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WO1993013788A1 - Derives conjugues de peptides contenant un site d'acceptation de glycosylation et utilisations therapeutiques - Google Patents

Derives conjugues de peptides contenant un site d'acceptation de glycosylation et utilisations therapeutiques Download PDF

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Publication number
WO1993013788A1
WO1993013788A1 PCT/US1993/000043 US9300043W WO9313788A1 WO 1993013788 A1 WO1993013788 A1 WO 1993013788A1 US 9300043 W US9300043 W US 9300043W WO 9313788 A1 WO9313788 A1 WO 9313788A1
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Prior art keywords
compound
substituted
amino acid
alkyl
aralkyl
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James E. Rothman
Manfred Weigele
Richard D. Klausner
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Ariad Pharmaceuticals Inc
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Ariad Pharmaceuticals Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to conjugated peptide derivatives containing an acceptor site for glycosylation, and use of such peptide derivatives for concentration of active agents in the lumen of the endoplasmic reticulum. Therapeutic methods based on such concentration are also provided.
  • Ribosomes are complexes that carry out protein synthesis within the cell by reading the three letter genetic code (codon) of each messenger RNA.
  • the endoplasmic reticulum (ER) is an interconnected series of flattened, generally layered, sacs within the cell. Ribosomes that are synthesizing secretory and integral membrane (ER, Golgi, and plasma membrane) proteins are tightly bound to the membrane of the ER (which is termed the rough ER with such bound ribosomes) .
  • Secretory proteins are transported across the ER membrane into the lumen, or cisterna, of the ER during synthesis; membrane proteins become inserted into the ER membrane during synthesis.
  • a signal sequence characteristically near the N-terminus of the newly synthesized protein and consisting of one or more positively charged amino acids followed by 6-12 continuous hydrophobic residues, directs a protein to the ER, and inserts itself into the ER membrane, with the aid of the signal recognition particle.
  • the signal sequence is cleaved off by signal peptidase, localized in the lumen of the ER.
  • Other topogenic sequences within membrane proteins e.g., stop- transfer membrane anchor sequences, function to orient the protein within the membrane.
  • the protein traverses the ER membrane in an unfolded state.
  • the newly synthesized proteins can undergo additional maturation modifications in the ER lumen, including formation of disulfide bonds and proper folding of the protein, formation into oligomers, and addition and modification of carbohydrates.
  • Disulfide bonding stabilizes the tertiary structure of proteins, and is important for proper maturation and activity of the protein.
  • Formation of multi-chain oligomeric proteins from their subunit constituents also occurs in the ER. Polypeptides that are misfolded are prevented from moving out of the ER and along their normal maturation pathway; such proteins either accumulate or are degraded in the ER via an active degradative pathway (Stafford and Bonifacino, 1991, J. Cell Biol.
  • glycosylation of proteins can be classified as O-linked (linked to the hydroxyl group oxygen of serine, threonine, and in collagen, hydroxylysine) and N-linked (linked to the amide nitrogen of asparagine) .
  • Glycosyltransferases are enzymes that catalyze the transfer of sugar to newly synthesized proteins; a different type of glycosyltransferase catalyzes the addition of different sugars. All known glycosyltransferases are integral membrane proteins with their active sites within the lumen of the ER or Golgi, where sugar transfer thus occurs.
  • N-linked oligosaccharides are synthesized from a common precursor in the ER.
  • the complete branched oligosaccharide consisting of three glucose, nine mannose, and two N-acetylglucosamine molecules, is transferre ⁇ by the enzyme oligosaccharyltransferase from oligosaccharylpyrophosphoryldolichol to an asparagine residue in an -Asn-X-Ser/Thr- acceptor site (where X is any amino acid except proline) on the nascent protein (Czichi et al., 1977, J. Biol. Chem. 252:7901-7904; Hart et al., 1979, J.
  • Oligosaccharyltransferase is a luminally oriented integral membrane protein of the ER, and the glycosylated protein formed by transfer of the oligosaccharide is sequestered within the endoplasmic reticulum (Hanover and Lennarz, 1980, J. Biol. Chem. 255:3600-3604).
