Protection, restoration, and enhancement of erythropoietin-responsive cells, tissues and organs
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- 1Anvendelse av et erytropoietm, valgt fra gruppen bestående av i) et erytropoietm med minst ingen sialmsyrehalvdeler, n) et erytropoietm med minst ingen N-forbundne eller O-forbundne karbohydrater, ni) et erytropoietm med minst et redusert karbohydratmnhold i kraft av behandling av naturlig erytropoietm med minst én glykosidase, iv) el erytropoietm med en karbohydratdel av erytropoielinmolekylet som har minst et ikke-mammalsk glykosylenngsmønster i kraft av ekspresjonen av et rekombinant erytropoietm i ikke-mammalske celler, v) et erytropoietm med minst ett eller flere oksiderte karbohydrater som også kan være kjemisk reduserte, vi) et erytropoietm med minst én eller flere modifiserte arginmrester, vii) et erytropoietm med minst én eller flere modifiserte lysmrester eller en modifikasjon av den N-termmale ammogruppe av erytropoielinmolekylet, viu)ct erytropoietm med minst en modifisert tyrosmrest, ix) et erytropoietm med minst en modifisert aspargmsyre- eller en glutammsyrerest, x) ct erytropoietm med minst en modifisert tryptofanrest, xi) et erytropoietm som har minst én ammogruppe fjernet, xii) ct erytropoietm med minst en åpning ved minst én av cystemforbmdelsene i crytropoietmmolekylet, xui) et erytropoietm tilveiebrakt som har minst én substitusjon av minst én aminosyre, og xiv) el avkortet erytropoietm, for fremstillingen av en farmasøytisk sammensetning for å beskytte, opprettholde, styrke eller gjenoppbygge funksjonen eller viabiliteten av erytropoietinresponsive pattedyrceller og deres assosierte celler, vev og organer 2 Anvendelse ifølge krav l, der nevnte erytropoietm er asialoerytropoietm 3 Anvendelse ifølge krav 2, der nevnte asialoerytropoietm er humant asialoerytropoietm 4 Anvendelse ifølge krav 1, der nevnte erytropoietin ikke har N-forbundne karbohydrater 5 Anvendelse ifølge krav 1, der nevnte erytropoietin ikke har 0-forbundne karbohydrater 6 Anvendelse ifølge krav 1, der at nevnte erytropoietin er behandlet med minst 6n glykosidase 7 Anvendelse ifølge krav 1, der nevnte erytropoietin er uttrykt i en insekt- eller plantecelle 8 Anvendelse ifølge krav 1, der nevnte erytropoietin er periodatoksidert erytropoietin 9 Anvendelse ifølge krav 8, der nevnte penodatoksiderle erytropoietin er kjemisk redusert med natriumeyanoborhydnd 10 Anvendelse ifølge krav 1, der nevnte erytropoietin omfatter en R-glyoksal halvdel og den ene eller flere argimnrester, hvor R er aryl eller alkylhalvdcl 11 Anvendelse ifølge krav 10, der nevnte erytropoietin er fenylglyoksal-erytropoietin 12 Anvendelse ifølge krav 1, der en arginmrest av nevnte erytropoietin er modifisert ved reaksjon med ct tilstøtende diketon, valgt fra gruppen bestående av 2,3-butandion og sykloheksandion 13 Anvendelse ifølge krav 1, der en arginmrest av nevnte erytropoietin er reagert med 3-deoksyglukoson 14 Anvendelse ifølge krav 1, der nevnte erytropoietinmolekyl har minst ett biotinylert lysin eller N-termmal ammogruppe 15 Anvendelse ifølge krav 14, der nevnte erytropoietinmolekyl er biotinylert erytropoietm 16 Anvendelse ifølge krav 1, der nevnte erytropoietm er glusilolyllysinerytropoietin eller fruktosyllysmerytropoietin 17 Anvendelse ifølge krav 1, der en lysinrest av nevnte erytropoietm er karbamylert 18 Anvendelse ifølge krav 1, der en lysinrest av nevnte erytropoietm er aeylert 19 Anvendelse ifølge krav 18, der en lysinrest av nevnte erytropoietm er acctylert 20 Anvendelse ifølge krav 1, der en lysinrest av nevnte erytropoietm er suksmylert 21 Anvendelse ifølge krav 1, der en lysinrest av nevnte erytropoietm er modifisert ved 2,4,6-lnnitrobenzensulfonatnatnum eller et annet salt derav 22 Anvendelse ifølge krav 1, der en lyrosmrest av nevnte erytropoietm er nitrert eller jodert 23 Anvendelse ifølge krav 1, der en aspargmsyre- eller glutaminsyrerest av nevnte erytropoietm er reagert med et karbodnmid etterfulgt av reaksjon med et amin 24 Anvendelse ifølge krav 21, karakterisert ved at nevnte amm er glysinamid 25 Anvendelse ifølge krav 1, der den erytropoietinresponsive celle eller vev er neuronale, retinale, muskel, hjerte, lunge, lever, nyre, tynntarm, binyrebark, bmyremarg, kapillære cndotel, testikkel, ovarie eller endometnumceller eller vev 26 Farmasøytisk sammensetning, karakterisert ved at den omfatter en effektiv erytropoietmresponsiv cellebeskyttende, opprettholdende, styrkende, eller gjenoppbyggende mengde av et erytropoietm valgt fra gruppen bestående av i) et erytropoietm med minst ingen siahnsyrehalvdeler, n) et erytropoietm med minst ingen N-forbundne eller O-forbundne karbohydrater, in) et erytropoietm med minst et redusert karbohydratmnhold i kraft av behandling av naturlig erytropoietm med minst cn glykosidase, iv) et erytropoietm med en karbohydratdel av erytropoietmmolekylet som har minst et ikke-mammalsk glykosylenngsmønster i kraft av ekspresjonen av et rekombinant erytropoietm i ikkc-mammalske celler, v) et erytropoietm med minst ett eller flere oksiderte karbohydrater som også kan være kjemisk reduserte, vi) et erytropoietm med minst én eller flere modifiserte argmmrester, vii) et erytropoietm med minst én eller flere modifiserte lysinrester eller en modifikasjon av den N-tcrminale ammogruppe av erytropoietmmolekylet, vm)et erytropoietm med minst en modifisert tyrosinrest, ix) et erytropoietm med minst en modifisert aspargmsyre- eller cn glutammsyreresl, x) et erytropoietm med minst en modifisert tryptofanrest, xi) et erytropoietm som har minst én ammogruppe fjernet, xii) et erytropoietm med minst en åpning ved minst én av cystemforbmdelsene i erytropoietmmolekylet, xm)et erytropoietm tilveiebrakt som har minst én substilusjon av minst én aminosyre, og xiv) et avkortet erytropoietm 27 Farmasøytisk sammensetning ifølge krav 26, karakterisert ved at nevnte erytropoietm er asialoerytropoietin eller fenylglyoksalerytropoietm 28 Anvendelse av et erytropoietin, for fremstillingen av en farmasøytisk sammensetning for å beskytte, opprettholde, styrke eller gjenoppbygge funksjonen eller viabihteten av en erytropoietmresponsiv celle eller dens assosierte celler, vev eller organer, hvor nevnte celler vev eller organer er ikke-eksiterbare celler, vev eller organer, eller omfatter hovedsakelig ikke-eksiterbare celler eller vev 29 Anvendelse ifølge krav 28, der nevnte erytropoietin er et erytropoietin eller et naturlig erytropoietin, eller en erytropoietmanalog, en erytropoielmetterlikner og erytropoietinfragmcnt, ct hybnd erytropoietinmolekyl, et erytropoietinresptorbmdende molekyl, en erytropoietinagomst, et nyreerytropoietin, et hjemeerytropoietin, en oligomer derav, en multimer derav, et mutein derav, en beslektet derav, en naturlig forekommende form derav, en syntetisk form derav, en rekombinant form derav, en glykosyleringsvanant derav, en deglykosylert variant derav eller en kombinasjon derav 30 Anvendelse ifølge krav 28, der nevnte erytropoietin er fenylglyoksalerytropoietin 31 Fremgangsmåte for beskyttelse, opprettholdelse eller styrking av viabihteten av en celle, et vev eller et organ, isolert fra en pattedyrskropp, karakterisert ved at den omfatter å eksponere nevnte celle, vev eller organ for en farmasøytisk sammensetning omfattende et erytropoietin 32 Fremgangsmåte ifølge krav 31, karakterisert ved at nevnte erytropoietin er et erytropoietin eller et naturlig erytropoietin, eller en erytropoietmanalog, en erytropoietinetterhkner og erytropoictmfragment, et hybnd erytropoietinmolekyl, et erytropoietinresptorbmdende molekyl, en erytropoietinagomst, et nyreerytropoietin, et hjemeerytropoietin, en oligomer derav, en multimer derav, et mutein derav, en beslektet derav, en naturlig forekommende form derav, en syntetisk form derav, en rekombinant form derav, en glykosylenngsvanant derav, en deglykosylert variant derav eller en kombinasjon derav 33 Fremgangsmåte ifølge krav 32, karakterisert ved at nevnte erytropoietin er i) et erytropoietin med minst ingen siahnsyrehalvdeler, II) et erytropoietin med minst ingen N-forbundne eller O-forbundne karbohydrater, III) et erytropoietin med minst et redusert karbohydratinnhold i kraft av behandling av naturlig erytropoietin med minst én glykosidase, iv) et erytropoietin med en karbohydratdel av erytropoietinmolekylet som har minst et ikke-mammalsk glykosylenngsmønster i kraft av ekspresjonen av et rekombinant erytropoietin i ikke-mammalske celler, v) et erytropoietin med minst ett eller flere oksiderte karbohydrater som også kan være kjemisk reduserte, vi) et erytropoietin med minst én eller flere modifiserte argininrester, vn) et erytropoietin med minst én eller flere modifiserte lysinresler eller en modifikasjon av den N-terminale ammogruppe av erytropoietinmolekylet, vin)et erytropoietin med minst en modifisert tyrosmrest, ix) et erytropoietin med minst en modifisert aspargmsyre- eller en glutammsyrerest, x) et erytropoietin med minst en modifisert tryptofanrest, xi) et erytropoietin som har minst én ammogruppe fjernet, xii) et erytropoietin med minst en åpning ved minst én av cysteinforbmdelsene i erytropoietinmolekylet, xiii) et erytropoietin tilveiebrakt som har minst én substitusjon av minst én aminosyre, eller xiv) et avkortet erytropoietin 34 Fremgangsmåte ifølge krav 31, karakterisert ved at nevnte erytropoietin er humant erytropoietin 35 Fremgangsmåte ifølge krav 31, karakterisert ved at nevnte erytropoietin er fenylglyoksalerytropoietin 36 Anvendelse av et erytropoietin valgt fra gruppen bestående av et erytropoietin eller naturlig erytropoietin, eller en erytropoietinanalog, en erytropoietinettcrlikncr og erytropoietinfragment, et hybrid erytropoietinmolekyl, et erytropoietinrcsptorbmdendc molekyl, en erytropoietinagomst, et nyreerytropoietm, et hjemeerytropoietin, en ohgomer derav, en multimer derav, et mutem derav, en beslektet derav, en naturlig forekommende form derav, en syntetisk form derav, en rekombinant form derav, en glykosylenngsvanant derav, en deglykosylert vanant derav eller en kombinasjon derav for fremstillingen av en farmasøytisk sammensetning for gjenoppbygging av kognitiv dysfunksjon i et pattedyr 37 Anvendelse ifølge krav 36, der den kognitive dysfunksjonen er et resultat av skade forårsaket ved anfallssykdom, multippel sklerose, slag, lavt blodtrykk, hjertestans, ischemi, myokardialt infarkt, inflammasjon, aldersrelatert tap av kognitiv funksjon, strålingsskade, cerebral palsi, neurodegenererende sykdom, Alzheimer’s sykdom, Parkinson’s sykdom, Leigh’s sykdom, AIDS-demens, hukommelsestap, amyotrofisk lateral sklerose, alkoholisme, humørforstyrrelser, angstforstyrrelser, forstyrrelse av manglende oppmerksomhet, autisme, Creutzfeld-Jakob’s sykdom, hjerne- eller ryggmargstraume eller ischemi, hjerte-lunge bypass, kronisk hjertesvikt, makulær degenerenng, diabetisk neuropali, diabetisk retmopati, glaukom, retinal ischemi eller retinal traume 38 Anvendelse ifølge krav 36, der nevnte erytropoietin er et fenylglyoksalerytropoietm 39 Anvendelse ifølge krav 36, der nevnte erytropoietin er i) et erytropoietin med minst ingen sialinsyrehalvdeler, u) et erytropoietin med minst mgen N-forbundne eller O-forbundne karbohydrater, ni) et erytropoietin med minst et redusert karbohydratmnhold i kraft av behandling av naturlig erytropoietin med minst én glykosidase, iv) et erytropoietin med en karbohydratdel av erytropoietinmolekylet som har minst et ikke-mammalsk glykosylenngsmønster i kraft av ekspresjonen av et rekombinant erytropoietin i ikke-mammalske celler, v) et erytropoietin med minst ett eller flere oksiderte karbohydrater som også kan være kjemisk reduserte, vi) et erytropoietin med minst en eller flere modifiserte argininrester, vii) et erytropoietin med minst én eller flere modifiserte lysinrester eller en modifikasjon av den N-terminale ammogruppe av erytropoietinmolekylet, vin)et erytropoietin med minst en modifisert tyrosmrest, ix) et erytropoietin med minst en modifisert aspargmsyre- eller en glutammsyrerest, x) et erytropoietin med minst en modifisert tryptofanrest, xi) et erytropoietin som har minst én ammogruppe fjernet, xn) et erytropoietin med minst cn åpmng ved minst én av cysteinforbmdelsene i erytropoietmmolekylet, xm)et erytropoietin tilveiebrakt som har minst én substitusjon av minst én aminosyre, eller xiv) et avkortet erytropoietin 40 Fremgangsmåte for å fremme transeytosen av et molekyl over en endotelcellebamere i et pattedyr, karakterisert ved at den omfatter å admmi strere til nevnte pattedyr en sammensetning som omfatter nevnte molekyl i assosiasjon med et erytropoietin valgt fra gruppen bestående av i) et erytropoietin med minst ingen sialinsyrehalvdeler, n) et erytropoietin med minst ingen N-forbundne eller O-forbundne karbohydrater, 111. et erytropoietin med minst et redusert karbohydratmnhold i kraft av behandling av naturlig erytropoietin med minst én glykosidase, iv) et erytropoietin med en karbohydratdel av erytropoietmmolekylet som har minst et ikke-mammalsk glykosylenngsmønster i kraft av ekspresjonen av et rekombinant erytropoietin i ikke-mammalske celler, v) et erytropoietin med minst ett eller flere oksiderte karbohydrater som også kan være kjemisk reduserte, vi) et erytropoietin med minst én eller flere modifiserte arginmrester, vii) et erytropoietin med minst én eller flere modifiserte lysmrester eller en modifikasjon av den N-termmale aminogruppe av erytropoietmmolekylet, vin)et erytropoietin med minst cn modifisert tyrosmrest, ix) et erytropoietin med minst en modifisert asparginsyre- eller en glutammsyrerest, x) et erytropoietin med minst en modifisert tryptofanrest, xi) et erytropoietin som har minst én aminogruppe (jernet, xii) et erytropoietin med minst en åpning ved minst cn av cysteinforbmdelsene i erytropoietmmolekylet, xm)et erytropoietin tilveiebrakt som har minst én substitusjon av minst én aminosyre, og xiv) et avkortet erytropoietin 41 Fremgangsmåte ifølge krav 40, karakterisert ved at nevnte assosiasjon er cn labil, kovalent binding, en stabil, kovalent binding eller en ikke-kovalent assosiasjon med et bindingssete for nevnte molekyl 42 Fremgangsmåte ifølge krav 40, karakterisert ved at nevnte ' endotelcellebamere er valgt fra gruppen bestående av blod-hjeme bameren, blod-øye barrieren, blod-lestikkcl barrieren, blod-ovarie bameren og blod-placenta bameren 43 Fremgangsmåte ifølge krav 40, karakterisert ved at nevnte molekyl er et reseptoragorusthormon eller reseptorantagonisthormon, en neurotrofisk faktor, et antimikrobielt middel, et radioaktivt legemiddel, et antisens ohgonukleotid, et antistoff, et lmmunforsvarsdempende middel eller et anti-cancerlegemiddel 44 Sammensetning for transport av et molekyl via transeytose over en endotelcellebamere, karakterisert ved al den omfatter nevnte molekyl i assosiasjon med et erytropoietin valgt fra gruppen bestående av i) et erytropoietin med minst ingen sialmsyrehalvdeler, li) et erytropoietin med minst ingen N-forbundne eller O-forbundne karbohydrater, ni) et erytropoietin med minst et redusert karbohydratinnhold i kraft av behandling av naturlig erytropoietin med minst én glykosidasc, iv) et erytropoietin med en karbohydratdel av erytropoietmmolekylet som har minst ct ikke-mammalsk glykosyleringsmønster i kraft av ekspresjonen av ct rekombinant erytropoietin i ikke-mammalske celler, v) el erytropoietin med minst ett eller flere oksiderte karbohydrater som også kan være kjemisk reduserte, vi) et erytropoietin med minst én eller flere modifiserte argininrester, vn) et erytropoietin med minst én eller flere modifiserte lysmrester eller en modifikasjon av den N-terminale aminogruppe av erytropoietmmolekylet, vm)et erytropoietin med minst en modifisert tyrosinrest, ix) ct erytropoietin med minst en modifisert aspargmsyre- eller en glutaminsyrerest, x) et erytropoietin med minst en modi fisert tryptofanrest, xi) et erytropoietin som har minst én aminogruppe fjernet, xii) et erytropoietin med minst en åpning ved minst én av cysteinforbmdelsene i erytropoietmmolekylet, xm)et erytropoietin tilvciebrakt som har minst en substitusjon av minst én aminosyre, og xiv) et avkortet erytropoietin 45 Sammensetning ifølge krav 44, karakterisert ved at nevnte assosiasjon er en labil, kovalent binding, en stabil, kovalent binding eller en ikke-kovalent assosiasjon med et bindingssete for nevnte molekyl 46 Sammensetning ifølge krav 44, karakterisert ved at nevnte molekyl er et reseptoragonisthomion eller reseptorantagomsthormon, en neurotrofisk faktor, et antimikrobielt middel, et radioaktivt legemiddel, et antisens oligonukleotid, et antistoff, et immunforsvarsdempende middel eller et anti-cancerlegemiddel 47 Anvendelse av et erytropoietm valgt fra gruppen bestående av i) et erytropoietm med minst ingen sialmsyrehalvdeler, ii) et erytropoietm med minst ingen N-forbundne eller O-forbundne karbohydrater, ni) et erytropoietm med minst et redusert karbohydratmnhold i kraft av behandling av naturlig erytropoietm med minst 6n glykosidase, iv) et erytropoietm med en karbohydratdel av erytropoietmmolekylet som har minst et ikke-mammalsk glykosylenngsmønster i kraft av ekspresjonen av et rekombinant erytropoietm i ikke-mammalske celler, v) et erytropoietm med minst ett eller flere oksiderte karbohydrater som også kan være kjemisk reduserte, vi) ct erytropoietm med minst én eller flere modifiserte argmmrcster, vii) et erytropoietm med minst én eller flere modifiserte lysmrester eller en modifikasjon av den N-tcrminale ammogruppe av erytropoietmmolekylet, vin) et erytropoietm med minst en modifisert tyrosmrest, ix) et erytropoietm med minst en modifisert aspargmsyre- eller en glutammsyrerest, x) el erytropoietm med minst en modifisert tryptofanrest, xi) et erytropoietm som har minst én ammogruppe fjernet, xn) et erytropoietm med minst en åpning ved minst én av cystemforbmdelsene i erytropoietmmolekylet, xm)et erytropoietm tilveiebrakt som har minst én substitusjon av minst én aminosyre, og et avkortet erytropoietm assosiert med et molekyl for fremstillingen av en farmasøytisk sammensetning for transport av nevnte molekyl via transeytose over en endotelcellebamere 48 Anvendelse ifølge krav 47, der nevnte assosiasjon er en labil, kovalenl binding, en stabil, kovalent binding eller en ikke-kovalent assosiasjon med et bindingssete for nevnte molekyl 49 Anvendelse ifølge krav 47, der nevnte molekyl er et reseptoragomsthormon eller reseptorantagonisthormon, en neurotrofisk faktor, et antimikrobiell middel, et radioaktivt legemiddel, et antiscns oligonukleolid, et antistoff, et immunforsvarsdempende middel eller et anti-cancerlegemiddel 50 Sammensetning, karakterisert ved at den omfatter penodatoksidert erytropoietin 51 Sammensetning, karakterisert ved at den omfatter glysitolyllysinerytropoietin 52 Sammensetning, karakterisert ved at den omfatter fruktosyll ys i nery tropoi eli n 53 Sammensetning, karakterisert ved at den omfatter 3-deoksyglukosonerytropoietin 54 Sammensetning, karakterisert ved at den omfatter karbamylerl asialoerytropoictin 55 Sammensetning, karakterisert ved at den omfatter biotmylert asialoerytropoietin 56 Sammensetning, karakterisert ved at den om fatter suksmylert asialoerytropoietin 57 Sammensetning, karakterisert ved at den omfatter acetylert asialoerytropoietin
241 paragraphs in 5 sections, as filed
PROTECTION, REBUILDING AND STRENGTHENING OF CELLS ERYTROPOIETINRESPONSIVE, tissues and organs
Priority of temporary Application No. 60/259 245, filed December 29, 2000, entirely incorporated by reference hen, the requirement under 35 USC § 119 (e) (l) BACKGROUND OF THE INVENTION For many years the only, clear physiological role of erythropoietin been its control of the production of red blood cells Lately suggests several directions for evidence that erythropoietin as a member of cytokmsuperfamilien, performs other important physiologic functions which are mediated through interactions with erythropoietin (erytropoietm-R) These virkmnger include mitogenesis, modulating kalsiuminfluks to smooth muscle cells and nerve cells and effects of intermedicrt metabohsme It is believed that erythropoietin tilveiebnnger compensation responses that serve to improve hypoxic cellular microenvironment as well as modulate programmed cell death caused by metabolic stress Although studies have proven that erythropoietin injected into the skull protects neurons against hypoxic neuronal injury, intrakramell administration cr an impractical to unacceptable way of administration for therapeutic use, particularly for normal mdivider Moreover, previous studies of anemic patients given erythropoietin have concluded that peripherally administered erythropoietin is not transported into the brain ( Marti et al, 1997 Kidney Int 51 416-8, Juul et al, 1999, Pediatr Res 46 543-547, Buemi et al, 2000, Nephrol Dial Transplanl 15 422-433)
