NO870008L - USE OF TUMOR CROSS FACTOR AS WEIGHT CONTROLLER. - Google Patents
USE OF TUMOR CROSS FACTOR AS WEIGHT CONTROLLER.Info
- Publication number
- NO870008L NO870008L NO870008A NO870008A NO870008L NO 870008 L NO870008 L NO 870008L NO 870008 A NO870008 A NO 870008A NO 870008 A NO870008 A NO 870008A NO 870008 L NO870008 L NO 870008L
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Description
Foreliggende oppfinnelse er gjennomført med støtte fra USA's regjering under bevilgning GM 25821, gitt av det nasjonale he 1 se ins t i tut t. De Forente Staters regjering har visse rettigheter i forbindelse med oppfinnelsen. The present invention has been carried out with the support of the United States government under grant GM 25821, given by the national he 1 se ins t i tut t. The United States government has certain rights in connection with the invention.
Oppfinnelsen angår kontroll av lipidmetabolismen ved bruk av tumornekrosefaktor TNF. Spesielt angår oppfinnelsen en fremgangsmåte for å kontrollere sterk fedme eller fettsyke ved administrering av TNF. The invention relates to control of lipid metabolism using tumor necrosis factor TNF. In particular, the invention relates to a method for controlling severe obesity or obesity by administering TNF.
Kontroll av sterk fedme er blitt et problem i utviklede kulturer på tross av det spektrum av sult som ofte dominerer deler av den mindre teknologisk fremskredne verden. Hovedtil-nærmelsen for de mindre enn vellykkede tilnærmelser som har vært benyttet i et forsøk på å kontrollere for høy kroppsvekt, er utvilsomt velkjente for de fleste. Løsningene ligger fra redusert næringsforbruk til ofte en ukritisk bruk av farmasøytika ment primært for andre formål, men som synes å ha en bivirkning med en noe ugjennomsiktlig mekanisme som resulterer i vekttap. Kort sagt forblir det millioner av mennesker som ønsker å redusere kroppsvekten uten deprivasjon og uten risiko for uønskede og eventuelt usunne bivirkninger. Control of severe obesity has become a problem in developed cultures despite the spectrum of hunger that often dominates parts of the less technologically advanced world. The main approach to the less than successful approaches that have been used in an attempt to control excessive body weight are undoubtedly well known to most. The solutions range from reduced nutrient consumption to the often uncritical use of pharmaceuticals intended primarily for other purposes, but which seem to have a side effect with a somewhat opaque mechanism that results in weight loss. In short, there remain millions of people who want to reduce their body weight without deprivation and without the risk of unwanted and possibly unhealthy side effects.
Selvfølgelig er ikke alle vekttap nødvendigvis ønskelige. Således er vekttapet som skyldes kronisk katabolisk tilstand, kalt kakeksi, utviklet under infeksiøse eller ondartede sykdommer, et handikap å overvinne og er ofte direkte fatalt. Generelt er imidlertid kakeksi antatt å være en normal respons på infeksjon, og er uønsket kun når den tillates å skride frem uten den riktige kontroll. Katabolismen karakte-riseres ved en netto nedbrytning av lipider i adiposeceller, og det er antatt at denne uønskede balanse i det minste delvis er et resultat i cellenes manglende evne til å syntetisere tilstrekkelige mengder lipogene enzymer. Of course, not all weight loss is necessarily desirable. Thus, the loss of weight due to a chronic catabolic state, called cachexia, developed during infectious or malignant diseases, is a handicap to overcome and is often directly fatal. In general, however, cachexia is thought to be a normal response to infection, and is undesirable only when it is allowed to progress without proper control. The catabolism is characterized by a net breakdown of lipids in adipose cells, and it is assumed that this undesirable balance is at least partly a result of the cells' inability to synthesize sufficient amounts of lipogenic enzymes.
Et råproteinekstrakt fra media av endotoksin-stimulerte makrofager, kalt "kakektin", har vist seg å indusere indisier på kakeksi i vevkultur (Torti, F., et al. "Science" (1985) 229: 867-869), og antistoffer dyrket derimot har vist seg å beskytte mus fra noen av virkningene av E. coli lipopolysakk-aridendotoksin (Beutler, B., et al. ibid, s. 869-871). A crude protein extract from the media of endotoxin-stimulated macrophages, termed "cachectin", has been shown to induce evidence of cachexia in tissue culture (Torti, F., et al. "Science" (1985) 229: 867-869), and antibodies cultured however, has been shown to protect mice from some of the effects of E. coli lipopolysacchar-aridendotoxin (Beutler, B., et al. ibid, pp. 869-871).
Det er nu vist at tumornekrosefaktor, TNF, spesifikt endrer adipocyters karakteristika i vevkultur på en måte som antas å være en modell for minst en del av kakeksi-prosessen. Senere arbeider har antydet at kakektin og nativ TNF som opprinnelig ble oppnådd fra sera av endotoksinbehandlede mus, og som nu er tilgjengelige i rekombinant form, har den samme primære struktur. I henhold til dette er TNF et brukbart materiale for vektkontroll ved stimulering av denne katabolske reaksjon under kontrollerte tilstander og under omstendigheter der slik stimulering er ønskelig. Det er også mulig å kontrollere uønsket vekttap ved nøytralisering av TNF. It has now been shown that tumor necrosis factor, TNF, specifically changes the characteristics of adipocytes in tissue culture in a way that is believed to be a model for at least part of the cachexia process. Subsequent work has suggested that cachectin and native TNF, originally obtained from the sera of endotoxin-treated mice, and now available in recombinant form, have the same primary structure. Accordingly, TNF is a useful material for weight control by stimulating this catabolic reaction under controlled conditions and under circumstances where such stimulation is desirable. It is also possible to control unwanted weight loss by neutralizing TNF.
Oppfinnelsen tilveiebringer en definert farmasøytisk tumor-nekrosef aktor , TNF, som er istand til å stimulere den fysiologiske tilstand i forbindelse med kakeksi. Kontrollen av vekt hos adipøse personer gjennomføres derved ved bruk av et materiale som stimulerer en naturlig fysiologisk respons mot infeksjoner under tilstander der slik stimulering kan reguleres og kontrolleres. The invention provides a defined pharmaceutical tumor necrosis factor, TNF, which is capable of stimulating the physiological state associated with cachexia. The control of weight in obese people is thereby carried out using a material which stimulates a natural physiological response against infections under conditions where such stimulation can be regulated and controlled.
