SE200080C1 - - Google Patents

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Publication number
SE200080C1
SE200080C1 SE200080DA SE200080C1 SE 200080 C1 SE200080 C1 SE 200080C1 SE 200080D A SE200080D A SE 200080DA SE 200080 C1 SE200080 C1 SE 200080C1
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SE
Sweden
Prior art keywords
kallikrein inactivator
derivative
kallikrein
inactivator
acid
Prior art date
Application number
Other languages
Swedish (sv)
Publication date
Publication of SE200080C1 publication Critical patent/SE200080C1/sv

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)

Description

KLASS INTERNATIONELLSVENSK A 61 k30 h:2/04 PATENT- OCH REGISTRERINGSVERKET Ans. 1699/1963 inkom den 15/2 1963 utlagd den 1/3 196 FARBENFABRIKEN BAYER AG, LEVERKUSEN-BAYERWERK, FORBUNDSREPUBLIKEN TYSKLAND Sat att framstalla ett svirlosligt derivat av kallikrein-inaktivatorn Upplinnare: F Schultz Prioritet begeird frau den 19 februari 1962 (Forbundsrepubliken Tgskland) Det ãr kant att man kan framstalla fran aggvita fria derivat av kallikrein-inaktivatorn (KI) frau djurkortlar genom att man. omsatter losningar av sadana kortlar genom tillsats av fallningsmedel for figgvita, sasom sulfosalicylsyra och triklorattiksyra. Kallikrein-inaktivatorn utfalles salunda icke genom tillsats av dessa medel (jmfr. tyska patentskriften 1 084 433). CLASS INTERNATIONAL SWEDISH A 61 k30 h: 2/04 PATENT AND REGISTRATION AGENCY Ans. 1699/1963 was received on 15/2 1963 issued on 1/3 196 FARBENFABRIKEN BAYER AG, LEVERKUSEN-BAYERWERK, FEDERAL REPUBLIC OF GERMANY Set to produce a virulence derivative of the kallikrein inactivator It is possible to produce aggregate-free derivatives of the kallikrein inactivator (KI) from animal cards by. reactes solutions of such cards by the addition of precipitants for white matter, such as sulfosalicylic acid and trichloroacetic acid. The Kallikrein inactivator is thus not precipitated by the addition of these agents (cf. German Patent Specification 1,084,433).

Enligt uppfinningen har det nu befunnits, att man kan framstalla ett svarlosligt derivat av kallikrein-inaktivatorn genom att omsatta densamma med ,metafosforsyra i vattenhaltig losning. Det i form av en fallning erhallna derivatet avskiljes fran reaktionsblandningen. According to the invention, it has now been found that a responsive derivative of the kallikrein inactivator can be prepared by reacting it with metaphosphoric acid in aqueous solution. The derivative obtained in the form of a precipitate is separated from the reaction mixture.

Man kan harvid ocksh. ga. tillvaga pa sa satt, att man omsatter losningar av kallikrein-inaktivatorn med vattentosliga salter av metafosforsyra, varvid nagon fanning icke uppkommer, och darefter genom surgoring utskiljer det svarlosliga derivatet. F8r detta andamal lampliga salter av metafosforsyra Oro frainfor alit alkali- och ammoniumsalterna. You can harvid ocksh. ga. prepared in such a way that solutions of the kallikrein inactivator are reacted with water-soluble salts of metaphosphoric acid, whereby no formation arises, and then the acid-soluble derivative is separated by acidification. For this purpose suitable salts of metaphosphoric acid Oro frainfor alit the alkali and ammonium salts.

Fallningen i enlighet med uppfinningen genomfores ocksa i fran aggvita fria losningar av kallikrein-inaktivatorn, t. ex. i sadana, mlka dessforinnan kvantitativt befriats frail aggvita med hjalp av sulfosalicylsyra eller triklorattiksyra. Harav framgar det, att det mid de i enlighet med foreliggande uppfinning foreslagna atgarderna fake rora sig om nagon fanning av aggvita. Fastmer mOjliggores i enlighet med forfarandet enligt uppfinningen utvinning av ett aggvitefritt, svarlosligt derivat av kallikrein-inaktivatorn. The precipitate according to the invention is also carried out in aggregate free solutions of the kallikrein inactivator, e.g. in such, milk may previously be quantitatively freed from agglomerates by means of sulfosalicylic acid or trichloroacetic acid. From this it appears that the measures proposed in accordance with the present invention are false about any formation of aggvita. However, in accordance with the process of the invention, it is possible to recover an agglite-free, unresponsive derivative of the kallikrein inactivator.

