DK149094B

DK149094B - PROCEDURE FOR PREPARING THYMOSINE ALFA1 OR PHYSIOLOGICALLY ACCEPTABLE SALTS THEREOF WITH ACIDS OR BASES FROM THYMOSIN FRACTION 5

Info

Publication number: DK149094B
Application number: DK478277AA
Authority: DK
Inventors: Allan L Goldstein; Teresa L K Low; Chun-Yen Lai; Su-Sun Wang
Original assignee: Hoffmann La Roche; Univ Texas
Priority date: 1976-10-28
Filing date: 1977-10-27
Publication date: 1986-01-20
Also published as: NZ185519A; YU40314B; SE7712071L; FR2369248A1; FI56317C; MC1167A1; PT67204B; AR214903A1; IL53218A; NL188699B; NO143346B; NO773681L; ATA765877A; JPS5411220A; GB1590457A; PT67204A; CH633258A5; ES463588A1; FI56317B; DK478277A

Description

149094149094

Den foreliggende opfindelse angår en fremgangsmåde til fremstilling af det hidtil ukendte thymosin eller fysiologisk acceptable salte deraf med syrer eller baser ud fra thymosin fraktion 5.The present invention relates to a process for preparing the novel thymosin or physiologically acceptable salts thereof with acids or bases from thymosin fraction 5.

Fremgangsmåden ifølge opfindelsen er ejendommelig ved det i kravets 5 kendetegnende del angivne.The process according to the invention is characterized by the characterizing part of claim 5.

Det er i vore dage en alment anerkendt kendsgerning, at thymus har én central betydning for udviklingen og ældningen af immunsystemet hos mennesker og dyr. Selv om der endnu kun eksisterer ringe viden om den molekylære mekanisme, hvorved thymus styrer T-cellernes 10 udvikling, tyder det på, at en væsentlig del af denne mekanisme styres hormonalt. Thymus producerer en gruppe af polypeptider, som kaldes thymosin, og måske et antal andre thymushormoner og/eller thymusfaktorer, som spiller en vigtig rolle for T-cellernes modning, differentiering og funktion. Det har vist sig, at thymosinet inducerer 15 T-cellernes differentiering og forstærker immunologiske funktioner hos genetisk athymiske mus, hos fuldvoksne thymektomerede mus, hos NZB-mus med stærke autoimmunreaktioner, hos mus med tumorer og hos mus med en casein-induceret amyloidose.It is nowadays a widely accepted fact that thymus has one central importance for the development and aging of the immune system in humans and animals. Although there is still little knowledge of the molecular mechanism by which the thymus controls the development of T cells 10, it suggests that a substantial part of this mechanism is hormonally controlled. The thymus produces a group of polypeptides called thymosin, and perhaps a number of other thymus hormones and / or thymus factors, which play an important role in the maturation, differentiation and function of T cells. It has been found that the thymosin induces 15 T cell differentiation and enhances immunological functions in genetically athymic mice, in adult thymectomized mice, in NZB mice with strong autoimmune reactions, in mice with tumors, and in mice with a casein-induced amyloidosis.

Det er kendt, at thymusfraktion 5 er et virksomt immunpotenserende 20 præparat, som kan virke ligesom thymus ved genoprettelsen af immunfunktioner hos individer uden thymus og/eller uden immunsystem. Vedvarende kliniske forsøg med thymusfraktion 5 synes at begrunde . formodningen om, at thymosinet forhøjer T-cellernes antal både hos børn, som lider af en thymus-afhængig primær immundefekt, og ved 25 kræftsygdomme med indskrænket immunforsvar.It is known that thymus fraction 5 is an effective immune potentiating preparation which may act like thymus in restoring immune functions in individuals without thymus and / or without immune system. Ongoing clinical trials with thymus fraction 5 appear to be justified. the presumption that the thymosin increases the number of T cells both in children suffering from a thymus-dependent primary immunodeficiency and in 25 cancers with a reduced immune system.

Ved analytiske metoder kan det vises, at thymusfraktion 5 består af 10-15 hoved- og 20 eller flere bibestanddele med en molekylvægt på mellem 1000 og 15000.By analytical methods, it can be shown that thymus fraction 5 consists of 10-15 major and 20 or more bib constituents with a molecular weight of between 1000 and 15000.

