Trp Gin Gin Het Glu Glu Leu Gly Het Ala ProGin Leu Asp Val Ala As? Phe Ala Thr Thr Ile 15 Trp Gin Gin Het Glu Glu Leu Gly Het Ala Pro
Ala Leu Gin Pro Thr Gin Gly Ala Het Pro AlaAla Leu Gin Pro Thr Gin Gly Ala Has Pro Ala
Phe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly , Val Leu Val Ala Ser His Leu Gin Ser Phe Leu 20 i Glu Val Ser Tyr Arg Val Leu Arg His Leu AlaPhe Ala Ser Ala Phe Gin Arg Arg Ala Gly Gly, Val Leu Val Ala Ser His Leu Gin Ser Phe Leu 20 in Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala
Gin Pro (forudsat, at m er 0 eller 1, og n er 0 eller 1) og mindst et materiale valgt blandt farmaceutisk accep-25 table overfladeaktive midler, saccharider, proteiner og forbindelser med høj molekylvægt, som anført i et eller flere af kravene 1-5.Gin Pro (assuming m is 0 or 1 and n is 0 or 1) and at least one material selected from pharmaceutically acceptable surfactants, saccharides, proteins and high molecular weight compounds, as claimed in one or more of the claims 1-5.
Pra GB-A 2 016 477 er det kendt at anvende albumin til stabilisering af et HGI (human granulocytindu-30 cerende) glycoprotein under oprensningen af dette.Pra GB-A 2 016 477 is known to use albumin to stabilize an HGI (human granulocyte-inducing) glycoprotein during its purification.
EP-A-0 162 332 viser i eksemplerne, at y-inter-feron (iFN-y) kan stabiliseres ved anvendelse af en modificeret gelatine.EP-A-0 162 332 shows in the examples that γ-interferon (iFN-γ) can be stabilized using a modified gelatin.
EP-A-0 178 576 beskriver en fremgangsmåde til at 35 hindre erythropoietin (EPO) i at blive adsorberet på den indvendige overflade af en beholdervæg. Der er in- 6 DK 171308 B1 gen beskrivelse af stabilisering af en aktiv substans.EP-A-0 178 576 discloses a method of preventing erythropoietin (EPO) from being adsorbed on the inner surface of a container wall. There is no description of stabilization of an active substance.
I alle tre tilfælde er der tale om substanser, der afviger fra G-CSF, der stabiliseres ifølge opfindelsen, på så væsentlige punkter som f.eks. molekyl-5 vægt, polysaccharidindhold, aminosyresammensætning, herunder tilstedeværelse af frie SH-grupper, renhed og lignende, at det G-CSF-holdige stabliserede præparat ifølge opfindelsen ikke kan anses for nærliggende for en fagmand på baggrund af denne kendte teknik.In all three cases, substances which differ from the G-CSF stabilized according to the invention are as important as e.g. molecular weight, polysaccharide content, amino acid composition, including the presence of free SH groups, purity and the like, that the G-CSF-containing stabilized composition of the invention cannot be considered obvious to one of ordinary skill in the art.
10 G-CSF'en til inkorporering i det farmaceutiske præparat ifølge opfindelsen kan opnåes ved en vilkårlig fremgangsmåde, som f.eks. dem der er beskrevet i beskrivelsen til de japanske patentansøgninger nr. 153273/1984, 269455/1985, 269456/1985 (de to sidstnævn-15 te jfr. dansk patentansøgning nr. 4432/86), 270838/1985 og 270839/1985. En human G-CSF kan f.eks. fremstilles enten ved dyrkning af en cellestamme (CNCM Accessions nr. 1-315 eller 1-483) opsamlet fra tumorceller fra patienter med mundhulecancer eller ved eksprime-20 ring af et rekombinant DNA (som var fremstillet ud fra et humant G-CSF-kodende gen) i en passende værtscelle (f.eks. E.coli, C 127-celler eller ovarieceller fra en kinesisk hamster).The G-CSF for incorporation into the pharmaceutical composition of the invention can be obtained by any method such as e.g. those disclosed in the disclosure to Japanese Patent Applications Nos. 153273/1984, 269455/1985, 269456/1985 (the latter two cf. Danish Patent Application No. 4432/86), 270838/1985 and 270839/1985. A human G-CSF can e.g. are prepared either by culturing a cell strain (CNCM Accession Nos. 1-315 or 1-483) collected from tumor cells from oral cancer patients or by expression of a recombinant DNA (which was prepared from a human G-CSF coding gene) in a suitable host cell (e.g., E. coli, C127 cells or ovarian cells from a Chinese hamster).
En hvilken som helst G-CSF med de ovenfor anfør-25 te karakteristika, der er oprenset til en høj grad af renhed, kan anvendes som G-CSF til inkorporering i det farmaceutiske præparat ifølge opfindelsen. Foretrukne humane G-CSF'er er dem der opnås ved isolation fra su-pernatanten fra en kultur af en human G-CSF-dannende 30 celle, og polypeptider eller glycoproteiner med human G-CSF-aktivitet, der opnås ved transformation af en vært med en rekombinant vector, hvori der er inkorope-reret et gen, der koder for et polypeptid med human G-CSF-aktivitet.Any G-CSF having the above-mentioned characteristics purified to a high degree of purity can be used as G-CSF for incorporation into the pharmaceutical composition of the invention. Preferred human G-CSFs are those obtained by isolation from the supernatant from a culture of a human G-CSF-forming cell, and polypeptides or glycoproteins with human G-CSF activity obtained by transformation of a host. with a recombinant vector incorporating a gene encoding a polypeptide with human G-CSF activity.
35 For detaljer vedrørende fremgangsmåden til frem stilling af ovennævnte to typer G-CSF, se beskrivelser- 7 DK 171308 B1 ne til Japansk patentansøgning nr. 153273/1984, 269455/1985, 269456/1985, 270838/1985 og 270839/1985, der alle er indleveret af ansøgeren til den foreliggende ansøgning.35 For details of the process for preparing the above two types of G-CSF, see the descriptions of Japanese Patent Application Nos. 153273/1984, 269455/1985, 269456/1985, 270838/1985 and 270839/1985. all have been filed by the applicant for the present application.
5 En anden fremgangsmåde, der kan anvendes, består i udførelse af fusion af en G-CSP-dannende celle med en selvprolifererende malign tumorcelle og dyrkning af den resulterende hybridoma i eller uden tilstedeværelse af mytogen.Another method that can be used is to perform fusion of a G-CSP-forming cell with a self-proliferating malignant tumor cell and culturing the resulting hybridoma in or without the presence of mitogen.
10 Den opnåede opløsning indeholdende humant G-CSF10 The obtained solution containing human G-CSF
kan opbevares i frossen tilstand efter udførelse af yderligere rensning og koncentrering efter behov ved en vilkårlig kendt metode. Alternativt kan opløsningen opbevares efter dehydratisering ved metoder, såsom fryse-15 tørring.can be stored in the frozen state after further purification and concentration as required by any known method. Alternatively, the solution may be stored after dehydration by methods such as freeze-drying.
Alle de således fremstillede humane G-CSF'er kan oparbejdes i henhold til den foreliggende opfindelse til opnåelse af stabile G-CSF-holdige farmaceutiske præparater.All the human G-CSFs thus prepared can be worked up according to the present invention to obtain stable G-CSF-containing pharmaceutical compositions.