  • An in vitro study has shown that amino-ter inal derivatives of Asn-Leu-Thr can act as substrates for oligosaccharyltransferase, while asparagine derivatives of the tripeptide were inactive as substrates or inhibitors of the enzyme ( elply et al., 1983, J. Biol. Chem.
  • oligosaccharide Immediately after transfer of the oligosaccharide to the protein, catalyzed by oligosaccharyltransferase, certain sugar residues are removed by different enzymes. Further processing of the N-linked oligosaccharide, to the high-mannose or complex form, is completed in the Golgi vesicles.
  • the glycoprotein is transported via transport vesicles from the cis Golgi to the trans Golgi to the trans Golgi reticulum, from where it is sorted to lysosomes or to transport vesicles, or secretory vesicles which eventually fuse with the plasma membrane.
  • the present invention relates to conjugated peptide derivatives containing an acceptor site for glycosylation by oligosaccharyltransferase, and the use of such peptide derivatives for concentration of active agents in the lumen of the endoplasmic reticulum (ER) .
  • Therapeutic methods and compositions based on such delivery are also provided.
  • the conjugates of the invention comprise (a) an amino- terminal blocked derivative of the tripeptide Asn-X-Y, in which X is any amino acid except Pro, and Y is Ser or Thr; and (b) an active agent conjugated to the derivative.
  • the conjugates are capable of being glycosylated by oligosaccharyl transferase, and are permeable to cell membranes until glycosylated by oligosaccharyltransferase in the lumen of the ER.
  • the active agent in the conjugate of the invention is a thiol-oxidizing agent, e.g. , a diazene dicarbonyl compound.
  • the active agent in the conjugate is a sulfhydryl-alkylating agent such as maleimide or a maleimide derivative.
  • the invention provides methods of treating cystic fibrosis, by administering a conjugate of the invention.
  • the present invention relates to conjugated peptide derivatives containing an acceptor site for glycosylation by oligosaccharyltransferase, and the use of such peptide derivatives for concentration of active agents in the lumen of the endoplasmic reticulum (ER) .
  • Therapeutic methods and compositions based on such delivery are also provided.
  • the conjugates of the invention comprise (a) an amino- terminal blocked derivative of the tripeptide Asn-X-Y, in which X is any amino acid except Pro, and Y is Ser or Thr, which derivative is an acceptor substrate for glycosylation by oligosaccharyltransferase; and (b) an active agent conjugated to the tripeptide derivative.
  • Such conjugation is preferably by direct derivatization of the tripeptide derivative with the active agent.
  • an active agent exerts a biological effect by virtue of its interaction with other molecule(s) in the lumen of the ER, by chemical reaction or by noncovalent or covalent binding with such molecule(s).
  • the conjugate is biocompatible (nontoxic and not highly immunogenic) .
  • the conjugate is permeable to cell membranes until glycosylated by oligosaccharyltransferase within the lumen of the ER, at which point it becomes impermeable to cell membranes and thus localized within the ER lumen.
  • the active agents present in the conjugates of the invention are compounds which, by performing their activity in the lumen of the ER, exert a therapeutic effect. It also follows that the active agent is not an inhibitor of oligosaccharyltransferase.
  • the tripeptide derivatives in the conjugates of the invention are substrates of oligosaccharyltransferase that contain an acceptor site for glycosylation by the enzyme.
  • the conjugates of the invention are preferably such tripeptide derivatives which have incorporated into their structure an active agent.
  • the active agent is a thiol-oxidizing agent.
  • the active agent is a sulfhydryl- alkylating agent, such as a maleimide or a maleimide derivative.
  • the thiol-oxidizing agents are preferably diazene dicarbonyl compounds, containing the following structural feature:
  • the active agent is an agent that prevents the abnormal misfolding, assembly or increased levels of degradation in the ER lumen of a defective lysosomal or secretory or cell membrane protein associated with a disease or disorder, thus allowing the protein to continue along its normal maturation pathway to secretion or to the plasma membrane or a lysosome.
  • an agent is an oxidizing agent.
