WO2012054822A1 - Conjugués polymère-glp 1 pharmacologiquement actifs - Google Patents

Conjugués polymère-glp 1 pharmacologiquement actifs Download PDF

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WO2012054822A1
WO2012054822A1 PCT/US2011/057267 US2011057267W WO2012054822A1 WO 2012054822 A1 WO2012054822 A1 WO 2012054822A1 US 2011057267 W US2011057267 W US 2011057267W WO 2012054822 A1 WO2012054822 A1 WO 2012054822A1
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glp
composition
polymer
moiety
water
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PCT/US2011/057267
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Mary J. Bossard
Dennis G. Fry
Xiaofeng Liu
John Zhang
Steven O. Roczniak
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Nektar Therapeutics
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    • 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

  • the present invention relates generally to
  • Glucagon-like peptide- 1 is a peptide proteolytically processed from pro-glucagon and is secreted from intestinal L cells. GLP-1 indirectly controls blood glucose levels by regulating insulin sensitivity and production, decreasing food intake, and restoring pancreatic beta cell functions.
  • the biological functions of GLP-1 have therapeutic benefit for the treatment of diabetes and numerous variants or formulations of the peptide have been approved for clinical use or are in late-stage clinical trials.
  • the in vivo circulation half-life of the peptide is very short, typically only a few minutes, and most efforts to improve the therapeutic properties of GLP-1 have focused on increasing its in vivo half-life.
  • GLP-1 conjugate Covalently attaching one or more water-soluble polymers to GLP-1 to thereby form a GLP-1 conjugate represents one approach proposed to increase the in vivo half-life of this peptide.
  • Various GLP-1 conjugates are described in the literature and in, for example, International Publication Nos. WO 99/43707, WO 2004/093823, WO 2007/075534 and WO 2010/033207 and U.S. Patent Application Publication Nos. 2005/0009988 and
  • a pharmaceutical comprising a dose of polymer-GLP-1 conjugates and a pharmaceutically acceptable excipient, wherein (i) each conjugate in the dose of polymer-GLP-1 conjugates comprises a water-soluble, non-peptidic polymer having a molecular weight of greater than 5,000 Daltons covalently attached through an
  • the pharmaceutical composition upon administration to a mammal, has a glucose-lowering effect
  • the dose of polymer-GLP-1 conjugates achieving the glucose-lowering effect is less than an amount of the GLP-1 moiety in an unconjugated form required to achieve the glucose lowering effect.
  • a polymer-GLP-1 conjugate is provided, wherein— in an in vivo model of unfed db/db mice—the polymer-GLP-1 conjugate provides a glucose level at that is at least 50% lower at four hours following intraperitoneal administration than an equimolar amount of the GLP-1 in unconjugated form.
  • a method comprising administering to a patient a dose of polymer-GLP-1 conjugates, wherein following administration, the dose of polymer-GLP-1 conjugates provides at least a 25% lower glucose level after eight hours compared to the expected glucose level at that time absent administration of the therapeutically effective dose of pharmacologically active GLP-1 conjugates.
  • a method comprising administering to a patient a dose of polymer-GLP-1 conjugates, wherein following administration, the dose of polymer-GLP-1 conjugates provides preprandial capillary plasma glucose within the range of 90- 180 mg/dl.
  • FIG. 1 is a typical SP-HP cation exchange purification profile of
  • FIG. 2 is a reverse phase HPLC chromatogram of a purified composition of
  • FIG. 3 is a typical SP-HP cation exchange purification profile of
  • FIG. 4 is a reverse phase HPLC chromatogram of a purified composition of
  • FIG. 5 is a typical SP-HP cation exchange purification profile of PEG-30K-
  • FIG. 6 is a reverse phase HPLC chromatogram of a purified composition of
  • FIG. 7 is a plot of the in vivo glucose lowering activity of saline, 75 ⁇ g of GLP
  • an and “the” include plural referents unless the context clearly dictates otherwise.
  • reference to “a polymer” includes a single polymer as well as two or more of the same or different polymers; reference to “an optional excipient” or to “a pharmaceutically acceptable excipient” refers to a single optional excipient as well as two or more of the same or different optional excipients, and the like.
  • peptide refers to polymers comprised of amino acid monomers linked by amide bonds.
  • peptide refers to polymers comprised of amino acid monomers linked by amide bonds.
  • Peptides may include the standard 20 a-amino acids that are used in protein synthesis by cells (i.e. natural amino acids), as well as non-natural amino acids (non-natural amino acids may be found in nature, but not used in protein synthesis by cells, e.g., ornithine, citrulline, and sarcosine, or may be chemically synthesized), amino acid analogs, and peptidomimetics.
  • the amino acids may be D- or L-optical isomers.
  • Peptides may be formed by a condensation or coupling reaction between the a-carbon carboxyl group of one amino acid and the amino group of another amino acid.
  • the terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other end of the chain (carboxy terminal) has a free carboxyl group.