2020
Typiske eksempler på det overfladeaktive middel, der anvendes til opnåelse af det stabile G-CSF-holdige farmaceutiske præparat ifølge opfindelsen er opremset i det følgende: ikke-ioniske overfladeaktive 25 midler med HLB på 6-18, såsom estere af sorbitan og alifatiske syrer (f.eks. sorbitanmonocaprylat, sorbi-tanmonolaurat og sorbitanmonopalmitat), estere af glycerol og alifatiske syrer (f.eks. glycerolmonoca-prylat, glycerolmonomyristat og glycerolmonostearat), 30 estere af polyglycerol og alifatiske syrer (f.eks. de-caglycerylmonostearat, decaglyceryldistearat og deca-glycerylmonolinoleat), polyoxyethylen-sorbitanestere af alifatiske syrer (f.eks. polyoxyethylen-sorbitanmono-laurat, polyoxyethylen-sorbitanmonooleat, polyoxyethy-35 len-sorbitanmonostearat, polyoxyethylensorbitanmonopal-mitat, polyoxyethylen-sorbitantrioleat, og polyoxyethy- 8 DK 171308 B1 len-sorbitantristearat), polyoxyethylen-sorbitolestere af alifatiske syrer (f.eks. polyoxyethylen-sorbitolte-trastearat og polyoxyethylen-sorbitoltetraoleat), po-lyethylenglycerolestere af alifatiske syrer (f.eks. po-5 lyoxyethylenglycerylmonostearat), polyethylenglycole-stere af alifatiske syrer (f.eks. polyethylenglycoldi-stearat), polyoxyethylenalkylethere (f.eks. polyoxy-ethylenlaurylether), polyoxyethylen-polyoxypropylenal-kylethere (f.eks. polyoxyethylen-polyoxypropylenglycol-10 ether, polyoxyethylen-polyoxypropylenpropylether og po-lyoxyethylen-polyoxypropylencetylether), polyoxyethyle-nalkylphenylethere (f.eks. polyoxyethylennonylphenyl-ether), polyoxyethyleret castorolie, polyoxyethyleret hærdet castorolie (polyoxyethyleret, hydrogeneret ca-15 storolie), polyoxyethylerede bivoksderivater (f.eks. polyoxyethyleret sorbitolbivoks), polyoxyethylenlano-linderivater (f.eks. polyoxyethylenlanolin), og polyo-xyethylen(alifatisk syre)amider, (f.eks. polyethylen-stearinsyreamid); ikke-ioniske overfladeaktive mid-20 ler, såsom alkylsvovlsyresalte med en C10_18 alkyl-gruppe (f.eks. natriumcetylsulfat, natriumlaurylsulfat og natriumoleylsulfat), polyoxyethylenalkylether-svovlsyresalte, hvori middelmoltallet for ethylenoxid--addition er 2-4, og alkylgruppen har 10-18 carbonato-25 mer (f.eks. polyoxyethylennatriumlaurylsulfat), salte af alkylsulfosuccinatestere, hvori alkylgruppen har 8-18 carbonatomer (f.eks. natriumlaurylsulfosuccinat-estere); og naturlige overfladeaktive midler, såsom lecithin, glycerophospholipider, sphingophospholipid 30 (f.eks. sphingomyelin), og sucroseestere af alifatiske syrer, hvori den alifatiske syre indeholder 12-18 carbonatomer. Disse overfladeaktive midler kan selvfølgelig anvendes enten enkeltvis eller i blanding.Typical examples of the surfactant used to obtain the stable G-CSF-containing pharmaceutical composition of the invention are listed below: nonionic surfactants with HLB of 6-18, such as esters of sorbitan and aliphatic acids ( e.g., sorbitan monocaprylate, sorbitan monolaurate and sorbitan monopalmitate), esters of glycerol and aliphatic acids (e.g. and deca-glyceryl monolinoleate), polyoxyethylene sorbitan esters of aliphatic acids (e.g., polyoxyethylene sorbitan monauralate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan - sorbitan tristearate), polyoxyethylene sorbitol esters of aliphatic acids (e.g. olyoxyethylene sorbitol tetraoleate), polyethylene glycerol esters of aliphatic acids (e.g. polyoxyethylene glyceryl monostearate), polyethylene glycol sterees of aliphatic acids (e.g. polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g. polyoxyethylene lauryl ether), polyoxyethylene polyoxypropylene alkyl ether (e.g. polyoxyethylene polyoxypropylene polyoxypropylene polyoxyethylene polyoxypropylene propyl ether and polyoxyethylene polyoxypropylene ethyl ether), polyoxyethylene alkyl phenyl ether (e.g. polyoxyethylenonylphenyl ether), polyoxyethylated castor oil, polyoxyethylated hardened castor oil (polyoxyethylated, hydrogenated caustic oil, e.g. , polyoxyethylene lanolin derivatives (eg polyoxyethylene lanolin), and polyoxyethylene (aliphatic acid) amides (eg polyethylene stearic acid amide); nonionic surfactants such as alkyl sulfuric acid salts having a C10-18 alkyl group (e.g., sodium cetyl sulfate, sodium lauryl sulfate and sodium oleyl sulfate), polyoxyethylene alkyl ether sulfuric acid salts wherein the average molar number of ethylene oxide addition is 10-4 and the alkyl group 18 carbon atoms (e.g. polyoxyethylene sodium lauryl sulfate), salts of alkyl sulfosuccinate esters wherein the alkyl group has 8-18 carbon atoms (e.g. sodium lauryl sulfosuccinate esters); and natural surfactants such as lecithin, glycerophospholipids, sphingophospholipid 30 (e.g., sphingomyelin), and sucrose esters of aliphatic acids, wherein the aliphatic acid contains 12-18 carbon atoms. Of course, these surfactants can be used either individually or in admixture.
De overfladeaktive midler opremset ovenfor an-35 vendes fortrinsvis i mængder på 1-10.000 vægtdele pr. vægtdel G-CSF.The surfactants listed above are preferably used in amounts of 1-10,000 parts by weight per day. weight portion G-CSF.
9 DK 171308 B19 DK 171308 B1
Saccharidet til anvendelse ved fremstilling af det stabile G-CSF-holdige farmaceutiske præparat ifølge opfindelsen kan vælges blandt monosaccharider, oligo-saccharider og polysaccharider og phosphatestere og nu-5 cleotidderivater deraf, for så vidt de er farmaceutisk acceptable. Typiske eksempler er opremset i det følgende : trivalente og højere sukkeralkoholer, såsom glycerol, erythritol, arabitol, xylitol, sorbitol, og mannitol; sukkersyrer, såsom glucuronsyre, iduronsyre, neu-10 raminsyre, galacturonsyre, gluconsyre, mannuronsyre, ketoglycolsyre, ketogalactonsyre og ketogulonsyre; hya-luronsyre og salte deraf, chondroitinsulfat og salte deraf, heparin, inulin, chitin og derivater deraf, chi-tosan og derivater deraf, dextrin, dextran med en mid-15 delmolekylvægt på 5.000-150.000, og alginsyre og salte deraf. Alle disse saccharider kan anvendes med fordel enten enkeltvis eller i blanding.The saccharide for use in preparing the stable G-CSF-containing pharmaceutical composition of the invention may be selected from monosaccharides, oligosaccharides and polysaccharides and phosphate esters and nucleotide derivatives thereof, as far as they are pharmaceutically acceptable. Typical examples are listed below: trivalent and higher sugar alcohols such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol; sugar acids such as glucuronic acid, iduronic acid, neuraminic acid, galacturonic acid, gluconic acid, mannuronic acid, ketoglycolic acid, ketogalactonic acid and ketogulonic acid; hyaluronic acid and its salts, chondroitin sulfate and its salts, heparin, inulin, chitin and its derivatives, chitosan and its derivatives, dextrin, dextran having an average molecular weight of 5,000-150,000, and alginic acid and salts thereof. All of these saccharides can be advantageously used either individually or in admixture.