  • the tripeptide derivatives in the conjugates of the invention are N-blocked derivatives of the tripeptide Asn-X-Y (written in the amino- to carboxy- terminal direction) , in which X is any amino acid except Pro, and Y is Ser. or Thr.
  • the amino terminus is derivatized, preferably to contain a lipophilic group, since unblocked tripeptides are not acceptors of glycosylation by oligosaccharyltransferase (Hart et al. , 1979, J. Biol. Chem. 254:9747-9753).
  • the side chain group of Asn should not be derivatized since such derivatization abolishes oligosaccharide acceptor activity ( elply et al., 1983, J. Biol. Chem. 258:11856-11863).
  • the tripeptide can also be derivatized at its C-terminus by groups including, but not limited to, amino and alkylamino groups. Tripeptide derivatives with N- or C-terminal groups that increase lipophilicity of the* molecule are preferred.
  • R is H or methyl.
  • R l , R 2 , R 3 , and R 4 can be an active agent, to form a conjugate of the invention.
  • the active agent is R 1 , R 1 being a diazene dicarbonyl compound of structure
  • R 5 -C-N N-C-
  • R S -C-N N-C-NH-(CH 2 ) n -C- in which R 5 is a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl; a mono- or di-substituted amino; or an alkoxy, aryloxy, or aralkoxy; and n is an integer of 1 or more.
  • R 2 is H or a lower alkyl
  • R 3 is the side chain of any natural amino acid, or a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl
  • R 4 is OH
  • R 1 and R 2 together form a ring so that the conjugate has the following structure:
  • R 3 has the following structure:
  • n is an integer, of 1 or more, preferably in the range of 1-4;
  • R 6 is H or a lower alkyl;
  • R 7 is a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl, a mono- or di- substituted amino, or an alkoxy, aryloxy, or aralkoxy; or R 6 and R 7 can together form a ring structure.
  • R 1 is an acyl group, including but not limited to acetyl, alkanoyl (e.g., octanoyl) , benzoyl, benzyloxycarbonyl, tert-butoxycarbonyl, and the like.
  • the acyl group is preferably in the range of C 2 to C 10 , most preferably C 2 to C 8 .
  • R 2 is H.
  • R 1 and R 2 can together form a ring structure, e.g., such that
  • R 4 is OH, NH 2 , a mono- or di- substituted amino, or alkoxy or arylkoxy.
  • R 4 has the following structure:
  • R 8 and R 9 are each independently H, a lower alkyl, aryl, or aralkyl;
  • R 10 is a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl, mono- or di-substituted amino, or an alkoxy, aryloxy, or aralkoxy; or
  • R 8 and R 9 can together form a ring structure; or R 9 and R 10 can together form a ring structure, As one example where R* and R ⁇ u form a ring:
  • R 1 is an acyl group
  • R 2 is H
  • R 1 and R 2 can together form a ring structure
  • R 3 is the side chain of a natural amino acid, or a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl.
  • the active agent in compound (I) is a maleimide derivative formed of R 1 and R 2
  • the compound has the structure of (III) or (IV) :
  • R n and R 12 are each independently H or a lower alkyl
  • R 3 is the side chain of a natural amino acid, or a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl
  • R 4 is OH, NH 2 , a mono- or di-substituted amino, or alkoxy or aralkoxy
  • n is an integer of 1 or more.
  • R 3 in compound (I) is a maleimide derivative
  • R 3 has the following structure:
  • n is an integer of 1 or more, preferably in the range of 1-4 , and R 11 and R 12 are each independently H or a lower alkyl.
  • R l is an acyl group;
  • R 2 is H; or
  • R 1 and R 2 together form a ring structure;
  • R 4 is OH, NH 2 , a mono- or di-substituted amino, or alkoxy, or aralkoxy.
  • R 4 in compound (I) is a maleimide derivative
  • R 4 has the following structure:
  • n is an integer of 1 or more, preferably in the range of 1-4;
  • R 13 is H, alkyl, aryl, or aralkyl;
  • R u and R 12 are each independently H or a lower alkyl;
  • R l is an acyl group;
  • R 2 is H; or
  • R 1 and R 2 together form a ring structure;
  • R 3 is the side chain of a natural amino thacid, or a straight chain or branched, substituted or unsubstituted, alkyl, aryl, or aralkyl.
