WO1994005332A2 - Glycolation de macromolecules glycosylees - Google Patents

Glycolation de macromolecules glycosylees Download PDF

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Publication number
WO1994005332A2
WO1994005332A2 PCT/US1993/008196 US9308196W WO9405332A2 WO 1994005332 A2 WO1994005332 A2 WO 1994005332A2 US 9308196 W US9308196 W US 9308196W WO 9405332 A2 WO9405332 A2 WO 9405332A2
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WIPO (PCT)
Prior art keywords
macromolecule
glycosylated
glycolated
glycol
polypeptide
Prior art date
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Ceased
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PCT/US1993/008196
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English (en)
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WO1994005332A3 (fr
Inventor
Thabiso M'timkulu
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Berlex Laboratories Inc
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Berlex Laboratories Inc
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Publication date
Application filed by Berlex Laboratories Inc filed Critical Berlex Laboratories Inc
Priority to AU50981/93A priority Critical patent/AU5098193A/en
Publication of WO1994005332A2 publication Critical patent/WO1994005332A2/fr
Publication of WO1994005332A3 publication Critical patent/WO1994005332A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol

Definitions

  • polypeptides in circulatory systems for the purpose of engendering a particular physiological response
  • insulin which is used in the treatment of diabetes.
  • Another group of polypeptides to which great therapeutic potential has been attributed are various enzymes.
  • a principal factor which has severely limited the use in therapeutics of polypeptides is that most of these com ⁇ pounds elicit an immunogenic response in body fluids, evidenced by changes in the composition of the circulatory system, i.e., the production of antibodies to the polypeptides. This effect has one or both of two secondary consequences: first, neutralization of the polypeptides by the antibodies thus produced; second, and more seriously, the development of an allergic response.
  • Neutralization of polypeptides by antibodies is be- lieved to be responsible for the rather low residence time of insulin in the human circulatory system; hence, persons afflicted with diabetes are forced to inject themselves fairly frequently with fresh doses of insulin.
  • parenterally administered enzymes not only is there the problem of neutralization of the pclypep.tide and the subsequent negation of its physiological activity, but also the extremely undesirable elicitation of an allergic reaction.
  • a limitation to the potential therapeutic benefit derived from the clinical use of polypeptides is their potential for eliciting such immune response in the circulatory system.
  • This immune response may be caused by aggregates in the material prior to injection as described by R. Illig (1970), j. ciin. Endocr..31. 679-688, W. Moore (178), . ciin. Endroc ⁇ 'noi. Hetab., 51, 691-697.
  • the antibody production may decrease or eliminate the desired bio ⁇ logical function of the polypeptide, sometimes by causing reduced residence time in the circulatory system (reduced half-life) or by modifying the molecule by virtue of the antibody-polypeptide interaction.
  • polypeptides include the modification of proteins with substantially straight- chain polymers such as polyethylene (PEG) or polypropylene glycol (PPG) .
  • PEG polyethylene
  • PPG polypropylene glycol
  • U.S. Patent No. 4,055,635 discloses pharmaceutical compositions comprising a water-soluble complex of a proteolytic enzyme linked covalently to a polymeric substance such as polysaccharides.
  • U.S. Patent No. 4,088,538 discloses a reversibly soluble, enzymatically active polymer enzyme product comprising an enzyme covalently bonded to an organic polymer such as polyethylene glycol.
  • U.S. Patent No. 4,415,665 discloses a method of conjugating an organic ligand containing at least one primary or secondary amino group, at least one thiol group, and/or at least one aromatic hydroxy group (described in column 3, lines 19-36) to a polymeric carrier with at least one hydroxyl group (described in column 2, lines 42-66).
  • U.S. Patent No. 4,496,689 discloses a covalently attached complex of ⁇ -1-proteinase inhibitor with a polymer such as PEG or methoxypolyethylene glycols.
  • U.S. Patent No. 3,788,948 discloses use of organic cyanate compounds to bind proteins to polymers.
  • U.S. Patent No. 4,055,635 discloses pharmaceutical compositions of a proteolytic enzyme linked covalently to a polymeric substance.
  • JP 57-92435 published November 26, 1982, discloses modified polypeptides, where all or part of the amino groups are substituted with a polyethoxyl moiety.
  • DE 2312615 published September 27, 1973, discloses conju ⁇ gating of polymers to compounds containing hydroxy or amino groups.
  • EP 147,761 discloses a covalent conjugate of ⁇ -1- proteinase inhibitor and a water-soluble polymer, where the polymer may be polyethylene glycol.