Genom fallningeii i enlighet med uppfinningen ager flagon denaturering av kallikreininaktivatorn icke rum, vilket utgor ett ytterligare bevis f8r, all det i foreliggande fall joke rot sig om flagon fallning av aggvita, eftersom aggvitautfallningar i allmanhet atfoljas av denatureringar av den. utfallda aggvitan, vilket ger sig tillkanna i en forlust i den biologiska verkan for ifragavarande substans. By precipitation in accordance with the invention, flaccid denaturation of the kallikreinin activator does not take place, which constitutes further proof that all the present case is rooted in flaccid precipitation of aggvite, since aggvite precipitates are generally followed by denaturations of it. precipitated aggvitan, which manifests itself in a loss in the biological effect of the substance in question.

Vid det uppfinningsenliga forfarandet utskiljes kallikrein-inaktivatorn kvantitativt i svarlOslig form. I detta preparat innehalles he-la den biologiska aktiviteten. PA grund av den erhallna produktens svarlOslighet ar forfarandet ocksa lampligt for rening av fororenad kallikrein-inaktivator. In the process according to the invention, the kallikrein inactivator is separated quantitatively in a soluble form. This preparation contains all the biological activity. Due to the responsiveness of the product obtained, the process is also suitable for the purification of contaminated kallikrein inactivator.

Suspensionerna av fallningsprodukten i vat-ten aro icke dialyserbara, under det att kallikrein-inaktivatorn i sin losliga form tail later sig dialyseras, aven frau landningar. The suspensions of the precipitation product in the water are not dialyzable, while the kallikrein inactivator in its loose form allows itself to be dialyzed, even from landings.

Den i enlighet med forfarandet enligt foreliggande uppfinning erhallbara produkten_ visa" vid anvandning in vivo en depaverkan. Den ar joke toxisk, utpraglat vavnadsvanlig och ay denna anledning val lampad for terapeutisk anvandning, amen fOr injektioner. The product obtainable in accordance with the method of the present invention exhibits a deposition effect when used in vivo. It is joke toxic, distinctly conventional and for this reason is preferred for therapeutic use, amen to injections.

Exempel 1. 114 mg kallikrein-inaktivator (1 enhet bunden till 0,38 y substans), motsvarande 300000 KIE, lostes i 5 ml omdestillerat vat-ten och glides med 2,5 ml 10-procentig metafosforsyra. Fallningen avcentrifugerades, tvattades tre ganger med omdestillerat vatten och suspenderas i 10 ml omdestillerat vatten. Suspensionen inneholl c:a 100 % av kallikreininaktivatoraktiviteten. Example 1. 114 mg of kallikrein inactivator (1 unit bound to 0.38 μl of substance), corresponding to 300,000 KIE, were dissolved in 5 ml of redistilled water and slid with 2.5 ml of 10% metaphosphoric acid. The precipitate was centrifuged off, washed three times with redistilled water and suspended in 10 ml of redistilled water. The suspension contained about 100% of the kallikrein inactivator activity.

Exempel 2. 114 mg kallikrein-inaktivator (1 enhet bunden till 0,38 substans), motsvarande 300000 KIE, tastes i 2,5 ml omdestillerat vatten. 10-procentig metafosforsyra installdes pa pH 6 (Lyphan-papper) med hj alp av NaOH och 2,5 ml av denna Na-metafosforsyralosning inrOrdes i kallikrein-inaktivatorlosningen. El-ter tillsats av 1 ml utspadd (1:10) isattika uppstod en fanning, som avcentrifugerades. Fallningen tvattades tre ganger med omdestil- Dupl. kl. 30 h: 2/ 2— ' — lerat vatten och suspenderades darefter i 10 ml omdestillerat vatten. Suspensionen baneholl c:a 100 % av kallikrein-inaktivatoraktiviteten. Example 2. 114 mg of kallikrein inactivator (1 unit bound to 0.38 substance), corresponding to 300,000 KIE, are diluted in 2.5 ml of redistilled water. 10% metaphosphoric acid was adjusted to pH 6 (Lyphan paper) with the aid of NaOH and 2.5 ml of this Na metaphosphoric acid solution was stirred into the kallikrein inactivator solution. After the addition of 1 ml of diluted (1:10) glacial acetic acid, a mixture was formed which was centrifuged off. The fall was washed three times with redistill- Dupl. at 30 h: 2/2 - '- clay water and then suspended in 10 ml of redistilled water. The suspension orbits about 100% of the kallikrein inactivator activity.