Various modified forms of erythropoietin have been described with activities directed towards improving the erythropoietic activity of the molecule, such as those with altered amino acids at the carboxy end, disclosed in US Patent 5,457,089 and in U.S. Patent 4,835,260, erytropoietimsoformer with different number of siahnsyrerester per molecule, as described in U.S. Patent 5,856,292, polypeptides described in US Patent 4,703,008, agonists described in U.S. Patent 5,767,078, peptides that bind to the erythropoietin receptor as described in U.S. Patents 5,773,569 5,830,851, and small molekylelterliknere, as described in U.S. Patent 5,835,382
The present invention is directed to the use of an erythropoietin for protecting, maintaining, enhancing or rebuild erytropoietmresponsive cells and associated cells, tissues and organs in situ as well as ex vivo, and to deliver an erythropoietin through a endotelcellebamere for use of protecting and strengthening of erytropoietmresponsive cells and associated cells, tissues and organs dislale to the vasculature, or to carry associated molecules
BRIEF DESCRIPTION OF THE INVENTION
In one aspect, the present invention is directed to anvendelseh of erythropoietins for the preparation of pharmaceutical compositions for rebuilding the function or viability of erytropoietinresponsive mammalian cells and their associated cells, tissues and organs In a specific aspect, the erytropoietinresponsive mammalian cells and their associated cells, tissues and organs distal to the vasculature by virtue of a tight endotelcellebamere In a annct particular aspect, the cells, tissues, organs or other body parts isolated from a mammalian body, such as those intended for transplant, using non-limiting examples, the erytropoietinresponsive cell or tissue be neuronal, retmale, muscular, heart, lung, liver, kidney, small intestine, adrenal, bmyremarg, kappilærendotel, testis, OVAN, pancreas or endometriumceller or tissues These examples of erytropoietinresponsive cells is illustrative only one one aspect, the cell or its erytropoietinresponsive associated cells, tissues or organs, not excitable cells, tissues, or organs, or do not include mainly excitable cells or tissues 1:01 particular utførclscsform mammalian cell, tissue or organ in which a previously mentioned crylropoietindenval used, those used or will benefit from a period of time under at least one condition counteracting the viability of the cell, tissue or organ Such conditions include traumatic m situ hypoxia or mctabolsk dysfunction, surgically induced in situ hypoxia or metabolic dysfunction, or in situ toksineksponenng, the latter may be associated with chemotherapy or radiation therapy In one embodiment, the countervailing conditions resulting from kardipulmonal by-pass (hjcrte-lung machine), which is used for certain surgical procedures
Erythropoietins are useful for the therapeutic or prophylactic treatment of human diseases of the central nervous system or the reception buffers nervous system which have primarily neurological or psykiatnske symptoms, as well as eye diseases, cardiovascular diseases, cardiopulmonary diseases, respiratonske diseases, kidney, unnveis and reproductive diseases, gastromlestinale diseases and metabolic abnormalities and endoknne
The invention is also directed to pharmaceutical compositions comprising certain erytropoietindcnvater for administration to a mammal, fortnnnsvis a human Such pharmaceutical compositions can be formulated for oral, intranasal or parcnteral administration, or in the form of a pcrfusjonsløsnmg for maintaining viability of cells, tissues or organs ex vivo
Erytropoietmdenvater useful for the aforementioned purposes may be any natural erytropoielin or an erythropoietin analog, one erytropoietinetterlikner and erytropoietinfragment, one hybnd erythropoietin molecule, an erythropoietin-reseptorbmdende molecule, one erytropoietmagonist, a renal erytropoietm, a hjemc erythropoietin, an oligomer thereof , one multimer thereof, a mutein thereof, a cognate thereof, a naturally occurring form thereof, a synthetic form thereof, a recombinant form thereof, one glykosylenngsvariant thereof, one deglycosylated Vanant thereof or a combination thereof Any kind of erytropoietm able to be in favor of erytropoietmresponsive cells, are included in this aspect of the invention
Other erytropoietindenvater useful for the aforementioned purposes and pharmaceutical compositions include both natural erythropoietin, as well as erytropoietmer modified with at least one modification as compared with natural erytropoietm, and preferably as compared to natural human erytropoietm The at least one modification may be a modification of at least one amino acid of the erythropoietin molecule, or a modification of at least one carbohydrate at erytropoietmmolekylet course erytropoietmmolekyler useful for the purposes is here, have a plurality of modifications compared to the part the natural molecule, such multiple modifications of ammosyrcdclcn of the molecule, multiple modifications of the carbohydrate part of the molecule or at least one modification of amoinosyredelen of the molecule and at least one modification of the carbohydrate portion of the molecule The modified erytropoietmmolekylet retains its ability to protect, maintain, enhance or restore function or viabihteten of erytropoietmresponsive pattedyrccller, still other properties of erytropoietmmolekylet not related to the previously mentioned desirable properties, may be absent, as compared to the natural molecule In a preferred embodiment seen form erytropoietindenvatet non-erythropoietic In a utførelscsform erythropoietin of the invention at least no siahnsyrehalvdeler In a preferred embodiment, the modified erythropoietin asialoerytropoietm, and most preferably human asialoerytropoietin 1 another embodiment, the modified erythropoietin 1.2, 3, 4.5, 6, 7, 8, 9,10,11,12 or 13 siahnsyrehalvdeler In a second embodiment, the modified erythropoietin has at least no N-linked or no O-linked carbohydrates 1 a third embodiment, the modified erythropoietin has at least a reduced karbohydratmnhold virtue of treatment of erytropoietm with its native carbohydrates with at least one glycosidase 1 a fourth embodiment, the carbohydrate portion of the modified erylropoictinmolekylet least a non-mammalian glykosylenngsmønster virtue of expression of a recombinant erythropoietin in non-mammalian cells In a preferred embodiment, the modified erythropoietins expression msektceller or plant cells 1 a fifth embodiment, the modified erytropoietmet least one or more oxidized carbohydrates which also may be chemically reduced In a preferred embodiment, the modified erytropoietmet penodatoksidert erythropoietin, in another embodiment penodatoksidert erythropoietin chemically reduced with borhydndsalt such as natnumklorhydnd or natnumeyanborhydnd 1 a sixth embodiment, the modified erytropoietmet for the application mentioned earlier, at least one or more modified arginine residues In one embodiment, the modified erytropoietmet a glyoksalhalvdel the one or more arginine residues, such as a arylglyoksal- or alkylglyoksalhalvdel In another embodiment, at least one argimnrest modified by reaction with el adjacent dikcton, such as, but not limited to 2,3-butanedione or cyclohexanedione, 1 seventh embodiment, the modified erytropoietmet least one or more modified lysine residues or a modification of the N-terminal ammo group of erytropoielmmolckylel, such modifications are those resulting from the reaction of glysmresten or the N-term male ammo group with an ammo group modifier The modified lysine residue may also be chemically produced in a preferred embodiment, an erythropoietin biotinylcrt or carbamylated via one or more lysine groups In another preferred embodiment, the lysine is reacted with the aldehyde or reducing sugar to form a lmin which can be stabilized by reduction with natnumeyanborhydnd to form an N-alkylated lysine such as glucitolyllysin or as in the case of reducing sugars may be stabilized by amadon or Heyns rearrangement to form an alpha-deoxy alpha-amino sugar such as alpha-dcoksy alpha fruktosyllysm 1 another preferred embodiment, the lysine carbamylated (carbamoylating), such as by virtue of reaction with cyanation, alkyl-carbamylated, aryl-carbamylated, or aryl-tiokarbamylert with an alkyl-isocyanate, aryl-isocyanate, or aryl-lsotiocyanat, or it may be aeylert by a reactive alkylcarboxylic or arylkarboksylsyredenvat, such as by reaction with eddiksyreanhydnd , ravsyreanhydnd or ftalsyreanhydnd At least one lysine group may also be tnnitrofenylmodifisert by reaction with a tnnitrobenzensulfonsyre or fortrmnvis its salts In another embodiment lysmrestene be modified by reaction with an glyoksaldenvat, such as reaction with glyoxal, methyl glyoxal or 3 dioksyglukoson to form the corresponding alpha-karboksyalkyldenvatene In an eighth embodiment, at least one tyrosmrest of erythropoietin be modified in an aromatic nngposisjon by eleklrofil reagent, such as by nitrenng or jodinenng In a ninth embodiment, at least one aspargmsyre or glutamic acid residue of an erythropoietin be modified, such as by reaction with an karbodnmid followed by reaction with a amm such as, but not limited to glysmamid In a tenth embodiment, at least a tryptophan residue of an erythropoietin modified such as by reaction with n-bromosuccinimide or n-klorsuksimmid 1 an eleventh embodiment, a modified erythropoietin molecule provided with at least one erytropoietmamingruppe pond, such as by reaction with ninhydnn followed by reaction of the resulting carbonyl group by reaction with borhydnd In a twelfth embodiment, a modified erythropoietin is provided having at least an opening of at least one of cysleinforbmdclsene in erytropoietmmolekylet by reaction with ct reducing agent such as dithiothreitol followed by leaksjon the subsequent sulhydryllene with jodacetamm, iodoacetic acid or another electrophile to prevent reformation of disulfide compounds In a thirteenth embodiment, a modified erythropoietin is provided having at least one substitution of any one of a number of amino acids, such as a leucine with at least one of lysine, arginine, tryptophan, tyrosm or eystemrester of erythropoietin, using molecular biological techniques In a fourteenth embodiment, a modified erythropoietin subjected to a limited chemical proteolysc targeted to specific residues, for example, to column after tryptophan residues Such resulting erytropoietmfragmentcr covered herein
As described above, the erythropoietin useful for use herein have at least one of the aforementioned modifications, but may have more than one of the modifications above By example of a modified erythropoietin with one modification to the carbohydrate portion of the molecule and one modification to the ammo acid portion, is a modified erythropoietin asialoerytropoictin and has its lysine residues biotmylert or carbamylated The present invention also includes compositions including pharmaceutical compositions comprising one or more of the aforementioned erytropoielinene In another aspect of the invention there is provided a method for protecting, maintaining, enhancing or restore function or viabihteten of erytropoietmresponsive palledyrceller and their associated cells, tissues and organs, by administering an effective amount of any or more of the aforementioned erythropoietins In a particular aspect of the method is the erytropoietmresponsive mammalian cells and their associated cells, tissues or organs distal to the vasculature by virtue of a tight endotelcellebamere one another particular aspect, the cells, tissues, organs or other body parts isolated from a pattcdyrkropp, such as those intended for transplant by non-limiting examples, the dc erytropoietmresponsive cells or tissues be neuronal, retinal , muscular, heart, lung, liver, kidney, small intestine, adrenal, bmyremarg, kappilærendotel, testis, ovary or endometnumceller or tissues These examples of erytropoietmresponsive cells is illustrative only In a particular embodiment, the erytropoietmresponsive cell or its associated cells, tissues, or means not excitable cells, tissues, or organs, or do not include mainly excitable cells or tissues In another particular embodiment, the mammalian cell, tissue or organ as previously mentioned erytropoietindenvat be supplied to, the one who has consumed or will consume a period of time during at least one state opposing to viabihteten of cell, tissue or organ Such conditions may include traumatic m situ hypoxia or metabolic dysfunction, surgically induced in situ hypoxia or metabolic dysfunction, or tn situ toksineksponermg, the latter may be associated with chemotherapy or strålmgsterapi In one embodiment protects invention against the counteracting conditions of kardipulmonal bypass In another aspect of the invention, any of the foregoing erythropoietins as well as any other erylropoietinmolekyler which includes natural, human crytropoietm used in the preparation of a pharmaceutical composition for ex vivo treatment of cells , tissues and organs for the purpose to protect, maintain, enhance or restore function or viabihteten of erytropoietmresponsive pattedyrcellcr and their associated cells, tissues and organs Such ex vivo treatment is useful, for example for storage of cells, tissues or organs for transplantation, whether autotransplantation or xenotransplantation cells, tissue or organ may be bathed in a solution comprising erytropoietm or perfusate instilled into the organ through the vasculature or other means for maintaining cellular function during the period wherein the cells, tissue or organ is not integrated with the vasculature of the donor or the recipient Administration of the perfusate can be made to a donor prior organhøstmgen, as well as to the HoStel means and to the receiver Moreover, the aforementioned use of any erytropoietm useful when a cell, tissue or organ is isolated from the vasculature of the individual and thus essentially existing ex vivo for a period of time, the term isolated referring to restricting or compressing the vasculature of or to the cell, tissue, organ or body part, such as it can be accomplished by surgery, which specifically include kardipulmonal by-pass surgery, bypassing the vasculature of the cell , tissue, organ or body part, removing the cell, tissue, organ or bodily part from the mammalian body, which can be performed before xenotransplantation or prior to or during autotransplantation or traumatic amputation of a cell, tissue, organ or body thus relates to this aspect of the invention, both perfusion with an erythropoietin in situ and ex vivo Ex vivo can erytropoietmet provided in cell, tissue or organ in oppbevanngsløsmng For both aspects exposure can be by means of continuous perfusion, pulsatile perfusion, infusion, bathing, injection or katetensenng In a still further aspect, the invention directed to a method to protect, maintain, enhance or restore viabihteten a mammalian cell, tissue, organ or body part which includes a erytropoietinresponsiv cell or tissue, wherein the cell, tissue, organ or bodily part is isolated from the mammalian body method includes at least exposing the isolated mammalian cell, tissue, organ or body of an amount of erythropoietin of a side that is effective to protect, maintain, enhance or restore the aforesaid viabihteten one non-limiting examples the isolated to narrow or compress the vasculature of or to the cell, tissue , organ or body part, such as may be performed during surgery, particularly when cardiopulmonary bypass surgery, by-pass of the vasculature of the cell, tissue, organ or body part, removing the cell, tissue, organ or body part from the mammalian body, which can be performed before xenotransplantation or prior to or during auto transplant, or traumatic amputation of a cell, tissue, organ or body therefore relates to this aspect of the invention both the perfusion with an erythropoietin in situ and ex vivo Ex vivo can erytropoietmet provided in cell, tissue or organ in oppbevanngsløsmng For both may exposure be by means of continuous perfusion, pulsatile perfusion, infusion, bathing, injection, or katetensenng 1 the aforementioned isolation or ex-vivo embodiment, a useful erythropoietin may be any of the aforementioned erytropoietinenc, includes any natural erythropoietin, or one erytropoietmanalog, one erytropoietmetterhkner and erytropoietinfragmenl, a hybnderytropoietmmolekyl, a erytropoietinreseptorbindende molecule, one erylropoietmagonist, etc renal erythropoietin, a hjemeerytropoietm, one ohgomer thereof, one multimer thereof, a mutein thereof, a cognate thereof, a naturally occurring form thereof, a synthetic form thereof, a recombinant form thereof, one glykosylenngsvanant thereof, one deglycosylated variant thereof, or a combination thereof Any form of erythropoietin capable of being in favor of erytropoietmresponsive cells are included in this aspect of the invention Other erythropoietins include, but are not limited to asialoerytropoietin , N-deglycosylated erythropoietin, 0-deglycosylated erythropoietin, erythropoietin with reduced karbohydratiimhold, erythropoietin with altered glykosylenngsmønster, erythropoietin with carbohydrates oxidized then reduced, arylglyoksal-modified erythropoietin, alkylglyoksal-modified erythropoietin, 2,3-butanedione-modified erythropoietin, cyclohexanedione modifiscrt erythropoietin, biotinylated erythropoietin, N-alkylated-lysyl-erythropoietin, glusitolyllysm erythropoietin, alpha-deoxy-alpha-fruktosyllysm-crytropoietin, carbamylated erythropoietin, acetylated erythropoietin, suksmylert erythropoietin, alpha-carboxyalkyl erythropoietin, mtrcrt erythropoietin, iodinated erythropoietin to name some representative however, non-limiting examples based on the teachings here A human erythropoietin is preferred, native human erythropoietin is most preferred 1 cn another embodiment, human asialoerytropoietin preferably In another embodiment human phenylglyoxal erythropoietin is preferred
In a non-limiting example, the aforementioned ex νινο erytropoietmresponsivc cell or tissue, be or comprise neuronal, retinal, muscle, heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, kappilærendotel, testis, ovary or endometnumceller or tissues These examples of erytropoietinresponsive cells are only lllustrative