  • R 1 or R 2 is not the active agent
  • R 2 is H
  • R l is an acyl group, in the range of C 2 to C, 0 , preferably C-_ to C g , including but not limited to, one of the following:
  • R 3 is not the active agent, preferably R 3 is the side chain of any natural amino acid, and is most preferably
  • R 4 is preferably NH 2 .
  • active agents can be used, as will be known to one skilled in the art.
  • the selection of an active agent will depend on the /5 therapeutic effect it is desired to achieve (see Section 4.4, infra) .
  • any peptide portion of the conjugate can be prepared in brief, by solid phase peptide synthesis which consists of coupling the carboxyl group of the C-terminal amino acid to a resin and successively adding N-alpha-protected amino acids.
  • the protecting groups may be any known in the art or those described infra. Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed.
  • the coupling of amino acids to appropriate resins is described by Rivier et al., U.S. Patent No. 4,244,946.
  • Such solid phase syntheses have been described, for example, by Merrifield, 1964, J. Am. Chem. Soc. 85:2149; Vale et al., 1981, Science 213:1394-1397;
  • tripeptide derivatives can be synthesized as described ( elply et al., 1983, J. Biol. Chem. 258:11856-11863), using various protected amino acids and either the mixed anhydride procedure (Anderson et al., 1967, J. Am. Chem. Soc. 89:5012- 5017) or the p-nitrophenyl ester method (Bodanski, M. , 1979, in The Peptides: Analysis. Synthesis, Biolo ⁇ v. Gross and Meienhofer, eds.. Vol. 1, Academic Press, New York, pp. 105-196) .
  • peptides are synthesized as described in Section 6, infra, prior to derivatization with the active agent.
  • the active agent is preferably conjugated directly to the tripeptide derivative, i.e., the i f tripeptide derivative is derivatized by the active agent, rather than through a polyfunctional linker molecule.
  • any available reactive group on the molecule is used, or else one can be incorporated into the tripeptide derivative or active agent, for use in conjugating the active agent and tripeptide derivative.
  • the term "reactive group” refers to a functional group that can react with a second functional group so as to form a covalent bond between the active agent and tripeptide derivative.
  • the term "functional group” retains its standard meaning in organic chemistry. Typical functional groups are thiol groups and amino groups.
  • Protecting groups for use in synthesis can be any of the large number of protecting groups known in the art.
  • an acetyl group can be added to a free amino group by treatment with acetic anhydride.
  • a carbobenzoxy group can be added by treatment with carbobenzoxy chloride.
  • Other N-protecting groups that are useful include the formyl, L-butoxycarbonyl, trifluoroacetyl, tosyl, p- nitrocarbobenzoxy, cyclopentyloxycarbonyl, and phenoxycarbonyl groups.
  • the active agent is a diazene dicarbonyl compound
  • the following compounds for use in the synthesis of the conjugates of the invention can be obtained or synthesized as described below:
  • Maleimides can be synthesized by methods known in the art (see, e.g., U.S. Patent No. 4,623,734 granted November 18, 1986 by Kita et al.) or purchased from a commercial vendor.
  • a conjugate of the present invention having the formula (V) in which the active agent comprises a diazene dicarbonyl oxidizing agent can be prepared by the method described below.
  • the groups R and R 3 have the meanings described previously, above.
  • a tripeptide having a primary amino group such as compound (VI) , illustrated below, is allowed to react with an
  • a diazene dicarboxylic acid ester such as dimethyl azodicarboxylate or diethyl azodicarboxylate
  • an inert solvent at a reaction temperature ranging from about 0°C to about room temperature.
  • the inert solvent is dioxane.
  • the diazene monoester (VII) is then allowed to react with an excess amount of a preselected nucleophile, Nu, such as ammonia, dimethylamine, piperidine, pyrrolidine and the like, in an inert solvent, at a reaction temperature ranging from about 0°C to about room temperature.
  • Nu such as ammonia, dimethylamine, piperidine, pyrrolidine and the like
  • the inert solvent is dioxane.