De ovenfor opremsede saccharider anvendes fortrinsvis i mængder på 1-10.000 vægtdele pr. vægtdel G-CSF. 20 Typiske eksempler på proteinet, der kan anvendes til fremstilling af det stabile G-CSF-holdige farmaceutiske præparat ifølge opfindelsen, omfatter humant serumalbumin, humant serumglobulin, gelatine, syrebehandlet gelatine (middelmolvægt 7.000-100.000), basebe-25 handlet gelatine (middelmolvægt 7.000-100.000), og collagen. Disse proteiner kan selvfølgelig anvendes enten enkeltvis eller i blanding.The saccharides listed above are preferably used in amounts of 1-10,000 parts by weight per day. weight portion G-CSF. Typical examples of the protein that can be used to prepare the stable G-CSF-containing pharmaceutical composition of the invention include human serum albumin, human serum globulin, gelatin, acid-treated gelatin (average molecular weight 7,000-100,000), base-treated gelatin (average molecular weight 7,000). -100,000), and collagen. Of course, these proteins can be used either individually or in admixture.
Proteinerne opremset ovenfor anvendes fortrinsvis i mængder på 1-20.000 vægtdele pr. vægtdel G-CSF.The proteins listed above are preferably used in amounts of 1-20,000 parts by weight per day. weight portion G-CSF.
30 Typiske eksempler på forbindelser med høj mole kylvægt til anvendelse ved fremstilling af det stabile G-CSF-holdige farmaceutiske præparat ifølge opfindelsen omfatter: naturlige polymerer, såsom hydroxypropylcel-lulose, hydroxymethylcellulose, natriumcarboxymethyl-35 cellulose og hydroxyethylcellulose; og syntetiske polymerer, såsom polyethylenglycol (molvægt = 300-6.000), 10 DK 171308 B1 polyvinylalkohol (molvægt = 20.000-100.000), og poly-vinylpyrrolidion (molvægt = 20.000-100.000). Det er klart, at disse forbindelser med høj molekylvægt kan anvendes enten enkeltvis eller i blandig.Typical examples of high molecular weight compounds for use in the preparation of the stable G-CSF-containing pharmaceutical composition of the invention include: natural polymers such as hydroxypropyl cellulose, hydroxymethyl cellulose, sodium carboxymethyl cellulose and hydroxyethyl cellulose; and synthetic polymers such as polyethylene glycol (molecular weight = 300-6,000), polyvinyl alcohol (molecular weight = 20,000-100,000), and polyvinylpyrrolidione (molecular weight = 20,000-100,000). It is to be understood that these high molecular weight compounds may be used either singly or in admixture.
5 Forbindelserne med høj molekylvægt opremset ovenfor anvendes hensigtmæssigt i mængder på 1-20.000 vægtdele pr. vægtdel G-CSF.The high molecular weight compounds listed above are conveniently used in amounts of 1-20,000 parts by weight per unit weight. weight portion G-CSF.
Foruden det overfladeaktive middel, saccharidet, proteinet eller forbindelsen med høj molekylvægt beskre-10 vet ovenfor kan mindst en komponent valgt blandt aminosyrer, svovlholdige reduktionsmidler og antioxidanter også inkoporeres ved fremstilling af det G-CSF-holdige farmaceutiske præparat ifølge opfindelsen. Eksempler på aminosyrer omfatter glycin, threonin, tryptofan, lysin, 15 hydroxylysin, histidin, arginin, cystein, cystin og methionin. Eksempler på svovlholdige reduktionsmidler omfatter: N-acetylcystein, N-acetylhomocystein, thioc-tinsyre, thiodiglycol, thioethanolamin, thioglycerol, thiosorbitol, thioglycolsyre og salte deraf, natrium-20 thiosulfat, natriumhydrogensulfit, natriumpyrosulfit, natriumsulfit, thiomælkesyre, dithiothreitol, gluta-thion og et mildt svovlholdigt reduktionsmiddel med en sulfhydrylgruppe, såsom en C^y-thioalkansyre. Eksempler på antioxidanter omfatter erythorbinsyre 25 (isoascorbinsyre), dibutylhydroxytoluen, butylhydroxya-nisol, dl-a-tocopherol, tocopherolacetat, L-ascorbin-syre og salte deraf, L-ascorbinsyrepalmitat, L-ascorbinsyrestearat, triamylgallat, propylgallat og chelateringsmidler, såsom dinatriumethylendiamintetra-30 cetat (EDTA), natriumpyrophosphat og natriummetaphos-phat.In addition to the surfactant, saccharide, protein or high molecular weight compound described above, at least one component selected from amino acids, sulfur-containing reducing agents and antioxidants may also be incorporated in the preparation of the G-CSF-containing pharmaceutical composition of the invention. Examples of amino acids include glycine, threonine, tryptophan, lysine, hydroxylysine, histidine, arginine, cysteine, cystine and methionine. Examples of sulfur-containing reducing agents include: N-acetylcysteine, N-acetylhomocysteine, thioc -tinic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate thio sulfite, sodium hydrogen sulfite, sodium hydrogen sulfite, sodium hydrogen sulfite, sodium hydrogen sulfite, sodium hydrogen sulfite, sodium hydrogen sulfite, sodium hydrogen sulfite a mild sulfur-containing reducing agent with a sulfhydryl group, such as a C 1-6 thioalkanoic acid. Examples of antioxidants include erythorbic acid (isoascorbic acid), dibutyl hydroxytoluene, butyl hydroxyanisole, dl-α-tocopherol, tocopherol acetate, L-ascorbic acid and ethyl saline, l-ascorbic acid, 30 cetate (EDTA), sodium pyrophosphate and sodium metaphosphate.
De ovenfor opremsede aminosyrer, svovlholdige reduktionsmidler og antioxidanter eller blandinger deraf anvendes fortrinsvis i mængder på 1-10.000 vægtdele 35 pr. vægtdel G-CSF.The above-mentioned amino acids, sulfur-containing reducing agents and antioxidants or mixtures thereof are preferably used in amounts of 1-10,000 parts by weight per gram. weight portion G-CSF.
Med henblik på formulering af det stabile G-CSF- 11 DK 171308 B1 holdige præparat ifølge opfindelsen i en passende dosisform, kan et eller flere af de følgende midler inkorporeres: et fortyndingsmiddel, et solubiliserings-hjælpestof, et isotonisk middel, en excipient, et pH-5 modificerende middel, et beroligende middel og en puffer.In order to formulate the stable G-CSF-containing preparation according to the invention in a suitable dosage form, one or more of the following agents may be incorporated: a diluent, a solubilizing auxiliary, an isotonic agent, an excipient, an pH-5 modifier, sedative and buffer.