I henhold til dette angår oppfinnelsen i et aspekt en fremgangsmåte for å kontrollere for sterk fedme ved administrering av TNF. I et annet trekk angår oppfinnelsen kontroll av kakeksi i cancer- eller infeksjonssyke pasienter ved å tilveiebringe antistoffer for nøytralisering av TNF. I andre trekk angår oppfinnelsen farmasøytiske preparater til bruk ved vektreduskjon og som inneholder TNF som en aktiv bestand-del, og immunoglobulinpreparater brukbare ved nøytralisering av TNF. Figur 1 viser nukleotidsekvensen som koder humanmaturt TNF og den derfra avledede aminosyresekvens. Figur 2 viser akkumuleringen av adiposeinduserbare mRNA i maturerende adiposeceller og reguleringen av syntesen av slik mRNA i nærvær og fravær av TNF. Figur 3 viser depresjonen av eksprimer ingen av adipose-stimulerte gener på grunn av TNF. Figur 4A og 4B er fotografier av mature adiposeceller med og uten TNF. Accordingly, the invention relates in one aspect to a method for controlling severe obesity by administering TNF. In another aspect, the invention relates to the control of cachexia in patients suffering from cancer or infection by providing antibodies for neutralizing TNF. In other aspects, the invention relates to pharmaceutical preparations for use in weight reduction and which contain TNF as an active ingredient, and immunoglobulin preparations usable for neutralizing TNF. Figure 1 shows the nucleotide sequence encoding human mature TNF and the amino acid sequence derived therefrom. Figure 2 shows the accumulation of adipose-inducible mRNA in maturing adipose cells and the regulation of the synthesis of such mRNA in the presence and absence of TNF. Figure 3 shows the depression of expression of none of the adipose-stimulated genes due to TNF. Figures 4A and 4B are photographs of mature adipose cells with and without TNF.
Def inis. 1 onerDefinitions. 1 oner
Som bruk heri, henviser "tumor nekrosefaktor", TNF, til en aminosyresekvens som typifiseres ved det som vises i fig.l, og som er istand til selektiv cytotoksisitet mot tumorceller. Sekvensen i fig. 1 er avledet fra et human-cDNA, men TNF kodet av andre pattedyrarter kan vise den krevede aktivitet. Gjenvinning og reduksjon av denne sekvens er beskrevet i detalj av Wang, et. al. "Science" (1985) 228:149. As used herein, "tumor necrosis factor", TNF, refers to an amino acid sequence typified by that shown in Fig. 1, which is capable of selective cytotoxicity against tumor cells. The sequence in fig. 1 is derived from a human cDNA, but TNF encoded by other mammalian species may exhibit the required activity. Recovery and reduction of this sequence is described in detail by Wang, et. eel. Science (1985) 228:149.
En TNF aminosyresekvens må, for å passe til denne definisjon, være aktiv ved in vitro cytotoksisistetanalyse basert på den kontinuerlige murine konnektive vevcellelinje L-929 som beskrevet nedenfor. Det erkjennes at denne definisjon for TNF-aktivitet ikke er nøyaktig den samme som det som er gitt i beskrivelsen som fastslår denne term av Carswell, et al. "Proe Nati Acad Sei" (USA) ( 1975) 72:3666. Imidlertid gir denne aktivitet som bekreftet ved in vitro cytotoksisitets-analyse mot humantumorceller en tilstrekkelig forsikring på brukbarhet at kvalifiseringen som en tumornekrosefaktor for mennesker ved bruk av denne analyse, er rettferdiggjort; cytotoksisitet mot L-929 generaliserer mot humantumorer. Det er også forventet at det er en vesentlig overlapping mellom faktorer som er aktive i den spesifiserte cytotoksisitets-analyse og den in vivo-analyse som trekkes opp av Carswell. TNF-proteinet ifølge oppfinnelsen kan, avhengig av pH-verdiene i omgivelsene hvis det foreligger suspendert eller i oppløsning, eller avhengig av omgivelsene hvis det foreligger krystallisert eller utfelt i fast form, foreligge i form av farmasøytisk akseptable salter eller kan være i nøytral form. De frie aminogrupper i proteinene er selvfølgelig i stand, til å danne syreaddisjonssalter med f.eks. uorganiske syrer som salt-, fosfor- eller svovelsyre; eller med organiske syrer slik som f.eks. eddik-, glykol-, rav- eller mandelsyre. De frie karboksylgrupper er istand til å danne salter med baser inkludert uorganiske baser slik som natrium-, kalium- eller kalsiumhydroksyder, og slike organiske baser som piperidin, glukosamin, trimetylamin, kolin og kaffein. I tillegg kan proteinet modifiseres ved kombinasjon med andre biologiske stoffer slik som lipider og sakkarider, eller ved side-kjedemodifi sering slik som acetylering av aminogrupper, fosforlering av hydroksylsidekjeder eller oksydasjon av sulfhydrylgrupper. Alle disse modifikasjoner ligger innenfor definisjonens ramme så lenge TNF-aktiviteten bibeholdes. A TNF amino acid sequence must, to fit this definition, be active in an in vitro cytotoxicity assay based on the continuous murine connective tissue cell line L-929 as described below. It is recognized that this definition for TNF activity is not exactly the same as that given in the description defining this term by Carswell, et al. "Proe Nati Acad Sei" (USA) ( 1975) 72:3666. However, this activity as confirmed by the in vitro cytotoxicity assay against human tumor cells provides sufficient assurance of utility that the qualification as a human tumor necrosis factor using this assay is justified; cytotoxicity against L-929 generalizes against human tumors. It is also expected that there is a significant overlap between factors active in the specified cytotoxicity assay and the in vivo assay raised by Carswell. The TNF protein according to the invention can, depending on the pH values in the environment if it is suspended or in solution, or depending on the environment if it is crystallized or precipitated in solid form, exist in the form of pharmaceutically acceptable salts or can be in neutral form. The free amino groups in the proteins are of course able to form acid addition salts with e.g. inorganic acids such as hydrochloric, phosphoric or sulfuric acid; or with organic acids such as e.g. acetic, glycolic, succinic or mandelic acid. The free carboxyl groups are capable of forming salts with bases including inorganic bases such as sodium, potassium or calcium hydroxides, and such organic bases as piperidine, glucosamine, trimethylamine, choline and caffeine. In addition, the protein can be modified by combination with other biological substances such as lipids and saccharides, or by side chain modification such as acetylation of amino groups, phosphorylation of hydroxyl side chains or oxidation of sulfhydryl groups. All these modifications are within the framework of the definition as long as the TNF activity is maintained.
Til slutt skal det være klart at den primære aminosyresekvens som vises i figur 1 kun er illustrerende og at tilsvarende sekvens resulterer i proteiner som har i det vesentlige ekvivalent eller forbedret aktivitet sammenlignet med det som angis i figur 1. Disse modifikasjoner kan være tilsiktede, slik som f.eks. oppnådd ved sete-rettet mutagenese, eller kan være tilfeldige slik som de som oppnås ved muteringer i verter som er TNF-produsenter. Alle disse modifikasjoner er inkludert sålenge TNF-aktiviteten som angitt ovenfor, bibeholdes. Finally, it should be understood that the primary amino acid sequence shown in Figure 1 is illustrative only and that corresponding sequence results in proteins having substantially equivalent or improved activity compared to that shown in Figure 1. These modifications may be intentional, such as like for example. obtained by site-directed mutagenesis, or may be random such as those obtained by mutations in TNF-producing hosts. All of these modifications are included as long as the TNF activity as indicated above is maintained.
