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DE2039061A1 - Partially amidated l-asparaginases - with chemotherapeutic action - Google Patents

Partially amidated l-asparaginases - with chemotherapeutic action

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Publication number
DE2039061A1
DE2039061A1 DE19702039061 DE2039061A DE2039061A1 DE 2039061 A1 DE2039061 A1 DE 2039061A1 DE 19702039061 DE19702039061 DE 19702039061 DE 2039061 A DE2039061 A DE 2039061A DE 2039061 A1 DE2039061 A1 DE 2039061A1
Authority
DE
Germany
Prior art keywords
asparaginases
partially amidated
asparaginase
carbodiimide
amino
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
DE19702039061
Other languages
German (de)
Inventor
Alfred Dr Arens
Klaus Dr Bauer
Robert Dr Bierling
Eckart Dr Irion
Johann Dr Puetter
Erich Dr Rauenbusch
Otto Dr Wagner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bayer AG
Original Assignee
Bayer AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bayer AG filed Critical Bayer AG
Priority to DE19702039061 priority Critical patent/DE2039061A1/en
Publication of DE2039061A1 publication Critical patent/DE2039061A1/en
Pending legal-status Critical Current

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/78Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/82Asparaginase (3.5.1.1)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y305/00Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01001Asparaginase (3.5.1.1)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Partially amidated L-asparaginases, which can be used for the same symptoms and in the same dosages as the known chemotherapeutic L-asparaginases but have a higher isoelectric point and behave differently in vivo (e.g. the half-life is altered), are prepared by reacting L-asparaginase (e.g. E. coli or Serratia marcescens L-asparaginase) with nucleophilic substances (e.g. amino-sugars, amines or, especially, amides or esters of amino-acids) in the presence of a carbodiimide. A degree of amidation of up to ca 20% is expedient.

Description

Partiell amidierte L-Asparaginasen Es ist bekannt, die Carboxylgruppen von Proteinen in Gegenwart eines wasserlöslichen Carbodiimids mit nucleophilen Substanzen umzusetzen. Hierbei entstehen die entaprechenden Amidderivate mit neuen physiko-chemischen Eigenschaften fj. Biol. Chem. 242 (1967) 24477. Partially amidated L-asparaginases It is known to have carboxyl groups of proteins in the presence of a water-soluble carbodiimide with nucleophilic substances to implement. This creates the corresponding amide derivatives with new physico-chemical ones Properties fj. Biol. Chem. 242 (1967) 24477.

Enzyme sind jedoch, soweit aus der Fachliteratur bekannt, bisher noch nicht unter Erhalt ihrer enzymatischen Aktivität derart modifiziert worden. Bei der hohen Spezifität und Instabilität der meisten Enzyme fUhren bereits leichte chemische Eingriffe zu einer irreversiblen Denaturierung und damit auch zu einem Verlust der enzymatischen Aktivität.However, so far as is known from the specialist literature, enzymes are still used has not been so modified while retaining their enzymatic activity. at The high specificity and instability of most enzymes lead to even slight reactions chemical interventions to an irreversible denaturation and thus also to a Loss of enzymatic activity.

Gegenstand der Erfindung sind nun partiell amidierte L-Asparaginasen. Diese werden erfindungsgemäß dadurch hergestellt, daß man B-Asparaginasen in Gegenwart eines Carbodiimids mit einer nucleophilen Komponente umsetzt.The invention now relates to partially amidated L-asparaginases. According to the invention, these are prepared by adding B-asparaginases in the presence a carbodiimide reacts with a nucleophilic component.

Gegenstand der Erfindung sind ferner Arzneimittel mit einem Gehalt an den genannten partiell amidierten L-Asparaginasen.The invention also relates to medicaments with a content on the partially amidated L-asparaginases mentioned.

L-Asparaginase (L-Aaparaginamidohydrolase, EC 3.5.1.1) hat kürslich Bedeutung als Chemotherapeutikum erlangt [Recent Results in Cancer Research, Vol. 33, 1970]. Die Applikation erfolgt in erater Linie durch intravenöse Injektion. Dabei verteilt sich das Enzym im extrazellulären Raum und wird nach einer gewissen Zeit abgebaut und ausgeschieden.L-asparaginase (L-aaparaginamidohydrolase, EC 3.5.1.1) has been purchased Gaining importance as a chemotherapeutic agent [Recent Results in Cancer Research, Vol. 33, 1970]. The application takes place in the erater line by intravenous injection. The enzyme is distributed in the extracellular space and becomes one degraded and eliminated for a certain period of time.

Die erfindungsgemäß hergestellten, neuen Derivate können teilweise kristallisiert werden und unterscheiden sich von den als Ausgangsprodukten dienenden L-Asparaginasen durch einen höheren isoelektrischen Punkt und somit durch eine geringere Wanderungsgeschwindigkeit zur Anode bzw. durch eine Wanderung zur Kathode. Die unterschiedlichen physiko-chemischen Eigenschaften dieser partiell amidierten L-Asparaginasen bewirken nun auch in vivo ein verändertes Verhalten, welches unter anderem durch eine veränderte Halbwertszeit zum Ausdruck kommt.The novel derivatives prepared according to the invention can in part are crystallized and differ from those used as starting products L-asparaginases by a higher isoelectric point and thus by a lower one Migration speed to the anode or due to a migration to the cathode. The different effect the physico-chemical properties of these partially amidated L-asparaginases now also in vivo a changed behavior, which among other things was changed by a Half-life is expressed.