Det stabiliserede, farmaceutiske G-CSF-præparat ifølge opfindelsen kan formuleres enten til oral eller parenteral administrering, som f.eks. ved injektion på 10 forskellige måder, og en mangfoldighed af dosisformer kan anvendes afhængigt af den specifikke administreringsmetode. Typiske dosisformer omfatter: dem der er beregnet til oral administrering, såsom tabletter, piller, kapsler, granulatpræparater og suspen-15 sionspræparater; opløsninger, suspensionspræparater og frysetørrede præparater, primært beregnet til intravenøs injektion, intramuskulær injektion, subcutan injektion eller intracutan injektion, og de former, der er beregnet til trasmucosal administrering, såsom rectal-20 suppositorier, nasale medikamenter og vaginialsupposi-torler.The stabilized pharmaceutical G-CSF composition of the invention can be formulated either for oral or parenteral administration, e.g. by injection in 10 different ways, and a variety of dosage forms can be used depending on the specific method of administration. Typical dosage forms include: those intended for oral administration, such as tablets, pills, capsules, granular preparations and suspension preparations; solutions, suspension preparations and lyophilized preparations primarily for intravenous injection, intramuscular injection, subcutaneous injection or intracutaneous injection, and the forms intended for trasmucosal administration, such as rectal suppositories, nasal drugs and vaginal suppositories.
Ifølge den foreliggende opfindelse sættes mindst et materiale valgt fra den angivne gruppe bestående af overfladeaktive midler, saccharider, proteiner og for-25 bindeiser med høj molekylvægte til et G-CSF-holdigt farmaceutisk præparat, således at det hindres i at blive adsorberet på væggen af beholderen eller sprøjten, hvori det befinder sig, idet det samtidig forbliver stabilt over langt tid.According to the present invention, at least one material selected from the indicated group consisting of surfactants, saccharides, proteins and high molecular weight compounds is added to a G-CSF-containing pharmaceutical composition so as to prevent it from being adsorbed on the wall of the container or syringe in which it is located, while remaining stable over a long period of time.
30 Den detaljerede mekanisme, ved hvilken materia lerne nævnt i det foregående stabiliserer G-CSF eller hindrer det i at blive adsorberet, er endnu ikke afklaret. I tilstedeværelse af et overfladeaktivt middel, vil overfladen af G-CSF, som er et hydrofobt pro-35 tein, blive dækket med det overfladeaktive middel, der bliver solubiliseret, således at G-CSF'et, det er til 12 DK 171308 B1 stede i spormængder, effektivt hindres i at blive ad-sorberet på væggen af beholderen eller sprøjten, hvori det befinder sig. Et saccharid eller en hydrofil forbindelse med høj molekylvægt vil danne et hydratiseret 5 lag mellem G-CSF og den adsoberende overflade af væggen af beholderen eller sprøjten og derved effektivt hindre adsorption af G-CSF. Et protein vil konkurrere med G-CSF om adsorption på væggen af beholderen eller sprøjten og derved effektivt hindre adsorption af G-10 CSF.The detailed mechanism by which the materials mentioned above stabilize G-CSF or prevent it from being adsorbed has not yet been clarified. In the presence of a surfactant, the surface of G-CSF, which is a hydrophobic protein, will be covered with the surfactant being solubilized, so that the G-CSF, which is present at 12 in trace amounts, effectively prevented from being adsorbed on the wall of the container or syringe in which it is located. A high molecular weight saccharide or hydrophilic compound will form a hydrated layer between G-CSF and the adsorbing surface of the wall of the container or syringe, effectively preventing adsorption of G-CSF. A protein will compete with G-CSF for adsorption on the wall of the container or syringe, effectively preventing adsorption of G-10 CSF.
Foruden forebyggelsen af G-CSF-adsorption, vil materialerne omtalt i det foregående også bidrage til forebyggelse af association eller polymerisation af G-CSF-molekylerne. I tilstedeværelse af et overfladeak-15 tivt middel, saccharid, protein eller en forbindelse med høj molekylvægt, spredes de enkelte G-CSF-molekyler i disse materialer, og interaktionen mellem G-CSF-molekylerne reduceres tilstrækkeligt til fremkaldelse af en væsentlig formindskelse af sandsynligheden for 20 association eller polymerisation af disse molekyler. Desuden vil disse materialer forsinke autooxidation af G-CSF, der accelereres ved høj temperatur eller fugtighed, eller hindre G-CSF i at blive associeret eller po-lymeriseret som et resultat af dets autoxidation. Disse 25 virkninger med forsinkelse af autoxidation af G-CSF eller hindring af G-CSF i association eller polymerisation vil blive forstærket yderligere ved tilsætning af en aminosyre, et svovlholdigt reduktionsmiddel eller en antioxidant.In addition to the prevention of G-CSF adsorption, the materials mentioned above will also contribute to the prevention of association or polymerization of the G-CSF molecules. In the presence of a surfactant, saccharide, protein or a high molecular weight compound, the individual G-CSF molecules are dispersed in these materials and the interaction between the G-CSF molecules is sufficiently reduced to produce a substantial reduction in the probability. for association or polymerization of these molecules. In addition, these materials will delay the auto-oxidation of G-CSF, which is accelerated at high temperature or humidity, or prevent the G-CSF from being associated or polymerized as a result of its autoxidation. These 25 effects of delaying G-CSF autoxidation or inhibiting G-CSF in association or polymerization will be further enhanced by the addition of an amino acid, sulfur-containing reducing agent or antioxidant.
30 Problemerne beskrevet i det foregående træder særligt frem i opløsninger til injektion og i suspensioner, men de forekommer også under processen til formulering af G-CSF i andre dosisformer, såsom tabletter. Tilsætningen af overfladeaktive midler, saccharider, 35 proteiner eller forbindelser med høj molekylvægt er også virksom i det sidste tilfælde.The problems described above are particularly apparent in solutions for injection and in suspensions, but they also occur during the process of formulating G-CSF in other dosage forms, such as tablets. The addition of surfactants, saccharides, proteins or high molecular weight compounds is also effective in the latter case.
13 DK 171308 B113 DK 171308 B1
Som det vil blive vist i eksemplerne, der følger, stabiliseres G-CSF i høj grad og dets aktivitet bevares i lang tid ved tilsætningen af mindst ét materiale valgt blandt overfladeaktive midler, saccharider, 5 proteiner og forbindelser med høj molekylvægt. Til opnåelse af disse resultater er mængden af hver af disse materialer, især de nedre grænser, afgørende, og de følgende mængdeområder er ønskelige: 1-10.000 vægtdele overfladeaktivt middel, 1-10.000 vægtdele saccharid, 10 1-20.000 vægtdele protein og 1-20.000 vægtdele forbindelse med højmolekylvægt pr. 1 vægtdel G-CSF.As will be shown in the examples that follow, G-CSF is highly stabilized and its activity is retained for a long time by the addition of at least one material selected from surfactants, saccharides, proteins and high molecular weight compounds. To achieve these results, the amount of each of these materials, especially the lower limits, is crucial and the following ranges of quantities are desirable: 1-10,000 parts by weight of surfactant, 1-10,000 parts by weight of saccharide, 10 1-20,000 parts by weight of protein and 1-20,000 parts by weight compound with high molecular weight per 1 part by weight of G-CSF.