Exempel 3. 300 mg kallikrein-inaktivator (1 enhet bunden till 0,6 y substans), motsvarande 500000 KIE, fraanstalld frau pankreas, tes i 10 ml omdestillerat vatten oeh falldes med 7,2 ml 10-procentig metafosforsyra. ningen avcentrifugerades, tvattades tre ganger med omdestillerat vatten och suspenderades i 22,5 ml aqua redest. Utbytet uppgick till 90 dvs. 1 ml air suspensionen inn.eholl 20000 KIE. Example 3. 300 mg of kallikrein inactivator (1 unit bound to 0.6 μl of substance), corresponding to 500,000 KIE, prepared from the pancreas, was taken up in 10 ml of redistilled water and precipitated with 7.2 ml of 10% metaphosphoric acid. The mixture was centrifuged off, washed three times with redistilled water and suspended in 22.5 ml of aqua redest. The exchange amounted to 90, ie. 1 ml air suspension inn.eholl 20000 KIE.

Exempel 4. 100 ml av en saltfri, vattenhaltig lOsning av kallikrein-inaktivator (1 enhet bunden till 0,42 y substans), erhallen frail parotis, innehallande 250000 KIE, falldes med 0,2 ml 10-procentig metafosforsyra. Fanning-en tvattades efter avcentrifugering tva ganger med aqua redest, varefter Lerstoden suspenderades i 20 ml oandestillerat vatten. 1 ml av suspensionen inneholl 11250 KIE, utbytet uppgick salunda till 90 %. Example 4. 100 ml of a salt-free, aqueous solution of kallikrein inactivator (1 unit bound to 0.42 μl of substance), obtained frail parotis, containing 250,000 KIE, was precipitated with 0.2 ml of 10% metaphosphoric acid. After centrifugation, the Fanning was washed twice with aqua redest, after which the Lerstoden was suspended in 20 ml of undistilled water. 1 ml of the suspension contained 11250 KIE, the yield was thus 90%.

Exempel 5. 150 ml av en saltfri, vattenhaltig .losning air kallikrein-inaktivator (1 enhet bunden till 0,3 7 substans), erhallen frail ‘lunga, innehallande 9,9 miljoner KIE, falldes 'tiled 10 ml av en 10-procentig metafosforsyra losning. Fallningen avcentrifugerades och tvattades tva ganger med omdestillerat vatten. Example 5. 150 ml of a saline, aqueous solution of air kallikrein inactivator (1 unit bound to 0.3 7 substance), obtained frail 'lung, containing 9.9 million KIE, fell' tiled 10 ml of a 10% metaphosphoric acid solution. The precipitate was centrifuged off and washed twice with redistilled water.

Darefter suspenderades aterstoden i 330 ml omdestillerat -vatten. Den harvid uppkommande suspensionen inneholl hela mangden insatt kallikrein-inaktivator. Utbytet uppgick salunda till 100 %. 1 KIE av denna suspension ar bunden till 0,22 y. The residue was then suspended in 330 ml of redistilled water. The resulting suspension contained the full amount of kallikrein inactivator used. The yield thus amounted to 100%. 1 KIE of this suspension is bound to 0.22 y.

Claims (2)

Patentansprik:Patent claim: 1. Salt att framstalla ett svarlasligt derivat air kallikrein-inaktivatorn, kannetecknat daratt kallikrein-inaktivatorn omsattes med metafosforsyra i vattenhaltig losning och alt det i form av en fallning erhallna derivatet avskiljes fran reaktionsblandningen.Salt to produce a responsive derivative of the kallikrein inactivator, characterized in that the kallikrein inactivator is reacted with metaphosphoric acid in aqueous solution and all the derivative obtained in the form of a precipitate is separated from the reaction mixture. 2. Modifikation av sattet enligt patentanspraket 1, kanneteeknad darav, att metafos, forsyran tillfores i form av vattenlosliga salter och att derivatet utfalles genom att reaktionsblandningen surgares. Anforda publikationer:Modification of the method according to claim 1, characterized in that metaphos, the formic acid is supplied in the form of water-soluble salts and that the derivative is precipitated by acidifying the reaction mixture. Request publications:
SE200080D SE200080C1 (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
SE200080T

Publications (1)

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SE200080C1 true SE200080C1 (en) 1965-01-01

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