Grundsätzlich sollen die neuen erfindungagemäßen Verbindungen als Arzneimittel für die gleichen Indikationen und in gleichen Dosierungen verwendet werden wie L-Asparaginase.In principle, the new compounds according to the invention as Medicines used for the same indications and in the same dosages become like L-asparaginase.

FUr das erfindungsgemäße Verfahren können nach beliebigen Methoden erhaltene L-Asparaginasen Verwendung finden, z.B.Any methods can be used for the process according to the invention L-asparaginases obtained can be used, e.g.

solche aus-den Mikroorganismen Escherichia coli oder Serratia sareeecan.those from the microorganisms Escherichia coli or Serratia sareeecan.

Zur Aktivierung der Carboxylgruppen der L-Asparaginasen kommen die in den betreffenden Lösungsmitteln löslichen Carbodiimide, wie z.B. 1-Äthyl-3-dimethylaminopropylcarbodiimid, 1-Cyclohexyl-3-(2-morpholinoäthyl)carbodiimid oder entsprechende Analoga, in Betracht.To activate the carboxyl groups of the L-asparaginases come the carbodiimides soluble in the solvents concerned, such as 1-ethyl-3-dimethylaminopropylcarbodiimide, 1-Cyclohexyl-3- (2-morpholinoethyl) carbodiimide or corresponding analogs, into consideration.

Als nucleophile Reaktionspartner eignen sich hauptsächlich die Amide oder Ester der Aminosäuren, jedoch können gane allgemein sämtliche Verbindungen mit nucleophilem Charakter eingesetzt werden, wobei hier z B. Aminozucker, Amine usw. genannt seien.The amides are mainly suitable as nucleophilic reactants or esters of amino acids, but generally all compounds can be used be used with nucleophilic character, here, for example, amino sugars, amines etc. may be mentioned.

Die Umsetzung erfolgt in v'äßriger Lösung, gegebenenfalls auch in mit Wasser mischbaren Lösungsmitteln, wie z.-B. Aceton, Alkohol, Dioxan usw. Der pH-Wert soll etwa zwischen 3 und 7 liegen.The reaction takes place in aqueous solution, if necessary also in water-miscible solvents such as Acetone, alcohol, dioxane, etc. The The pH should be between 3 and 7.

Als zweckmäßig hat es sich erwiesen, L-Asparaginase in einer Konzentration zwischen 0,1 und 5 ffi und die nucleophile Somponente in einer Konzentration zwischen 0,01 und 1 molar vorzulegen und die Reaktion mit dem Carbodiimid zu starten (Endkonzentration zwischen 0,01 und 0,1 molar). Durch Variation der Temperatur, der Zeit sowie der Konzentration der Reaktionspartner kann die Geschwindigkeit sowie der Grad der Umsetzung beeinflußt werden. Zweckmäßig ist ein Amidierungsgrad bis zu etwa 20 %. Die Reaktion kann durch Zusatz von organischen Säuren, wie z.B. Ameisensäure, Essigsäure usw.It has proven to be useful to use L-asparaginase in one concentration between 0.1 and 5 ffi and the nucleophilic component in a concentration between 0.01 and 1 molar and start the reaction with the carbodiimide (final concentration between 0.01 and 0.1 molar). By varying the temperature, the time and the Concentration of the reactants can affect the speed as well as the degree of implementation to be influenced. A degree of amidation of up to about 20% is expedient. The reaction can be achieved by adding organic acids such as formic acid, acetic acid, etc.

oder deren Salzen abgestoppt werden. Die anschließende Isolierung der bei der Reaktion entstandenen partiell amidierten L-Asparaginasen erfolgt nach den üblichen Methoden der Proteinchemie, also beispielsweise durch Ausfällen mit Polyäthylenglykol, organischen Lösungsmitteln oder Salzen, durch Adsorption an Hydroxylapatit oder Cellulosen oder aber auch durch Gefriertrocknung, gegebenenfalls nach vorhergehender. Entsalzung mittels Dialyse oder Gelchromatographie.or their salts are stopped. The subsequent isolation the partially amidated L-asparaginases formed in the reaction takes place after the usual methods of protein chemistry, for example by precipitating with Polyethylene glycol, organic solvents or salts, by adsorption on hydroxyapatite or celluloses or else by freeze-drying, if appropriate after the preceding. Desalination using dialysis or gel chromatography.