Ifølge den foreliggende opfindelse anvendes et overfladeaktivt middel, et saccharid, et protein og/eller en forbindelse med høj molekylvægt i en speci-15 ficeret koncentration, og dette er ikke kun effektivt til hindring af G-CSF i at blive adsorberet på væggen af beholderen eller sprøjten, hvori den befinder sig, men også til forbedring af stabiliteten af et G-CSF-holdigt farmaceutisk præparat. Som et resultat heraf 20 bliver det muligt at sikre administrering af en lille, men meget præcis dosis af G-CSF til patienter. Idet G-CSF er dyrt, vil dets effektive udnyttelse føre til lavere omkostninger ved fremstilling af G-CSF-holdige farmaceutiske præparater.According to the present invention, a surfactant, saccharide, protein and / or a high molecular weight compound is used at a specified concentration, and this is not only effective in preventing G-CSF from being adsorbed on the wall of the container. or the syringe in which it is located, but also to improve the stability of a G-CSF-containing pharmaceutical composition. As a result, it will be possible to ensure administration of a small but very accurate dose of G-CSF to patients. Because G-CSF is expensive, its effective utilization will result in lower costs of producing G-CSF-containing pharmaceuticals.
25 De følgende eksempler gives med henblik på nær mere belysning af opfindelsen og må på ingen måde opfattes som begrænsende. I disse eksempler bestemtes restaktiviteten af G-CSF ved en af de følgende metoder.The following examples are provided with a view to more elucidation of the invention and should in no way be construed as limiting. In these examples, the residual activity of G-CSF was determined by one of the following methods.
30 (a) Blød agarmetoden med anvendelse af museknoglemarvs-celler: 0,4 ml hesteserum, 0,1 ml af prøven, 0,1 ml 35 C3H/He (hun)museknoglemarvscellesuspension (0,5- 1 x 105 nuclearceller), og 0,4 ml af en modificeret McCoy's 14 DK 171308 B1 5A-kulturopløsning indeholdende 0,75% agar blandedes, hældtes i en plastskål til vævsdyrkning (35 mm i diameter) , koaguleredes og dyrkedes i 5 dage ved 37 °C i 5% C02/95% luft ved 100% fugtighed. Antallet af dannede 5 kolonier taltes (en koloni bestående af mindst 50 celler) og aktiviteten bestemtes med en enhed som aktiviteten til dannelse af en koloni.(A) Soft agar method using mouse bone marrow cells: 0.4 ml of horse serum, 0.1 ml of the sample, 0.1 ml of 35 C3H / He (female) mouse bone marrow cell suspension (0.5-1 x 105 nuclear cells), and 0.4 ml of a modified McCoy's 14 DK 171308 B1 5A culture solution containing 0.75% agar was mixed, poured into a tissue culture dish (35 mm in diameter), coagulated and cultured for 5 days at 37 ° C in 5% CO 2 / 95% air at 100% humidity. The number of 5 colonies formed was counted (a colony of at least 50 cells) and the activity was determined by a unit such as the colony-forming activity.
Den modificerede McCoy's 5A-kulturopløsning anvendt ved metoden (a) fremstilledes ved den følgende 10 fremgangsmåde.The modified McCoy's 5A culture solution used by method (a) was prepared by the following procedure.
Modificeret McCoy's 5A-kulturopløsning (dobbelt koncentration).Modified McCoy's 5A culture solution (dual concentration).
15 12 g af McCoy's 5A kulturopløsning (Gibco), 2,55 g MEM-aminosyre-vitaminmedium (Nissui Seiyaku Co., Ltd.), 2,18 g natriumhydrogencarbonat og 50.000 enheder kaliumpenicillin G opløstes 2 gange i 500 ml destilleret vand, og opløsningen filtreredes aseptisk gennem et 20 0,22 ym Milliporefilter.12 g of McCoy's 5A culture solution (Gibco), 2.55 g of MEM amino acid vitamin medium (Nissui Seiyaku Co., Ltd.), 2.18 g of sodium bicarbonate and 50,000 units of potassium penicillin G were dissolved twice in 500 ml of distilled water, and the solution was aseptically filtered through a 0.22 µm Millipore filter.
(b) Revers fase HPLC:(b) Reverse phase HPLC:
Restaktiviteten af G-CSP (injiceret i en mængde 25 svarende til lyg) bestemtes ved anvendelse af en revers fase C8-kolonne (4,6 mm x 300 mm; 5 ym) og en n-propanol/trifluoreddikesyreblanding som mobil fase under de følgende gradientbetingelser; 1 2 3 4 5 6The residual activity of G-CSP (injected in an amount corresponding to lyse) was determined using a reverse phase C8 column (4.6 mm x 300 mm; 5 µm) and an n-propanol / trifluoroacetic acid mixture as mobile phase during the following gradient conditions; 1 2 3 4 5 6
Tid (sek.) Opløsn.middel(A) Opløsn♦middel(B) Gradient 2 0 100% 0% 3 lineær 4 15 0% 100% 5 lineær 6 25 100% 0% 15 DK 171308 B1Time (sec) Solvent (A) Solvent ♦ Mean (B) Gradient 2 0 100% 0% 3 linear 4 15 0% 100% 5 linear 6 25 100% 0% 15 DK 171308 B1
Opløsningsmiddel (A): 30% n-propanol og 0,1 tri- fluoreddikesyre,Solvent (A): 30% n-propanol and 0.1 trifluoroacetic acid,
Opløsningsmiddel (B): 60% n-propanol og 0,1% tri- fluoreddikesyre.Solvent (B): 60% n-propanol and 0.1% trifluoroacetic acid.
5 Detektion udførtes ved en bølgelængde på 210 nm, og den procentvise rest-G-CSF-aktivitet beregnes ved hjælp af den følgende formel:Detection was performed at a wavelength of 210 nm and the percent residual G-CSF activity calculated by the following formula:
Rest-G-CSF- restmængden af G-CSF efter 10 aktivitet (%) = et givet tidsrum_ x 100Residual G-CSF - residual amount of G-CSF after 10 activity (%) = given time_ x 100
startmængden af G-CSFthe starting amount of G-CSF
Restmængden af G-CSF bestemt ved denne fremgangsmåde svarerede meget godt til resultaterne opnået 15 ved måling ved blødagarmetoden (a) med anvendelse af museknoglemarvsceller.The residual amount of G-CSF determined by this method corresponded very well with the results obtained by measurement by the soft agar method (a) using mouse bone marrow cells.