All of the foregoing methods and uses are preferably applicable to humans, but is also useful for any mammal, such as but not limited to, pets, domestic animals, livestock and animals in zoo supply routes of the aforementioned pharmaceutical compositions include oral, intravenous, intranasal , topical, intraluminal, inhalation or parenteral administration, the latter including intravenous, intraarterial, subcutaneous, intramuscular, mtraperitoneal, submucosal or mtradermal For ex vivo application is a perfusate or badeløsnmg preferred This includes perfusing an isolated portion of the vasculature m situ In a further aspect of the invention any of the aforementioned erytropoietmene useful in preparing a pharmaceutical composition for the reconstruction of one dys functional cell, tissue or organ when administered after the onset of the disease or condition responsible for the dysfunction By non-limiting examples rebuilds administration of a pharmaceutical composition comprising erythropoietin, cognitive function in animals previously had home trauma, even when administered long after (e.g., three days, five days, one week, one month or longer) trauma has subsided Erythropoietins useful for such applications includes any of the particular aforementioned erytropoietmene or any natural erythropoietin, or an erythropoietin analog, one erytropoietinetterhkner and erytropoietinfragment, a hybrid erytropoietmmolekyl, an erythropoietin reseptorbindcnde molecule, one erytropoietinagomst, a renal erythropoietin, a hjcme-erytropoietm, one ohgomer thereof, one multimer thereof, a mutein thereof, a related form thereof, a naturally occurring form thereof, a synthetic form thereof, a recombinant form thereof, cn glykosylenngsvarianl thereof, one deglycosylated variant thereof, or a combination thereof Any form of erythropoietin capable to be benefit of erytropoielinresponsive cells, are included in this aspect of the invention Other erytropoietindenvater useful for the aforementioned purposes and pharmaceutical compositions include both natural erythropoietins as well as erythropoietins that have been modified by at least one modification as compared with natural erytropoielin and preferably when compared with natural human erytropoietm The at least one modification may be a modification of at least one amino acid of the erythropoietin molecule, or a modification of at least one carbohydrate of the erythropoietin molecule course erytropoietmmolekyler useful for the purposes is here, have a plurality of modifications compared to the native molecule, such as multiple modifications of the ammo acid portion of the molecule, multiple modifications of the carbohydrate portion of the molecule or at least one modification of ammo acid portion of the molecule and at least one modification of the carbohydrate portion of the molecule The modified erythropoietin molecule retains its ability to protect, maintain, enhance or restore function or viabilitelen of erytropoielinresponsive mammalian cells, yet other properties of the erythropoietin molecule unrelated to the aforementioned desirable property, be absent, as compared to the natural molecule A human erytropoietm are preferred, natural human erytropoietm is most preferable In another embodiment, human, asialoerytropoietin preferred In another embodiment tilveiebnnger invention relates to methods for using the aforementioned erytropoietmet to rebuild a dysfunctional cell, tissue or organ when administered after the onset of the disease or condition responsible for the dysfunction By non-limiting examples rebuilds methods for administration of a pharmaceutical composition comprising erytropoietm, cognitive function in animals previously had home trauma, even when administered long after (e.g., three days, five days, one week, one month or longer) trauma has subsided Erythropoietins useful for such applications include any of the particular aforementioned erytropoietmene, or any natural erytropoietm, or erytropoietmanalog, one erytropoietmetterlikner and erytropoietmfragment, a hybrid erytropoietmmolekyl, a erytropoietm receptor binding molecule, one erytropoietmagonist, a renal erytropoietm, a home-erythropoietin, one ohgomer thereof, one multimer thereof, a mutein thereof, a related form thereof, a naturally occurring form thereof, a synthetic form thereof, a recombinant form thereof, one glykosylermgsvanant thereof, one deglycosylated variant thereof, or a combination thereof Any kind of erytropoietm able to benefit for erytropoielinresponsive cells, are included in this aspect of the invention Other erytropoietindenvater useful for the aforementioned purposes and pharmaceutical compositions include both natural erytropoteliner as well as erythropoietins that have been modified by at least one modification as compared with natural erythropoietin, and preferably when compared with natural human erythropoietin The at least one modification may be a modification of at least one amino acid of erytropoietmmolekylet, or a modification of at least one carbohydrate of erytropoietinmolckylet course, erythropoietin molecules useful for the purposes is here, have a plurality of modifications compared to the native molecule such as multiple modifications of the amino acid moiety of the molecule, multiple modifications of the carbohydrate portion of the molecule or at least one modification of the amino acid moiety of the molecule and at least one modification of the carbohydrate portion of the molecule The modified erytropoielinmolekylel retains its ability to protect, maintain, enhance or restore the function or viability of erytropoictmresponsive mammalian cells, yet other properties of erytropoietmmolekylet not related to the previously mentioned desirable properties, may be absent, as compared to the natural molecule A human erythropoietin is preferred, native human erythropoietin is most preferred In another embodiment, human, asialoerytropoietm preferably 1 ct still further aspect of the present invention are methods provided for promoting transeytosen of molecules of endolelcellebarneren in a mammal by administration of a composition of a molecule in association with an erythropoietin such as a erythropoietin having at least no sialmsyrehalvdeler, an erythropoietin having at least no N -forbundne or no O-linked carbohydrates, an erythropoietin having at least a reduced carbohydrate content by virtue of treatment of natural erythropoietin with at least one glycosidase, an erythropoietin with a carbohydrate moiety of erytropoietmmolekylet, having at least a non-mammalian glykosylermgsmønster virtue of the expression of a recombinant erythropoietin in non-mammalian cells, an erythropoietin has at least one or more oxidized carbohydrates which also may be chemically reduced, a erythropoietin having at least one or more modified arginmrester, an erythropoietin having at least one or more modified lysmrester or a modification of the N-terminal amino group of erytropoietmmolekylet, an erythropoietin having at least one modified tyrosine residue, an erythropoietin having at least a modified aspartic acid or glutamic acid residue, an erythropoietin having at least a modified tryptophan residue, an erythropoietin having at least one annnogruppe removed, an erythropoietin least has an opening of at least one of cystemforbindelsene in erytropoietmmolekylet, an erythropoietin is provided having at least one substitution of at least one amino acid, or a truncated erythropoietin
The association between the molecule to be bh transported and erylropoietmel may be an unstable covalent bond, a stable covalent bond, or a non-covalent association with a binding site for the molecule Endotelcellebarnérer can be blood-home barrier, blood-eye barnere, Blood testicular Bamer, blood-OVAN banners and the blood-placenta barrier Suitable molecule for transport by the method of the present invention include hormones, such as growth hormone, antibiotics and anti-cancer agents
It is a further aspect of the present invention to provide a composition for promoting the transcytosis of a molecule across a cndotelccllebamere in a mammal, said composition comprising said molecule in association with a erytropoietm, such as a erytropoietm having at least no siahnsyrehalvdeler, a erytropoietm that having at least no N-linked or no O-linked carbohydrates, a erytropoietm having at least a reduced carbohydrate waistband distance by virtue of treatment of natural erytropoietm with at least one glykosidasc, a erytropoietm with a carbohydrate moiety of part modified erytropoietmet having at least a non-mammalian glykosylenngsmønstcr by virtue of the expression of a recombinant erytropoietm in non-mammalian cells, a erytropoietm has at least one or more oxidized carbohydrates which also may be chemically reduced, ct erytropoietm having at least one or more modified arginmrester, a erytropoietm having at least one or more modified lysmrester or a modification of the N-term male ammo group of erytropoietmmolekylet, a erytropoietm with at least one modified tyrosmrest, a erytropoietm having at least a modified aspargmsyre or glutammsyrerest, a erytropoietm having at least a modified tryptophan residue, a erytropoietm having at least one amino group removed , a erytropoietm having at least an opening of at least one of cystemforbindelsene in erytropoietmmolekylet, a erytropoietm is provided having at least one substilusjon of at least one amino acid, or a truncated erytropoietm
The association may be an unstable covalent bond, a stable covalent bond, or a non-covalent association with a binding site for the molecule Endotelccllebamerer can be blood-home barrier, blood-eye barrier, blood-testis Bamer, blood-OVAN children rising and the blood-placental barrier Suitable molecule for transport by the method of the present invention include hormones, such as growth hormone, antibiotics and anti-cancer agents 1 ct further aspect of the present invention is any of the aforementioned erytropoietmene useful in the preparation of a pharmaceutical composition for promote the transcytosis of a molecule across a endotelcellebamcre in ct mammal, said composition comprising said molecule in association with ct erytropoietm, such as a erytropoietm having at least no siahnsyrehalvdeler, a erytropoietm having at least no N-linked or no O-linked carbohydrates, a erythropoietin having at least a reduced karbohydratmnhold virtue of treatment of natural erythropoietin with at least one glycosidase, an erythropoietin with a carbohydrate moiety of the modified erythropoietin molecule having at least ct non-mammalian glykosylenngsmønster virtue of the expression of a recombinant erythropoietin in non-mammalian cells, an erythropoietin having at least one or more oxidized carbohydrates which also may be chemically reduced, a erythropoietin having at least one or more modified argminrester, an erythropoietin having at least one or more modified lysine residues or a modification of the N-term male amino group of the erythropoietin molecule, a erythropoietin having at least one modified tyrosine residue, an erythropoietin having at least a modified asparagmsyre or glutamic acid residue, an erythropoietin having at least a modified tryptophan residue, an erythropoietin having at least one amino group removed, an erythropoietin having at least an opening of at least one of cystemforbmdelsene in erythropoietin molecule, an erythropoietin is provided having at least one substitution of at least one amino acid, or a truncated erythropoietin
The association may be an unstable covalent bond, a stable covalent bond, or a non-kovalcnt association with a binding site for the molecule Endotelcellebarricrer may be b lod-home barrier, blood-eye Bamer, blood-testis barrier, blood-OVAN Bamer and blood-placental barrier Suitable molecule for transport by the method of the present invention include hormones, such as growth hormone, antibiotics and anti-cancer agents
These and other aspects of the present invention will be better understood by reference to the following figures and detailed description BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts the translocation of parenterally administered to the cerebrospinal fluid erylropoietm
Figure 2 shows the protection of the myocardium from ischemic damage by erylropoietm after short term vascular occlusion
Figure 3 shows the maintenance of the function of a heart prepared for transplantation by erytropoictm
Figure 4 compares the in vitro effectiveness of erylropoietm and viabihteten of serum-starved P19-cellcr
Figure 5 is another experiment which compares in vitro effectiveness of crytropoietin and asialoerytropoietin on viabihteten of serum-starved P19 cells
Figure 6 compares m vitro effectiveness of erytropoietm and phenylglyoxal-modified erylropoietm on viabihteten of serum-starved P19 cells
Figure 7 shows protection of erytropoictm and asialoerytropoietin in a rat focal cerebral ischemia model
Figure 8 shows a dose response comparing the efficacy of human crytropoietin and human asialoerytropoietin in middle cerebral artcncokklusjon in a model of ischemic stroke
Figure 9 shows the effect of biotmylert erytropoietm and asialoerytropoietin in P19 test
Figure 10 shows the activity of iodinated erytropoietm in P19 test
Figure 11 depicts the effect of erytropoietinbehandlmg in a rottcglaukommodell
Figure 12 shows the extent of the maintenance of retinalfunksjon by erytropoietm in rotteglaukommodel 1:01
Figure 13 depicts the reconstruction of cognitive function after trauma home by supplying crytropoietin which started five days after trauma
Figure 14 depicts the reconstruction of cognitive function after home trauma by administration of erythropoietin which started 30 days after trauma
Figure 15 depicts the efficacy of human asialoerytropoietin in a kainatmodell of cerebral toxicity
DETAILED DESCRIPTION OF THE INVENTION
"Erytropoielinresponsiv cell" refers to a paltedyrcelle whose function or viability may be maintained, promoted, enhanced, regenerated, or in any other way benefit from exposure to an erythropoietin Non-limiting examples of such cells include ncuronalc retinal muscular heart, lung, liver, kidney, small intestine, adrenal cortex, adrenal medulla, kappilærendotel, testis, ovary and endometnumceller addition such erytropoietinresponsive cells and the benefits provided by an erythropoietin be extended to provide protection or stabilization, indirectly to other cells that are not directly erytropoietinresponsive or of tissues or organs which contain such non-erytropoietinresponsive cells These other cells, tissues or organs which benefit indirectly from the enhancement of erytropoietinresponsive cells, presents as part of the cells, tissue or organ as "associated" cells, tissues and organs Benefits of an erythropoietin as described herein may thus be provided as a result of the presence of a small number or ratio of erytropoietinresponsive cells in a tissue or organ, such excitabelt or neuronal tissue present in such tissue or Leyding cells in the testes, which produce testosterone in one aspect, the erytropoietinresponsive cell or its associated cells, tissues or organs not excitable cells, tissues or organs or comprises substantially non excitable cells or tissues
Invention methods tilveiebnnger the local or systemic protection or strengthening of cells, tissues and organs in a mammal's body, under a wide range of normal and countervailing conditions or protection of those who are determined to relocate to another mammalian body In addition, reconstruction or regenerermg of dysfunction Also provided As mentioned above, an erythropoietin ability to cross a tight endotelcellebamere and exert its positive effects on erytropoietinresponsive cells (as well as other cell types) distal to the vasculature, and have the potential to prevent as well as treat a wide variety of conditions and diseases which otherwise cause significant ccllulær- and tissue damage in an animal including human, and moreover to permit success of heretofore not proven surgical procedures where the risk traditional set outweigh the benefits Duration and extent of important countervailing factor induced for ultimate benefit, such as high dose chemotherapy, stråhngsterapi, prolonged ex νινο transplant survival and extended periods of surgically induced ischemia, may be carried out by taking advantage of the present invention However, the invention is not so limited, but includes "an aspect methods or compositions wherein the targeted erytropoietmresponsive cells are distal of the vasculature by virtue of a endotelcellebamerc or dense endotelforbmdelser In general, the invention is directed to any erytropoietmresponsive cells and associated cells, tissues and organs that can benefit exposure to erytropoietm Furthermore cellulær-, tissue or organ dysfunction rebuilt or regenerated after an acute countervailing event ( such as trauma) by exposure to a erytropoietm
The invention is therefore generally directed to the use of erylropoietiner for preparing pharmaceutical compositions for the aforementioned purposes in which cellular function is maintained, promoted, enhanced, regenerated, or in any other manner that may be beneficial The invention is also directed to methods for maintaining, enhancing, promoting or regenerate cellular function by administering to mammals an effective amount of a erytropoietm as described herein The invention is further directed to methods for maintaining, enhancing, promoting or regenerate cellular function ex vivo by exposing the cells, tissue or organ of a erytropoietm invention is also directed to a perfusatsammensetmng comprising a erytropoietm for use for organ or vevsoppbevanng
The various methods of the present invention utilizes a pharmaceutical composition including at least a erytropoietm in an effective amount for the particular route and duration of eksponenng for exercising positive effects or benefits on erytropoietmresponsive cells in or removed from a mammalian body wherein the targeted cell, tissue or means for the intended therapy require erytropoietm to cross an endothelial cellebamere include the pharmaceutical composition one erytropoietmkonsentrasjon which is able to exert the desired effect on the erytropoietmresponsive cells after crossing endotelcellebameren Molecules that can mteragere with the erythropoietin receptor and modulating rcseptoraktivitcten , referred to herein as erytropoietm or crytropoietmreseptoraktivitetsmodulatorer, useful in the context of the present invention these molecules may be naturally occurring or synthetic rekombmante forms of erytropoietmmolekylene, as described above, or other molecules which may not necessarily resemble erytropoietm in any way, with Except for modulating erytropoietmresponsiv cell activity as described herein
Erytropoietm is a glykoprotemhormon which in humans has a molecular weight of about 34 kDa The mature protein comprises 165 amino acids, and the glycosyl residues comprise about 40% of the molecular weight forms of erythropoietin useful o the embodiment of the present invention include naturally occurring, synthetic and rekombmante forms of the following human and other mammalian erytropoietm-related molecules erythropoietin asialoerytropoietm, deglycosylated erythropoietin erytropoietmanaloger, erytropoietinctterliknere, erytropoietmfragmenter, hybriderytropoietmmolekyler, erytropoietmreseptorbindende molecules, erytropoietinagomster, renal erythropoietin, home-erythropoietin oligomercr and multimers thereof, mutcincr thereof and related thereof, in addition includes erytropoietmformer, useful in the methods of the present invention, proteins that represent functionally equivalent gene products Such equivalent erylropoietingenprodukt include mutant erythropoietins, which may contain deletions, including internal deletions, additions, including additions that provide fusjonsprotciner or conservative substitutions of amino acid residues within and / or adjacent to the amino acid sequence, but that result in a "silent" change, in that the change produces electric functional equivalent erythropoietin Such amino acid substitutions may be made on the basis of polaritetshkhet, charge, dissolve! speeD, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues involved Ikkcpolare (hydrophobic) amino acids include, for example alanine, leucine, isoleusin, valine, proline, phenylalanine, tryptophan and methionine, polar neutral amino acids include glysm, sedge, threonine, cysteine, tyrosine, asparagm and glutamm, positively charged (basic) amino acids include arginine , lysine, and histidine, and negatively charged (acidic) amino acids include aspartic acid and glutamic acid Alternatively, non-conservative aminosyreendnnger and larger insertions and deletions used to create functionally altered erytropoietinmutanter Such mutants used to change erytropoietmegenskaper the desired manner one one embodiment, for such as an erythropoietin useful for the present invention embodiment be a mutant erythropoietin altered in one or more amino acids within the four functional domains of erythropoietin which affect receptor binding VLQRY and / or TKVNFYAW and / or SGLRSLTTL and / cller SNFLRG In another embodiment, the used erythropoietins containing mutations in the surrounding areas of the molecule which affect the kinetics or reseptorbindmgsegenskaper of the molecule
The term "erythropoietin", as well as a "erythropoietin" used alternating or closely connected, and the various analogs, fragments, hybndmolekyler, agomster, Mutema and other forms, as described above, the Vanant in extent and sites of glykosylenng of erythropoietin, which includes natural, deglycosylated, asialylert and other partially glycosylated forms of erythropoietin Non-begrensendc examples of such variants are described in Tsuda et al, 1990, Eur J Biochem, 188 405-411, which is incorporated by reference hen baked Kitchener, yeast, insect , plant, mammalian, including human Additionally, a large variety of host systems used for expression and production of recombinant erytropoielm, including but not limited to, baked thousands, yeast, insect, plants and mammals, including human cell systems Recombinant erytropoietm produced in baked thousands who glycosylated or sialated product, for example, may be used for the production of LKKC-glycosylated forms of erytropoietm Alternatively, recombinant erytropoietm produced in other systems like glycosylated, such plants including human cells