  • the peptides may be non-linear, branched peptides or cyclic peptides.
  • the peptides may optionally be modified or protected with a variety of functional groups or protecting groups, including on the amino and/or carboxy terminus.
  • Leucine is Leu or L; Isoleucine is He or I; Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gin or Q; Asparagine is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine is Cys or C;
  • Tryptophan is Tip or W; Arginine is Arg or R; and Glycine is Gly or G.
  • therapeutic peptide fragment refers to a peptide that comprises a truncation at the amino-terminus and/or a truncation at the carboxyl-terminus of a therapeutic peptide as defined herein.
  • therapeutic peptide fragment or “fragments of therapeutic peptides” also encompasses amino-terminal and/or carboxyl-terminal truncations of therapeutic peptide variants and therapeutic peptide derivatives.
  • Therapeutic peptide fragments may be produced by synthetic techniques known in the art or may arise from in vivo protease activity on longer peptide sequences. It will be understood that therapeutic peptide fragments retain some or all of the therapeutic activities of the therapeutic peptides.
  • therapeutic peptide variants or “variants of therapeutic peptides” refer to therapeutic peptides having one or more amino acid
  • substitutions including conservative substitutions and non-conservative substitutions, amino acid deletions (either internal deletions and/or C- and/or N- terminal truncations), amino acid additions (either internal additions and/or C- and/or N- terminal additions, e.g., fusion peptides), or any combination thereof.
  • Variants may be naturally occurring (e.g. homologs or orthologs), or non-natural in origin.
  • the term "therapeutic peptide variants" may also be used to refer to therapeutic peptides incorporating one or more non-natural amino acids, amino acid analogs, and peptidomimetics.
  • therapeutic peptide fragments retain some or all of the therapeutic activities of the therapeutic peptides.
  • PEG polyethylene glycol
  • poly(ethylene glycol) as used herein, are interchangeable and encompass any non-peptidic water-soluble poly(ethylene oxide).
  • PEGs for use in accordance with the invention comprise the following structure M -(OCH 2 CH 2 )n-" where (n) is 2 to 4000.
  • PEG also includes
  • PEG poly(ethylene glycol)
  • M -(OCH 2 CH 2 ) n O-
  • PEG poly(ethylene glycol)
  • the term “PEG” includes structures having various terminal or “end capping” groups and so forth.
  • the term “PEG” also means a polymer that contains a majority, that is to say, greater than 50%, of -OCH 2 CH 2 - repeating subunits.
  • the PEG can take any number of a variety of molecular weights, as well as structures or geometries such as “branched,” “linear,” “forked,” “multifunctional,” and the like, to be described in greater detail below.
  • end-capped and “terminally capped” are interchangeably used herein to refer to a terminal or endpoint of a polymer having an end-capping moiety.
  • the end-capping moiety comprises a hydroxy or Ci_ 20 alkoxy group, more preferably a CMO alkoxy group, and still more preferably a C 1-5 alkoxy group.
  • examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxy and benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and the like. It must be
  • the end-capping moiety may include one or more atoms of the terminal monomer in the polymer [e.g., the end-capping moiety "methoxy" in CH 3 0(CH 2 CH 2 0) n - and CH 3 (OCH 2 CH 2 ) n -].
  • the end-capping group can also be a silane.
  • the end-capping group can also advantageously comprise a detectable label.
  • the amount or location of the polymer and/or the moiety (e.g., active agent) to which the polymer is coupled can be determined by using a suitable detector.
  • suitable detectors include photometers, films, spectrometers, and the like.
  • phospholipids include, without limitation, those selected from the class of phospholipids called phosphatidylcholines.
  • Specific phospholipids include, without limitation, those selected from the group consisting of dilauroylphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine, behenoylphosphatidylcholine, arachidoylphosphatidylcholine, and lecithin.
  • Non-naturally occurring with respect to a polymer as described herein, means a polymer that in its entirety is not found in nature.
  • a non-naturally occurring polymer may contain one or more monomers or segments of monomers that are naturally occurring, so long as the overall polymer structure is not found in nature.
  • water soluble as in a “water-soluble polymer” is any polymer that is soluble in water at room temperature. Typically, a water-soluble polymer will transmit at least about 75%, more preferably at least about 95%, of light transmitted by the same solution after filtering. On a weight basis, a water-soluble polymer will preferably be at least about 35% (by weight) soluble in water, more preferably at least about 50% (by weight) soluble in water, still more preferably about 70% (by weight) soluble in water, and still more preferably about 85% (by weight) soluble in water. It is most preferred, however, that the water-soluble polymer is about 95% (by weight) soluble in water or completely soluble in water.
  • Molecular weight in the context of a water-soluble polymer can be expressed as either a number average molecular weight or a weight average molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the weight average molecular weight. Both molecular weight determinations, number average and weight average, can be measured using gel permeation chromatography or other liquid chromatography techniques.