  • EP 154,316 published September 11, 1985, discloses and claims chemically modified lymphokines, such as IL-2 containing PEG bonded directly to at least one primary amino group of a lymphokine.
  • U.S. Patent No. 4,414,147 describes rendering inter- feron less hydrophobic by conjugating it to an anhydride of a dicarboxylic acid, such as poly(ethylene succinic anhydride) .
  • glycol couples most likely through an amino group on the protein, but also discloses an embodiment where the terminal hydroxy group of the glycol is converted to an amino group, e.g., with a sulfonating agent or a halogenating agent, and the resultant halide or tosylate is coupled with a carboxyl group of the polypeptide by known methods.
  • a sulfonating agent or a halogenating agent e.g., a sulfonating agent or a halogenating agent
  • the resultant halide or tosylate is coupled with a carboxyl group of the polypeptide by known methods.
  • One aspect of the invention is a process for the glycolation of a glycosylated macromolecule, comprising activating a polyalkylene glycol, reacting the activated polyalkylene glycol with a diamino compound, whereby the activated polyalkylene glycol is coupled to the diamino compound through one of its amino groups, 93/08196
  • the invention preferably comprises a process for the
  • PEGylation of a glycosylated macromolecule comprising:
  • n is preferably about 2-500.
  • x is preferably about 1-20.
  • Macromolecules usable in the invention include virtually any bioactive macromolecule bearing glyco ⁇ sylations (regardless of how bonded, e.g., covalently, etc.) or which can be glycosylated, e.g., polypeptides and/or proteins, nucleic acids, lipids, or carbohydrates.
  • Preferred peptides include those comprising an antigen binding region, a cytokine, a receptor, an antithrom- botic, a growth factor, or an angiohypotensive reagent.
  • the polypeptide is an immunoglobulin, an interferon, a receptor tyrosine kinase, a thrombomodulin, a transforming growth factor, an endothelin, or an analog of the above.
  • One especially preferred protein is the monoclonal antibody TAb-250 or its chimeric analog BACh 250 ("BACh-250" or "C-erb-B2") . See Molecular Oncology as a Basis for New Strateties in Cancer Therapy: Efficacy of an Anti-c-erbB-2 Mouse/Human Chimeric Antibody Alone and in Combination with cis-
  • CDDP Diammedichloroplatinum
  • oxidoreductases such as Urate:oxygen- oxidoreductase, Hydrogen-peroxide:hydrogen-peroxide oxidoreductase, Cholesterol-reduced-NADP:oxygen oxidoreductase (20-3-hydroxylating) ; transferases, such as UDP glucuronate glucuronyl-transferase (acceptor unspecific) , UDP glucose: ⁇ -D-Galactose-1-phosphate; hydrolases, such as Mucopeptide N-acetyl- muramyl-hydrolase, Trypsin, L-asparagine aminohydrolase; lyases, such as Fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate-lyase; isomerases, such as D-Xylose ketol-isomerase; and ligases, such as L-Citrulline
  • peptide hormones examples include insulin, ACTH, Glycagon, Somatostatin, Somatotropin, Thymosis, Parathyroid hormone, Pigmentary hormones,
  • Macromolecules which have no sugars may be glyco ⁇ sylated by means which are well known in the art, e.g. , as disclosed in Creighton, proteins, W.H. Freeman & Co., New York, 1983. Virtually any sugar which is reactive to oxidation is suitable. Examples include galactose, man- nose, glucose, N-acetylglucosamine, N-acetylgalactos- amine, sialic acids, fucose, and/or xylose.
  • the length of the carbohydrate chains of the sugar may vary widely, i.e., poly- and oligosaccharides may be used. Normally, the glycosylation will be that which is indigenous to the species producing the macromolecule, e.g. , mammalian.
  • the glycol is preferably polyethylene glycol, poly- propylene glycol, or a mixture thereof, as well as a mixed polyethylene-polypropylene glycol.
  • Polyethylene glycols (PEG's) are most preferred, especially ono- methoxyethylene glycol.
  • Preferred polyethylene glycols have the formula CH 3 0-(CH 2 CH 2 0) n -H, wherein n is 2-500, more preferably 20-400.
  • the diamino compound preferably has the formula H 2 N- R-NH 2 , wherein R is an organic moiety.
  • R may be, for example, a C ⁇ -aliphatic or C 3.20 -cycloaliphatic moiety or a C 5 . 20 -aryl moiety.
  • Aliphatic moieties include straight- or branched-chain or cyclic alkyl, alkenyl, dienyl, and alkynyl groups.
  • Preferred aliphatic moieties are C 2 . 12 - alkyl groups.
  • Preferred aryl moieties are heterocyclic, i.e., containing one or more O, S, or N atoms, and aromatic, e.g., phenyl groups.
  • Activation of the glycol with the addition of the diamino compound preferably occurs as in Veronese et al. , Applied Biochemistry-Biotechnology, Vol. 11, pp. 141-152 (1985) .
  • the glycol is activated by reaction with 2,4,5- trichlorophenyl-chloroformate or p-nitrophenylchloro- formate and triethylamine to yield a glycolphenylcar- bonate, which is then reacted with the diamino compound in high excess so that only one amino group of the diamino compound reacts with the activated glycol.