  • the product, which is the therapeutic oxidizing agent of the formula (V) is then isolated using standard methods well known in the art. kf
  • the therapeutic conjugate of the formula (VIII) in which R can be an H or methyl and n can have the values described previously, above (e.g., or 4) , can be prepared by the following method.
  • a tripeptide having a primary amino group such as compound (IX) , illustrated below, is allowed to react with an
  • diazene dicarboxylic acid ester such as dimethyl azodicarboxylate or diethyl azodicarboxylate
  • inert solvent at a reaction temperature ranging from about 0°C to about room temperature.
  • the inert solvent is dioxane.
  • the diazene monoester (X) is then allowed to react with an excess amount of a preselected nucleophile, such as ammonia, dimethylamine, piperidine, pyrrolidine and the like, in an inert solvent, at a reaction temperature ranging from about 0°C to about room temperature.
  • a preselected nucleophile such as ammonia, dimethylamine, piperidine, pyrrolidine and the like
  • the inert solvent is dioxane.
  • the product, which is the therapeutic oxidizing agent of the formula (VIII) is then isolated using standard methods well known in the art.
  • the conjugate is made as set forth in Section 5.3 hereof, it is preferably tested in vitro to ensure that it is permeable to cell membranes and can act as an acceptor for glycosylation by oligosaccharyltransferase.
  • Such assays can be carried out by any method known in the art. In preferred aspects, the assay is carried out by exposing intact cells or rough microsomes to the conjugate, and detecting glycosylation of the conjugate within the lumen of the ER. Such assays can be carried out as described in Section 7, infra (see also Welply et al., 1983, J. Biol. Chem.
  • Rough microsomes are small closed vesicles formed by fragments of the rough ER produced upon homogenization of cells; microsomes have the same orientation (ribosomes on the outside of the vesicles) as that of the ER within the cell (Darnell et al., 1990, Molecular Cell Biology. 2d Ed., .H. Freeman & Co. , New York, p. 646).
  • the conjugates of the invention can be administered therapeutically, where a therapeutic effect is mediated by the active agent upon concentration in the ER lumen by virtue of the ability of the conjugate to be glycosylated therein by oligosaccharyltransferase. Such glycosylation renders the conjugate impermeable to cell membranes such that the active agent is thereby concentrated in the ER lumen, where it performs its chemical activity(ies) or binding.
  • the therapeutic methods of the invention are carried out by administration to a subject of an effective amount of the conjugates of the invention.
  • the subject is preferably a mammal, including but not limited to animals such as cows, pigs, etc., and is most preferably human. Methods for prevention of disorders, by administering a therapeutic conjugate of the invention, are also provided.
  • the active agent is an oxidizing agent
  • the conjugate is administered to a patient for treatment of a disorder involving a genetically mutated lysosomal or secretory or plasma, ER or Golgi membrane protein.
  • a disorder involving a genetically mutated lysosomal or secretory or plasma, ER or Golgi membrane protein e.g., a genetically mutated lysosomal or secretory or plasma, ER or Golgi membrane protein.
  • cystic fibrosis is associated with a mutation in the transmembrane protein CFTR.
  • the major genetic cause of emphysema and difficulty in breathing is due to a mutation in the secretory protein ⁇ ,-antiprotease ( ⁇ ,- antitrypsin) (Darnell et al. , 1990, Molecular Ceil Biology. 2d Ed., .H. Freeman & Co., New York, p. OS
  • Tay-Sachs disease is caused by a defect in the lysosomal enzyme beta-N-hexosaminidase A (id., p. 671) .
  • Other lysosomal storage diseases are caused by defective lysosomal enzymes.
  • Insulin receptor deficiency results from a mutant (plasma membrane) insulin receptor while familial hypercholesterolemia results from a mutant LDL (low density lipoprotein) (plasma membrane) receptor.
  • Hunter's syndrome and Hurler's syndrome are caused by genetic defects in the lysosomal enzymes which catabolize sulfated mucopolysaccharides (Darnell et al., supra at p. 671).