  • the polymers of the invention are typically polydisperse (i.e., number average molecular weight and weight average molecular weight of the polymers are not equal), possessing low polydispersity values of preferably less than about 1.2, more preferably less than about 1.15, still more preferably less than about 1.10, yet still more preferably less than about 1.05, and most preferably less than about 1.03.
  • active when used in conjunction with a particular functional group refers to a reactive functional group that reacts readily with an electrophile or a nucleophile on another molecule. This is in contrast to those groups that require strong catalysts or highly impractical reaction conditions in order to react (i.e., a "non-reactive” or “inert” group).
  • spacer moiety refers to an atom or a collection of atoms optionally used to link interconnecting moieties such as a terminus of a polymer segment and a therapeutic peptide or an electrophile or nucleophile of a therapeutic peptide.
  • the spacer moiety may be hydrolytically stable or may include a physiologically hydrolyzable or enzymatically degradable linkage. Unless the context clearly dictates otherwise, a spacer moiety optionally exists between any two elements of a compound (e.g., the provided conjugates comprising a residue of a therapeutic peptide and a water-soluble polymer that can be attached directly or indirectly through a spacer moiety).
  • Alkyl refers to a hydrocarbon, typically ranging from about 1 to 15 atoms in length. Such hydrocarbons are preferably but not necessarily saturated and may be branched or straight chain, although typically straight chain is preferred. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, 2-methylbutyl, 2-ethylpropyl, 3-methylpentyl, and the like. As used herein, "alkyl” includes cycloalkyl as well as cycloalkylene-containing alkyl.
  • “Lower alkyl” refers to an alkyl group containing from 1 to 6 carbon atoms, and may be straight chain or branched, as exemplified by methyl, ethyl, n-butyl, /-butyl, and t- butyl.
  • Cycloalkyl refers to a saturated or unsaturated cyclic hydrocarbon chain, including bridged, fused, or spiro cyclic compounds, preferably made up of 3 to about 12 carbon atoms, more preferably 3 to about 8 carbon atoms.
  • Cycloalkylene refers to a cycloalkyl group that is inserted into an alkyl chain by bonding of the chain at any two carbons in the cyclic ring system.
  • Alkoxy refers to an -O-R group, wherein R is alkyl or substituted alkyl, preferably C 1-6 alkyl ⁇ e.g., methoxy, ethoxy, propyloxy, and so forth).
  • substituted refers to a moiety (e.g., an alkyl group) substituted with one or more noninterfering substituents, such as, but not limited to: alkyl; C 3-8 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g., fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl; substituted phenyl; and the like.
  • “Substituted aryl” is aryl having one or more noninterfering groups as a substituent. For substitutions on a phenyl ring, the substituents may be in any orientation (i. e. , ortho, meta, or para).
  • Noninterfering substituents are those groups that, when present in a molecule, are typically nonreactive with other functional groups contained within the molecule.
  • Aryl means one or more aromatic rings, each of 5 or 6 core carbon atoms.
  • Aryl includes multiple aryl rings that may be fused, as in naphthyl or unfused, as in biphenyl. Aryl rings may also be fused or unfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings. As used herein, "aryl” includes heteroaryl.
  • Heteroaryl is an aryl group containing from one to four heteroatoms, preferably sulfur, oxygen, or nitrogen, or a combination thereof. Heteroaryl rings may also be fused with one or more cyclic hydrocarbon, heterocyclic, aryl, or heteroaryl rings.
  • Heterocycle or “heterocyclic” means one or more rings of 5-12 atoms, preferably 5-7 atoms, with or without unsaturation or aromatic character and having at least one ring atom that is not a carbon.
  • Preferred heteroatoms include sulfur, oxygen, and nitrogen.
  • Substituted heteroaryl is heteroaryl having one or more noninterfering groups as substituents.
  • Substituted heterocycle is a heterocycle having one or more side chains formed from noninterfering substituents.
  • An "organic radical” as used herein shall include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, and substituted aryl.
  • Electrophile and "electrophilic group” refer to an ion or atom or collection of atoms, which may be ionic, having an electrophilic center, i.e., a center that is electron seeking, capable of reacting with a nucleophile.
  • Nucleophile and nucleophilic group refers to an ion or atom or collection of atoms that may be ionic having a nucleophilic center, i.e., a center that is seeking an electrophilic center or with an electrophile.
  • a "physiologically cleavable” or “hydrolyzable” or “degradable” bond is a bond that reacts with water (i.e., is hydrolyzed) under physiological conditions.
  • the tendency of a bond to hydrolyze in water will depend not only on the general type of linkage connecting two central atoms but also on the substituents attached to these central atoms.
  • Appropriate hydrolytically unstable or weak linkages include but are not limited to carboxylate ester, phosphate ester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides.
  • Releasably attached e.g., in reference to a therapeutic peptide releasably attached to a water-soluble polymer, refers to a therapeutic peptide that is covalently attached via a linker that includes a degradable linkage as disclosed herein, wherein upon degradation (e.g., hydrolysis), the therapeutic peptide is released.