  • the molar ratio of diamino compound to activated glycol is at least 2:1, and more preferably at least
  • the reaction proceeds rapidly for a time prefer ⁇ ably from about 1 minute to 2 hours, more preferably from about 5 minutes to an hour.
  • temperatures from 4°C to 100°C will be utilized, preferably between 10° and 60°, more preferably near ambient, i.e., room temperature.
  • the glycol may be activated as in
  • oxidation is accomplished by reaction of the macromole ⁇ cule with sodium periodate (NaI0 ) in a preferred molar ratio of sugar to NaIO of 1000:1 to 10:1.
  • the concentra ⁇ tion of NaI0 4 is preferably 10-1000 mM.
  • the reaction may preferably proceed at temperatures of 0°C to 50°C, and preferably for 1 minute to 4 hours, more preferably about 30 minutes to 2 hours, most preferably about 30 minutes. Adjustment of the reaction time may be made to control the amount of glycols per protein, since longer incuba ⁇ tion results in greater oxidation of the sugars and, accordingly, more points on the macromolecule available for glycol attachment.
  • the macromolecule is coupled with the amino group-bearing glycol, by simple mixing, at temperatures preferably of about 4°C to 100°C, and for times preferably from about 1 minute to 5 hours, more typically between 3 minutes and 1 hour, and more preferably between 5 minutes and 30 minutes. Isolation, if desired, and work-up for biological applications is conventional, as disclosed in, e.g., U.S. Patent No. 4,179,337 or as described below.
  • the macromolecules may be used as diagnostic reagents, therapeutic reagents, test samples, etc. , as known in the art and dependent on their disclosed biological utilities.
  • the triethylammonium chloride is then filtered off using a sintered glass funnel. 200 ml of ethyl ether is added, and the solution is left to crystallize overnight at 4°C. The product is filtered, washed with ether to remove all of the yellow color, and recrystallized from acetonitrile-ether. The yield is 75%. The product is then assayed spectrophotometrically by the release of p-nitrophenol by e-amino-n-caproic acid (ACA) .
  • ACA e-amino-n-caproic acid
  • the purity of the product is further verified spectrophotometrically.
  • TAb-250 5 mg is dialyzed extensively into 50 iruM sodium borate buffer pH 8.3. A lower pH is used in order to ensure that only the very reactive epsilon amino groups of lysine are PEGylated.
  • To the 2 ml dialyzed sample 3 mg of the activated mPEG is added, a 5 molar excess. Every 30 minutes, 5 ⁇ l is removed and mixed with 5 ⁇ l of 25 iruM ACA. Immediate ⁇ ly, 3 mg of activated mPEG is added and incubated at room temperature with shaking for a further 30 minutes.
  • the reaction is stopped after 2 hours; final molar excess is 20-fold, by loading the sample on a NAP 25 (Pharmacia) desalting column and eluting it with 50 i ⁇ M NaP0 4 buffer, pH 6.8.
  • the desalted sample is loaded on Superose 6 column (1 x 30 cm BioRad Econocolumn ® ) and eluted with 50 mM NaPO ⁇ buffer, pH 6.8.
  • Four resultant peaks from the Superose column and the 30-minute time point samples are assayed by SDS-PAGE.
  • the Superose peaks are further assayed by Radial Immuno-diffusion (RID) from Tago Immuno, Inc. for the quantitation of mouse IgG lr more specifically a kappa light chain. See Table 1.
  • RID Radial Immuno-diffusion
  • Tago Standards IgG 5.5 0.16/mg/mL Tago Immuno, Inc. (Cone, on vial 0.156 mg/mt )
  • Immunoglobulin G contains approximately 3% carbohydrate by weight linked to the F c region of the protein.
  • mPEG- ⁇ -p-nitrophenyl 0.5 g is slowly added to 5 mL of 50 mM Na-borate buffer, pH 9.0, containing 44.25 mg (100 mmoles) of 1, 4-aminobutane.
  • the reaction is incu ⁇ bated at room temperature with shaking for 3 hours.
  • the reaction is stopped by passing it through an NAP 25 desalting column and eluted with water and dialyzed into milli-Q H 2 0.
  • the dialyzed material is lypophylized and weighed.
  • 1,4-diaminobutane is in high excess to guard against reacting both amino groups.
  • Immunoglobulin G., (IgG.,) contains approximately 3% carbohydrate by weight linked to the F c region of the protein.
  • Coupling Buffer 0.05 M sodium acetate
  • TAb-250 0.5 mg is buffer exchanged into the coupling buffer using an NAP-10 (Pharmacia) desalting column.
  • NAP-10 Pulacia
  • the solution is mixed gently, and the sealed reaction vial is shielded from light and incubated at room temperature for 30 minutes.
  • To stop the reaction the sample is passed through a NAP-10 desalting column and is equilibrated with wash buffer. The column is eluted with the conjugation buffer.
  • oxidized TAb-250 is added 5 mg of PEG- ⁇ -butamine.
  • the reaction vial is overlayed with nitrogen and is tumbled gently overnight at 4°C.
  • the molar ratio of TAb-250 to PEG- ⁇ -butamine is 1:100.
  • the sample is then loaded following optional reduction of the TAb-250 onto the same Superose 6 column.
  • the IgG peaks are pooled and are concentrated on an amicon stirred cell concentrator.