F.eks. er det funnet at et mutein som mangler de første fire aminosyrer ved N-terminus i sekvensen som vises i fig. 1 (Val-Arg-Ser-Ser ) har en spesifikk aktivitet som er flere ganger høyere enn TNF med den viste struktur. I henhold til dette, inkluderer definisjonen av TNF spesifikt denne av-kappede form. I tillegg har muteiner som mangler de N-terminale ti eller færre aminosyrer, tilsvarende forbedret aktivitet, og det synes som om mangler på opptil 10 amino syrer fra N-terminus ikke ødelegger men tvert imot forbedrer den biologiske aktivitet. E.g. it has been found that a mutein lacking the first four amino acids at the N-terminus in the sequence shown in fig. 1 (Val-Arg-Ser-Ser) has a specific activity that is several times higher than TNF with the structure shown. Accordingly, the definition of TNF specifically includes this truncated form. In addition, muteins lacking the N-terminal ten or fewer amino acids have correspondingly improved activity, and it seems that deficiencies of up to 10 amino acids from the N-terminus do not destroy but, on the contrary, improve the biological activity.
Derfor inkluderer definisjonen av TNF slik den her gjelder, spesifikt proteiner med en aminosyresekvens i det vesentlige ekvivalent med den som vises i figur 1, men som mangler 1-10 aminosyrer ved N-terminalsekvensen som vist i figuren. Therefore, the definition of TNF as applied herein specifically includes proteins having an amino acid sequence substantially equivalent to that shown in Figure 1, but lacking 1-10 amino acids at the N-terminal sequence as shown in the figure.
Også effektive er cysteinfattig muteiner av TNF i figur 1. Generelt resulterer nøytralaminosyreerstatninger av cysteinet i posisjon 69 eller 101 eller begge deler, i aktive TNF-proteiner. Nøytralaminosyreerstatning inkluderer ala, ser, val o.l., fortrinnsvis ser. Disse muteiner kan også modifiseres for å oppnå avstumpede former som kan bibeholde TNF-aktivitet og som kan ha forbedret spesifikk aktivitet in vitro og in vivo. Also effective are cysteine-poor muteins of TNF in Figure 1. In general, neutral amino acid substitutions of the cysteine in position 69 or 101 or both result in active TNF proteins. Neutral amino acid substitutions include ala, ser, val and the like, preferably ser. These muteins can also be modified to obtain truncated forms that can retain TNF activity and that can have improved specific activity in vitro and in vivo.
Som en bemerkning skal det opplyses at når spesielle peptider spesifiseres vil proteinet som har aminosyresekvensen metallene 1-157 i figur 1 benyttes som referanse og kalles, muligens tilfeldig valgt, mTNF (matur-TNF). Alle andre spesifikke proteiner med homologitet med NTF og som viser NTF biologisk aktivitet, vil kalles "muteiner" av NTF, og vil angis med henblikk på deres forskjeller fra mTNF ved bruk av nummereringen av restene som er vist i figuren. For eksemplel vil muteiner som har erstatninger for cystein i posisjon 69 angis ved bruk av den substituerte rest og posisjonsnummeret, f.eks. peptider med et serin i stedet for cystein i posisjon 69, kalles ser^g TNF. Muteiner som f.eks. mangler tre N-terminale aminosyrer sammenlignet med proteinet vist i figur 1, kalles V3TNF. Der begge de foregående endringer er gjennomført, kalles muteinet V3ser£,gTNF. As a note, it should be stated that when special peptides are specified, the protein that has the amino acid sequence metals 1-157 in Figure 1 will be used as a reference and called, possibly randomly chosen, mTNF (food TNF). All other specific proteins with homology to NTF and exhibiting NTF biological activity will be termed "mutens" of NTF, and will be designated in terms of their differences from mTNF using the residue numbering shown in the figure. For example, muteins that have substitutions for cysteine at position 69 will be indicated using the substituted residue and the position number, e.g. peptides with a serine instead of cysteine at position 69 are called ser^g TNF. Mutes such as e.g. lacking three N-terminal amino acids compared to the protein shown in Figure 1, is called V3TNF. Where both of the preceding changes are carried out, the mutein is called V3ser£,gTNF.
Spesielt foretrukne utførelsesformer av oppfinnelsens TNF inkluderer T2ser6gTNF, V2ser10iTNF, V2ser6gser101<T>NF, og de tilsvarende V3-, V4-, V5-, V6-, V7-, V8-, og V10-cystein-manglende muteiner. Spesielt foretrukket er VSser^gTNF, Particularly preferred embodiments of the TNF of the invention include T2ser6gTNF, V2ser10iTNF, V2ser6gser101<T>NF, and the corresponding V3, V4, V5, V6, V7, V8, and V10 cysteine-lacking muteins. Particularly preferred are VSser^gTNF,
V8ser101TNF, VSser^gser ±0l og v^se<r>^<g>TNF, T4ser10lTNF°S v"4ser5gserioiTNF • V8ser101TNF, VSser^gser ±0l and v^se<r>^<g>TNF, T4ser10lTNF°S v"4ser5gserioiTNF •
Ikke alle muteiner av mTNF fremstilles rekombinant eller tilsiktet. Således har nativ HL-60 cellesekretert TNF mindre forskjeller fra mTNF i de kjente posisjoner av primærstruk-turen, selv om begge proteiner viser TNF-aktivitet. Spesifikt har den deduserte sekvens fra fiur 1 et ytterligere par serinrester efter serinet i posisjon 1 sammenlignet med HL-60 avledet TNF før homologien opptas som vist mellom posisjonene 4-12 i det HL-60 avledede proteinet og posisjonene 6 til 14 i den deduserte sekvens. I tillegg er posisjonene 13 of 14 i det HL-60-avledede protein Val-Ser; de tilsvarende posisjoner 15 og 16 til den deduserte sekvens er His-Val. Not all muteins of mTNF are produced recombinantly or intentionally. Thus, native HL-60 cell-secreted TNF has minor differences from mTNF in the known positions of the primary structure, even though both proteins show TNF activity. Specifically, the deduced sequence from figure 1 has an additional pair of serine residues after the serine at position 1 compared to HL-60 derived TNF before the homology is taken up as shown between positions 4-12 of the HL-60 derived protein and positions 6 to 14 of the deduced sequence . In addition, positions 13 of 14 in the HL-60-derived protein are Val-Ser; the corresponding positions 15 and 16 of the deduced sequence are His-Val.