Den foreliggende opfindelse angår en fremgangsmåde til fremstilling af 30 et i thymosinfraktion 5 forekommende surt polypeptid, som kaldes thymosin a^, og hvis aminosyresekvens har kunnet opklares, ved isolering og renfremstilling ud fra denne thymusfraktion 5. Thymosin 2 149094 a.j viser ved forskellige in-vitro- og /n-wVo-tests, som giver et mål for T-celledifferentieringen og -funktionen, i sammenligning med thymosinfraktion 5 en 10 - 1000 gange så stor aktivitet.The present invention relates to a process for preparing an acidic polypeptide present in thymosin fraction 5, called thymosin α, and whose amino acid sequence has been resolved, by isolation and purification from this thymus fraction 5. Thymosin 2 in vitro and / n-wVo tests, which provide a measure of T-cell differentiation and function, in comparison with thymosin fraction 5 a 10 - 1000 times greater activity.

Thymosin har en molekylvægt på 3108, en pl-værdi i området 5 4,0-4,3 (bestemt ved isoelektrisk fokusering i en gel ved en pH-værdi i området 3-5) og den følgende aminosyresekvens: (N-Acetyl)-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-lle-Thr-Thr-Thymosin has a molecular weight of 3108, a pI value in the range 5 4.0-4.3 (determined by isoelectric focusing in a gel at a pH in the range 3-5) and the following amino acid sequence: (N-Acetyl) -Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-

Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH.Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH.

Isoleringen og renfremstillingen af thymosin ud fra thymosinfrakti-10 on 5 sker ved fremgangsmåden ifølge opfindelsen ved en kombination af ionbytterchromatografi og gelfiltrering på følgende måde: a) Den lyofiliserede thymosinfraktion 5 chromatograferes på en kolonne med carboxymethylcellulose i en natriumacetat/2-mercaptoethanol-puffer, som er 10 millimolær med hensyn til natriumacetat og 1,0 15 millimolær med hensyn til 2-mercaptoethanol, og som har en pH-værdi på 5,0, hvorhos kolonnen først vaskes med denne pufferopløsning og derefter elueres med en lineær gradient af denne puffer og en 1:1 (vol/vol) blanding af denne puffer og pufferen med et indhold på 1,0M natriumchlorid.The isolation and purification of thymosin from thymosin fraction 5 is carried out by the method of the invention by a combination of ion exchange chromatography and gel filtration as follows: a) The lyophilized thymosin fraction 5 is chromatographed on a column of carboxymethyl cellulose in a sodium acetate / 2-acetate / 2-acetate / 2 which is 10 millimolar with respect to sodium acetate and 1.0 15 millimolar with respect to 2-mercaptoethanol, having a pH of 5.0 at which the column is first washed with this buffer solution and then eluted with a linear gradient of this buffer and a 1: 1 (v / v) mixture of this buffer and the buffer containing 1.0M sodium chloride.

20 b) Den første i trin a) vundne "proteintop" fraktioneres på en dex-trangelkolonne (Sephadex G-25) under anvendelse af sterilt vand.B) The first "protein peak" obtained in step a) is fractionated on a dex-trang column (Sephadex G-25) using sterile water.

c) Den anden i trin b) vundne "proteintop" chromatograferes på en kolonne med 2-diethylaminoethyl-cellulose (DE-32), i tris(hydroxyme-thyl)aminomethan(tris)/2-mercaptoethanol-pufferopløsning, der er 50 25 mM med hensyn til tris og 1,0 mM med hensyn til 2-mercaptoethanol og med pH-værdi 8,0. Der elueres først med denne puffer og derpå med en lineær gradient af denne puffer og denne puffer med et indhold på 0,8M natriumchlorid.c) The second "protein peak" obtained in step b) is chromatographed on a column of 2-diethylaminoethyl cellulose (DE-32), in tris (hydroxymethyl) aminomethane (tris) / 2-mercaptoethanol buffer solution which is 50 mM for Tris and 1.0 mM for 2-mercaptoethanol and pH 8.0. This buffer is first eluted and then with a linear gradient of this buffer and this buffer containing 0.8M sodium chloride.

d) De fraktioner, som indeholder den første sjettedel af de i trin c) 30 vundne "proteintoppe", forenes og renses yderligere på en dextran- 3 149094 / gelkolonne (Sephadex G-75) i en guanidinhydrochlorid/tris-puffer, der er 6,OM med hensyn til guanidinhydrochlorid og 10 milliM med hensyn til tris og med en pH-værdi på 7,5.d) The fractions containing the first sixth of the "protein peaks" obtained in step c) are combined and further purified on a dextran (Sephadex G-75) column in a guanidine hydrochloride / tris buffer which is 6, OM with respect to guanidine hydrochloride and 10 milliM with respect to Tris and having a pH of 7.5.

e) Til slut afsaltes de midterste fraktioner af de i trin d) vundne 5 "proteintoppe" på en dextrangelkolonne (Sephadex G-10) under anvendelse af sterilt vand.e) Finally, the middle fractions of the 5 "protein peaks" obtained in step d) are desalted on a dextran column (Sephadex G-10) using sterile water.