  • the active agent is a diazene dicarbonyl compound (I) such as diamide, or a thiol-oxidizing agent such as a maleimide derivative, and the conjugate of the invention is administered to treat cystic fibrosis.
  • CFTR cystic fibrosis transmembrane conductance regulator
  • CFTR is an integral membrane protein that appears to act as a chloride channel (Anderson et al., 1991, Cell 67:775- 784; Rich et al., 1990, Nature 347:358-363; Drumm et al., 1990, Cell 62:1277-1233).
  • the present invention provides for treatment of cystic fibrosis by exposure of mutant CFTR in the lumen of the ER, to the oxidizing or alkylating agent that is the active agent in the conjugate of the invention.
  • the oxidizing or alkylating agent inhibits degradation and/or promotes the correct folding/assembly in the ER lumen of the mutant CFTR that otherwise *1would be abnormally processed or degraded and never reach the plasma membrane, thus achieving proper processing of CFTR to the cell membrane.
  • the conjugate is administered so as to allow, or preferably target, delivery to the in vivo cellular location of CFTR, (Crawford et al. , 1991, Proc. Natl. Acad. Sci. USA 88:9262-9266), namely epithelial cells, such as. those lining sweat ducts, small pancreatic ducts, and intestinal crypts, and in the kidney, and in the lung.
  • Suitable in vitro and n vivo assays can be used to demonstrate therapeutic utility of the conjugates of the invention.
  • any animal model system known in the art may be used prior to administration to humans.
  • An animal model system for rheumatoid arthritis is that consisting of animals of the autoimmune MRL/1 mouse strain (Murphy, E.D. and Roths, J.B., 1978, in Genetic Control of Autoimmune Disease. Rose, N.R. , et al., eds. , Elsevier/North-Holland, New York, pp. 207-219) , that develop a spontaneous rheumatoid arthritis-like disease (Hang et al. , 1982, J. Exp. Med. 155:1690-1701).
  • ком ⁇ онент ком ⁇ онент ⁇ THERAPEUTIC ADMINISTRATION AND COMPOSITIONS
  • Various delivery systems are known and can be used to administer the conjugates of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, etc.
  • encapsulation in liposomes or other type of lipid layer is preferred.
  • Other methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes.
  • the compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active compounds.
  • epithelial or mucocutaneous linings e.g., oral mucosa, rectal and intestinal mucosa, etc.
  • compositions comprise a therapeutically effective amount of a compound of the invention, and a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • the conjugate of the invention is formulated as an inhalant.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the conjugates of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.
  • the amount of the conjugate of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
  • the invention also provides a pharmaceutical pack comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Tripeptide derivatives of the form N-acyl- Asn-Tyr-Thr-NH 2 are synthesized as follows (Wieland, et al., 1987, Cell 50:289-300):
  • the extract is dried by lyophilization, and the peptides are further purified by high performance liquid chromatography (HPLC) .
  • HPLC high performance liquid chromatography
  • the following conditions are used: 5% acetonitrile in 0.1% trifluoroacetic acid (TFA) for 10 min after injection; thereafter, a linear gradient of 5%-65% acetonitrile in a 0.1% is applied at 1 ml/min.
  • the NH 2 -Asn-Tyr-Thr-NH 2 eluted at about 21% acetonitrile.
  • a reversed phase RP18 column (Lichrosorb, Merck) is used with a Beckman controller and delivery system, and the effluent is monitored in a flow cell at 280 nm.
  • the purity of the resulting peptides is confirmed by amino acid analysis after acid hydrolysis (8 N HCl, 18 hr, 105°C) .
  • Acetylation is performed with the resin-linked, boc-protected peptides according to Stewart and Young (1984, Solid Peptide Synthesis, 2d Ed., Pierce Chemical Co.,
  • the samples After addition of 5 vol of 70% methanol, the samples are treated with an equimolar amount of NaOH for 20 min at 37°C. Thereafter they are purified by chromatography on AG 1 x 8 ion exchange resin (acetate form) in 70% methanol. The eluants are chromatographed on Dowex AG 50 (H + form) in 70% methanol, and the flow-throughs are lyophilized in a
  • the N-acetyl derivative elutes at about 23% acetonitrile, the N-octyl derivative at about 35% acetonitrile.