Fremsti11ingsmåteMethod of progress
De TNF som kan benyttes ved fremgangsmåten ifølge oppfinnelsen kan mest hensiktsmessig fremstilles ved å benytte rekombinante metoder. Detaljerte beskrivelser av måter for fremstilling av rekombinant TNF er angitt i Wang, et al. Science (supra). I dette henseende er forskjellige DNA-sekvenser som koder TNF deponert i "American Type Culture Collection", Rockville, MD". Disse DNA-ekvenser inkluderer de som er inneholdt i pE4 som rommer human cDNA-innskuddet ATCC #39894); pAW711 som er en eksprimeringsvektor egnet for prokarioter som koder TNF i figur 1 (ATCC #39918) og pAW736, en ekspr imer ingsvektor som koder V4-muteinet av mTNF (ATCC #53092). Vektorer egnet for eksprimering av andre TNF muteiner (V10, V9, V6, W og V8) er deponert som ATCC nr. 53161, 53162, 53163, 53164 henholdsvis 53165. The TNFs that can be used in the method according to the invention can most conveniently be produced by using recombinant methods. Detailed descriptions of methods for the preparation of recombinant TNF are provided in Wang, et al. Science (supra). In this regard, various DNA sequences encoding TNF are deposited in the "American Type Culture Collection", Rockville, MD". These DNA sequences include those contained in pE4 which harbors the human cDNA insert ATCC #39894); pAW711 which is an expression vector suitable for prokaryotes encoding TNF in Figure 1 (ATCC #39918) and pAW736, an expression vector encoding the V4 mutein of mTNF (ATCC #53092).Vectors suitable for expression of other TNF muteins (V10, V9, V6 , W and V8) have been deposited as ATCC No. 53161, 53162, 53163, 53164 and 53165 respectively.
I tilegg til rekombinant reduserte stoffer kan TNF ekstrah-eres fra naturlige kilder slik som human- eller annet pattedyrvev, eller fra human- eller andre pattedyravledede cellelinjer. Kilden for proteinet er selvfølgelig uvesentlig for oppfinnelsens gjennomføring, bortsett fra at den kan ha innflytelse på spesifikke doseringsnivåer og administrerings-ruter. In addition to recombinantly reduced substances, TNF can be extracted from natural sources such as human or other mammalian tissue, or from human or other mammalian derived cell lines. The source of the protein is of course immaterial to the practice of the invention, except that it may have an influence on specific dosage levels and routes of administration.
Formulering og administreringsmåteFormulation and method of administration
For å bevirke den ønskede lipidmobilisering som oppnås ved administrering av TNF, kan de aktive bestanddeler formuleres til et antall aksepterbare midler, velkjente i denne teknikk. Karakteristisk blir TNF administrert ved injeksjon, enten intraperitonialt eller intravenøst. Ved egnet formulering kan det imidlertid være mulig å oppnå preparater som kan administreres topisk eller oralt, eller som kan være istand til transmisjon gjennom mykøse membraner. To effect the desired lipid mobilization achieved by administration of TNF, the active ingredients can be formulated into a number of acceptable agents well known in the art. Typically, TNF is administered by injection, either intraperitoneally or intravenously. With a suitable formulation, however, it may be possible to obtain preparations which can be administered topically or orally, or which may be capable of transmission through mucous membranes.
Avhengig av arten av preparatet og administreringsmetoden kan preparatet foreligge i flytende eller fast form. For faste preparater inklukderer konvensjonelle bærere f.eks. farma-søytiske kvaliteter av manitol, laktose., stivelse, cellulose, magnesiumkarbonat o.l. TNF kan formuleres som et suppositorium f.eks. ved bruk av polyalkylenglykoler som bærer. Flytende preparater kan fremstilles ved oppløsning eller dispergering av TNF og eventuelle tilsetningsstoffer i en bærer, slik som f.eks. vann, saltoppløsning, vandig dekstrose osv. Hvis ønskelig kan preparatet også inneholde mindre mengder ikke-toksiske hjelpestoffer slik som fukte-eller emulger ingsmidler, pH-buf f ermidler o.l. Aktuelle metoder for fremstilling av doseringsformer er kjente eller vil være åpenbare for fagmannen. Preparatene vil i ethvert _tilfelle inneholde en mengde TNF effektiv til bevirkning av den ønskede lipidmobilisering. Depending on the nature of the preparation and the method of administration, the preparation may be available in liquid or solid form. For solid preparations, conventional carriers include e.g. pharmaceutical qualities of mannitol, lactose, starch, cellulose, magnesium carbonate etc. TNF can be formulated as a suppository e.g. using polyalkylene glycols as a carrier. Liquid preparations can be prepared by dissolving or dispersing TNF and any additives in a carrier, such as e.g. water, saline solution, aqueous dextrose, etc. If desired, the preparation can also contain smaller amounts of non-toxic excipients such as wetting or emulsifying agents, pH buffers, etc. Current methods for producing dosage forms are known or will be obvious to the person skilled in the art. The preparations will in any case contain an amount of TNF effective for effecting the desired lipid mobilization.
En foretrukket administreringsmetode av dette protein er ved injeksjon, oftest intraperitoneal eller intravenøs injeksjon. Stoffet som skal injiseres kan fremstilles enten som en flytende oppløsning eller en suspensjon eller i en form som er egnet for rekonstituering. A preferred method of administration of this protein is by injection, usually intraperitoneal or intravenous injection. The substance to be injected can be prepared either as a liquid solution or a suspension or in a form suitable for reconstitution.
Doseringen og administreringsmetoden vil selvfølgelig avhenge av nivå av den lipidmobilisering som ønskes, objektets natur, og legens bedømmelse. Generelt ligger imidlertid effektive doser innen området 0,2-2 jjg TNF pr. kg. kroppsvekt pr. dag så lenge administrering kreves. Dette området representerer selvfølgelig et bredt anslag da de ovenfor angitte faktorer er av stor betydning for bestemmelse av optimale doseringsnivåer. The dosage and administration method will of course depend on the level of lipid mobilization desired, the nature of the object, and the physician's judgment. In general, however, effective doses are within the range of 0.2-2 jjg TNF per kg. body weight per day as long as administration is required. This range of course represents a broad estimate as the factors stated above are of great importance for determining optimal dosage levels.
I det aspekt ved oppfinnelsen som angår nøytralisering av TNF i kakeksiske pasienter benytter foretrukne utførelsesformer for nøytralisering av aktiviteten til TNF polyklone eller monoklone antistoffer. Disse nøytraliserende antistoffer benyttes fortrinnsvis som en tilleggsbehandling sammen med andre cytotoksiske medikamenter hos kreftpasienter. F.eks. kan metotrexat administreres til en pasient for å drepe tumoren, fulgt av antistoffpreparatet for å nøytralisere TNF for å forhindre vekttap. In the aspect of the invention which concerns the neutralization of TNF in cachexic patients, preferred embodiments for neutralizing the activity of TNF use polyclonal or monoclonal antibodies. These neutralizing antibodies are preferably used as an additional treatment together with other cytotoxic drugs in cancer patients. E.g. methotrexate can be administered to a patient to kill the tumor, followed by the antibody preparation to neutralize TNF to prevent weight loss.