Det fremkomne thymosin kan derefter om ønsket omdannes til et fysiologisk acceptabelt salt deraf med en syre eller base.The resulting thymosin can then, if desired, be converted to a physiologically acceptable salt thereof with an acid or base.

Eksempler på sådanne salte er natrium- eller kaliumsaltet, salte med 10 stærke organiske baser såsom guanidin eller syreadditionssalte såsom hydrochlorid, hydrobromid, sulfat, phosphat, malat, maleat, acetat, citrat, succinat, benzoat og ascorbat.Examples of such salts are the sodium or potassium salt, salts with 10 strong organic bases such as guanidine or acid addition salts such as hydrochloride, hydrobromide, sulfate, phosphate, malate, maleate, acetate, citrate, succinate, benzoate and ascorbate.

Den således fremstillede, rensede proteinfraktion (udbytte 0,6%) identificeres som thymosin med den ovenfor angivne aminosyresek-15 vens. Præparatet er frit for carbonhydrater og nucleotider, Strukturopklaringen og bestemmelsen af aminosyresekvensen sker ved de almindelige sædvanlige metoder, dvs. ved enzymatisk nedbrydning med trypsin, chymotrypsin, thermolysin eller subtilisin, opspaltning af fragmenterne ved papirelektroforese og/eller chromatografi og 20 Edman-nedbrydning af de enkelte peptidfragmenter.The thus purified protein fraction (yield 0.6%) is identified as thymosin with the above amino acid sequence. The preparation is free of carbohydrates and nucleotides. The structure elucidation and determination of the amino acid sequence is by the usual conventional methods, ie. by enzymatic degradation with trypsin, chymotrypsin, thermolysin or subtilisin, cleavage of the fragments by paper electrophoresis and / or chromatography, and Edman degradation of the individual peptide fragments.

Af den nedenstående tabel fremgår den med faktoren 10-1000 gange større biologiske aktivitet af thymosin α^, sammenlignet med aktiviteten af thymusfraktion 5 ved tre kendte bioforsøg, nemlig mitogentes-ten på mus, lymphokin-testen (en in-vitro-test, hvorved produktionen 25 af makrophagmigrationshæmningsfaktoren, MIF, måles) og E-rosette-testen med menneskelige lymfocyter.The following table shows the factor of 10-1000 times greater biological activity of thymosin α 1, compared to the activity of thymus fraction 5 in three known bioassays, namely the mitogenesis test in mice, the lymphokine test (an in vitro test whereby production of the macrophage migration inhibitory factor, MIF, is measured) and the E-rosette test with human lymphocytes.

Claims

Thymosin α-j can be administered to humans and mammals parenterally, namely intravenously, subcutaneously or intramuscularly. In the case of intravenous administration, the daily dose is usually in the range of 1-100 µg / kg body weight. It goes without saying that it varies from case to case and depends on the nature of the disease, its strength and duration. A suitable pharmaceutical dosage unit is 1 mg of lyophilized thymosin ct., Which can be dissolved in sterile water or a saline solution shortly before use. Process for Preparing Thymosin with the Amino Acid Sequence 20 (N-Acetyl) -Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-III-Thr-Thr-Lys-Asp-Leu-Lys-Glu Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH or physiologically acceptable salts thereof with acids or bases from thymosin fraction 5, characterized in that a) the lyophilized thymosin fraction 5 is chromatographed on a column with carboxymethyl cellulose in a sodium acetate / 2-mercapto-ethanol buffer of 10 milliM with respect to sodium acetate and 1.0 milliM with respect to 2-mercaptoethanol and at pH 5.0 where the column is first washed with this buffer and then eluted with a 30 linear gradient of this buffer and a 1: 1 (v / v) mixture of this buffer and the buffer containing 1.0M sodium chloride,