  • Thr-NH 2 is synthesized as follows (Welply et al., 1983,
  • N ⁇ -Boc-Asn-Leu-Thr-NH-, — Boc-Leu-Thr-NH 2 (0.93 g. 2.8 mmol) is dissolved in CH 2 C1 2 - x j* trifluoroacetic acid (15 ml; 1:1, v/v), and the solution is let stand at room temperature for 30 min. The solvent is then evaporated j-n vacuo. and the residue is precipitated by the addition of ether. The trifluoroacetate salt is isolated and dried. It is then coupled with Boc-Asn-ONp (0.99 g, 2.8 mmol) in the presence of NMM (0.31 ml, 2.8 M) in DMF (5 ml).
  • N°-Ac-Asn-Leu-Thr-NH 2 The Boc-group from Boc-Asn-Leu-Thr-NH 2 (178 g, 0.4 mmol) is cleaved as described above. The resulting CF 3 COOH-Asn-Leu-Thr-NH 2 peptide is then coupled with p-nitrophenyl acetate (72 mg, 0.44 mmol) in the presence of NMM (0.044 ml, 0.4 mM) in DMF (1 ml) . After a reaction time of 20 h, ether (50 ml) is added to the mixture. The solid obtained is filtered, and washed with ether.
  • the compounds to be assayed for its ability to permeate cell membranes and be glycosylated by an oligosaccharyltransferase is labeled with 125 I.
  • the compound contains a tyrosine, the following procedure can be used (Wieland et al., supra) :
  • Iodination of the Acceptor Compound Up to 50 nmol of tyrosine containing compound in 50 ⁇ l of acetonitrile is added to 100 ⁇ l of 0.5 M NaP* (pH 7.5). Between 0.5 and 10 mCi of [ 125 I] Nal (carrier-free, ICN) is added. To this solution, 100 ⁇ l of chloramine T (Sigma) (2 mg/ml) in 0.05 M NaP* (pH 7.5) is added. After 2 min at room temperature, the reaction is stopped by addition of 400 ⁇ l of a solution of sodium bisulfite (2.4 mg/ml) in 0.05 M NaP, (pH 7.5).
  • CHO Cells or HepG2 cells are used. Wild-type CHO cells are grown in suspension cultures in ⁇ MEM (GIBCO) as described (Balch et al. , 1984, Cell 39:405-416). CHO clone 15B cells are grown in monolayer.
  • HepG2 cells are grown as described (Strous and Lodish, 1980, Cell 22:709-717). Media are usually changed the day after passing, and the cells are used 3 days later. Incubation of Cells With the Compound to be
  • CHO cells (2 x 10 7 total cells) are washed once with buffer B (25 mM Tris-HCl [pH 7.4], 137 mM NaCl, 5 mM KC1, 0.7 mM Na 2 HP0 4 ) and then resuspended in growth medium without serum (that is additionally buffered with 20 mM HEPES [pH 7.4]) at a density of 1 x 10 7 cells per ml. Then cycloheximide (100 ⁇ g/ml) is added.
  • buffer B 25 mM Tris-HCl [pH 7.4], 137 mM NaCl, 5 mM KC1, 0.7 mM Na 2 HP0 4
  • buffer B 25 mM Tris-HCl [pH 7.4], 137 mM NaCl, 5 mM KC1, 0.7 mM Na 2 HP0 4
  • resuspended in growth medium without serum that is additionally buffered with 20 mM HEPES [p
  • the 12S I compound is added from a stock solution in DMSO (not exceeding a final concentration of 1% DMSO) . Typically, between 5 and 50 ⁇ Ci is added per ml of suspension. Incubation is at 37°C with gentle stirring. Aliquots of 200 ⁇ l are removed after one hour and immediately chilled on ice and centrifuged in the cold in a Eppendorf centrifuge for 1 min. The supernatant media are separated from the cell pellets.
  • Each cell pellet is extracted with 200 ⁇ l of buffer A (10 mM Tris-HCl [pH 7.4], 0.15 M NaCl, l mM CaCl 2 , l mM MnCl 2 , 0.5% Triton X-100) , and the supernatant after centrifugation in the microfuge for 1 min can be saved for further analysis.