  • the therapeutic peptide thus released will typically correspond to the unmodified parent or native therapeutic peptide, or may be slightly altered, e.g., possessing a short organic tag.
  • the unmodified parent therapeutic peptide is released.
  • An "enzymatically degradable linkage” means a linkage that is subject to degradation by one or more enzymes.
  • a “hydrolytically stable” linkage or bond refers to a chemical bond, typically a covalent bond, which is substantially stable in water, that is to say, does not undergo hydrolysis under physiological conditions to any appreciable extent over an extended period of time.
  • hydrolytically stable linkages include, but are not limited to, the following: carbon-carbon bonds (e.g., in aliphatic chains), ethers, amides, urethanes, and the like.
  • a hydrolytically stable linkage is one that exhibits a rate of hydrolysis of less than about 1 -2% per day under physiological conditions. Hydrolysis rates of representative chemical bonds can be found in most standard chemistry textbooks.
  • linkages can be hydrolytically stable or hydrolyzable, depending upon (for example) adjacent and neighboring atoms and ambient conditions.
  • One of ordinary skill in the art can determine whether a given linkage or bond is hydrolytically stable or hydrolyzable in a given context by, for example, placing a linkage-containing molecule of interest under conditions of interest and testing for evidence of hydrolysis (e.g., the presence and amount of two molecules resulting from the cleavage of a single molecule).
  • Other approaches known to those of ordinary skill in the art for determining whether a given linkage or bond is hydrolytically stable or hydrolyzable can also be used.
  • compositions of the invention refer to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.
  • pharmaceutically acceptable amount refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.
  • physiologically effective amount refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.
  • therapeutically effective amount are used interchangeably herein to mean the amount of a polymer-(therapeutic peptide) conjugate that is needed to provide a desired level of the conjugate (or corresponding unconjugated therapeutic peptide) in the bloodstream or in the target tissue.
  • the precise amount will depend upon numerous factors, e.g. , the particular therapeutic peptide, the components and physical characteristics of the therapeutic composition, intended patient population, individual patient considerations, and the like, and can readily be determined by one skilled in the art, based upon the information provided herein.
  • Multi-functional means a polymer having three or more functional groups contained therein, where the functional groups may be the same or different.
  • Multi-functional polymeric reagents of the invention will typically contain from about 3-100 functional groups, or from 3-50 functional groups, or from 3-25 functional groups, or from 3-15 functional groups, or from 3 to 10 functional groups, or will contain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups within the polymer backbone.
  • a "difunctional” polymer means a polymer having two functional groups contained therein, either the same (i.e., homodifunctional) or different (i.e., heterodifunctional) .
  • subject refers to a vertebrate, preferably a mammal.
  • Mammals include, but are not limited to, murines, rodents, simians, humans, farm animals, sport animals, and pets.
  • substantially means nearly totally or completely, for instance, satisfying one or more of the following: greater than 50%, 51% or greater, 75% or greater, 80%) or greater, 90% or greater, and 95%> or greater of the condition.
  • a pharmaceutical comprising a dose of polymer-GLP-1 conjugates and a pharmaceutically acceptable excipient, wherein (i) each conjugate in the dose of polymer-GLP-1 conjugates comprises a water-soluble, non-peptidic polymer having a molecular weight of greater than 5,000 Dal tons covalently attached through an
  • the pharmaceutical composition upon administration to a mammal, has a glucose-lowering effect
  • the dose of polymer-GLP-1 conjugates achieving the glucose-lowering effect is less than an amount of the GLP-1 moiety in an unconjugated form required to achieve the glucose lowering effect.
  • each conjugate making up the dose having at least one (and in the majority of cases only one) water-soluble, non-peptidic polymer covalently attached to a GLP-1 moiety.
  • GLP-1 moiety refers to those peptides, polypeptides and proteins having GLP-1 activity (and includes GLP-1 activity- containing peptides derived through site-directed mutagenesis or other mutations), including (for example) GLP-1.
  • the GLP-1 moiety Prior to conjugation, has at least one electrophilic group or nucleophilic group suitable for reaction with a water soluble polymer.
  • GLP-1 moiety encompasses both the GLP-1 moiety prior to conjugation as well as the GLP-1 moiety residue following conjugation. It will be
  • the GLP-1 moiety when the GLP-1 moiety is covalently attached to a water-soluble polymer, the GLP-1 moiety is slightly altered due to the presence of one or more covalent bonds associated with linkage to the polymer (or linker that is attached to the polymer), due to reaction of one of more reactive groups of the GLP-1 moiety (e.g., an amino, carboxyl, etc.), with the water soluble polymer.
  • this slightly altered form of the GLP-1 moiety attached to another molecule, such as a water-soluble polymer is referred to as a "residue" of the GLP-1 moiety.