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  • Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
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  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
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Abstract

L'invention concerne un procédé de couplage de glycols à des macromolécules par glycosylations sur ces dernières et non par des groupes amino ou carboxyle sur le squelette de celles-ci. On produit ainsi des macromolécules à réponse immunogène réduite et à activité maintenue. Ledit procédé de glycosylation d'une macromolécule glycosylée consiste à activer un glycol de polyalkylène; à faire réagir le glycol de polyalkylène activé avec un composé diamino, le glycol de polyalkylène activé étant couplé au composé diamino par un de ses groupes amino; à oxyder la macromolécule afin d'activer au moins un groupe glycosyl qu'elle contient; et à faire réagir le glycol de polyalkylène couplé au composé diamino avec le groupe glycosyl oxydé dans la macromolécule. On obtient ainsi une macromolécule glycosylée glycolée dans laquelle le glycol est lié à la macromolécule par ses glycosylations.
PCT/US1993/008196 1992-09-01 1993-09-01 Glycolation de macromolecules glycosylees Ceased WO1994005332A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU50981/93A AU5098193A (en) 1992-09-01 1993-09-01 Glycolation of glycosylated macromolecules

Applications Claiming Priority (2)

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US93777992A 1992-09-01 1992-09-01
US07/937,779 1992-09-01

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WO1994005332A2 true WO1994005332A2 (fr) 1994-03-17
WO1994005332A3 WO1994005332A3 (fr) 1994-04-14

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US9187532B2 (en) 2006-07-21 2015-11-17 Novo Nordisk A/S Glycosylation of peptides via O-linked glycosylation sequences
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