  • the media is made 0.5% in Triton X-100, and 1 mM MnCl 2 and 1 mM CaCl 2 are added from stock solutions.
  • CHO cells and HepG2 cells are incubated for 1 or 5 hr at 37°c or 32°C, respectively, in the presence of 10 ⁇ g/ml tunicamycin (Sigma) .
  • the tunicamycin stock solution used is 1 mg/ml in 250 mM NaOH.
  • I-glycocompounds intended for further analysis are prepared similarly, but after the five washes with buffer A, five washes are performed with 1 ml each of buffer A without Triton X-100. Elution with ⁇ - methylmannoside is without Triton X-100 as well.
  • Oviduct Microsome Preparation The magnum portion of freshly killed laying hens is freed from connective tissue, minced, homogenized, and centrifuged as described by Pless and Lennarz (1977, Proc. Natl. Acad. Sci. USA 74:134-138). Prior to use, microsomes are stored at -70°C at a concentration of -30 mg protein/ml.
  • the oligosaccharyl ⁇ transferase assay can be carried out using newly synthesized oligosaccharide-lipid (see Welply et al., supra) or by quantitating the incorporation of labeled (radioactive) sugars into non-labeled acceptor substrates (Hart et al., 1979, J. Biol. Chem. 254:9747-9753; Hanover and Lennarz, 1990, J. Biol. Chem. 255:3600-3604).

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Abstract

L'invention se rapporte à des dérivés conjugués de peptides contenant un site d'acceptation de glycosylation par transférase d'oligosaccharyle, ainsi qu'à l'utilisation desdits dérivés de peptides dans l'administration d'agents actifs au lumen du réticulum endoplasmique. L'invention décrit également des procédés et des compositions thérapeutiques à base de l'administration desdits agents. Les conjugués décrits par l'invention comprennent: (a) un dérivé de tripeptide de formule R1-Àsn-X-Y dans laquelle X représente tout acide aminé sauf Pro et Y est Ser ou Thr ou un de leur dérivé et R1 représente un groupe à terminaison N ne représentant pas un ou plusieurs acides aminés; (b) un agent actif conjugué au dérivé de tripeptide. Lesdits conjugués peuvent être glycosylés par transférase d'oligosaccharyle et sont perméables à des membranes cellulaires jusqu'à leur glycosylation par transférase d'oligosaccharyle dans le lumen du réticulum endoplasmique. Dans un mode de réalisation spécifique de l'invention, l'agent actif du conjugué est un agent oxydant, par exemple, un dicarbonyle de diazène présentant une liaison covalente à l'amiloride. Dans un autre mode de réalisation, l'invention décrit des procédés de traitement de fibroses cystiques au moyen de l'administration d'un conjugué décrit par l'invention dans lequel l'agent actif est un agent oxydant.
PCT/US1993/000043 1992-01-06 1993-01-05 Derives conjugues de peptides contenant un site d'acceptation de glycosylation et utilisations therapeutiques Ceased WO1993013788A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995005810A1 (fr) * 1993-08-26 1995-03-02 National Research Council Of Canada Compositions et procedes de detection et de traitement des troubles des echanges entre proteines et d'accroissement des secretions proteiniques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 252, No. 22, issued 25 November 1977, CZICHI et al., "Localization of the Enzyme System for Glycosylation of Proteins Via the Lipid-Linked Pathway in Rough Endoplasmic Reticulum", pages 7901-7904. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 254, No. 19, issued 10 October 1979, HART et al., "Primary Structural Requirements for the Enzymatic Formation of the N-Glucosidic Bond in Glycoproteins", pages 9747-9753. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995005810A1 (fr) * 1993-08-26 1995-03-02 National Research Council Of Canada Compositions et procedes de detection et de traitement des troubles des echanges entre proteines et d'accroissement des secretions proteiniques
US5691306A (en) * 1993-08-26 1997-11-25 National Research Council Of Canada Methods of detection and treatment of protein trafficking disorders and increasing secretory protein production

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