  • a "residue" of the GLP-1 moiety As will be explained in further detail below, one of ordinary skill in the art can determine whether any given moiety has GLP-1 activity.
  • GLP- 1 itself (and not in a conjugate form), “GLP- 1 " shall be understood to designate the peptide having the truncated "7-36" amino acid sequence (SEQ ID NO: l): NH 2 -His 7 -Ala 8 -Glu 9 -G ⁇
  • GLP-1 (7-37)OH an exemplary GLP-1 moiety, shall be understood to designate the peptide having the following amino acid sequence (SEQ ID NO:l): NH 2 -His 7 - Ala 8 -Glu 9 -Gly 10 -Thr n -Phe ,2 -Tl ⁇
  • GLP-1 moieties for use in connection with the present invention include GLP-1 (1-36), GLP-1 analogs (such as those described in WO 91/1 1457), GLP-1 derivatives, GLP-1 moieties described in U.S. Published Patent Application No. 2004/0235710, GLP-1 biologically active fragments, extended GLP-1 (see, for example, WO 03/058203, in particular with respect to the extended glucagon-like peptide- 1 analogs described therein), N-terminal truncated fragments of GLP-1 (such as those described in EP 0 699 686, and exendins (including, for example, exendin-4 and analogs thereof).
  • exendins are peptides that were first isolated from the salivary secretions of the Gila-monster and the Mexican Beaded Lizard.
  • the exendins have a degree of similarity to several members of the GLP family, with the highest homology, 53%, to GLP-1 (7-36)NH 2 (Goke, et al, J. Biol. Chem., 268: 19650-55, 1993).
  • Particular exendins for use in the present invention include exendin-3 and exendin-4 (synthetic extendin-4 is also known as Exenatide).
  • Exendin-3 (1-39) has the following amino acid sequence (SEQ ID NO:3): His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val- Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 .
  • exendin-4 (1-39) corresponds to (SEQ ID NO:4): His-Gly-Glu- Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile- Glu-T -Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser wherein the C-terminus serine is amidated.
  • the GLP-1 moiety may be obtained from either non-recombinant methods or from
  • GLP- 1 moieties are commercially available, e.g., hGLP-1 , rExtendin-4, and rHuGLP-1 are available from ProSpecTany Techno Gene LTD (Rehovot, Israel); and (Ser8)GLP-l(7- 36)amide, hGLP-1 amide, and hGLP-1 (7-36)Lys(biotin)amide are available from American Peptide Co., Sunnyvale, CA. Methods for preparing GLP-1 moieties are well-known, and are described, e.g., in U.S. Patent Nos. 5,118,666; 5,120,712; and 5,523,549.
  • GLP-1 moieties can be prepared using standard methods of solution or solid phase peptide synthesis such as those described in Dugas H., Penny, C, Bioorganic Chemistry, Springer Verlag, New York, p. 54-92 (1981); Merrifield (1962) Chem Soc. 85:2149, and Stewart and Young, Solid Phase Peptide Synthesis, Freeman, San Francisco, p. 24-66 (1969).
  • Peptide synthesizers are available from, e.g., Applied Biosystems, Foster City, CA.
  • Solid phase synthesizers are typically used according to manufacturers' instructions for blocking interfering groups, protecting certain amino acids, coupling, decoupling, and capping unreacted amino acids.
  • BOC-amino acids and other reagents are commercially available from Applied Biosystems, Foster City CA. Sequential BOC
  • Arg tosyl
  • Asp cyclohexyl
  • Glu cyclohexyl
  • Ser benzyl
  • Thr benzyl
  • Tyr 4- bromocarbobenzoxy
  • BOC deprotection may be carried out with trifluoroacetic acid in methylene chloride.
  • the resulting peptide may be deprotected and cleaved from the resin using, e.g., anhydrous HF containing 10% meta-cresol.
  • a GLP-1 moiety as described herein is prepared by constructing the nucleic acid encoding the desired polypeptide or fragment, cloning the nucleic acid into an expression vector, transforming a host cell (e.g., plant, bacteria such as Escherichia coli, yeast such as Saccharomyces cerevisiae, or mammalian cell such as Chinese hamster ovary cell or baby hamster kidney cell), and expressing the nucleic acid to produce the desired polypeptide or fragment.
  • a host cell e.g., plant, bacteria such as Escherichia coli, yeast such as Saccharomyces cerevisiae, or mammalian cell such as Chinese hamster ovary cell or baby hamster kidney cell
  • the expression can occur via exogenous expression (when the host cell naturally contains the desired genetic coding) or via endogenous expression.
  • Methods for producing and expressing recombinant polypeptides in vitro and in prokaryotic and eukaryotic host cells are known to those of ordinary skill in the art. See, for example, U.S. Patent No. 4,868,122.
  • GLP-1 is typically expressed in E. coli (since it doesn't require glycosylation for activity).
  • nucleic acid sequences that encode an epitope tag or other affinity binding sequence can be inserted or added in- frame with the coding sequence, thereby producing a fusion protein comprised of the desired peptide and a polypeptide suited for binding.