Polyklone antistoffer fremstilles mot TNF ved å benytte konvensjonelle prosedyrer ved å injisere renset TNF til en egnet vert, slik som kaniner eller mus, og så å høste høytitersera. Fremstilling av polyklone preparater følger generelt de nu kjente prosedyrer i henhold til Kohler og Millstein. Polyclonal antibodies are prepared against TNF using conventional procedures by injecting purified TNF into a suitable host, such as rabbits or mice, and then harvesting high-titer sera. Production of polyclonal preparations generally follows the now known procedures according to Kohler and Millstein.
EksempelExample
De følgende eksempler er ment å illustrere oppfinnelsen uten å begrense den. The following examples are intended to illustrate the invention without limiting it.
Preparat A: Fremstilling av kakektinPreparation A: Preparation of cachectin
Kakektin ble benyttet som en kontroll i de følgende eksempler. Forbindelsen ble fremstilt fra de kondisjonerte media av endotoksinstimulerte makrofager som beskrevet av Kawaskami, M. , et al. Proe Nati Acad Sei (USA) (1982) 79:912; Pekala, P.H., et al. (ibid 1983) 80:2743. Cachectin was used as a control in the following examples. The compound was prepared from the conditioned media of endotoxin-stimulated macrophages as described by Kawaskami, M., et al. Proe Nati Acad Sei (USA) (1982) 79:912; Pekala, P.H., et al. (ibid. 1983) 80:2743.
Eksempel 1; Virkningen av TNF på adipose- induserbare mRNA-nivåer Example 1; The effect of TNF on adipose-inducible mRNA levels
En in vitro-model for studium av abnormal metabolisme til kakeksi er utviklet ved bruk av en stabil adipogen cellelinje, TA1 , som beskrevet av Chapman, A.B. J. Biol Chem An in vitro model for the study of abnormal metabolism leading to cachexia has been developed using a stable adipogenic cell line, TA1, as described by Chapman, A.B. J. Biol Chem
(1984) 259:15548-15555. Disse celler utvikler en adipocytisk morfologi omtrent 3 dager efter komfluens i vevkulturmono-sjikt, og viser, parallelt med denne morfologi, eksprimering av forskjellige gener. Visse proteiner og enzymer er tilstede kun i differensierte adipocyter og er ikke tilstede eller tilstede kun i upåvisbare nivåer i de ikke-differensierte forløpere. Disse proteiner ansees å være produkter av "adiposeinduserbare gener" og deres mellomliggende mRNAer. (1984) 259:15548-15555. These cells develop an adipocytic morphology approximately 3 days after confluence in tissue culture monolayers, and show, parallel to this morphology, expression of various genes. Certain proteins and enzymes are present only in differentiated adipocytes and are not present or present only at undetectable levels in the undifferentiated precursors. These proteins are thought to be the products of "adipose inducible genes" and their intermediate mRNAs.
Gener for hvilke ekprimering først er påviselig efter differensiering er identifisert. De er kalt klonene 1, 10, 28, 47 og GPD (Chapman, A. B. et al. supra). Deres eksprimering skyldes åpenbart transkripsjonen aktivering. Genes for which expression is only detectable after differentiation has been identified. They are named clones 1, 10, 28, 47 and GPD (Chapman, A. B. et al. supra). Their expression is obviously due to transcriptional activation.
Derfor er et mål av betydningen av forbindleser på adipocyter deres virkning på nivåene av adiposeinduserbar nRNA. Therefore, a measure of the importance of compounds on adipocytes is their effect on the levels of adipose-inducible nRNA.
I denne analyse ble den ovenfor angitte stabile adipogene cellelinje TA1 dyrket fra prekonfluens til 24 dager efter konfluens. TNF, fremstilt ved dyrking av pAW711 transformert E. coli og indusering av eksprimeringen av TNF som beskrevet i US-SN 760.661, supra, ble tilsatt til cellekulturene fra prekonfluens til 6 dager efter. In this assay, the above stable adipogenic cell line TA1 was cultured from preconfluence to 24 days post confluence. TNF, prepared by culturing pAW711 transformed E. coli and inducing the expression of TNF as described in US-SN 760,661, supra, was added to the cell cultures from preconfluence until 6 days post.
I større detalj, ble TAl-celler, avledet fra 5-azacytidin-behandling av 10 T1/2C18 celler (Resnikof. C, et al. Cancer Res (1973) 333231-3231-3238; Taylor, S.M., et al. Cell (1979) 17:771-779 )dyrket i Eagel's basalmedium supplert med 10$ varmeinaktiverte fetal kalveserum. 10~^- M deksametason var tilstede i media for de første 3 dager efter konfluens, og 5 jjg/ml storfeinsulin de første 6 dager efter konfluens. Rekombinant TNF (10-30 ng/ml) ble først tilsatt til preadipocytkulturer 2 dager før konfluens. (Tilsetning av denne mengde inhiberer 90 lipoproteinlipaseaktivitet i dyrkede adipocyter.) Cellene ble matet med resupplementering av TNA på dag 0 (konfluens) og dag 3. Cellene ble høstet dag 6. In greater detail, TAl cells, derived from 5-azacytidine treatment of 10 T1/2C18 cells (Resnikof. C, et al. Cancer Res (1973) 333231-3231-3238; Taylor, S.M., et al. Cell ( 1979) 17:771-779 ) grown in Eagel's basal medium supplemented with 10% heat-inactivated fetal calf serum. 10~^- M dexamethasone was present in the media for the first 3 days after confluence, and 5 jjg/ml bovine insulin for the first 6 days after confluence. Recombinant TNF (10-30 ng/ml) was first added to preadipocyte cultures 2 days before confluence. (Addition of this amount inhibits 90 lipoprotein lipase activity in cultured adipocytes.) Cells were fed with resupplementation of TNA on day 0 (confluence) and day 3. Cells were harvested on day 6.
Total RNA ble isolert i henhold til Chirgwin, J.M., et al, "Biochemistry (1979) 18:5294-5299, og påført på nitrocellulose i en flekk-blotapparatur (BRL). Nick translaterte cDNA-kloner av adiposeinduserbare gener (kalt klonene 1, 10 og 47) og glyserolfosfatdehydrogenese (GDH) ble benyttet for å probe filterne under hybridiseringsbetingelser som beskrevet av Chapman, A.B., et al, J. Biol Chem (1948) (supra). Filtrene ble vasket og eksponert til XAR 5 film ved -70° C med en intensiverende skjerm. Total RNA was isolated according to Chirgwin, J.M., et al, "Biochemistry (1979) 18:5294-5299, and blotted onto nitrocellulose in a blot blot apparatus (BRL). Nick translated cDNA clones of adipose-inducible genes (called the clones 1 , 10 and 47) and glycerol phosphate dehydrogenase (GDH) were used to probe the filters under hybridization conditions as described by Chapman, A.B., et al, J. Biol Chem (1948) (supra). The filters were washed and exposed to XAR 5 film at - 70° C with an intensifying screen.