As mentioned above, the present invention includes any and all erylropoietmreseptoraktivitetsmodulatormolekyler able to exercise positive activity on erytropoietinresponsive cells, irrespective of the molecule's structural relationship with erytropoietm Additionally erytropoietm itself modified tailored its activities for a spesifikt- or specific tissue More non-limiting strategies that may be performed to achieve the desired tissue specificity include modifications that shorten sirkulasjonshalvenngstidcn thus reducing the time erythropoietin can mteragere with erytroidforløpere or modification of pnmærstrukturen of erytropoietmmolekylet One way to reduce sirkulasjonshalvenngstiden is to remove or modify glykosyleringshalvdelene where erytropoietm has three N-linked and O-linked Such variants of glycosylated erytropoietm can be produced many ways For example sialmsyrene terminating sugar chain ends are removed by specific siahdascr depending on the chemical linkage connecting siahnsyren sugar chain Alternatively, the glycosylated structure breaks down in different ways using by other enzymes that cleave at specific bmdmger Techniques that modify pnmærstrukturen are diverse and include subslitusjon of specific amino acids, chemical modification of amino acids or addition of other structures which mterfererer with the interaction of erytropoietm with any of its receptors Use of such forms of erytropoietm are fully covered hen In a preferred embodiment halvenngstiden of the non-erythro poi in a liquid erytropoietm of the invention reduced by omknng 90% from the natural erytropoietm
Some of these molecules will nevertheless mimic the effects of erytropoietm itself in other tissues or organs For example, a 17-mer containing ammo acid sequence of 31-47 of native erytropoietm idle crytropoiese but fully active for NeuralC cells out vitro ( Campana & 0'Bnen 1998 Tnt J Mol Med 1 235-41)
Furthermore, derivative erythropoietin molecules desirable for the uses described herein, generated by guanidmenng, amidinenng, karbamylenng (karbamylenng), tnmtrofenylering, acetylenng, suksmilenng, nitrenng or modification of arginine, lysine, tyrosine, tryptophan, or cysteine residues or carboxyl groups, among other procedures, such as limited proteolysis, removal of ammonium groups and / cller mutasjonssubstilusjon of arginine, lysine, tyrosine, tryptophan, or cysteine residues by molecular biological techniques for the production of erytropoietmer which maintain an adequate level of activities for specific organs and tissues but not for others, such as erythrocytes ( for example (Satake et al, 1990, Biochim Biophys Acta 1038 125-9, incorporated stand at reference in its entirety) A non-limiting example, as described below, the modification of erytropoietm arginmresler by reaction with a glyoxal such as phenylglyoxal (according the protocol of Takahashi, 1977.7 Biochem 81 395-402) As seen below retains a erytropoietmmolekyl its neurotrophic effect completely Such erythropoietin molecules are fully covered for the various uses and compositions described hen
Synthetic and rekombmante molecules such as hjemeerytropoietin and renal erytropoietm, rekombmante mammalian forms of erytropoietm, as well as the naturally occurring tumordenverte and rekombmante isoforms, such as recombinantly expressed molecules and those prepared by homologous rekombmasjon, is provided herein Furthermore, the present invention molecules including peptides that bind to the erythropoietin receptor, as well as rekombmante structures or other molecules that have partially or all of the structural and / or biological properties of erytropoietm, including fragments and multimers of erytropoietm or its fragments Erytropoietm here comprises molecules with altered erytropoietinrcseptorbindingsaktiviteter, preferably with increased reseptoraffmitet, specifically relating to enhanced transport across endotelcellebamerene Muteins comprising molecules having additional or reduced numbers of glykosylenngsseter included herein As described above, the terms "erytropoietm" and "etterliknere" as well as other designations alternating used going to refer to the erytropoietmrcsponsive cell protective and restorative molecules related to erytropoietm as well as those molecules capable of crossing endotclcellebarncrer molecules produced by transgenic animals are also also encompassed herein It should be noted that erythropoietin molecules covered away, not necessarily similar erytropoietm structurally or otherwise manner, except for ability to mteragere with the erythropoietin receptor, modulate or activate erytropoietinaktiverte erytropoictmrescptoraklivitet signahsenngskaskader, shk described hen
By non-limiting examples include forms of erytropoietm, useful for the practice of the present invention, erytropoietmer such as those with altered amino acids at the karboksytermmale end, disclosed in U.S. Patents 5,457,089 and 4,835,260, asialoerytropoietin and erytropoietmisoformer with various numbers of sialic acid residues per molecule, as described in U.S. Patent 5,856,298, polypeptides described in US Patent 4,703,008, agomster described in U.S. Patent 5,767,078, peptides that bind to erytropoietmreseptoren as described in US patents 5,773,569 and 5,830,851, small molekyletterliknerc activating erytropoietmreseptoren as described in U.S. Patent 5,835,382, and crytropoietmanaloger described in WO 95/05465, WO 97/18318 and WO 98/181926 All of the above are incorporated henvisnmgene going to the extent that such designations refer to the different alternative forms and processes for preparing such forms of the erythropoietins of the present invention
Erytropoietm can be obtained commercially, for example under the trademarks "PROCRIT", available from Ortho Biotech Inc., Rantan, NJ, and "EPOGEN", available from Amgen, Inc., Thousand Oaks, CA
The activity (in units) of erytropoietm (erytropoietm) and erytropoietinhknende molecules are traditional defined based on its effectiveness in stimulating red blood cell production in gnagcrmodcller (and as derived by international standards of erytropoietm) One unit (U) of ordinary erytropoietm (MW of ~ 34000 ) is ~ 8 ng of protein (1 mg protein is approximately 125,000 U) Since the effect of erythropoiesis is incidental to the desired activities herein and may not be a detectable property of certain of the erythropoietins of the invention, however, the definition of activity based on erythropoietic activity not appropriate As used hen is therefore activity unit of erytropoietm or erytropoictmrelaterte molecules defined as the amount of protein required to elicit the same activity in neural or other erytropoietinresponsivc cellular systems induced by WHO international standard erytropoietm in the same system The trained scientist will easily determine the units of a non-erythropoietic erytropoietm or related molecule following the guidance hen In continuation of the above erytropoietinmodifikasjonene useful herein, the subsequent discussion in the various erytropoietmer invention
A erytropoietm invention may have at least no sialmsyrehalvdeler, referred to as asialoerytropoietin Preferably a erytropoietm inventive human asialoerytropoietin 1 alternative embodiments erytropoietmet invention have at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 sialic acid residues may be prepared by desialylermg of erytropoietm using a siahdase, as described in the manufacturer's packaging for Sialydase A from Prozyme Inc., San Leandro, California Usually "Prozyme" "GLYCOPRO" sekvensermgsgrid "SIALYDASE A "(N-acetylneuraminate glycohydro- lases, E.coli 3 2 1 18) used for cleavage of all non-reducing terminal siahnsyrerester from complex carbohydrates and glycoproteins such as erytropoietm It will then slit forgrenetc sialic acids (linked to an internal residue) Sialydase A is isolated from a clone of Arthrobacter ureafaciens
A erytropoietm can have at least a reduced number of N-linked carbohydrate order to remove N-linked carbohydrate can be treated with hydrazm erytropoietm, in accordance with for example the methods described by Hermentm et al, 1996, Glycobiology 6 (2) 217-30 As described above erytropoietm three N-linked carbohydrate halves, the present invention include those erythropoietins with two, one or no N-linked carbohydrates
A erytropoietm invention may have at least a reduced karbohydratmnhold virtue of treatment of natural erytropoietm with at least one glycosidase For example, the procedure of Chen and Evangelista, 1998, Electrophoresis 19 (15) 2639-44 followed Furthermore, removal of O-linked carbohydrate performed by following the methods described in Hokke et al, 1995, Eur J Biochem 228 (3) 981-1008
The carbohydrate portion of a erytropoietmmolekyl can have at least a non-mammalian glykosylenngsmønster virtue of the expression of a recombinant erytropoietm in Not-mammalian cells Preferably erythropoietins expressed in insect or plant cells In one non-limiting example, expression of erytropoietm in msektceller using a baculovirus ckspresjonssystem, carried out in accordance with Quelle et al, 1989, Blood 74 (2) 652-657 Another method is described in U.S. Patent 5,637,477 Expression in a plantcsystem can be performed according to the method of Matsumoto et al, 1993, Biosci Biotcch Biochem 57 (8) 1249-1252. Alternatively, expression in bacteria will result in non-glycosylated forms of erytropoietm These are just examples of methods useful for the production of a erytropoietm invention and are in no way limiting
A erytropoietm invention may have at least one or more oxidized carbohydrates which also may be reduced For example erytropoietmet be penodat oxidized erytropoietm, it penodat-oxidized erytropoietm can also chemically reduced with a borhydndsalt such as nalriumborhydrid or natnumeyanoborhydnd Pcnodatoksidermg of erytropoietm can be performed for example the methods described by Linslcy et al, 1994, Anal Biochem 219 (2) 207-17 Chemical reduction following penodatoksidermg can be conducted following procedures to Tonelh and Meint, 1978, J Supramol Struct 8 (1) 67 * 78
An erythropoietin for the aforementioned uses may have at least one or more modified arginine residues, for example, the modified erythropoietin may comprise a R-glyoksalhalvdel on the one or more arginine residues, where R may be an aryl, heteroaryl, lower alkyl, lower alkoxy or cycloalkyl, or an alpha-deoksyglycidylgruppe As used herein, the term lower "alkyl" means a straight or branched chain, saturated ahfatisk hydrocarbon group preferably containing 1 to 6 carbon atoms Representative of such groups are methyl, ethyl, isopropyl, isobutyl, butyl, pentyl, hexyl and the like The term "alkoxy" means a lower alkyl group as defined above, attached to the rest of the molecule by oxygen Exemplary alkoxy includes methoxy, ethoxy, propoxy, isopropoxy and the like The term "syktoalkyl" refers to sykhske alkyl groups with three up to about 8 carbon atoms, which includes for example cyclopropyl, cyclobutyl, cyclohexyl and the like The term aryl refers to phenyl and naphthyl The term heteroaryl refers to heterosykhske groups containing 4-10 nngmedlemmer and 1-3 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur Examples include, but are not limited to isoksalyl, fenylisoksazolyl, furyl, pyrimidinyl, quinolyl, tetrahydrokmolyl, pyndyl, imidazolyl, pyrrohdinyl, 1,2,4-tnazolyl, thiazolyl, thienyl and the like R gruppcn may be substituted, such as 4-trihydroksybutylgruppen of 3- deoksyglukoson Typical examples of R glyoksalsammensetmnger glyoxal, methyl glyoxal, 3-deoksyglukoson and phenylglyoxal Preferred R glyoksalsammensetmnger is methyl glyoxal or phenylglyoxal A eksemplansk method for such modification may be found in Werber et al, 1975, Isr J Med Sci 11 (11) 1169- 70, using phenylglyoxal In a further example, at least one argimnrest modified by reaction with an adjacent diketone, such as 2,3-butanedione or cyclohexanedione, preferably in omknng 50 milhmolar borate buffer of pH 9.8 A procedure for the latter modification with 2,3-butanedione may be carried out in accordance with Riordan, 1973, Biochemistry 12 (20) 3915 to 3923, and with cyclohexanone according Ul Patthy et al, 1975, J Biol Chem 250 (2) 565-9
An erythropoietin of the present invention may comprise at least one or more modified lysmrester or a modification of the N-terminal amino group of erytropoictmmolekylet, such modifications are those resulting from the reaction of lysmresten a aminogruppemodifiserende agent In another embodiment, lysmrester modified by reaction with glyoksalderivatcr , such as reaction with glyoxal, methyl glyoxal and 3-deoksyglukoson to form alpha-karboksyalkyldenvater
Examples are reaction with glyoxal to form karboksymetyllysm which Glomb and Monmer 1995, J Biol Chem 270 (17) 10017-26, or with methyl glyoxal to form (1-carboxyethyl) pylon which Degenhart ei al, 1998, Cell Mol Biol (Noisy-le-grand) 44 (7) 1139-1145 The modified lysmresten can be further reduced chemically For instance, erythropoietin is biotinylated through lysmgrupper, such as according to the method described in Example 5, wherein the D biotmyl e-aminokapromsyre N -hydroksysuksmimidester was reacted with erythropoietin, followed by removal of unreacted biotin by gel filtration on a Ccntncon-lo column as described in Wojchowski and Caslake, 1989, Blood 74 (3) 952-8 In this presentation uses authors three different methods for biotinylcre erythropoietin, where any of them can be used for preparation of erythropoietins for use hen Biotin may be added Itl (1) sialmsyrehalvdelene (2) carboxylate groups or (3) ammo groups In another preferred embodiment, the lysine is reacted with a aldchyd or a reducing sugar to form a lmin, which can be stabilized by reaction with natnum cyanoborhydnd to form an N-alkylcrt lysine, such as glusitolyllysm, or as in the case of reducing sukkcre can be stabilized by amadon or Heyns omgruppenng to form an alpha-deoxy alpha ammo sugar such as alpha-deoxy-alpha-fruktosyllysin As an example, preparation of a fruktosyllysmmodifisert protein by incubation with 0.5 M glucose in natnumfosfatbuffer pH 7.4160 days described Makila et al, 1992, J Biol Chem 267 5133-5138 In another example, lysmgruppen carbamylated, such as in kiaft of reaction with cyanate ion, or alkyl or arylkarbamylert or -liokarbamylert with an alkyl or aryl isocyanate or -isotiocyanat or may aeyleres by a reactive alkyl or arylkarboksylsyredenvat so which upon reaction with eddiksyreanhydnd or ravsyreanhydnd or ftalsyrcanhydnd Modification of lysmgrupper 4-sulfofenyhsoliocyanat or eddiksyreanhydnd are exemplary, both are described in Gao et al, 1994, Proc Natl Acad Sci USA 91 (25) 12027-30 Lysmgrupper Also trinitrofenylmodifisercs by reaction with tnnitrobenzensulfonsyre fortnnnsvis or with its salts Such methods are described below in Example 5
At least one tyrosine residue of erythropoietin may be modified in an aromatic position upon an electrophilic reagent, such as by mtrenng or jodenng Using non-limiting examples, the erythropoietin is reacted with tetranitromethane (Nestler et al, 1985, J Biol Chem 260 (12) 7316 -21), or iodinated as described in Example 5 At least one aspargmsyre- or a glutamic acid residue of an erythropoietin may be modified, such as by reaction with carbodiimide followed by reaction with el amine such as, but not limited to glysmamid Examples of such modifications are described in Example 5 In another example, a tryptophan residue of a erytropoictm modified such as by reaction with N-bromosuccinimide or N-klorsuksimmid by following the procedure as described in Josse et al, Chem Biol Internet, May 14 1999, 119-120 In yet another example, an erythropoietin molecule is prepared by removing at least one aminogmppe, obtainable by reaction with mnhydnd followed by reduction of the subsequent carbonyl group by reaction with borhydnd In yet a further example is a erytropoietm provided having at least one opening of one cysteinforbindelse the erythropoietin molecule by reaction with a reducing agent such as dithiothreitol, followed by reaction of the subsequent sulfhydryls with jodacetamm, iodoacetic acid or another electrophile one to prevent reformation of the disulfide linkages
A erytropoietm is provided having at least one substitution of any of a number of amino acids, such as leusm with at least one of lysine, arginine, tryptophan, tyrosine or Eystein residues erytropoietm using molecular biology techniques
A modified erytropoietm prepared by subjecting a erytropoietm for a limited chemical proteolysis that targets specific residues, for example by severing after tryptofanresler Such resulting erytropoietinfragmenter covered hen
As noted above, a erytropoietm useful for the purpose of going, at least one of the aforementioned modifisenngene, but may have more than one of the modifications above By example of a modified erytropoietm with a modification at karbohydratdelcn of the molecule and one modification at ammo acid portion, a modified erytropoietm be asialoerytropoictm and have their lysmrestcr biotmylert or carbamylated
Various erytropoietmmolekyler and pharmaceutical compositions containing them for the uses described hen is thus incorporated Such erytropoietmmolekyler include, but are not limited to asialoerytropoietm, N-deglycosylated erytropoietm, O-deglycosylated erytropoietm, erytropoietm reduced karbohydratmnhold, erytropoietm with altered glykosylermgsmønstrc, erytropoietm with carbohydrates oxidized , then reduced, arylglyoksalmodifisert erytropoictm, alkylglyoksalmodifisert erytropoietm, 2,3-bulandionmodifiscrt erytropoietm, sykloheksandionmodifisert erytropoietm, biotmylert erytropoietm, N-alkylated-lysyl crytropoietm, glusitolyllysin-erytropoietm, alpha-dcoksy alpha fruktosyllysm-erytropoietm, carbamylated erytropoietm, acetylated erytropoietm, suksinylert erytropoietm, alpha-carboxyalkyl erytropoietm, mtrert erytropoietm, iodinated erytropoietm, to name some representative, but non-limiting examples based on the teachings hen Preferred are the aforementioned modified forms based on human erytropoietm
Some of the aforementioned erythropoietins are also new, and the invention is directed to such compounds as well as pharmaceutical compositions as those covered by Using non-limiting examples include such new crytropoietiner pcnodatoksidert erytropoietm, glusitolyllysm erytropoietm, fruktosyllysin erytropoietm, 3 deoksyglukoson erytropoietm and carbamylated asialoerytropoietm
Various vertekspresjonsvektorsystemer can be used for the production of erythropoietins and the erytropoietinrelaterte molecules of the present invention Such vertekspresjonssystemer represent means where erythropoietins of interest may be produced and purified, but also represent cells that may, when a transformed or transfected with the appropriate nucleotide probe sequences, obtain the modified erytropoietmgenproduktet m situ These include, but are not limited to, bacteria, insect, plant, mammalian, including human host systems, such as, but not limited to, insect cell systems infected with rekombmante virusekspresjonsvektorer (for example, baculovirus) containing the modified erytropoietmproduktcne that encoding sequences, planteccllesystemer infected with rekombmante virusekspresjonsvektorer (for example, cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with rekombmante plasmid (for example Ti plasmid) containing erytropoietinrelaterte molekylkodende sequences, or pattcdyrccllesystemer as mkludcier human cell systems (for example, HT1080, COS, CHO , BHK, 293,3T3) containing rekombmante expression constructs containing promoters derived from pattedyrgenomets cells (such metallotionem promoter) or from mammalian viruses (eg adenovirus late promoter, vaccmiavirus 7 5K. promoter) In addition, a vertsccllestamme chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific manner desired Such modifications (for example glykocylcrmg) and processing (e.g., cleavage) of protemprodukter may be important for Protem function Possible host cells have karaktenstika and specific mechanisms for the post-translational processing and modification of proteins and gene products Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed 1 this regard, eukaryotic host cells with the cellular machine for correct processing of the primary transcript, glykocylermg and fosforylermg of the gene product may be used Such mammalian host cells include human host cells, include but are not limited to HT1080, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3 and WI38