Die in den Beispielen aufgeführten partiell amidierten L-Asparaginasen wurden nach der folgenden allgemeinen Vorschrift gewonnen: L-Asparaginase (210 bis 240 E/mg) und die nucleophile Eomponente werden in der gewünschten Konzentration in Wasser gelöst und mit verdünnter HC1 oder NaOH auf einen bestimmten pH-Wert eingestellt. Die Reaktion wird durch Zugabe einer wäßrigen Carbodiimidlösung (Endkonzentration s. Tabelle) gestartet, gegebenenfalls wird der pH-Wert korrigiert. Nach einer bestimmten Zeit wird die Reaktion durch HinzufUgen des doppelten Volumens 1 M Natriumacetatlösung abgestoppt. Diese Mischung wird gegen Wasser dialysiert und dann der nichtdialysierbare Rückstand gefriergetrocknet. Die Ausbeuten sind nahe zu quantitativ.The partially amidated L-asparaginases listed in the examples were obtained according to the following general procedure: L-asparaginase (210 to 240 U / mg) and the nucleophilic Eomponente are in the desired concentration dissolved in water and adjusted to a certain pH value with dilute HCl or NaOH. The reaction is stopped by adding an aqueous carbodiimide solution (final concentration see table), the pH value is corrected if necessary. After a certain Time the reaction by adding twice the volume of 1 M sodium acetate solution stopped. This mixture is dialyzed against water and then the non-dialyzable Freeze-dried residue. The yields are almost too quantitative.

T a b e l l e Bei- Proteinkons. Nucleophile Komponente Carbodiimid+ Reaktioinszeit pH Temp. spez. Akt. spiel (%) (Min.) (°C) (E/mg) Nr. 1 1 0,1 M Glycinamid 0,05 M EDC 10 4,8 4 150 2 1 " " " " " " 70 4,8 4 76 3 1 " " " " " " 5 4,8 22 120 4 1 0,02 " " 0,01 " " 10 4,8 4 200 5 1 " " " " " " 30 4,8 4 190 6 0,5 0,1 " " " " " 30 4,8 4 170 7 " " " " " " " 30 6,0 4 210 8 " " " Glycinmethyl- " " " 30 4,8 4 170 ester 9 " " " " " " CMC 30 4,8 4 165 10 " " " Anilin " " EDC 30 4,8 4 171 11 " " " NH4Cl " " " 30 4,8 4 182 12 " " " Glucosamin " " " 30 4,8 4 189 13 " " " Lysin " " " 30 4,8 4 165 +EDC = 1-Äthyl-3-dimethylaminopropylcarbodiimid CMC = 1-Cyclohexyl-3-(2-morpholinoäthyl)carbodiimidTabel At- protein cons. Nucleophilic component carbodiimide + reaction time pH temp. Spec. Act. game (%) (Min.) (° C) (E / mg) No. 1 1 0.1 M glycinamide 0.05 M EDC 10 4.8 4 150 2 1 """""" 70 4.8 4 76 3 1 """""" 5 4.8 22 120 4 1 0.02 "" 0.01 "" 10 4.8 4 200 5 1 """""" 30 4.8 4 190 6 0.5 0.1 """""30 4.8 4 170 7 """""""30 6.0 4 210 8 """Glycinemethyl-""" 30 4.8 4 170 ester 9 """""" CMC 30 4.8 4 165 10 """Aniline""EDC 30 4.8 4 171 11 """NH4Cl""" 30 4.8 4 182 12 """Glucosamine""" 30 4.8 4 189 13 """Lysine""" 30 4.8 4 165 + EDC = 1-ethyl-3-dimethylaminopropylcarbodiimide CMC = 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide

Claims (1)

Patentansprüche Partiell amidierte L-Asparaginasen.Claims partially amidated L-asparaginases. 2. Verfahren zur Herstellung von partiell amidierten L-Asparaginasen, dadurch gekennzeichnet, daß man L-Asparaginasen in Gegenwart eines Carbodiimids mit nucleophilen Substanzen umsetzt.2. Process for the production of partially amidated L-asparaginases, characterized in that L-asparaginases in the presence of a carbodiimide Reacts with nucleophilic substances. 5. Arzneimittel, gekennzeichnet durch einen Gehalt an L-Asparaginase-Amidderivaten.5. Medicinal products, characterized by a content of L-asparaginase amide derivatives.
DE19702039061 1970-08-06 1970-08-06 Partially amidated l-asparaginases - with chemotherapeutic action Pending DE2039061A1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998056410A1 (en) * 1997-06-09 1998-12-17 Childrens Hospital Los Angeles Utilization of wolinella succinogenes asparaginase to treat diseases associated with asparagine dependence
US6991788B1 (en) 1997-06-09 2006-01-31 Childrens Hospital Los Angeles Utilization of wolinella succinogenes asparaginase to treat diseases associated with asparagine dependence

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998056410A1 (en) * 1997-06-09 1998-12-17 Childrens Hospital Los Angeles Utilization of wolinella succinogenes asparaginase to treat diseases associated with asparagine dependence
US6991788B1 (en) 1997-06-09 2006-01-31 Childrens Hospital Los Angeles Utilization of wolinella succinogenes asparaginase to treat diseases associated with asparagine dependence

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