EKSEMPEL 1 20 Til 5 pg G-CSF sattes et af stabiliseringsmid lerne angivet i tabel l, og blandingen opløstes aseptisk i 20 mM pufferopløsning (indeholdende 100 mM na-triumchlorid, pH 7,4) til fremstilling af et farmaceutisk præparat indeholdende 5 pg G-CSF pr. ml., som der-25 næst frysetørredes. Den tidsafhængige ændring af G-CSF-aktiviteten måltes ved metoden (a), og resultaterne er vist i tabel l. Udtrykket "aktivitet (%)" i tabellen betegner restaktiviteten af G-CSF i forhold til startaktiviteten og er defineret ved den følgende formel: 30 aktivitet i enheder efterEXAMPLE 1 To 5 µg of G-CSF, one of the stabilizers listed in Table 1 was added and the mixture was aseptically dissolved in 20 mM buffer solution (containing 100 mM sodium chloride, pH 7.4) to prepare a pharmaceutical composition containing 5 µg -CSF pr. ml, which was then lyophilized. The time-dependent change of G-CSF activity was measured by method (a) and the results are shown in Table 1. The term "activity (%)" in the table denotes the residual activity of G-CSF relative to the starting activity and is defined by the following formula : 30 activity in units after
Aktivitet (%) = et givet tidsrum_x 100 startaktivitet i enheder o 35 Frysetørring udførtes ved følgende fremgangsmå de: 16 DK 171308 B1 G-CSF-opløsningen indeholdende et stabiliseringsmiddel fyldtes i et sterilt sulfabehandlet hætteglas, blev frosset til -40 eC eller til en lavere temperatur i 4 timer, underkastedes primær tørring ved op-5 varmning fra -40 °C til 0 °C i løbet af 48 timer, idet trykket øgedes fra 4 til 13,3 Pa, og dernæst sekundær ved opvarmning fra fra 0 °C til 20 °C i løbet af 12 timer med en trykstigning fra 4 til 10,7 Pa. Derefter fyldtes hætteglassets indre med en steril tør nitrogen-10 gas til opnåelse af atmosfæretryk, og hætteglasset lukkedes med en frysetørringsgummiprop og forsegledes dernæst med en aluminiumhætte.Activity (%) = a given time_x 100 initial activity in units o Freeze drying was carried out by the following procedure: 16 The 17-CSF solution containing a stabilizer loaded into a sterile sulphated vial was frozen to -40 ° C or lower. temperature for 4 hours, subjected to primary drying by heating from -40 ° C to 0 ° C over 48 hours, increasing the pressure from 4 to 13.3 Pa, and then secondary by heating from 0 ° C to 20 ° C over 12 hours with a pressure increase from 4 to 10.7 Pa. Then, the vial's interior was filled with a sterile dry nitrogen gas to obtain atmospheric pressure, and the vial was closed with a freeze-drying rubber stopper and then sealed with an aluminum cap.
DK 171308 B1 nDK 171308 B1 n
Tabel 1 Mængde Aktivitet (%) (vægtdele) Efter opbe- Efter opbe-Table 1 Amount of Activity (%) (parts by weight) After storage
Stabiliserincsmiddel . , . . · , " varing ved vanng ved 4°C i 6 370C i 1 _____måneder__måned._ xylitol 10,000 92 86 mannitol 10,000 91 85 10 glucuron-syre 10,000 86 82 hyaluron-éyre 2,000 92 89 dextran (m-v· 40,000) 2,000 95 90 heparin 5,000 85 80 15 chitosan 2,000 93 91 algininsyre 2', 000 90 90 hu&iant serumalbumin 1,000 98 99 humant serumglobulin 1,000 98 95 2o -->---- syrebehandlet gelatine 2,000 97 95 basebehandlet gelatine 1,000 99 96 collagen 2,000 95 90 25 polyethylenglycol (m.v. 4,000) 10,000 94 90 hydroxypropy^cellulose 1,000 98 94Stabilizing agent. ,. . ·, "Being at water at 4 ° C at 637 ° C for 1 _____ months__month. Xylitol 10,000 92 86 mannitol 10,000 91 85 10 glucuronic acid 10,000 86 82 hyaluronic acid 2,000 92 89 dextran (etc. · 40,000) 2,000 95 90 heparin 5,000 85 80 15 chitosan 2,000 93 91 alginic acid 2 ', 000 90 90 human serum albumin 1,000 98 99 human serum globulin 1,000 98 95 2o -> ---- acid-treated gelatin 2,000 97 95 base-treated gelatin 1,000 99 96 collagen 2,000 95 90 25 polyethylene glycol (etc. 4,000) 10,000 94 90 hydroxypropylcellulose 1,000 98 94
Tiatriumcarboxymethyl- . ... on cellulose 1,000 88 80 30 hydroxymethyl cellulose 5,000 92 90 polyvinyl alkphol (rø.v. 50,000) 2/000 96 95 polyvinylpyrrolidon 2 ...Tiatrium carboxymethyl-. ... on cellulose 1,000 88 80 30 hydroxymethyl cellulose 5,000 92 90 polyvinyl alcohol (roughly 50,000) 2/000 96 95 polyvinylpyrrolidone 2 ...
(m.v. 50,000) <2,000 95 94 35 ’ humant serumalbumin 2,000 mannitol 2,000 100 97 cystein 100 5 18 DK 171308 B1(e.g. 50,000) <2,000 95 94 35 'human serum albumin 2,000 mannitol 2,000 100 97 cysteine 100 5 18 DK 171308 B1
Tabel 1 (fortsat) aktivitet (%) . . ,, , Mæn^de Efter opbe-l Efter opbe-Table 1 (continued) activity (%). . ,, Men ^ after After-
Stabiliseringsmiddel (vægtdele) bevar<n| bevaring ved 4°C i ved 37 C i 10 6 måneder 1 måned humant serumalbumin 2,000 polyoxyethvlen 100 99 96 sbrbitanmonolaurat mannitol 2,000 humant serumalbumin 2,000 hydroxypropylcellulose 500 98 92 I dextran pn.v. 40,000) 2,000 polyoxyethylen- ,nn 20 sorbitanmonolaurat 98 96 sorbitol 2,000 polyoxyethyliéret-, hærdet castorolie 94 92 25 dextran ( m.v.40,000) 2,000 ikke tilsat__~__^__£8_ 1 35 DK 171308 B1 19 EKSEMPEL 2Stabilizer (parts by weight) preserves <n | preservation at 4 ° C at 37 C for 10 6 months 1 month human serum albumin 2,000 polyoxyethylene 100 99 96 sbrbitan monolaurate mannitol 2,000 human serum albumin 2,000 hydroxypropyl cellulose 500 98 92 In dextran pn.v. 40,000) 2,000 polyoxyethylene nn 20 sorbitan monolaurate 98 96 sorbitol 2,000 polyoxyethylated hardened castor oil 94 92 25 dextran (m.c. 40,000) 2,000 not added _ ~ __ ^ __ £ 8_ 1 35 DK 171308 B1 19 EXAMPLE 2
Til 10 yg G-CSF sattes et af stabiliseringsmidler angivet i tabel 2, og blandingen opløstes aseptisk 5 i 20 mM phosphatpufferopløsning (indeholdende 100 mM natriumchlorid, pH 7,4) til fremstilling af et farmaceutisk præparat indeholdende 10 yg G-CSP pr. ml. Præparatet fyldtes aseptisk i et sulfabehandlet hætteglas, som forsegledes, til opnåelse af en G-CSP-opløsning.To 10 µg G-CSF was added one of the stabilizers listed in Table 2 and the mixture was dissolved aseptically 5 in 20 mM phosphate buffer solution (containing 100 mM sodium chloride, pH 7.4) to prepare a pharmaceutical composition containing 10 µg G-CSP per ml. ml. The preparation was aseptically filled into a sulfurized vial which is sealed to obtain a G-CSP solution.
10 Den tidsafhængige ændring af G-CSP-aktiviteten i denne opløsning måltes ved den samme metode som anvendt i eksempel 1, og resultaterne er vist i tabel 2.The time-dependent change of G-CSP activity in this solution was measured by the same method as used in Example 1 and the results are shown in Table 2.