For long term høyutbytteproduksjon of recombinant proteins are stable expression is preferred For example, cell lines stably expressing part erytropoietmrelaterte molekylgenproduktet constructed 1 rather than using expression vectors containing viral origins of replication, cells can be transformed with DNA controlled by appropriate expression control (such as promoter, enhancer, sequences, transknpsjonstermmatorer, polyadenylermgsseter, etc.) and a selectable marker after infection of the foreign DNA, engineered cells may be allowed to grow 11.2 days in a annka medium, and changed then to el selective medium The selectable marker in part recombinant plasmid confer resistance for the selection and allows cells stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expand into cell lines, this method may be advantageously used for construction of cell lines expressing the erytropoietmrelaterte molekylgenproduktet Such engineered cell lines are particularly useful in screening and evaluation of compounds that affect the endogenous activity of the erytropoietmrelaterte molekylgenproduktet
Alternatively ekspresjonskaraktenslikken of an endogenous erytropoietm gene in a cell line or microorganism may be modified by inserting a heterologous DNA regulatory element in a stable ccllelmjes genomic or cloned microorganism such that the portion inserted regulatonske element operatively associated with the endogenous erytropoictin gene For example, , an endogenous erythropoietin gene which is normally "transcriptionally silent", i.e. an erythropoietin gene which is normally not expressed, or expressed only at very low levels in a cell line, is activated by inserting the electrical regulatonsk element able to promote expression of a normally expressed gene product in the cell line or microorganism Alternatively, a transknpsjonelt silent, endogenous erytropoietm activated by insertion of a promiscuous regulatonsk element that works across cell types
A heterologous regulatonsk element can be inserted into a stable cell line or a cloned microorganism, such that it is operatively connected to the electric endogenous erylropoietm gene using techniques such as targeted homologous recombination, which are well known to those with skill in the art, and described for example in French Patent 2,646,438 to Pasteunnstituttet, US Patent 4,215,051 to Chappel, US Patent 5,578,461 to Sherwin et al, International application PCT / US92 / 09627 (WO 93/09222) by Selden et al, and International application PCT / US90 / 06436 (WO 91/06667) by Skoultchi et al, all of which are incorporated away in its entirety by reference In an embodiment of the present invention, a erytropoietmrelatert molecule that partially lacking sialrester, or completely lacking sialrester produced in mammalian cells including a human cell Such cells can be constructed as deficient in, or lacking the enzymes that add siahnsyre, ie β-galactosidase α 2,3 sialyltransferase ("a 2,3 sialyltransferase") and β-galactosidase α 2,6 sialyltransferase ("a 2,6 sialyltransferase ") activity 1:01 embodiment, a pattcdyrcelle used where one or both α 2,3 sialyltransferasegenet and / or α 2,6 sialyltransferasegenet is Yemen Such deletions may be constructed using gene knock-leknikker well known in the art In another embodiment, dihydrofolate reductase (DHFR) deficient Chinese Hamster Ovary (CHO) cells used as the host cell for the production of recombinant molecules erylropoietinrelaterte CHO cells not expressing? 2,6-sialyltransferase enzyme, adding therefore not siahnsyre 12.6 compound to N -forbundne oligosakkander of glycoproteins produced in these cells As a result lacks recombinant proteins produced in CHO cells in 2.6 siahnsyre compound to galactose (Sasaki et al (1987, Takeuchi et al supra, Mutsaers et al Eur J Biochem 156, 651 ( 1986), Takeuchi et al J Chromotgr 400207 (1987) In an embodiment for producing a host cell for the production of asialoerytropoietm, the gene coding for alpha-2,3 sialyltransferase in CHO cells removed Such alpha-2,3 sialyltransferase knockout CHO cells completely lacking sialyltransferaseaktivitet, and as a result, they are useful for the recombinant expression and production of asialoerytropoietm In another embodiment asialoerytropoietinet produced by mterferering with siahnsyretransport to Golgi apparatus, for example, (Eckhart et al, 1998, J Biol Chem 273 20189-95) Using methods well known to those skilled in the art (for example, Oelmann et al, Biol Chem 276 1 26291-300) may mulagenese of nukleotidsukkeret CMP sialmsyre conveyor implemented for the production of mutanler Chinese hamster ovary cells These cells can not add siahnsyrerester to glycoproteins such as erythropoietin and produce only asialoerytropoietm Transfektcrte mammalian cells producing erythropoietin also produce cytosohsk siahdase as if it leaks into dyrkmgsmediet degrades asialoerytropoietm with high efficiency (e.g., Gramer et al, 1995, Biotechnology 13 692-698) Using methods well known to those of knowledge in the art (for example, from information provided in Ferrari et al, 1994, Glycobiology 4 367-373), cell lines can be transfected, mutated or otherwise operated in constitutive production of siahdase On this way asialoerytropoietm produced during the manufacture of asialoerytropoietm In the practice of one aspect of the present invention may cn pharmaceutical composition as described above containing an erythropoietin administered to a mammal by any route that tilveiebnnger a sufficient level of an erythropoietin in the vasculature to permit translocation across a endotelcellebamere and have beneficial effects on erytropoietinresponsive cells When used for the purpose of perfusing a tissue or an organ, is similar results desirable In the case where erytropoictmet used ex vivo perfusion, the erythropoietin may be any form of erythropoietin, such as the aforementioned erythropoietins but not limited thereto, and can include natural erythropoietins including human erythropoietin In the case where the cells or tissue is non-vaskulansert and / or the administration is by bathing the cells or tissue with the composition of the invention, tilveiebnnger the pharmaceutical composition an effective erylropoietinresponsiv cell beneficial amount of an erythropoietin Endotelcellebamerene as erythropoietin can be translocated across includes dense compounds perforated compounds, compounds with small windows and any other types of endotelbamerer present in a mammal A preferred barrier is an endotelcelletett compound, but the invention is not so limiting
The aforementioned erythropoietins are useful generally for the therapeutic or prophylactic treatment of human diseases of the central nervous system or peripheral nervous system which have primarily neurological or psychiatric symptoms, eye diseases, cardiovascular diseases, cardiopulmonary diseases, respiratonske diseases, kidney, urinary and reproductive diseases, gastromtestinale diseases and endoknne and metabolic abnormalities Specifically include such conditions and diseases hypoxic conditions, which adversely affect excitable tissues, such as excitable tissues in sentralnervesystemvev, peripheral nervesystemvev, cardiac tissue or retinal tissue such as, for example, homes, heart or retina / eye Therefore the invention used to treat or prevent damage to cxcitabelt tissue resulting from hypoxic conditions in a variety of conditions and circumstances Non-limiting examples of such conditions and circumstances are provided in the table below in the example for the protection of ncrvevevpatologier that can be treated in accordance with the present invention include those palologier, those resulting from reduced oksygenenng of nerve tissue Any condition which reduces the availability of oxygen to neuronal tissue, resulting in stress, damage, and finally cell death, can be treated by the methods of the present invention Generally referred to as hypoxia and / or ischenn, arises these conditions the basis of or include, but are not limited to, stroke, vascular occlusion, prenatal or postnatal oxygen deprivation, difficulty breathing, choking, near drowning, karbonmonooksidforgiftnmg, røykmhalasjon, trauma, including surgery and radiotherapy, asphyxia, epilepsy, hypoglycemia, chronic obstructive pulmonary disease, emphysema, respiratonsk disorder syndrome, hypotensive shock, septic shock, anafylaklisk shock, lnsulmsjokk, sickle cell anemia, cardiac arrest, dysrhythmia, nitrogen narcosis, and neurological deficits caused by heart-lung bypass procedures in one embodiment, for example, the specific EPO compositions admimstreres to prevent injury or tissue damage resulting from risk of injury or tissue damage during surgical procedures, such as, for example, tumor resection or aneunsmereparasjon Other pathologies caused by or resulting from hypoglycemia which are treatable by the methods described herein, msulinoverdose, also referred to as lalrogen hyperinsuhnemi, msulmom, vcksthormonmangler, hypokortisohsme, drug overdose and certain tumors
Other pathologies resulting from excitable nervevevsskade include seizure disorders such as epilepsy, convulsions, or chronic seizure disorders Other conditions and diseases that can be treated include diseases such as stroke, multiple sclerosis, hypotension, cardiac arrest, Alzheimer's disease, Parkinson's disease, cerebral palsy, brain or spinal cord trauma, AIDS dementia, age-related loss of cognitive function, memory loss, amyotrophic lateral sclerosis, seizure disorders, alcoholism, rctinal ischemia, optic nerve damage resulting from glaucoma and nervetap
The specific compositions and methods of the invention can be used for treating conditions of, and damage on retmalt tissues Such disorders include, but are not limited to retinal ischemia, macular degeneration, retinal detachment, retmitis pigmentosa, atenosklerotisk retmopati, hypertensive retmopati, retinal arteneblokkermg , retinal vein blockage, hypolensjon and diabetic retmopati In another embodiment, the method principles of the invention used for the protection or treatment of injury resulting from radiation damage to excitable tissue An additional application of the methods of the present invention is in the treatment of neurotoksinforgiflmng, such as domoic acid shellfish poisoning, neurolatyrisme and Guam disease, amyotrophic lateral sclerosis and Parkinson's disease
As mentioned above, the present invention also directed to a method for enhancing excitabel tissue function in a mammal by penfer administration of a erytropoietm as described above Various diseases and conditions can be treated using this method, and further, this method useful for enhancing cognitive function in the absence of any condition or disease These uses of the present invention are described in further detail below and include enhancement of learning and training in both human and non-human mammals
Conditions and diseases treatable by the methods of this aspect of the present invention is directed to the central nervous system include, but are not limited bl, humørforstyrrclser, anxiety disorders, depression, autism, attention lack hyperactive disorder and cognitive dysfunction These conditions benefit from enhancement of neuronal function Other disorders treatable in accordance with the teachings of the present invention include sleep disruption, for example, sleep apnea and reiscrelaterte disorders, sub arachniode and aneunsmale blødmnger, hypotensive shock, jarring injury, septic shock, anaphylactic shock, and sequelae of various enccfahtter and menmgittcr such bindevcvsykdomsrelaterte ccrebnter such as lupus Other uses include prevention of or protection from poisoning by neurotoksmer such as domomsyre skalldyrforgjftnmg, neurolatyrisme and Guam disease, amyotrophic lateral sclerosis and Parkinson's disease, post-surgical treatment embohsk or ischcmisk damage, whole hjemestråhng, sigdcelleknse and eclampsia
A further group of conditions treatable by the methods of the present invention include mitokondnell dysfunction of either a hereditary or acquired nature, which are the cause of a variety of neurological diseases represented by neuronal injury and death For example Leigtfs disease (subacute necrotizing cncefalopati) characterized in progressive visual loss and encephalopathy, due to neuronal loss and myopathy In these cases fails defective mitokondnell metabolism to supply necessary high energy substrate for supplying metabolism of excitable cells A erytropoietinreseptoraktivitetsmodulator optimizes failing function in a variety of mitokondnelle diseases As mentioned above, hypoxic assistance adversely affect excitable tissues The excitable tissues include, but are not limited to sentralnervcsystemvev, peripheral nervesystemvev and heart In addition to the conditions described above, the methods of the present invention are useful in the treatment of inhalenngsforgiftmng such as of carbon monoxide and smoke, severe asthma, respiratory hdelsessyndrom in adults, and choking and near drowning Further conditions which create hypoxic conditions or by other means induce damage to the tissue excitabelt include hypoglycemia that may occur tissues unfortunate dosage of insulin or by insuhnproduserendc neoplasms (insulinoma)
Various neurophysiological disorders are thought to stem from damage excitabelt tissue, can be treated by the instant methods Chronic disorders in which nerve damage is involved and for which treatment by the present invention is provided include disorders relating to the central nervous system and / or peripheral nervous system including age-related loss of cognitive function and senile dementia, chronic seizure disorders, Alzheimer's disease,
Parkmson's disease, dementia, memory loss, amyotrophic lateral sclerosis, multiple sclerosis, tuberous sclerosis, Wilson's Disease cerebral and progressive supranuclear palsy, Guam disease, Lewy body dementia, pnonsykdommer such as spongiform encephalopathies, such as Creutzfeldt-Jakob disease, Huntmgton's disease, myotonic dystrophy , Freidnch's ataxia and other ataxias, as well as Gilles de la Tourelte's syndrome, seizure disorders such as epilepsy and chronic seizure disorder, stroke, brain or spinal cord trauma, AIDS dementia, alcoholism, autism, retinal ischemia, glaucoma, autonomic function disorders such as hypertension and sleep disorders and neuropsykiatnske disorders include, but are not limited to schizophrenia, schizoaffective disorder, attention missing disorder, dysthymic disorder, major depressive disorder, mania, anxiety disorder, psychoactive subslansbruksforstyrrelse, anxiety, panic disorders as well as unipolar and bipolar affective disorders Further neuropsykiatnske and neurodegenerative disorders include, for example, the opphstet in Amencan Psychiatnc Association's Diagnostic and Statistical Manual of Mental Disorders (DSM), the most current version of which is incorporated hen by reference in its entirety In another embodiment rekombmante chimeric toxin molecules comprising erytropoietm used for therapeutic delivery of toxins for the treatment of proliferative disorders such as cancer, or viral disorder, such as subacute forkalkendc panencefahtt
The following table lists the further examples, non-limiting indications as to the various conditions and diseases amenable to treatment by the aforementioned erytropoietmene
Cell, tissue dysfunction or condition or type or organ pathology disease
Cardiac Ischemia coronary Acute, chronic disease species Stable, unstable
Myocardial infarction Angina Dresslers syndrome
Congenital Valvular heart disease Cardiomyopathy
Prmzmetal angina
Or cardiac Aneunsmatisk Septal perforation
Cell, tissue dysfunction in condition or disease type or pathology or organ
Heart Set
Arrhythmia Tachy-, bradyarrhythmia Stable, unstable
Supraventnkulær, Hyperscnsitiv carotid sinus node ventnkulær
Ledningsabnormiteter
Congestive Left, right, ICardiomyopatier, such as heart defects biventnkulær ldiopatisk familial, infectious, metabolic, lagnngssykdom, missing, bmdevevsforstyrrelse, infiltration and granuloma, neurovascular
Myocarditis Autoimmune, contagious, ldiopatisk
Cor pulmonary
Butt and penetrating trauma
Toxins Cocaine
Vascular Hypertension Primary, secondary
Decompression sickness fibromuscular hyperplasia
Aneunsme Dissecting, torn, enlarged
Congestion Obstructive Asthma
Chronic bronchitis and emphysema Airway Obstruction
Ischemic Pulmonary embolism Pulmonary Disease Pulmonary thrombosis
Fetl embolism
Ambient lung disease
Ischemic Pulmonary embolism Pulmonary Disease Pulmonary thrombosis
Mellomroms- ldiopatisk, pulmonary lung fibrosis
Congenital Cystic Fibrosis
Cell, tissue dysfunction in condition or disease type or organ or pathology
Lungs Cor pulmonary
Trauma
Pneumom and Contagious, parasitic, pneumonitis toxic, traumatic, burns, aspiration
Sarcoidosis
Pancreas Endoknn Diabetes melhlus type I beta cell failure, dysfunction and type II Diabetic neuropathy
Other endoknn cell failure of the pancreas
Eksoknn Eksoknn pacretic Pancreatitis
Bone Osteopenia Primary Secondary Hypogonadism immobilisenng Postmenopause Age-related Hypcrparatyroidisme Hyperthyroidism Calcium, magnesium, phosphorus and / or vitamin D deficiency
Osteomyelitis Avascular necrosis Trauma Paget's disease
Skin Alopecia Areata totalis Pnmær
Secondary
Baldness in men
Vitiligo Localised generalized Pnmær
Secondary
Diabetic ulceration Pen fer vascular disease Burns
Cell, tissue or dysfunction or pathology in condition or disease type organ
Autoimmune Lupus erythematosus, Sjogren disturbance,
Rheumatoid arthritis,
Glomerulonefntt
Entered
Langerhans histiocytosis's Eye Optical neuntt _ penetrating trauma, infections, sarkoid, sigdccllcsykdom, retinal detachment, Temporal artentt
Embryonic Asphyxia ogføtale Ischemia disturbances CNS Chronic fatigue syndrome, acute and chronic hypoosmolare and hyperosmolar syndrome, AIDS dementia, electrical killing
Encephalitis Rabies, Herpes
Meningitis
Subdural hematoma
Nikotmavhengighet
Drug Abuse and Cocaine, heroin, crack, confiscation marijuana, LSD, PCP, poly- drug abuse, eestasy, opioids, sedative hypnotics, amphetamines, caffeine
Angstforslyrrel see Spinal slenose, transverse myelitis, Guilhan Barre, trauma, nerverotskompresjon, tumoral compression, heat stroke
Cell, tissue or dysfunction or condition or type organ pathology disease ENT Tinnitus, Meniere's syndrome, hearing loss ENT Traumatic injury, barotrauma
Kidney Renal failure Acute, chronic vascular / ischemic, intermediate disease, diabetic kidney disease, neurotic syndromes, infections
Henoch S Purpura
Striped muscle Autoimmune Myastema gravis, disorders dermatomyositis
Polymyositis
Myopathies Hereditary metabolic, and toxic endokrm
Heatstroke
Squeeze Damage
Rabdomylose
Mitokondnal disease
Necrotizing fasciitis infection
Sexually Central and penfer Impotence dysfunction secondary to medication
Liver Hepatitis Viral, bacterial, parasitic
Ischemic disease Cirrhosis, fatty liver Lnfiltrcrcnde / metabolic disease
Gastrointestinal Ischemic bowel disease
Inflammatonsk bowel disease Necrotizing enlerokolilt
Cell, tissue or organ dysfunction or condition or disease type pathology
Organ Transplantation Treatment of donor and recipient
Propagation System Infertility Vascular
Autoimmune
Livmorabnomnteter
Tmplantasjonsforstyrrelser
Endocrine Premiums and function of the gland
As mentioned above, these diseases, disorders or conditions only lllustrative for reach and benefits provided by the erythropoietins of the present invention Accordingly tilveiebnnger this invention generally therapeutic or prophylactic treatment of the consequences of mechanical trauma or of human diseases Therapeutic or prophylactic treatment for diseases, disorders or conditions CNS and / ellcr the peripheral nervous system are preferred Therapeutic or prophylactic treatment for diseases, disorders or conditions which have a psychiatric component is provided Therapeutic or prophylactic treatment for diseases, disorders or conditions including, but not limited to those having a oflalmisk- , cardiovascular, kardiopulmonar-, respiratonsk-, kidney unnveis-, forplantmngs-, gastroinlestinal-, endocrine or mctabolsk component is provided In one embodiment, such a pharmaceutical composition of an erythropoietin administered systemically to protect or strengthen the targeted cells , tissue or organ Such administration can be parenteral via inhalation or transmucosal, e.g., oral, nasal, rectal, intravaginal, sublmgual, submucosal or transdermal Preferably administration parenterally, for example via intravenous or intraperitoneal injection, and includes, but is not limited to intraartenell, mtramuskulær, intradermal and subcutaneous administration
For other routes of administration, such as by use of a perfusate or injection into an organ or Annel local administration, a pharmaceutical composition is provided which results in similar levels of an erythropoietin as described above a level of about 15 pM-30 nM is preferred.