Resultatene er vist i venstre spalte i figur 2, merket med "stabil tilstand". Det er klart fra disse resultater at TNF-behandlingen forhindrer akkumulering av adiposeinduserbar mRNA. Disse resultater er sammenlignbare med de som ble oppnådd når 10 pl/ml kakektin ble benyttet for TNF. The results are shown in the left column of Figure 2, labeled "steady state". It is clear from these results that the TNF treatment prevents the accumulation of adipose-inducible mRNA. These results are comparable to those obtained when 10 µl/ml cachectin was used for TNF.
Lipidakkumulering ble også fullstendig inhibert av kakektin eller TNF, og TAl-cellekulturer behandlet med disse forbind-elser er opprettholdt i helt opptil 23 dager uten opptreden av nøytralt lipid, påvist ved olje rød 0 farving. Ved fjerning av kakektin eller TNF fra mediet, vendte dog adipocytmorfologien tilbake slik det skjer ved eksprimering av adiposeinduserbare gener. Det ble også vist at TNF- eller kakektinbehandling av preadipocytkulturer ikke påvirket celleveksten eller levedyktigheten, bestemt ved celletelling og<3>H-tymidininnarbeiding, såvel som ved trypanblå eksklusjon og klonalvekstanalysé i henhold til Ham. R.G., et al. "Cell Culture Methods for Molecular and Cellular Biology" Barnes, New York (1984) 1:3-21. Lipid accumulation was also completely inhibited by cachectin or TNF, and TAl cell cultures treated with these compounds have been maintained for up to 23 days without the appearance of neutral lipid, as detected by oil red 0 staining. However, when cachectin or TNF was removed from the medium, the adipocyte morphology reverted, as occurs when adipose-inducible genes are expressed. It was also shown that TNF or cachectin treatment of preadipocyte cultures did not affect cell growth or viability, as determined by cell counting and <3>H-thymidine incorporation, as well as by trypan blue exclusion and clonal growth analysis according to Ham. R.G., et al. "Cell Culture Methods for Molecular and Cellular Biology" Barnes, New York (1984) 1:3-21.
I henhold til dette viser resultatene at både kakektin eller TNF undertrykker den totale mengde adiposeinduserbare mRNA som er tilstede i adipocytene eller preadipocytene, uten negativt å påvirke resten av cellens metabolisme. Accordingly, the results show that both cachectin and TNF suppress the total amount of adipose-inducible mRNA present in the adipocytes or preadipocytes, without negatively affecting the rest of the cell's metabolism.
Eksempel 2: Inhibering av transkriberingExample 2: Inhibition of transcription
At mRNA akkumuleringsinhibering transkripsjonelt reguleres ble vist ved nukleær transkripsjonsanalyser gjennomført som beskrevet av Vannice, et al, Proe Nati Acad Sei (USA) (1984) 81:4241-4245 og ved Israel, J. , et al. "J Biol Chem" (1984) 259:5400-5402, modifisert i henhold til Knight, et al (submitted) for adiposeceller. That mRNA accumulation inhibition is transcriptionally regulated was shown by nuclear transcription analyzes carried out as described by Vannice, et al, Proe Nati Acad Sei (USA) (1984) 81:4241-4245 and by Israel, J., et al. "J Biol Chem" (1984) 259:5400-5402, modified according to Knight, et al (submitted) for adipose cells.
På hvert datapunkt ble celler dyrket og behandlet i eksempel 1 avkjølt til 4°C og mediet ble aspirert og vasket med fosfatbuffret saltoppløsning. En ml hypotonisk buffer (20 nM Tris(HCl. pH, 4 mM MgCl2, 6 mM CaCl2, 0,5 mM ditiotreitol) ble tilsatt til platene. Efter 5 min. ble 1 ml lyseringsbuf-fer 0,6 M sukrose, 0,256 NP 40, og 0,5 mM ditiotreitol) tilsatt og cellene ble skrapet fra vevkulturplåtene. Efter Dounce homogenisering ble kjerner pellitisert ved 500 g, vasket en gang i resuspensj onsbuffer, repelletisert og så resupendert i buffer inneholdende 0,4 ml mM hver av ATP, CPT, GTP, 10% glyserol og lOjjg/ml BSA. Kjernene ble inkubert med en a-32P-UTP (600 Ci/mmol) ved en konsentrasjon på 2 pCi/ml i 40 minutter ved 45° under mild omrøring. At each data point, cells cultured and treated in Example 1 were cooled to 4°C and the medium was aspirated and washed with phosphate buffered saline. One ml of hypotonic buffer (20 nM Tris(HCl. pH, 4 mM MgCl2, 6 mM CaCl2, 0.5 mM dithiothreitol)) was added to the plates. After 5 min, 1 ml of lysis buffer 0.6 M sucrose, 0.256 NP 40, and 0.5 mM dithiothreitol) were added and the cells were scraped from the tissue culture plates. After Dounce homogenization, nuclei were pelletized at 500 g, washed once in resuspension buffer, repelletized and then resuspended in buffer containing 0.4 ml mM each of ATP, CPT, GTP, 10% glycerol and 10 µg/ml BSA. The nuclei were incubated with an α-32 P-UTP (600 Ci/mmol) at a concentration of 2 pCi/ml for 40 minutes at 45° with gentle agitation.
RNA ble høstet fra kjernene som beskrevet av Smith, et al. Cell (1978) 15:615-626 modifisert av Knight, et al (supra) og hybridisert til lineæriserte cDNAer som var påført på nitrocellulosefiltre og behandlet i 2 timer ved 80°C i en vakuumovn. Filterprehybridisering- og hybridiseringsbeting-elsene var i henhold til Friedman, R.L., et al, Cell (1984) 38:745-755, og hybridiseringene ble gjennomført i 3 dager ved 42°C ved ca. 15 x 10 26 cpm pr. reaksjonsblanding påført i 150 Ml volum til dotter av lineariserte enkeltstrengadipose cDNAer for klon 28, og cDNAet som koder P-aktin og det ikke- tilsvarende nøytrale plasmid pEMBL ble benyttet som sammen-ligninger, p-aktin cDNA-klonen var en gave fra P. Gunning og L. Kedes. RNA was harvested from the nuclei as described by Smith, et al. Cell (1978) 15:615-626 modified by Knight, et al (supra) and hybridized to linearized cDNAs which were applied to nitrocellulose filters and treated for 2 hours at 80°C in a vacuum oven. The filter prehybridization and hybridization conditions were according to Friedman, R.L., et al, Cell (1984) 38:745-755, and the hybridizations were carried out for 3 days at 42°C at approx. 15 x 10 26 cpm per reaction mixture applied in 150 ml volume to the daughter of linearized single-stranded adipose cDNAs for clone 28, and the cDNA encoding β-actin and the non-corresponding neutral plasmid pEMBL were used as comparisons, the β-actin cDNA clone was a gift from P. Gunning and L. Kedes.