DK 171308 B1 20DK 171308 B1 20
Tabel 2Table 2
Aktivitet (%) Mængde Efter , Bfter Efter 5 Stabiliseringsmiddel (vægt- opbeva- opbeva- opbeva- dele) rincf ved rina ved rina ved 4C i 4°C i RT„ i 7 dage 2 mån. 1 mån.Activity (%) Quantity After, After After 5 Stabilizer (weight storage storage) rincf at rina at rina at 4C at 4 ° C in RT for 7 days 2 months. 1 mo.
mannitol 5,000 91 87 82 hyaluronayre * 2,000 93 87 70 10 —---—^------- dextran (m.v. 40,000) 2,000 96 95 85 glycerol 10,000 90 90 88 neuraminsyre 5,000 93 91 84 15 chitin 2,000 95 92 86 dextrin 2,000 90 92 87 humant serumalbumin 1,000 99 95 92 humant serumglobulin 1,000 98 94 90 20 syrebehandlet gelatine 2,000 97 96 87 basebehandlet gelatine 500 99 95 92 collagen 2,000 99 94 88mannitol 5,000 91 87 82 hyaluronic acid * 2,000 93 87 70 10 - - - - - - - dextran (etc. 40,000) 2,000 96 95 85 glycerol 10,000 90 90 88 neuraminic acid 5,000 93 91 84 15 chitin 2,000 95 92 86 dextrin 2,000 90 92 87 human serum albumin 1,000 99 95 92 human serum globulin 1,000 98 94 90 20 acid-treated gelatin 2,000 97 96 87 base-treated gelatin 500 99 95 92 collagen 2,000 99 94 88
polyethylenglycol 10 000 94 89 gQpolyethylene glycol 10,000 94 89 gQ
25 (m. V. 4,000) ιυ,υυυ *4 ay yu hydroxypropyicellulose 2,000 98 95 9225 (m. V. 4,000) ιυ, υυυ * 4 ay yu hydroxypropyicellulose 2,000 98 95 92
Natriumcarboxymethyl- cellulose 2,000 92 91 80 30 hydroxyethylcellulose 4,000 92 94 90 polyvinylalkohol 4,000 97 93 90 \ · m. v. 3 u ·υ u u i pol winy lpyrrolidon 95 95 92 l m.v.bu,uuu) 35 sorbitarfnonolaurat 400 97 96 95 polyoxyet hylen- 400 100 96 94 sorbitaimonolaurat 4UU ιυυ y4 21 DK 171308 B1Sodium carboxymethyl cellulose 2,000 92 91 80 30 hydroxyethyl cellulose 4,000 92 94 90 polyvinyl alcohol 4,000 97 93 90 µm 3 u · υ uui pol viny lpyrrolidone 95 95 92 l mvbu, uuu) sorbitan phenolaurate 400 97 96 95 polyoxyethylene 400 400 96 94 sorbitan monolaurate 4UU ιυυ y4 21 DK 171308 B1
Tabel 2 (fortsat)Table 2 (continued)
Aktivitet (%) Mængde Efter Efter Efter 5 Stabiliserinasmiddel °?beva' opbeva- opbeva- dele) ring ved ring ved r^na ved '* 4°C i 4°C ‘i ÉT' i 7 dage 2 mån. 1 måned polyoxyethylen- 400 98 97 94 scrbitanmonostearat 10----- polyoxyethylen- polyoxypropylen- 400 100 94 93 glycolether polyoxyethyleret, 400 99 98 90 hærdet castorolieActivity (%) Amount After After After 5 Stabilizing agent ° preserve storage) ring by ring at r ^ na at '* 4 ° C at 4 ° C' in ONE 'for 7 days 2 months. 1 month polyoxyethylene 400 98 97 94 scrbitan monostearate 10 ----- polyoxyethylene polyoxypropylene 400 100 94 93 glycol ether polyoxyethylated 400 99 98 90 cured castor oil
Natriumlaurylsulfat 2/000 97 93 87 lecithin 2,000 97 94 90 humant serumalbumin 2 000 mannitol 2,000 100 99 97 20 cystein 100 humant serumalbumin 2,000 polyoxyethylen- 100 99 97 95 sorbitanmonolåurat ±υυ ** 3 25 mannitol 2,000 humant serumalbumin 1,000 hydroxypropylcellulose 500 99 97 95 dextran (m.v. 40,000) 2,000 polyoxyethylen- lonSodium lauryl sulfate 2/000 97 93 87 lecithin 2,000 97 94 90 human serum albumin 2,000 mannitol 2,000 100 99 97 20 cysteine 100 human serum albumin 2,000 polyoxyethylene 100 99 97 95 sorbitan monolaurate ± υυ ** 3 25 mannitol 2,000 human serum albumin 1,000 hydroxypropyl cellulose 500 99 dextran (etc. 40,000) 2,000 polyoxyethylene lon
30 sorbitanmonopalmitat ASorbitan monopalmitate A
96 96 93 sorbitol 2,000 polyoxyethylated, inn96 96 93 sorbitol 2,000 polyoxyethylated, inn
hærdet castorolie iUUhardened castor oil iUU
95 92 92 35 dextran (m.v. 40,000) 2,000 ikke tilsat - 72 61 47 22 DK 171308 B1 EKSEMPEL 395 92 92 35 dextran (etc. 40,000) 2,000 not added - 72 61 47 22 DK 171308 B1 EXAMPLE 3
Til 10 yg G-CSF sattes et af stabiliseringsmidlerne angivet i tabel 3, og blandingen opløstes asep-5 tisk i 20 mM phosphatpufferopløsning (indeholdende 100 mM natriumchlorid, pH 7,4) til opnåelse af et farmaceutisk acceptabelt præparat indeholdende 10 yg G-CSP pr. ml. En milliliter af dette præparat fyldtes i et sulfa-behandlet siliconeovertrukket hætteglas, som man lod 10 henstå ved 4 °C . Effektiviteten af hvert stabiliseringsmiddel til forebyggelse af G-CSF-adsorption bedømtes ved måling af rest-G-CSF-aktiviteten i opløsningen efter 0,5, 2 og 24 timer. Målingerne udførtes ved metode (b) ved anvendelse af revers-fase hplc. Resultater-15 ne er vist i tabel 3.To 10 µg of G-CSF was added one of the stabilizers listed in Table 3, and the mixture was dissolved aseptically in 20 mM phosphate buffer solution (containing 100 mM sodium chloride, pH 7.4) to obtain a pharmaceutically acceptable preparation containing 10 µg of G-CSP per. ml. One milliliter of this preparation was filled into a sulfa-treated silicone-coated vial which was allowed to stand at 4 ° C. The efficacy of each stabilizer for G-CSF adsorption prevention was assessed by measuring residual G-CSF activity in the solution after 0.5, 2 and 24 hours. The measurements were performed by method (b) using reverse phase hplc. The results are shown in Table 3.