Dc pharmaceutical compositions of the invention may comprise a therapeutically effective amount of a compound and a pharmaceutically acceptable carrier In
a specific embodiment, the term "pharmaceutically acceptable" approved by a regulatory agency of forbunds- or state government or listed in the US Pharmacopeia or other generally recognized, foreign apotekerbok for use in animals, and more particularly in humans The term "carrier" refers to a diluent, adjuvant, binder or transport in which the therapeutic is administered at Such pharmaceutical carriers can be sterile liquids, such as saltoppløsnmger in water and oils, including those of petroleum, animal, vegetable or synthetic oppnnnelse such as pcanøttolje, soybean oil, mineral oil, sesame oil and the like A saltoppløsmng is a preferred carrier when the pharmaceutical composition is administered intravenously Saltoppløsnmger and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, preferably for injectable solutions Suitable pharmaceutical bmdemidler include starch, glucose, laktosc, sucrose, gelatin, malt, ns, flour, chalk, silica gel , sodium stearate, glycerol monostearate, talc, natnumklond, dried skim milk, glycerol, propylene, glycol, water, methanol and the like The composition can, if desired, also contain minor amounts of wetting or emulsifying agents, or pH buffering agents These compositions may take the form of solutions , suspensions, emulsions, tablets, pills, capsules, powders, formulations with sustained frigivnmg and similar composition may be formulated as a suppository with traditional binders and carriers, such as tnglysender compounds of the invention may be formulated neutral or salt forms Pharmaceutically acceptable salts include the which is formed with fne ammonium groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric, etc., and those in the form of free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, jemhydroksider, lsopropylamm, trietylamm, 2 elylaminoetanol, histidm, procaine, etc. Examples of suitable pharmaceutical carriers are described in "Remmgton's Pharmaceutical Sciences" by EW Martin Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of a carrier providing Fonn for proper administration to the patient Formulenngen to fit admimstrasjonsmåten Pharmaceutical compositions adapted for oral administration may be provided as capsules or tablets, powders or granules, solutions, syrups or suspensions (in aqueous or non-aqueous liquids), edible foams or creams, or as emulsions Tablets or hard gelatine capsules may comprise lactose, starch or denvater thereof, magnesium stearate, natnumsakkann, cellulose, magnesium carbonate, stcannsyre or salts thereof Soft gelatine capsules may comprise vegetable oils, waxes, fats, semi-solid substances or væskepolyoler etc. Solutions and syrups may comprise water , polyols and sugars
An active agent intended for oral administration may be coated with or admixed with a material that retards dissolution and / or absorption of the active agent in the digestive tract (for example, glyceryl monostearate or glyceryl used) Thereby, the sustained release of an active agent is achieved over many hours and, if necessary, the active agent is protected from degradation in the stomach, pharmaceutical compositions for oral administration may be formulated to promote the release of an active agent at a particular location because gastromtestinal specific pH or enzymatic conditions
Pharmaceutical compositions adapted transdermal administration may be provided in discrete increments, to remain in close contact with the recipient's epidermis over a longer time period Pharmaceutical compositions adapted topical administration may be provided as ointment, cream, suspension, lotion, powder, solution, paste, gel, spray, aerosol or oil for topical administration to the skin, mouth, eye or other external tissues used preferably a topical ointment or cream When formulated in an ointment, the active ingredient employed with either a paraffin or a water-miscible ointment base Alternatively, the active ingredient formulated in a cream with an oil-in-water base or a water-in-oljc base Pharmaceutical compositions adapted topical administration to the eye include eye drops 1 these compositions, the active ingredient is dissolved in or suspended in a suitable carrier, for example in a aqueous solvent Pharmaceutical compositions adapted topical administration in the mouth include lozenges, pastilles and softener mouth
Pharmaceutical compositions adapted for nasal and pulmonary administration may comprise solid carriers such as powders (preferably having parlikkelstørrclse in the range 20 to 500 microns) powder can be applied in the same way as snuff, ie by rapid inhalation through the nose from a container of powder held lightly against the nose Compositions adapted for nasal administration may comprise liquid carriers, for example, nasal spray or nasal drops inhalation directly into the lungs may alternatively be performed by deep inhalation or placement through a mouthpiece into the oropharynx These compositions may comprise vandige- or oljeløsmnger of the active ingredient Compositions for administration by inhalation can supplied in specially adapted devices including, but not limited to aerosol on tap box atomizers or pusteanordnmger, which is constructed to provide predetermined dosages of the active ingredient In a preferred ulførelsesform administered pharmaceutical compositions of the invention to the nostrils directly or into the lungs via the nostril or oropharynx
Pharmaceutical compositions adapted rectal administration may be provided as suppositories or enemas Pharmaceutical compositions adapted vaginal administration may be provided as pessaries, tampons, creams, gels, paste, foam or sprayformulennger
Pharmaceutical compositions adapted parenteral administration include aqueous and non-aqueous sterile injectable solutions or suspensions, which may contain antioxidants, buffers, baktenostater and solvents, making the compositions substantially isotonic with the recipient's blood Other components which may be present in such compositions include, for example, water , alcohols, polyols, glycerine and vegetable oils Compositions adapted parenteral administration may be presented in unit-dose or multi-dose containers, for example sealed vials and tubes, can be stored in a freeze dried (lyofihsert) condition requiring only the addition of a sterile liquid carrier, for example sterile saltoppløsnmg for injections immediately prior to use Extemporaneous mjeksjonsløsninger and suspensions may be prepared from sterile powders, granules and tablets In one embodiment, an autoinjector comprising an mjiserbar solution of a erytropoietm provided for use in emergency ambulance, emergency room and battlefield situations, and even for self- -tilførsel in a home situation, especially when the possibility of traumatic amputation may occur, such as by careless use of the lawnmower likelihood that cells and tissues in an injured foot or toe will survive after reforbmdelse may be increased by applying a erytropoietm to multiple locations in the damaged part as quick as possible, even before the arrival of medical personnel to the site, or arrival of the rammete individual with damaged toe to damage living In a preferred embodiment, the compositions are formulated in accordance with routine procedures as a pharmaceutical composition adapted to intravenous administration to humans Generally compositions for intravenous administration solutions in sterile isotonic aqueous buffer Where necessary, the composition may also include a solubilizing agent and a local anastetikum such as lidokam to reduce pain at the site of injection Generally, the ingredients are supplied separately or mixed together in enhetsdosermgsform, for such as a dry, lyofihsert powder or vannfntt concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent when the composition is to be supplied ve infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saltoppløsnmg Where the composition is to be administered by injection, an ampoule of stenl saltoppløsnmg brought provide, shk that the ingredients can be mixed prior to administration
Suppositories generally contain active ingredient in the range of from 0.5 to 10 weight percent, oral formulations preferably contain 10% to 95% active ingredient
A perfusate composition may be provided for placement in transplanted organ baths for m situ perfusion, or for administration to an organ donor vasculature before organ harvesting Such pharmaceutical compositions may comprise levels of an erythropoietin or a form of a erytropoietm not suitable for acute or chronic, local or systemic administration to a subject, but will provide the functions intended here in a cadaver, organ bath, organperfusat or in situ perfusate prior to f noble deed or reducing the levels of erythropoietin that they contain before exposure or returning the treated organ or tissue to regular circulation erythropoietin this aspect of the present invention may be any erytropoietm, such as naturally occurring forms such as human erytropoietm, or any of erytropoietmene described above, such as asialoerytropoietin and phenylglyoxal-erytropoietmer, as non-limiting examples
The invention tilveiebnnger also provides a pharmaceutical pack or a set, comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention Optionally associated with such container (s) can be a notice in the prescribed form of a government office that regulates the manufacture, use or sale of pharmaceutical or biological products, where the message reflects approval from the office for manufacture, use or sale for human administration 1 another utførclsesform For example, a erytropoietm delivered in a controlled fngjørmgssystem polypeptide may for example be administered using intravenous infusion, a implantable osmotic pump, a transdermal means, liposomcr or other means of administration In one embodiment, a pump is used (see Langer, supra, Sefton, 1987, CRCCnt Ref Biomed Eng 14 201, Buchwald et al, 1980, Surgery88 507, Saudek / af, 1989, N Engl J Med 321 574) In another embodiment, the compound is delivered in a vesicle, in particular a liposome (see Langer, Science 249 1527-1533 (1990) Treat et al, Liposomes m in the Therapy of Infcctious Disease and Cancer, Lopez-Beresford Vote and Fidler (eds), Liss, New York, pp 353-365 (1989), WO 91/04014, U.S. Patent 4,704,355, Lopez-Beresford Lein, ibid, pp 317-327, see generally ibid) In another embodiment, polymeric materials are used [see Medical Applications of Controlled Release, Langer and Wise (eds), CRC Press Boca Raton, Florida, 1974, Controlled Drug Bioavailabihty, Drug Product Design and Performance, Smolen and Ball (eds) , Wiley New York (1984), Ranger and Peppas, J Macromol Sci Rev Macromol Chem 23 61.1953, see also Levy et al, 1985, Science 228 190, During et al, 1989, Ann Neurol 25 351, Howard et al, 1989, J Neurosurg 71105) In another embodiment, a controlled fngivelsessystem placed near the therapeutic target, ie the targeted cells, tissue or organ, thereby requiring only a faction of the systemic dose (see, for example, Goodson, pp 115 -138 in Medical Applications of Controlled Release, Vol 2, supra, 1984) Other controlled fngjøringssystemer are discussed in the review to Langer (1990, Science 249 1527-1533) In another embodiment, crytropoietin, suitably formulated, administered by nasal, oral, rectal or vaginal administration or by administration under the tongue 1 a specific embodiment, the part may be desirable to add erytropoictinsammensetnmgene invention locally to the area in need of treatment, this can be achieved by, for example, without limitation, local infusion during surgery, topical application, e.g. in conjunction with a wound dressing after surgery, by injection, by means of a catheter, a suppository, an implant, said implant can be porous, non-porous or gelatinous material, including membranes such as silastic membranes or fibers
Selection of the preferred effective dose determined by one skilled in the art, based on consideration of several factors known to those of ordinary skill in the art Such factors include the particular form of erytropoietm and its farmakokmetiske parameters such as bioavailability, metabohsme, half-life etc., which are established during the usual utvikhngsprosedyrene, usually used to obtain regulatory approval for a pharmaceutical composition further factors, the dose, inkludcicr condition or disease to be treated or the benefits to be achieved in a normal individual, patient's body mass, the supply route, whether the supply is acute or chronic, concomitant medication collector and other factors known to affect the effectiveness of injected pharmaceuticals Therefore the precise dosenngen determined according to physician judgment and each patient's circumstances, for example, depending on the condition and the immune status of the individual patient, according to standard clinical techniques In another aspect of the invention, a perfusate or perfusjonsløsning brought provided for perfusion and storage of organs for transplant, perfusjonsløsningcn include an amount of a erytropoietm, effective to protect erytropoietmresponsive cells and associated cells, tissues or organs Transplant includes, but is not limited to xenotransplantation, where a organ (including cells, tissue or other kroppsdelcr) is harvested from one donor and transplanted into a different recipient, and autotransplantation, where an organ is taken from one part of a body and set back by another part of the body, include surgical procedures in which an organ may be removed, and while ex vivo operated away, repaired, or otherwise manipulated, such as by tumorfjemmg, and then returned to the original place In one embodiment, perfusjonsløsmngen The Umveisity of Wisconsin (UW) solution (U.S. Patent 4,798,824) which contains from about 1 to about 25 U / ml erytropoietm, 5% hydroxyethyl starch (having a molecular weight from about 200,000 to about 300,000 and substantially free of ethylene glycol, etylenklorhydnn , natnumklond and acetone), 25 mM ΚΉ2ΡΟ4, 3 mM glutathione, 5 mM adenosine, 10 mM glucose, 10 mM HEPES buffer, 5 mM magnesmmglukonat, 1.5 mM CaCl2, 105 mM natnumglukonat, 200,000 units of penicillin, 40 units of insulin , 16 mg dexamethasone, 12 mg phenol red and has a pH of 7.4-7.5 and an osmolality of 320 mOsm omknng / 1 The solution is used to maintain kadavemyrer and pancreas before transplantation Using this solution can be extended beyond storage the 30-hour limit recommended for kadavernyreoppbevanng This perfusalet is only lllustrativt for a number of such solutions that can be adapted for the present use by inclusion of an effective amount of a erytropoietm 1 a further embodiment, perfusatløsnmgen from omknng 5 to about 35 U / ml erytropoietm, or from 10 to omknng omknng U / ml erytropoietm As mentioned above, any form of erytropoietm used one aspect of the invention this
While the preferred recipient of a erytropoietm for purposes going all the way cr a human, the methods of hen used one level on other mammals, particularly in domestic animals, livestock, pets and animals 1 zoo invention cr not so limiting and the benefits can be used any mammal one further aspects of the ex v / vo-invention is any erytropoietm, such as, but not limited to erytropoietmet described above, as well as natural crytropoietmer, as well as an analog thereof, one erylropoietmetterlikner and erytropoietmfragment, a hybnd erytropoietmmolekyl, a erytropoietmreseptorbindende molecule, one erytropoietmagonist, a Renall erytropoietm, etc hjemeerytropoietm and ohgomer thereof, one multimer thereof, a mutein thereof, a cognate thereof, a naturally occurring form thereof, a synthetic form thereof, a recombinant form thereof, one glykosylcringsvanant thereof, one deglycosylated variant thereof, or a combination thereof one another aspect of the invention, methods and compositions for enhancing viabihteten to cells, tissues or organs which are not isolated from the vasculature at a endolelcellebamere provided by exposing the cells, tissue ellet means direct high for a pharmaceutical composition comprising a erytropoietm, or administering to or contacting a pharmaceutical composition erytropoietmholdig 1 contact vasculature 1 tissue or organ Strengthened activity of erytropoietmresponsive cells 1 treated tissue or organ is responsible for the positive effects that unfolds
As described above, the invention is partly based on the discovery that erytropoietmmolekyler can be transported from the lummale surface to the basement membrane surface of endothelial cells 1 capillaries bodies endotelcelletette compounds, such as including brain, retina and testes Thus erytropoietmresponsive cells on the other side of the barrier susceptible targets for the beneficial effects of erytropoietm, and other cell types or tissues or organs that contain and depend in whole or in part by erytropoietmresponsive cells, are targets for the methods of the invention Although it is not desired to be bound to any particular TEONA may erytropoietm after transcytosis create interactions with an erythropoietin receptor on a erytropoietmresponsiv cell, for example, neuronal, retinal, muscle, heart, lung, liver, kidney, small intestine, adrenal, bmyremarg, capillary endothelial, testes, OVAN or endometnumcelle and reseptorbmding can start a signaltransduksjonskaskade that resulting in aktivenng a genekspresjonsprogram in the erytropoietmresponsive cell or tissue, resulting in the protection of cell, tissue or organ from damage, such as by toxins, chemotherapeutic agents, strålmgsterapi, hypoxia, etc. In this manner, methods for protecting erytropoietmresponsiv cell mneholdende tissue from injury or hypoxic stress, and enhancing the function of such tissue, described in detail below, in the process of another embodiment of the invention, it was planned to harvest various organs of a victim of a traffic accident for transplant into a number of recipients, some of them required transport for an extended distance and period of time before organhøslmg was the victim mfusert with a pharmaceutical composition comprising a erytropoietm as described here Høstedc organs for shipment were perfused with a perfusate containing erytropoietm as described herein, and stored in a bath containing erytropoietm Certain organs were continuously perfiisert with a pulsating perfusjonsanordnmg using a perfusate containing a erytropoietm in accordance with the present invention Minimal deterioration of organ function occurred during the transport and during implantenng and reperfusion of the organs in situ