Resultatene er vist i høyre kolonne, i figur 2. De fire datapunkter er prekonfluens (PC), 4 og 24 timer efter TNF-tilsetning samt "kotroll", som representerer TA1 ubehandlet med TNF. Kolonnene A og E er respektive p<->aktin- og pEMBL-sammenligningsdotprobene; kolonne 28 representerer klon 28 dotproben. Disse resultater viser at transkripsjonssystemet inneholdt i kjernen undertrykkes med henblikk på kodingssekvensene for klon 28, som er karakteristisk for adiposeceller, men upåvirket med henblikk på kodingssekvensene for aktin. Tilsvarende resultater ble oppnådd da TAl-cellene ble behandlet med 10 jjI/ml kakektin. Derfor opererer TNF på samme måte som kakektin direkte på genomet for å undertrykke transkripsjon av adiposerelaterte sekvenser, men avbryter ikke andre genefunksjoner. The results are shown in the right column, in Figure 2. The four data points are preconfluence (PC), 4 and 24 hours after TNF addition and "control", which represents TA1 untreated with TNF. Columns A and E are the p<->actin and pEMBL comparison dot probes, respectively; column 28 represents the clone 28 dot probe. These results show that the transcription system contained in the nucleus is repressed with respect to the coding sequences of clone 28, which is characteristic of adipose cells, but unaffected with respect to the coding sequences of actin. Similar results were obtained when the TAl cells were treated with 10 µl/ml cachectin. Therefore, similarly to cachectin, TNF operates directly on the genome to repress transcription of adipose-related sequences, but does not interrupt other gene functions.
Eksempel 3: Virkning av TNF på mature adiposecellerExample 3: Effect of TNF on mature adipose cells
Da adipocyter hos voksne mennesker undergår liten eller Ingen proliferering, var det ønskelig å studere virkningen av TNF på mature adiposeceller. En prøve på 10-30 ng/ml TNF ble tilsatt til dag 6 adipocyt TAl-kulturer differensiert som beskrevet i eksempel 1. Total RNA ble isolert fra cellene på forksjellige tidspunkter efter TNF-eksponeringen, og påført på nitrocellulose med en dot-blot-apparatur. Filtrene ble probet med cDNAer med henblikk på GPD, klon 47 og klon 1, vasket, autoradiografert og avsøkt ved bruk av et Hoeffner GS300 densitometer festet til en rapporterende integrator. De oppnådde punkter ble normalisert for differanser i mengden påført RNA ved bruk av en cDNA-probe som totaliserte cellu-lært RNA. Figur 3 viser at maksimaleksprimering av GPD, eller klonene 1 eller 47, alvorlig inhiberes efter de første 6 timer i nærvær av TNF. Resultatene er tilsvarende for kakektin. As adipocytes in adult humans undergo little or no proliferation, it was desirable to study the effect of TNF on mature adipose cells. A sample of 10-30 ng/ml TNF was added to day 6 adipocyte TAl cultures differentiated as described in Example 1. Total RNA was isolated from the cells at various times after the TNF exposure, and applied to nitrocellulose with a dot-blot- apparatus. The filters were probed with cDNAs for GPD, clone 47 and clone 1, washed, autoradiographed and probed using a Hoeffner GS300 densitometer attached to a reporter integrator. The points obtained were normalized for differences in the amount of applied RNA using a cDNA probe that totaled cellular RNA. Figure 3 shows that maximal expression of GPD, or clones 1 or 47, is severely inhibited after the first 6 hours in the presence of TNF. The results are similar for cachectin.
Nothern-blots, probet med klon 47 på forskjellige tidspunkter efter TNF behandlingen, viste at RNA hybridiserbart til denne klon forsvant hurtig efter TNF-administrering. For disse analyser ble 12 pg tilsammen av RNA bragt til en sluttkonsentrasjon på 2,2 M formaldehyd, 30% formamid, 10 mM NaH2P0I4, pH 7 og oppvarmet i 15 min. ved 56°C. Prøver ble elektrofor-sert i en 1% agaroseformaldehydgel med en sluttkonsentrasjon på 2,2 M formaldehyd, 20 mM MOPS, pH 7,0, 5,0 mM natriumace-tat og 1 mM EDTA. Gelene ble vasket i destillert vann i 3 minutter fulgt av to 30 minutters vaskinger i 10 mM NaP04, pH 7,4, og 1 mM EDTA, før overføring til cellulose, og hybridi-sering med lineærisert cDNA fra klon 47. Også her viste kakektinbehandlede celler tilsvarende resultater. Northern blots, probed with clone 47 at various times after TNF treatment, showed that RNA hybridizable to this clone disappeared rapidly after TNF administration. For these analyses, a total of 12 pg of RNA was brought to a final concentration of 2.2 M formaldehyde, 30% formamide, 10 mM NaH 2 POI 4 , pH 7 and heated for 15 min. at 56°C. Samples were electrophoresed in a 1% agarose formaldehyde gel with a final concentration of 2.2 M formaldehyde, 20 mM MOPS, pH 7.0, 5.0 mM sodium acetate and 1 mM EDTA. The gels were washed in distilled water for 3 minutes followed by two 30-minute washes in 10 mM NaPO 4 , pH 7.4, and 1 mM EDTA, before transfer to cellulose, and hybridization with linearized cDNA from clone 47. Here, too, cachectin-treated cells corresponding results.
Det ble også vist at efter 4-6 dager TNF-eksponering mistet de fleste celler sitt nøytrale lipid. Efter at 10-30 ng/ml TNF var tilsatt, var 70-80 av cellene fylt med store lipid-dråper; 6 dager senere hadde mindre enn 10% celler identifi-serbare triglycerider på olje rød 0 flekker. Disse resultater er vist i figr 4A og 4B, som representerer ubehandlede og TNF-behandlede celler. Olje rød 0 flekken er så og si fullstendig forsvunnet fra cellene i figur 4B. It was also shown that after 4-6 days of TNF exposure, most cells lost their neutral lipid. After 10-30 ng/ml TNF was added, 70-80 of the cells were filled with large lipid droplets; 6 days later, less than 10% of cells had identifiable triglycerides on oil red 0 staining. These results are shown in Figures 4A and 4B, which represent untreated and TNF-treated cells. The oil red 0 stain has almost completely disappeared from the cells in Figure 4B.