DK 171308 B1 23DK 171308 B1 23
Tabel 3 ----—- <- I-“T-Table 3 ----—- <- I- “T-
Restaktivitet (%)Residual activity (%)
Stabiliseringsmiddel Mæn9de (vægtdele initial 0.5 h 2 h 24 h mannitol 5/000 100 93 90 91 hyaluronsyre 2,000 100 97 92 92 dextran (m.v. 40,000) 2,000 100 98 95 96 10 glycerpl 10,000 100 94 91 90 heparin 2,000 100 92 90 90 glucuronsyre 5,000 100 96 90 91 ketoglycolsyre 5,000 100 92 88 90 15 -------- humant serumalbumin 1,000 100 100 101 99 ^ humant serumglobulin 1,000 100 98 100 98Stabilizer Amount (parts by weight initial 0.5 h 2 h 24 h mannitol 5/000 100 93 90 91 hyaluronic acid 2,000 100 97 92 92 dextran (etc. 40,000) 2,000 100 98 95 96 10 glycerop 10,000 100 94 91 90 heparin 2,000 100 92 90 90 glucuronic acid 5,000 100 96 90 91 ketoglycolic acid 5,000 100 92 88 90 15 -------- human serum albumin 1,000 100 100 101 99 ^ human serum globulin 1,000 100 98 100 98
Basebehandlet gelatine 500 100 99 98 99 20 .Syrebehandlet gelatine 2,000 100 99 97 97 collagen 2,000 100 100 98 99 u.rtfooo")91^01 100 100 100 99 hydroxypropylcellulose 2,000 100 100 100 99 25 —---:----- ”lnu™ierbOXymethy1' 2'000 100 98 96 95 hydroxyethylpellulose _ 4,000 100 96 93 92 30 M°° 100 99 100 98 ffy^ffio)011*0" 4-000 100 98 98 96 sorbitarfnonocaprylat 400 100 100 100 98 1 polyoxyet hylen- 40n 100 10Q 10n 35 sorbitarfnonostearat 400 100 100 98 100 — ---- '______ - polyoxyet hyleret, 40Q 100 99 1M 99 hærdet castorolie____J____ DK 171308 B1 24Base-treated gelatin 500 100 99 98 99 20 .Acid-treated gelatin 2,000 100 99 97 97 collagen 2,000 100 100 98 99 u.rtfooo) 91 ^ 01 100 100 100 99 hydroxypropylcellulose 2,000 100 100 100 99 25 --- ---: ---- - "lnu ™ iberOXymethyl 1 2,000 100 98 96 96 95 hydroxyethyl pellulose 4,000 100 96 93 92 30 M 100 100 100 100 98 (ffio) 011 * 0" 4-000 100 98 98 96 sorbitan phenocaprylate 400 100 100 100 98 1 polyoxyethylene 40n 100 10Q 10n 35 sorbitan phenostearate 400 100 100 98 100 - ---- '______ - polyoxyethylene, 40Q 100 99 1M 99 hardened castor oil____J____ DK 171308 B1 24
Tabel 3 (forsat)Table 3 (continued)
Restaktivitet ^ Stabili seringsmiddel længde (vægtdele *n*^*a^· 0*5 h 2 h 24 h I " * ' '—" ----- natriumlaurylsulfat * 2,000 100 100 99 97 lecithin 2,000 100 99 . 100 98 10 humant serumalbumin 2,000 mannitol 2,000 100 100 100 101 cystein 100 humant serumalbumin 2,000 15 P°i£?£i®thyl®nrr“ 100 100 100 98 99 sorbitanmonolaurat mannitol 2,000 i " j humant serumalbumin 1,000 j hydroxypropylpellulose 500 100 101 99 100 20 dextran (m-v· 40,000) 2,000Residual activity ^ Stabilizer length (parts by weight * n * ^ * a ^ · 0 * 5 h 2 h 24 h In "*" - "----- sodium lauryl sulfate * 2,000 100 100 99 97 lecithin 2,000 100 99. 100 98 10 human serum albumin 2,000 mannitol 2,000 100 100 100 101 cysteine 100 human serum albumin 2,000 15 P ° i £? £ i®thyl®nrr “100 100 100 98 99 sorbitan monolaurate mannitol 2,000 i” human serum albumin 1,000 j hydroxypropyl pellulose 500 100 101 99 100 20 dextran (etc. · 40,000) 2,000
polyoxyethylen- 10Qpolyoxyethylene-10Q
so r b i t arhnbno laur at 100 100 99 99 sorbitol 2,000 ------------ *-- ---------- · - . _ - 1 r ------ ·> 25 polyoxyethyLeret, 100 hærdet-castorolie 100 100 98 97 dextran (m.v. 40,000) 2,000 ’ikke tilsat “ 100 91 72 73 30 35so r b i t arhnbno laur at 100 100 99 99 sorbitol 2,000 ------------ * - ---------- · -. 25 Polyoxyethylated, 100 cured castor oil 100 100 98 97 Dextran (m.s. 40,000) 2,000 'not added “100 91 72 73 30 35
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| US4929442A (en) * | 1986-09-26 | 1990-05-29 | Exovir, Inc. | Compositions suitable for human topical application including a growth factor and/or related materials |
| US4847325A (en) * | 1988-01-20 | 1989-07-11 | Cetus Corporation | Conjugation of polymer to colony stimulating factor-1 |
| NO179479C (en) * | 1988-03-11 | 1996-10-16 | Teikoku Seiyaku Kk | Process for the preparation of an intravaginal pharmaceutical preparation |
| US5981485A (en) | 1997-07-14 | 1999-11-09 | Genentech, Inc. | Human growth hormone aqueous formulation |
| US5349052A (en) * | 1988-10-20 | 1994-09-20 | Royal Free Hospital School Of Medicine | Process for fractionating polyethylene glycol (PEG)-protein adducts and an adduct for PEG and granulocyte-macrophage colony stimulating factor |
| GB8824591D0 (en) * | 1988-10-20 | 1988-11-23 | Royal Free Hosp School Med | Fractionation process |
| US6132763A (en) * | 1988-10-20 | 2000-10-17 | Polymasc Pharmaceuticals Plc | Liposomes |
| US5104651A (en) * | 1988-12-16 | 1992-04-14 | Amgen Inc. | Stabilized hydrophobic protein formulations of g-csf |
| EP0459516A1 (en) * | 1990-06-01 | 1991-12-04 | Kirin-Amgen, Inc. | Oral dosage form of biologically active proteins |
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1987
- 1987-07-08 GR GR871067A patent/GR871067B/en unknown
- 1987-07-10 IT IT67594/87A patent/IT1218927B/en active
- 1987-07-13 NL NL8701640A patent/NL192917C/en not_active IP Right Cessation
- 1987-07-14 AT AT0177587A patent/AT402259B/en not_active IP Right Cessation
- 1987-07-15 BE BE8700787A patent/BE1000253A3/en not_active IP Right Cessation
- 1987-07-15 DK DK368387A patent/DK171308B1/en not_active IP Right Cessation
- 1987-07-15 AU AU75665/87A patent/AU611856B2/en not_active Expired
- 1987-07-15 CA CA000542239A patent/CA1297007C/en not_active Expired - Lifetime
- 1987-07-16 PT PT85343A patent/PT85343B/en unknown
- 1987-07-16 NO NO872966A patent/NO171828C/en not_active IP Right Cessation
- 1987-07-16 CH CH2727/87A patent/CH671157A5/en not_active IP Right Cessation
- 1987-07-17 IE IE193387A patent/IE60290B1/en not_active IP Right Cessation
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- 1987-07-17 SE SE8702907A patent/SE503312C2/en not_active IP Right Cessation
- 1987-07-17 IL IL83220A patent/IL83220A/en not_active IP Right Cessation
- 1987-07-17 ES ES8702106A patent/ES2010226A6/en not_active Expired
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- 1987-07-17 YU YU134287A patent/YU47543B/en unknown
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- 1987-07-17 GB GB8716904A patent/GB2193631B/en not_active Expired - Lifetime
- 1987-07-18 CN CN87104963A patent/CN1033738C/en not_active Expired - Lifetime
- 1987-07-18 KR KR1019870007804A patent/KR930004597B1/en not_active IP Right Cessation
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1993
- 1993-05-18 SG SG64393A patent/SG64393G/en unknown
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Legal Events
| Date | Code | Title | Description |
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| B1 | Patent granted (law 1993) | ||
| PUP | Patent expired |