To repair a heart valve, according to another embodiment of the invention, required a surgical procedure temporarily cardioplegi and arteneokklusjon Before surgery, the patient mfusert with 500 U erytropoietm / kg body weight Such treatment prevented ccllulær damage from hypoxic ischemia, especially after reperfusion 1 any surgical procedure, according cn another embodiment of the invention, such as by kardiopulmonar bypass surgery, a naturally occurring erytropoietm or any erytropoietm invention may be used in the cn embodiment, administration of a pharmaceutical composition comprising a erytropoietm, as described above, carried out before, during and / or after bypass procedure, to protect the function of home, heart and other organs In the previous examples where erytropoietm invention include naturally occurring erytropoietm, is used for ex-vivo applications, or for treatment of erytropoietmresponsive cells, such as nerve tissue, retinalvev, heart, lung , liver, kidney, small intestine, adrenal, bmyremarg, capillary endothelial, testes, OVAN or endometnumceller or tissues, tilveiebnnger invention a pharmaceutical composition in dosenngenhetsform adapted for protection or enhancement of erytropoietinresponsive cells, tissues or organs distal to the vasculature which comprises per dosermgsenhct an effective, non-toxic amount within the range from about 50 000 to 500 000 units, 60 000 to 500 000 units, 70 000 to 500 000 units, 80 000 to 500 000 units, 90 000 to 500 000 units, 100 000 to 500 000 units, 150 000 to 500 000 units, 200 000 to 500 000 units, 250 000 to 500 000 units, 300 000 to 500 000 units, 350 000 to 500 000 units, 400 000 to 500 000 units or 450 000 to 500 000 units of erythropoietin, one erytropoietmreseptoraktivitetsmodulator or erytropoictinaktivert reseplormodulator and a pharmaceutically acceptable carrier In a preferred embodiment, the effective non-toxic amount of erythropoietin in the range from about 50 000 Ul 500,000 units In a preferred embodiment, the erythropoietin in the aforementioned composition does not -erytropoietisk In a further aspect of the invention was erytropoietinadmimstrasjon found to restore cognitive function in animals who had undergone home trauma After a delay of either 5 days or 30 days, administration of erythropoietin was still able to restore function as compared with the proforma-treated animals, indicates the ability of erythropoietin to regenerate or restore brain activity In this manner, the invention is also directed to the use of an erythropoietin for the preparation of a pharmaceutical composition for treatment of home trauma and other cognitive dysfunctions, including treatment for longer after injury (for example, 3 days , five days, a week, a month or longer) The invention is also directed to a method for the treatment of cognitive dysfunction following injury by administering an effective amount of an erythropoietin Any erythropoietin as described hen can be used for this aspect of the invention
Furthermore, this restorative aspect of the invention directed to the use of any of erytropoietmene hen for preparing a pharmaceutical composition for the restoration of cellular, tissue or organ dysfunction, wherein treatment has commenced or, and long after the original event responsible for the dysfunction Treatment using erytropoietmer invention can further extend over the course of the disease or condition at the acute phase as well as in a chronic phase In a preferred embodiment in which an erythropoietin of the invention has erythropoietic activity, the erythropoietin administered systemically at a dosage between omknng 300 and about 10,000 Units / kg body weight, preferably about 500 to 5,000 units / kg body weight, most preferably omknng 1000 units / kg body weight / supply This effective dose should be sufficient to achieve serum levels of erylropoietin greater than about 10,000 , 15,000, or 20,000 mU / ml of serum after erytropoietmtilførsel Such serum levels may be achieved by omknng 1.2, 3.4, 5.6, 7, 8, 9 or 10 hours or supply Such dosages may be repeated if necessary For example, supply repeated daily, as long as clinically necessary, or after an appropriate interval, such as every 1 to 12 weeks, preferably every 1 to 3 weeks County embodiment, the effective amount of erylropoietin and a pharmaceutically acceptable carrier packed in a enkeldoseampulle or other container In a Another embodiment is an erythropoietin useful for use hen non-erythropoietic, ie it is able to exert activity described going, but does not cause an increase in hemoglobin concentration or hematoknt Such a non-erythropoietic form of erythropoietin is preferred in instances where the methods of the present invention is intended to be chronic provided 1:01 another embodiment, an erythropoietin given at a dose greater than that necessary to maximally stimulate erythropoiesis As mentioned above, an erythropoietin of the invention does not necessarily erythropoietic activity, and therefore are dosenngene above expressed in hematopoietic units only examples of erytropoietmer that are erythropoietic, above molar equivalents for dosages are provided which are applicable to any erythropoietin
The present invention is further directed to a method to facilitate the transport of a molecule across a endotelcellebamere in a mammal by administering a composition which comprises the particular molecule in association with erythropoietin, as described above As described above, forming a tight relationship between endothelial cells in certain body organs one Bamer for the introduction of individual molecules for treatment of various conditions within enclosed bodies are means for promoting passage of pharmaceutical agents desirable An erythropoietin of the invention is useful as a carrier for delivering other molecules across the blood-home barrier and other similar barriers A composition comprising a molecule which is ivng after crossing the barrier with erythropoietin is prepared, and peripheral administration of the composition results in transeytose of the composition across the barrier association between the molecule to be transported across the barrier and the erythropoietin may be a labile covalent bond, where in this case the molecule is released from association with erytropoictmet after crossing barrier If the desired pharmacological activity molecule is maintained or unaffected by association with erythropoietin, such a complex bh managed
The trained scientist will be aware of various means for the association of molecules with an erythropoietin of the invention and the second agent described above, by covalent, non-covalent, and other additives, further, the evaluation of the effectiveness of the composition readily determined in an experimental system Association of molecules with erythropoietin may be accomplished by a number of means, including labile, covalent binding, cross-linking, etc. Biotin / avidin interactions may be used As mentioned above, a hybndmolekyl prepared by rekombmante or synthetic means, such as including both domain of the molecule with desired pharmacological Activity and domain responsible for erytropoietmreseptoraktivitetsmodulenng
A molecule can be conjugated to an erythropoietin through a polyfunctional molecule, i.e., a polyfunctional krysshnker As used herein the term "polyfunctional molecule" molecules having a functional group that can react more than one time in succession, such as formaldehyde, as well as molecules with more than one reactive group As used herein, the term "reactive group" to a functional group of krysslmkeren that reacts with a functional group on a molecule (e.g. peptide, protein, carbohydrate, nucleic acid, particularly a hormone, antibiotic or anti-cancer agents to be delivered over a endotelcellebamere) to form a covalent bond between krysslmkeren and the molecule The term "functional group" retains its standard meaning in organic chemistry The polyfunctional used here is preferably biocompatible lmkere, ie they are non-karsmogene, nontoxic and substantially non-immunogenic m vivo Polyfunctional krysshnkere such as those known in the art and described hen, can be tested in animal models to determine their biokompatibihtel The polyfunctional molecule is preferably bi-functional As used hen the term "bifunctional molecule" refers to a molecule with two reactive groups The bifunctional molecule may be heterobifunctional or homobifunksjonelt A heterobifunctional krysshnker allows vectorial konjugenng It is particularly preferred that the polyfunctional molecule is sufficiently soluble in water to kryssbindmgsreaksjonene should be conducted in aqueous solutions such as in aqueous solutions bufiret at pH 6 to 8, and that konjugatresultatet vannløsehg remains for more efficient biodistnbusjon Generally bind the polyfunctional molecule covalently with an amino group or an sulfidylfunksjonell Polyfunctional molecules are, however, reactive with other functional groups, such as carboxylic acids or hydroxyl groups considered in the present invention
The homobifunksjoncllc molecules having at least two equally reactive functional groups The reactive functional groups on a homobifunksjonelt molecule include, for example aldehydgruppcr and active ester groups homobifunctional molecules having aldehyde groups include, for example, glutaraldehyde and subaraldehyd
The use of glutaraldehyde as a crosslinking agent, was set by Poznansky et al, Science 223.1304 to 1306 (1984) homobifunctional molecules having at least two active ester units include esters of dikarboksylsyrcr and N-hydroxysuccinimide Some examples of such N-suksimmidylestere includes disuksimmidylsuberat and dithio-bis (suksimmidylpropionat), and their soluble bis-sulfonic acid and bis-sulfonate salts such as their sodium and calcium salts These homobifunctional reagents are available from Pierce, Rockford, Illinois
The heterobifunctional molecules have at least two different reactive groups the reactive groups react with different functional groups, such as present on erytropoietmet and molecule These two different functional groups that react with the reactive group on the heterobifunctional krysslmkeren is usually an amino group, such as epsilon -ammogruppen with lysine, one sulfidylgruppe, for example the thiol group of cysteine, one carboxylic acid, such as carboxylate at aspargmsyre, or a hydroxyl group, such as hydroxyl at Mountain
The various erythropoietin molecules of the invention may of course may not have suitable reactive groups similar provided for use with certain cross-linking agents, one with skill in the art will be aware of the choice of cross-linking agents based on the available groups for cross-linking in a erytropoietm invention When a reactive group on a heterobi functional molecule forms a covalent bond with an amino group, the covalent bond will usually be an amide or Lave-bindmg The reactive group that forms a covalent bond with an amino group, may be for example an activated carboxyl group, one halokarbonylgruppe or an ester group, the preferred halokarbonylgruppen is a klorkarbonylgruppc The ester groups are preferably reactive ester groups such as, for example, an N-hydroksysuksimmidestergruppe
The second functional group is usually either a thiol group, a group that can be converted to a thiol group, or a group forming a covalent bond with a thiol group The covalent bond will usually be a tioeterbindmg or disulfide The reactive group that forms a covalent bond with a thiol group may for example be a dobbeltbmding which reacts with thiol groups or an activated disulfide A reactive group containing a dobbeltbmding which is capable of reacting with a thiol group is the maleimide group, although others, shk as akrylnitnl also possible A reactive disulfide group may for example, a 2-pyndyIditiogruppe or a 5,5'-dithio-bis (2-mtrobenzosyre) group Some examples of heterobifunctional reagents containing reactive disulfidbmdinger include N-succinimidyl 3- (2-pyndyl-dilio) propionate (Carlsson, a al, 1978, J Biochcm, 173 723-737), sodium S-4- suksimmidyloksykarbonyl alpha metylbcnzyltiosulfat, and 4-suksimmidyloksykarbonyl-alpha-methyl- (2-pyndylditio) toluene N-suksmimidyl 3- (2-pyndylditio) propionate are preferred Some examples of heterobifunctional reagents comprising reactive groups with a dobbeltbmding which reacts with a thiol group include suksmimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate and
suksmimidyl m-maleimidobcnzoat Other heterobifunctional molecules include suksmimidyl 3- (maleimide) propionate, sulfosuksimmidyl 4- (p-maleimide phenyl) butyrate, sulfosuksmimidyl 4- (N-maleimidmetylsykloheksan) -l -carboxylate, maleimidobenzoyl-N-hydroxy-suksmimidester Natnumsulfonalsaltet of suksmimidyl m-maleimidobenzoat is preferred Many of the above-mentioned heterobifunctional reagents and their sulfonate salts are available from Pierce Chemical Co., Rockford, Illinois, USA
The need for the conjugate described above to be reversible or labile may be readily determined by the trained scientist A conjugate may be tested in vitro for both erytropoietmet and for the desirable pharmacological activity About the conjugate retains both properties, its suitability further tested m νινο About the conjugate molecule requires separation from erytropoietmet for activity, a labile bond or reversible association with erythropoietin be desirable Labihtetsegenskapene can also be tested using standard in vitro procedures before m vivo testing
Further information regarding the preparation and application of these, as well as other polyfunctional reagents may be obtained from the following publications or others available in the art
Carlsson, J. et al, 1978, Biochem J 173 723-737
Cumber, JA et al, 1985, Methods in Enzymology 112 207-224
Juc, R et al, 1978, Biochem 17 5399-5405
Sun, TT et al, 1974, Biochem 13 2334-2340
Blättler WA et al, 1985, Biochem 24 1517-152
Liu, FT et al, 1979, Biochem 18 690-697
Youle, RJ ogNeville, DM Jr, 1980, Proc Natl Acad Sci USA 77 5483-5486
Lemer, RA et al, 1981, Proc Natl Acad Sci USA 78 3403-3407
Jung, SM and Moroi, M, 1983, Biochem Biophys Acta 761 162
Caulfield, MP et al, 1984, Biochem 81 7772-7776
Staros, JV, 1982, Biochem 21 3950-3955
Yoshitake, S et al, 1979 Eur J Biochem 101 395-399
Yoshitakc, S et al, 1982, J Biochem 92 1413-1424
Pilch PF and Czech, MP, 1979 J Biol Chem 254 3375-3381
Novick, D et al, 1987, J Biol Chem 262 8483 to 8487 Lomant, AJ and Fairbanks, G, 1976 J Mol Biol 104 243-261 Hamada, H and Tsuruo, T, 1987, Anal Biochem 160 483-488 Hashida, S et al, 1984, J Applied Biochem 6 56-63
Methods of crosslinking is additionally reviewed by Means and Fceney, 1990, Bioconjugate Chem 12-12
Barriers which are crossed by the above-described methods and compositions of the present invention include, but are not limited to blood-home Bamer, blood-eye barrier, blood-tcstikkel Bamer, blood-ovanc barrier and blood-barrier hvmor
Candidate molecules for transport across a endotelcellebamere include, for example honnoncr, such as growth hormones, neurotrophic factors, antibiotics or anlisoppmidler such as those normally excluded from the brain and any other enclosed organs, peptide radiopharmaceuticals, antisensmidlcr, antibodies against biologically-active agents, pharmaceuticals, and anti-canccrmidler Non-bcgrensende examples of such molecules include growth hormone, nerve growth factor (NGF), home-derived ncurotrofisk factor (BDNF), ciliary ncurotrofisk factor (CNTF), basic fibroblast growth factor (bFGF), trans proliferating growth factor βΐ (TGFpl), transforming growth factor β2 ( TGFp2), transforming growth factor β3 (ΤΰΡβ3), mterleukin 1, mterleukin 2, mterleukin 3 and interleukin 6, AZT, antibodies against tumomekrose factor and immunosuppressive agents such as syklosponn
The present invention is also directed to a composition comprising a molecule to be transported via transeytose over a endotelcelletett forbmdelsesbamere and a erytropoictm as described above The invention is further directed to the use of a conjugate between el molecule and erythropoietin described above for the preparation of a pharmaceutical composition for delivery of the molecule across a barrier as described above
The present invention may be better understood by reference to the following non-limiting examples are provided as examples of the invention The following examples are presented to more fully illustrate the preferred embodiments of the present invention However, they must in no way be interpreted as limiting the scope of this invention
Example 1
Erythropoietin cross the blood cerebrospinal fluid CLOSE
BARRIER
Adult Sprague-Dawley rats were anesthetized and administered recombinant human erytropoietm intrapenlonealt Cerebrospmalvæske were collected from the cisterna magna at 30 minute intervals up to 4 hours and erytropoietinkonsenlrasjonen was determined using a sensitive and specific enzyme-linked immunoassay, as illustrated in Figure 1, is basalhnje-erytropoietinkonsentrasjonen in CSF 8 mU / ml after a stay of several hours beginning levels of erytropoietm measured in CSF to increase, and by 2.5 hours and later they are significantly different from basallinjekonsenlrasjonen at p <0.01 level The top level of about 100 mU / ml is within the range known to exert protective effects in vitro (0.1 to 100 mU / mL) Time to reach flea takes place after about 3.5 hours, which is significantly delayed from the peak levels in the serum (less than 1 hour) The results of this experiment illustrate significant levels of erytropoietm achieved over a dense cellular connection by bolus parentcral supply of erytropoietm at appropriate concentrations
Example 2
MAINTENANCE OF FUNCTION IN HEART PREPARED FOR
GRAFT
Male Wistar rats weighing 300 to 330 g were fed erytropoietm (5000 U / kg body weight) or conveyance 24 hours before removal of the heart for ex vivo studies in accordance with the protocol of Delcayre et al, 1992, Amer J Physiol 263 Hl 537-45 The animals were sacrificed with pentobarbital (0.3 ml), and intravenously hepann (0.2 mL) Hearts were initially allowed to equilibrated 115 minutes The left ventnkulære balloon was then inflated to a volume that produced a diastolic pressure of 8 mm Hg The left ventnkulære pressure-volume curve is constructed by growing mnblåsning of balloon volume by 0.02 ml ahkvoter Zero volume is defined as the point at which it left, ventnkulære sluttdiastohskc pressure is zero Upon completion of the pressure / volume curve, the left, ventnkulære balloon reduced to put it sluttdiastohske pressure back to 8mmHg and kontrollpcnoden was followed for 15 minutes after control of coronærstrømmng Dei after the heart was stopped with 50 mL Celsior + molecule to rest at 4 ° C at a pressure of 60 cm H2O The heart was then removed and stored 5 hours 1 at 4 ° C 1 a plastic container filled with the same solution and surrounded with crushed ice