  • Fusion proteins can be identified and purified by first running a mixture containing the fusion protein through an affinity column bearing binding moieties (e.g., antibodies) directed against the epitope tag or other binding sequence in the fusion proteins, thereby binding the fusion protein within the column. Thereafter, the fusion protein can be recovered by washing the column with the appropriate solution (e.g., acid) to release the bound fusion protein.
  • binding moieties e.g., antibodies
  • the GLP-1 moiety is not in the form of a fusion protein. See, for example, Dillon et al. (1993) Endocrinology 133: 1907- 1910.
  • GLP-1 activity Various assays may be used to assess bioactivity, including in-vitro and in- vivo assays that measure GLP-1 receptor binding activity or receptor activation. See, for example, EP 0 619 322 and U.S. Patent No. 5,120,712 for descriptions of assessing GLP-1 activity.
  • a receptor-signaling assay may also be used to assess GLP-1 activity, such as described in Zlokarnik et al. (1998) Science 279:84-88.
  • GLP-1 activity can also be used to determine whether a given moiety has GLP-1 activity. Such methods are useful for determining the GLP-1 activity of both the moiety itself (and therefore can be used as a "GLP- 1 moiety"), as well as that of the corresponding polymer-moiety conjugate. For example, one can determine whether a given moiety is an agonist of the human GLP-1 receptor by assessing whether that moiety stimulates the formation of cAMP in a suitable medium containing the human GLP-1 receptor. The potency of such moiety is determined by calculating the EC50 value from a dose response curve.
  • BHK cells baby hamster kidney cells
  • expressing the cloned human GLP-1 receptor can be grown in DMEM media containing penicillin, streptomycin, calf serum, and Geneticin.
  • the cells are then washed in phosphate buffered saline and harvested.
  • Plasma membranes are then prepared from the cells by homogenization, and the homogenate is then centrifuged to produce a pellet.
  • the resulting pellet is suspended by homogenization in a suitable buffer, centrifuged, and then washed.
  • the cAMP receptor assay is then carried out by measuring cyclic AMP (cAMP) in response to the test insulinotropic moiety.
  • cAMP can be quantified using the AlphaScreenTM cAMP Kit (Perkin Elmer).
  • Incubations are typically carried out in microtiter plates in buffer, with addition of, e.g., ATP, GTP, IBMX (3-isobutyl-l-methylxanthine, Tween-20, BSA, acceptor beads, and donor beads incubated with biotinylated cAMP.
  • Counting may be carried out, e.g., using the FusionTM instrument (Perkin Elmer). Concentration-response curves are then plotted for the individual insulinotropic moieties under evaluation, and their EC 50 values determined.
  • Biologically active fragments, deletion variants, substitution variants or addition variants of any of the foregoing that maintain at least some degree of GLP-1 activity can also serve as a GLP-1 moiety in the conjugates of the invention.
  • GLP-1 moieties for use in connection with the present invention include any of the GLP-1 moieties described herein modified via methylation, N-terminal modification and/or glycosylation.
  • a GLP-1 moiety may possess one or more methyl or other lower alkyl groups at one or more positions of the GLP-1 sequence.
  • groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, and so forth.
  • Sites of modification include residues corresponding to positions 7, 8, 9, and/or 10 [based on a GLP-1 (7-36) numbering convention], with the 7 and/or 9 positions being preferred.
  • DPP IV dipeptidyl peptidase IV
  • GLP-1 N-methylated GLP-1
  • alpha-methylated GLP-1 alpha-me-GLP-1
  • desamidated GLP-1 desamino-GLP-1
  • imidazole-lactic acid substituted GLP-1 imi-GLP-1
  • the GLP-1 moieties described herein may also contain one or more glycosides.
  • the GLP-1 moiety is preferably modified by introduction of a monosaccharide, a disaccharide or a trisaccharide.
  • any site on the GLP-1 moiety may be modified by introduction of a saccharide, preferably, the saccharide is introduced at a residue or residues corresponding to any one or more of positions 7, 8, or 9 [based on a GLP-l(7-36) numbering convention] to protect the peptide against DPP IV proteolysis.
  • additional glycosides may be introduced, e.g., at any one or more of positions 22, 23 and 24 [again, based on a GLP- 1(7-36) numbering convention] to increase the helicity through the central portion of the peptide, as well as provide additional resistance to proteolysis.
  • Glycosylated GLP-1 moieties are prepared using conventional Fmoc chemistry and solid phase peptide synthesis techniques, where the desired protected glycoamino acids are prepared prior to peptide synthesis and then introduced into the peptide chain at the desired position during peptide synthesis.
  • Preparation of amino acid glycosides is described in U.S. Patent No. 5,767,254. Briefly, alpha and beta selective glycosylations of serine and threonine residues are carried out using the Koenigs-Knorr reaction and Lemieux's in situ anomerization methodology with Schiff base intermediates. Deprotection of the Schiff base glycoside is then carried out using mildly acidic conditions or hydrogenolysis.