Sammenligning av disse resultater med de som er vist i figur 3, viser at endringer i adiposespesifiserte RNAer på grunn av TNF-behandling skjer hurtigere enn lipidmobilisering. Efter 6-24 timer efter tilsetning av TNF til mature TAl-lymfocyter observeres mer enn en 90%-ig reduksjon i disse RNAer; flere dager er nødvendig for at dette skal reflekteres i en reduksjon i lipidinnholdet. Comparison of these results with those shown in Figure 3 shows that changes in adipose-specific RNAs due to TNF treatment occur faster than lipid mobilization. After 6-24 hours after the addition of TNF to mature TAl lymphocytes, more than a 90% reduction in these RNAs is observed; several days are required for this to be reflected in a reduction in the lipid content.
Addendum: Cytotoksisk analyseprosedyreAddendum: Cytotoxic assay procedure
Definisjonen av et protein som TNF avhenger av dens aktivitet i L-929-analysen. Denne analyse skal derfor beskrives her. L-929-analysesystemet er en forbedret hensiktsmessig in vitro-analyse som tillater hurtig måling av TNF-aktiviteten. Korrelasjonsgraden med in vivo tumor nekroseanalysen til Carswell er idag ukjent; i og med at den imidlertid benytter murintumorceller spesifikt, antas korrelasjonen å være høy. Det protein som er kalt lymfotoksin i EPO-publ ikas j on 0100641, gir også aktiviteten i denne analyse. Analysen tilsvarer i konsept den som er beskrevet i US-PS 4.457.916 som benytter murin L-M-celler og metylen blå farving. Imidlertid har L-929 analysen vist seg å korrelere (for HL-60-avledet TNF) med humantumorcellelinjecytotoksisitet. The definition of a protein as TNF depends on its activity in the L-929 assay. This analysis will therefore be described here. The L-929 assay system is an improved convenient in vitro assay that allows rapid measurement of TNF activity. The degree of correlation with Carswell's in vivo tumor necrosis analysis is currently unknown; however, since it uses murine tumor cells specifically, the correlation is assumed to be high. The protein called lymphotoxin in EPO publication 0100641 also provides the activity in this analysis. The assay corresponds in concept to that described in US-PS 4,457,916 which uses murine L-M cells and methylene blue staining. However, the L-929 assay has been shown to correlate (for HL-60-derived TNF) with human tumor cell line cytotoxicity.
I det her beskrevne L-929 analysesystem, blir L-929 celler preparert overnatt som monosjikt i mikrotiterplater. Prøvene fortynnes to ganger på tvers av platen, UV-bestråles og tilsettes på de preparerte monosjikt. Kulturmediene i brønnene bringes så til 1 pg/ml aktinomycin D. Platene tillates inkubering i 18 timer ved 37° C og bedømmes så visuelt under mikroskop. Hver brønn gis en 25, 50 75 eller 100% bedømmelse som tilkjennegir graden av celledød i brønnen. En enhet TNF-aktivitet defineres som det resiproke av fortynningen der 50% avlivning skjer. In the L-929 analysis system described here, L-929 cells are prepared overnight as a monolayer in microtiter plates. The samples are diluted twice across the plate, UV-irradiated and added to the prepared monolayers. The culture media in the wells is then brought to 1 pg/ml actinomycin D. The plates are allowed to incubate for 18 hours at 37° C and then assessed visually under a microscope. Each well is given a 25, 50, 75 or 100% assessment which indicates the degree of cell death in the well. One unit of TNF activity is defined as the reciprocal of the dilution at which 50% killing occurs.
I tillegg ble en mer sensitiv versjon av denne analyse utviklet og som overvåker frigjøringen av<3>^S merkede peptider fra på forhånd merkede celler, når disse var behandlet med prøven og aktinomycin D. Denne versjon av analysen kan benyttes for å kvantifisere potensen, f.eks. for å bedømme den relative potens av oocyttranslatert materiale. Kort sagt blir aktivt voksende L-929-kulturer merket med<35>S metionin (200 pCi/ml) i 3 timer i metioninfrie media supplert med 2% dialysert fetal kalveserum. Cellene blir så vasket og bragt i 96 brønners plater, inkubert overnatt og behandlet den neste dag med to gangers fortynninger av prøver over lg/ml aktinomycin D. Kulturene ble .så inkubert ved 37°C i 18 timer. 100 pl supernatantmengder fra hver brønn ble så overført til en annen 96 brønners plate, syre (TCA) presipi-tert og høstet på glassfiberfiltre. Filtrene ble vasket med 95% etanol , tørket og tellet. En NP4ødetergenskontroli inkluderes I hver analyse for å måle maksimal frigivning av radioaktivitet fra cellene. Prosentualt<35>S-frigjøring blir så beregnet ved forholdet mellom differansen i telling mellom behandlede celler og ubehandlede kontroller, og differansen mellom NP4gbehandlede celler og ubehandlede kontroller, dvs. ved forholdet: In addition, a more sensitive version of this assay was developed which monitors the release of <3>^S labeled peptides from previously labeled cells, when these were treated with the sample and actinomycin D. This version of the assay can be used to quantify the potency, e.g. to assess the relative potency of oocyte-translated material. Briefly, actively growing L-929 cultures are labeled with<35>S methionine (200 pCi/ml) for 3 hours in methionine-free media supplemented with 2% dialyzed fetal calf serum. The cells are then washed and placed in 96-well plates, incubated overnight and treated the next day with two-fold dilutions of samples above 1g/ml actinomycin D. The cultures are then incubated at 37°C for 18 hours. 100 µl supernatant amounts from each well were then transferred to another 96 well plate, acid (TCA) precipitated and harvested on glass fiber filters. The filters were washed with 95% ethanol, dried and counted. An NP4 destruction agent control is included in each assay to measure the maximum release of radioactivity from the cells. Percent<35>S release is then calculated by the ratio between the difference in counts between treated cells and untreated controls, and the difference between NP4g-treated cells and untreated controls, i.e. by the ratio:
Høyere TNF-potens resulterer i høyere verdier av dette forhold. Higher TNF potency results in higher values of this ratio.
OppsummeringSummary
De foregående eksempler indikerer at TNF kan undertrykke metabolismen av adiposeceller. Denne aktivitet er også demonstrerbar for kakektin: se F.M. Torti, et al (supra). I henhold til dette forhindrer TNF eksprimering av gener som er ansvarlige for å produsere enzymer som er viktige med henblikk på lagring av fett og som kan benyttes for å kontrollere vekt. The preceding examples indicate that TNF can suppress the metabolism of adipose cells. This activity is also demonstrable for cachectin: see F.M. Torti, et al (supra). Accordingly, TNF prevents the expression of genes responsible for producing enzymes that are important for the storage of fat and that can be used to control weight.
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PCT/US1986/000952 WO1986006280A1 (en) | 1985-05-02 | 1986-05-01 | Use of tumor necrosis factor as a weight regulator |
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