After completion lagnng the heart was transferred to a Langendorff apparatus balloon catheter was again inserted one left ventnkkel and again inflated to the same volume during the pre-ischemic penode heart reperfused for at least 2 hours at 37 ° C Reperfusjonstrykket is set at 50 cm H20 in 15 minutes reflux and then back to 100 cm H201 the next 2 hours Heart rate (320 beats / mmutt) recovered isovolumetric measurements of contractile indexes and diastolic pressure are made in tnphkat at 25.45, 60 and 120 minutes of re-perfusion At this time any pressure-volume curves, and coronary effluent during the 45 mn reperfusion collected to measure keratinkmase leak The two treatment groups are compared using an unpaired t-read, and a linear regression using the sluttdiastohske pressure data used to construct the corresponding curves As shown in Figure 2 occurs significant improvement of left ventnkulære pressure developed after treatment with erythropoietin, as well as improved volume / pressure curve, decrease of left diastolic, ventnkulære pressure and reduction of leakage keratinkmase
Example 3 erythropoietin PROTECTS myocardium FRA ischemic injury Adult male rats were administered recombinant human erythropoietin (5000 U / kg body weight) 24 hours previously were anesthetized and prepared for coronary arteneokklusjon An additional dose of erythropoietin was administered at the start of the procedure and the left large coronary species blocked for 30 minutes and then frigiort The same dose erythropoietin was administered daily for one week after treatment animals were then studied for cardiac function, as illustrated in Figure 3 demonstrated animals receiving a proforma injection (saltoppløsmng) a greater increase in the left sluttdiastohske pressure, indicates a dilated, stiff heart to myocardial infarction In contrast, animals receiving erythropoietin lcd not of decrease in cardiac function, compared with pro forma operated controls (difference significant at p <0.01 level)
Example 4
Erythropoietin
Natural erythropoietin may be modified to tailor its activities for a specific tissue or specific tissues Several non-limiting strategies can be carried out to achieve this desired tissue specificity include modifications that remove or modify glykosylenngshalvdelene, erythropoietin has three Ν'-linked and O associated Such Vanant of glycosylated erythropoietin can be produced in many ways sialic terminating end of the sugar chains can for example be removed by specific siahdaser, depending on the chemical linkage connecting the sialic acid to the sugar chain Alternatively, the glycosylated structure dismantled in several ways by using other enzymes that clover at specific bindmger For confirmation of these principles, recombinant human erylropoietin desialisert using Siahdase A (Prozyme Inc.) according to manufacturer's protocol Successful chemical modification was confirmed by running the reaction product on an SDS-polyacrylamide gel and color the resultant bands which showed the chemically modified erythropoietin had an apparent molecular weight of ~ 31 kD as expected, compared to unmodified erythropoietin which was ~ 34 kD and by measuring the remaining sialmsyrerester by chemical means to be <0.1 mole / mole of erythropoietin In a another modification in which the ammo acid residues of erythropoietin are modified, was argminrester modified by using phenylglyoxal according to the protocol of Takahashi (1977, J Biochem 81 395-402) performed by varying lengths of time ranging from 0.5 to 3 hours at room temperature reaction was terminated by dialysis of the water reaksjonsblandmgen Use of such modified forms of erythropoietin is fully covered hen
Asialoerytropoietin and fenylglyoksalerytropoietin was as effective as natural erythropoietin for neural cells in vitro as shown Figures 4-6 In vitro testing was performed using nerve hknende embryonic carcinoma cells (P19) that undergo apoptosis upon removal of serum 24 hours before removal of the serum was 1-1000 ng / ml erythropoietin or a modified erythropoietin was added to the cultures the following day, the medium was removed, cells washed with fresh, non-serum containing medium, and medium containing the test substance (no serum) was returned to the cultures for an additional 48 hours To determine the number of viable cells, a tetrazolium reduction test performed (CellTiter 96, Promega, Inc.) As illustrated in Figure 4-5, showing asialoerytropoietin to be as effective as erythropoietin in preventing cell death It fenylglyoksalmodifiserte erythropoietin was tested using the neural-hknende P19 cell assay described above As illustrated in Figure 6 retains this chemically modified erythropoietin fully its neuroprotective effects
Retention of neuroprotective activity m vivo was confirmed using a rottefokal lschemimodell which a reversible lesion in the territory of the middle cerebral species is performed as described previously (Bnnes et al, 2000, Natl Acad Ptoc Set USA 97 10526-31) Adult Sprague -Dawley hannrolter was fed asialoerytropoietin or erythropoietin (5000 U / kg body weight mtrapentonealt) or conveyance at the start of the species or for the occlusion 24 hours later the animals were sacrificed and their brains removed for study tendon sections were cut and stained with tetrazohumsalter for ldentifisenng of living brain regions, as shown in Figure 7 was asialoerytropoietin as effective as natural erythropoietin in to exhibit neuroprotection from 1 hour of ischemia Figure 8 shows the results of another focal lschemimodell which a comparative dose response was performed with erythropoietin and asialoerytropoietin At the lowest dose of 250 U / kg, gave asialoerytropoietin protection while unmodified erythropoietin did not protect
Example 5 MODIFICATION OF PRIMARY STRUCTURE OF erythropoietin and EFFICIENCY BY neuronal PROTECTION A number of mutant erytropoietmmolekyler that does not bind to the erythrocyte erythropoietin receptor is described, and thus do not support erythropoiesis m νινο or in vitro Some of these molecules will nevertheless mimic the effects of erythropoietin in other tissues or organs For example, a 17-mer containing ammo acid sequence of 31-47 of natural erythropoietin inactive for erythropoiesis but fully active for neural cells in vitro (Campana & 0'Bnen, 1998 Int J Mol Med 1 235-41)
Derivative erythropoietins desirable for the uses described hen, can be produced by guamdmenng, amidmering, tnnitrofenylenng, acetylenng, suksmilenng, mtrermg or modification of argimnrester or carboxyl groups, among other procedures as mentioned above, to produce erythropoietins which maintain their activities for specific organs and tissues but not for others, such as erythrocytes When erythropoietin is subjected to the above reactions, it has generally been found that the resultant molecule lacks both in vivo and in vitro erylropoielisk activity (such as Satake et al, 1990, Biochim Biophys Acta 1038 125 -9) Some examples of the preparation of modified erythropoietins are described below
Biotinylation of free aminogruDDer of ervtroooietin 0.2 mg D-biotmyl-e-aminocaproic acid-N-hydroxysuccinimide ester (Boehnnger Mannheim No. 1418165) was dissolved in 1100 μΐ DMSO This solution was combined with 400 μΐ PBS containing approximately 0.2 mg erythropoietin in a foil covered tubes After incubation for 4hours at room temperature, the unreacted biotmet separated by gclfiltrermg on a Centncon 10 column as shown in Figure 10, protects this biotinylated erythropoietin P19 cells from serumfjeming 1 "Biolinylated recombinant human erythropoietin Bioaclivity and Utility as a receplor ligand" by Wojchowski et al, Blood, 1989.74 (3) 952-8, the authors uses three different methods to biotinylate erythropoietin Biotin is added to (1) siahnsyrehalvdelene (2) carboxylate groups (3) ammo groups authors employ a musemiltcelleproliferasjonstest to demonstrate that (1 ) the addition of biotin to siahnsyrehalvdelene do not inactivate the biological activity of erythropoietin (2) the addition of biotin to carboxylate groups results in a material biologically maktivenng of erytropoietm (3) the addition of biotin to ammogmppene resulted in complete biological maktivenng of erytropoietm These methods and modifications are fully covered herein Figure 9 shows the activity of biotinylated erytropoietm and asialoerytropoietin in serumsultetP19 test
Jodinerme of ervtroooietin
Procedure 1 - Jodkuler Enjodkule (Pierce, Rockford, IL) was incubated in 1100 μΐ PBS (20 mM natnumfosfat, 0.15 M NaCl, pH 7.5) containing 1 mCi free Na125! in 5 minutes 100 pg erytropoietm 1100 μΐ PBS was then added to the mixture after 10 minutes mkubasjonspenode at room temperature the reaction was quenched by the removal of 200 μΐ solution from the reaction tube (jodkulene remained in the tube) Jodoverskuddet was removed by gelfiltrermg on a Centncon 10 column, as shown in Figure 11 is iodine erytropoietm produced in this manner effective for protecting P19 cells from serumfjemmg
Method 2 - Kloramm T 100 pg erytropoietm 1100 μΐ PBS was added to 500 uCi Na125! and mixed together in an eppendorf 25 μΐ chloro frames T (2 mg / ml) was then added and the mixture was incubated for one minute at room temperature 50 μΐ kloramm T stop buffer (2.4 mg / ml sodium metabisulfite, 10 mg / ml tyrosine, 10% glycerol, 0.1% xylene in PBS) was then added and iodinated erytropoietm Jodtyrosmet was then separated by gelfiltrermg on a Centricon 10 column
Lysinmodifikasjoner Karbamylenng Erytropoietm (100 pg) was modified with kahumeyanat as described in Plapp et al ("Activity of bovinc pancreatic deoxyribonuclease A wilh modified amino groups" 1971, J Biol Chem 246, 939-845).
Tnnitrofenylenng Erytropoietm (100 pg) was modified with 2,4,6-tnmtrobcnzensulfonat as described in Plapp et al, ("Activity of bovine pancreatic deoxyribonuclease A wilh modified amino groups" 1971 J Biol Chem 246939-845).
Acetylermg Erytropoietm (100 pg) were incubated in 0.3 M phosphate buffered (pH 7.2) containing the same amount of eddiksyreanhydnd at 0 ° C for 1 hour The reaction was stopped by dialysis against distilled water
Suksinylermg Erytropoietm (100 pg) 10.15 M NaHCO> 3 (pH 8.0) was incubated a 15 molar excess of ravsyreanhydnd at 15 DC for 1 hour The reaction was stopped by dialysis against distilled water
Argminmodifikasjoner Erytropoietm was modified with 2,3-butanedione as described in Riordan ("Functional argmyl Residue m carboxypeptidase A Modification with bulanedione" Riordan JF, Biochemistry 1973.12 (20) 3915-3923)
Erytropoietm was modified with cyclohexanone as in Palthy et al, ('' Identification of functional residues in argmin nbonuclease A and lysozome "Patthy, L, Smith ELJ Biol Chem 1975 250 (2) 565-9)
Erytropoietm was modified with phenylglyoxal as described in Werber et al, ("Proceedmgs Carboxypeptidase B modification of functional arginyl residues" Werber, Μ M, Sokolovsky M Isr J Med Sci 1975 11 (11) 1169-70)
Tyrosine modifications Erytropoietm (100 pg) was incubated with tetranitromethane as previously described in Nestler et al, "Stimulation of rat ovanan cell steroidogenesis by high density lipoproteins modified with tetramtromethane" Nestler JE, Chacko GK, Strauss JF 3rd J Biol Chem 1985 June 25, 260 (12) 7316-21)
Glutammsyrc (and aspargmsyre) modifications order to modify carboxyl groups were erytropoietm (100 pg) was incubated with 0.02 M EDC 11 Mglysmamid with PFT 4.5 at room temperature for 60 minutes as described in Carraway et al "Carboxyl group modification in chymotrypsin and chymotrypsmogen" Carraway KL, Spoerl P, Koshland DE Jr Jmol Biol 1969 May 28.42 (1) 133-7
Tryptophan residumodifikasjoner Erytropoietm (100 pg) were incubated with 20 uM n-bromosuksimmid in 20 mM kahumfosfatbuffer (pH 6.5) at room temperature as described in Ali et al, J Biol Chem 1995 March 3, 270 (9) 4570-4 number of oxidized tryprofanresler was determined by the method described in Korotchkma (Korotchkma, LG et al Protein Expr Punf 1995 Feb, 6 (1) 79-90)
Removal of the ammo groups To remove ammo groups were erytropoietm (100 pg) was incubated in PBS (pH 7.4) containing 20 mM nmhydnn (Pierce Chemical, Rockford, IL) at 37 ° C for 2 hours as described in Kokkmi el al (Kokkini , G, et al "Modification of hemoglobin by ninhydrm" Blood, Vol 556, No 4 1980 701-705) Reduction of the resulting aldehyde was accomplished by reacting the product with natnumborhydrid or litiumalummiumhydnd Specifically erytropoietm (100 pg) was incubated with 0.1 M natnumborhydrid in PBS for 30 minutes at room temperature reduction was stopped by cooling the samples on ice for 10 minutes and at dialysis mole PBS three times overnight (Kokkini, G., Blood, Vol 556, No 4 1980 701-705) Reduction by using litiumalummiumhydrid was accomplished by incubating erythropoietin (100 pg) with 0.1M litiumaluminiumhydnd in PBS for 30 minutes at room temperature reduction was stopped by cooling the samples on ice for 10 minutes and dialysis against PBS three times overnight
Disulfidreduksjon and stabilization Erythropoietin (100 pg) were incubated with 500 mM DTT115 minutes at 60 ° C 20 mM iodoacetamide in water was then added to the mixture and incubated for 25 minutes at room temperature in the dark
Limited proteolysis Erythropoietin can be subjected to a limited chemical proteolysis that targets specific residues Erythropoietin was reacted with 2- (2-mtrofenylsulfenyl) -3-methyl-3'-bromindolenin which clover specifically by tryptophan residues in a 50-fold excess of 50 % acetic acid at 48 hours in the dark at room temperature for pipes put under nitrogen pressure reaction was stopped by repression with tryptophan and desalting
Example 6
PROTECTION OF retinal ischemia during peripheral MANAGED
Erythropoietin
Retinalceller are very sensitive to ischemia such that many will die after 30 minutes of ischemic stress Furthermore located subacute or chronic ischemia behind deterioration of vision, which follow a number of common human diseases, such as diabetes mcllitus, glaucoma and macular degeneration At the present time there is no effective therapies to protect cells against ischaemia A close endotelbamcrc exists between the blood and the retina that excludes most large molecules To test whether peripherally administered erythropoietin will protect cells sensitive to ischemia, an acute, reversible glaucoma rat model used as described by Rosenbaum et al, (1997, Vis Res 37 3443-51) Specifically saltoppløsnmg injected into the anterior chamber of the eye of adult male rats to a pressure above systennsk artenelt pressure and maintained for 10 minutes animals were fed saltoppløsnmg or 5000 U erytropoielin / kg body weight intrapenloneall 24 hours before the induction of ischemia, and continued as a daily dose for an additional 3 days Electroretinography was performed on dark-adapted rats one week after treatment Figure 11-12 illustrate that the administration of erythropoietin is associated with good preservation of the electroretinogram ( ERG) (Panel D), in contrast to animals treated with saltoppløsnmg alone (panel C), in which very little function remained Figure 11 compares elektroretinogram a- and b-wave amplitudes of the erytropoietinbehandlete groups and saltoppløsnmgsbehandlete groups, and shows significant protection provided by erythropoietin
Example 7
Replenishing EFFECTS of erythropoietin on REDUCED COGNITIVE FUNCTION ARISING FROM brain damage
In a study for demonstration of erythropoietin ability to rebuild reduced cognitive function in mice after they've gotten home trauma, were Balb / C mice exposed to dim home trauma, as described in Bnnes et al, PNAS 2000.97, 10295-10672, and 5 days later started daily mtrapentoneal erytropoietmadministrasjon with 5000 U / kg body weight 12 days after injury, animals were tested for cognitive function in the VAT vannlabynnt, with four trials per day While both treated and non-treated animals performed poorly in the test (with swim times on omknng 80 seconds of 90 possible), Figure 13 shows that erytropoietinbehandlete animals performed better (in this presentation is a negative value better) Although the start of crytropoietinbehandhng is delayed until 30 days after trauma (Figure 14), the reconstruction of cognitive function also seen
Example 8
Kainate MODEL
In kainate neurotoksisitelsmodellen asialoerytropoietm was administered according to the protocol of Bnnes et al, Proc Natl Acad Sci USA 2000, 97, 10295-10672 at a dose of 5000 U / kg body weight administered mtraperitonealt 24 hours before administration of 25 mg / kg kainate showing to be as effective as erythropoietin, as shown by time to death (Figure 15)
FIELD shall not be limited by the specific embodiments described which are intended as single illustrations of individual aspects of the invention, and functional equivalent methods and components are within the scope of the invention Of course, various modifications of the invention, in addition to those shown and described going, be apparent to those skilled in the art, from the aforementioned description and attached figures Such modifications are intended to fall within the scope of the appended claims
All references cited hen incorporated by reference in its entirety herein for all purposes
Contents5
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1 legal event, as the office reported them to INPADOC
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Titles2
- Norwegian
- Beskyttelse, gjenoppbygging og styrking av erytropoietinresponsive celler, vev og organer.
- English
- Protection, reconstruction and strengthening of erytropoietinresponsive cells, tissues and organs.
Classification
- CPC, 38
- A61K38/1816
- A61K45/06
- A61K38/22
- A61P1/04
- A61P1/16
- A61P11/00
- A61P13/12
- A61P15/00
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- A61P21/02
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- A61P25/08
- A61P25/14
- A61P25/16
- A61P25/18
- A61P25/20
- A61P25/22
- A61P25/24
- A61P25/28
- A61P25/32
- A61P27/02
- A61P27/06
- A61P29/00
- A61P3/00
- A61P35/00
- A61P3/08
- A61P43/00
- A61P5/38
- A61P7/00
- A61P7/04
- A61P9/00
- A61P9/02
- A61P9/04
- A61P9/10
- A61P9/12
- A61P3/10
- IPC, 30
- A61K38 18
- A61K38 00
- A61K45 00
- A61K38 16
- A61K38 19
- A61K38 24
- A61K38 27
- A61K45 06
- A61P3 10
- A61P7 00
- A61P9 00
- A61P9 04
- A61P9 10
- A61P21 04
- A61P25 00
- A61P25 02
- A61P25 08
- A61P25 14
- A61P25 16
- A61P25 18
- A61P25 22
- A61P25 28
- A61P25 32
- A61P27 02
- A61P27 06
- A61P29 00
- A61P43 00
- C07K14 505
- C07K14 52
- C07K14 575