  • Monosaccharides that may be used for introduction at one or more amino acid residues of GLP-1 include glucose (dextrose), fructose, galactose, and ribose. Additional monosaccharides suitable for use include glyceraldehydes, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose, xylose, ribulose, xylulose, allose, altrose, mannose, as well as others. Glycosides, such as mono-, di-, and trisaccharides for use in modifying a GLP-1 moiety, may be naturally occurring or may be synthetic.
  • Disaccharides that may be used for introduction at one or more amino acid residues of GLP-1 include sucrose, lactose, maltose, trehalose, melibiose, and cellobiose, among others.
  • Trisaccharides include acarbose, raffinose, and melezitose.
  • the water-soluble, non-peptidic polymer used in the polymer-GLP-1 conjugates in connection with the present invention is hydrophilic, non-peptidic, and biocompatible.
  • a substance is considered biocompatible if the beneficial effects associated with use of the substance alone or with another substance (e.g., an active agent such a therapeutic peptide) in connection with living tissues (e.g., administration to a patient) outweighs any deleterious effects as evaluated by a clinician, e.g., a physician.
  • a substance is considered nonimmunogenic if the intended use of the substance in vivo does not produce an undesired immune response (e.g. , the formation of antibodies) or, if an immune response is produced, that such a response is not deemed clinically significant or important as evaluated by a clinician.
  • the water-soluble polymer is hydrophilic, biocompatible and nonimmunogenic.
  • the water-soluble polymer is typically characterized as having from 2 to about 300 termini, preferably from 2 to 100 termini, and more preferably from about 2 to 50 termini.
  • poly(alkylene glycols) such as polyethylene glycol (PEG), poly(propylene glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefmic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly( vinyl alcohol), polyphosphazene,
  • PEG polyethylene glycol
  • PPG poly(propylene glycol)
  • copolymers of ethylene glycol and propylene glycol and the like poly(oxyethylated polyol), poly(olefmic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide
  • polyoxazoline poly(N-acryloylmorpholine), and combinations of any of the foregoing, including copolymers and terpolymers thereof.
  • the water-soluble polymer is not limited to a particular structure and may possess a linear architecture (e.g., alkoxy PEG or bifunctional PEG), or a non-linear architecture, such as branched, forked, multi-armed (e.g., PEGs attached to a polyol core), or dendritic (i.e. having a densely branched structure with numerous end groups).
  • the polymer subunits can be organized in any number of different patterns and can be selected, e.g., from homopolymer, alternating copolymer, random copolymer, block copolymer, alternating tripolymer, random tripolymer, and block tripolymer.
  • Preferred in connection with the present invention is a water-soluble, non-peptidic polymer that is branched and/or a homopolymer.
  • a PEG used to prepare a therapeutic peptide polymer conjugate of the invention is "activated” or reactive. That is to say, the activated PEG (and other activated water-soluble polymers collectively referred to herein as "polymeric reagents") used to form a conjugate comprises an activated functional group suitable for coupling to a desired site or sites on the therapeutic peptide.
  • a polymeric reagent for use in preparing a conjugate includes a functional group for reaction with the therapeutic peptide.
  • Representative polymeric reagents and methods for conjugating such polymers to an active moiety are known in the art, and are, e.g., described in Harris, J.M. and Zalipsky, S., eds, Poly (ethylene glycol), Chemistry and Biological Applications, ACS, Washington, 1997; Veronese, F., and J.M Harris, eds., Peptide and Protein PEGylation, Advanced Drug Delivery Reviews, 54(4); 453-609 (2002); Zalipsky, S., et al, "Use of Functionalized Poly(Ethylene Glycols) for Modification of Polypeptides" in Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, ed., Plenus Press, New York (1992); Zalipsky (1995) Advanced Drug Reviews .157-182, and in Roberts, et al, Adv. Drug Delivery Reviews, 54, 459 >-4 ⁇ '6 (2002).
  • PEG reagents suitable for use in the present invention are available from commercial sources and can be prepared synthetically. Descriptions of polymeric reagents, as well as methods for making polymeric reagents, can be found in, for example, U.S. Patent Nos. 5,252,714, 5,650,234, 5,739,208, 5,932,462, 5,629,384, 5,672,662, 5,990,237,
  • the weight- average molecular weight of the water-soluble polymer in the conjugate is from about 5,000 Daltons to about 150,000 Daltons.
  • Exemplary ranges include weight-average molecular weights in the range of from about 5,000 Daltons to about 80,000 Daltons, from 5,000 Daltons to about 80,000 Daltons, from about 5,000 Daltons to about 65,000 Daltons, from about 5,000 Daltons to about 40,000 Daltons, from 5,000 Daltons to about 40,000 Daltons, from greater than 5,000 Daltons to about 80,000 Daltons, from about 10,000 Daltons to about 80,000 Daltons, from about 15,000 Daltons to about 45,000 Daltons, from about 20,000 Daltons to about 45,000 Daltons, from about 30,000 Daltons to about 50,000 Daltons, and from about 35,000 Daltons to about 45,000 Daltons.
  • Exemplary weight-average molecular weights for the water-soluble polymer include about about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 55,000 Daltons, about 60,000 Daltons, about 65,000 Daltons, about 70,000 Daltons, and about 75,000 Daltons.
  • Branched versions of the water-soluble polymer e.g. , a branched 40,000
  • Dalton water-soluble polymer comprised of two 20,000 Dalton polymers or the like having a total molecular weight of any of the foregoing can also be used.
  • the conjugate is one that does not have one or more attached PEG moieties having a weight-average molecular weight of less than about 6,000 Daltons.
  • the number of repeat units typically comprises a number of (OCH 2 CH 2 ) monomers.
  • the number of repeat units is typically identified by the subscript "n" in, for example, "(OCH 2 CH 2 ) n .”
  • the value of (n) typically falls within one or more of the following ranges: from 113 to about 2050; from about 136 to about 2050, from about 225 to about 1930, from about 450 to about 1930, from about 1200 to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to about 2730, from about 795 to about 2730, from about 909 to about 2730, and from about 1,200 to about 1,900.
  • V the number of repeating units
  • a polymer for use in the invention may be end-capped, that is, a polymer having at least one terminus capped with a relatively inert group, such as a lower alkoxy group (i.e., a Ci_ 6 alkoxy group) or a hydroxyl group.
  • a relatively inert group such as a lower alkoxy group (i.e., a Ci_ 6 alkoxy group) or a hydroxyl group.
  • mPEG methoxy-PEG
  • -OCH 3 methoxy
  • the -PEG- symbol used in the foregoing generally represents the following structural unit: -CH 2 CH 2 0-(CH 2 CH 2 0) n -CH 2 CH 2 -, where (n) generally ranges from about zero to about 4,000.
  • Multi-armed or branched PEG molecules such as those described in U.S.
  • Patent No. 5,932,462 are also suitable for use in the present invention.
  • the PEG may be described generally according to the structure:
  • poly a and poly b are PEG backbones (either the same or different), such as methoxy poly(ethylene glycol); R" is a non-reactive moiety, such as H, methyl or a PEG backbone; and P and Q are non-reactive linkages.
  • the branched PEG molecule is one that includes a lysine residue, such as the following reactive PEG suitable for use in forming a therapeutic peptide conjugate.
  • lysine residue such as the following reactive PEG suitable for use in forming a therapeutic peptide conjugate.
  • the polymeric reagent (as well as the corresponding conjugate prepared from the polymeric reagent) may lack a lysine residue in which the polymeric portions are connected to amine groups of the lysine via a "-OCH 2 CONHCH 2 CO-" group.
  • the polymeric reagent (as well as the corresponding conjugate prepared from the polymeric reagent) may lack a branched water-soluble polymer that includes a lysine residue (wherein the lysine residue is used to effect branching).
  • Additional branched PEGs for use as polymeric reagents to prepare the polymer-GLP-1 conjugates include those polymer reagents described in U.S. Patent
  • branched polymers described therein include those having the following generalized structure:
  • POLY 1 is a water-soluble polymer
  • POLY 2 is a water-soluble polymer
  • (a) is 0, 1, 2 or 3
  • (b) is 0, 1, 2 or 3
  • (e) is 0, 1, 2 or 3
  • (f) is 0, 1 , 2 or 3
  • (g') is 0, 1, 2 or 3
  • (h) is 0, 1, 2 or 3
  • (j) is 0 to 20
  • each R 1 is independently H or an organic radical selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl
  • X when present, is a spacer moiety
  • X when present, is a spacer moiety;
  • X when present, is a spacer moiety; X , when present, is a spacer moiety; X , when present, is a spacer moiety; X s , when present, is a spacer moiety; R 5 is a branching moiety; and Z is a reactive group for coupling to a therapeutic peptide, optionally via an intervening spacer.
  • POLY 1 and POLY 2 in the preceding branched polymer structure may be different or identical, i.e., are of the same polymer type (structure) and molecular weight.
  • a exemplary branched construct used in a branched polymer corresponds to the following structure:
  • exemplary branched polymeric reagents can have the following structure:
  • n is from 113 to about 2050 and Z is an electrophile-containing
  • Branched polymers suitable for preparing conjugates useful in connection with the present invention also include those represented more generally by the formula R(POLY) y , where R is a central or core molecule from which extends 2 or more POLY arms such as PEG.
  • the variable y represents the number of POLY arms, where each of the polymer arms can independently be end-capped or alternatively, possess a reactive functional group at its terminus.
  • a more explicit structure in accordance with this embodiment of the invention possesses the structure, R(POLY-Z) y , where each Z is independently an end-capping group or a reactive group, e.g., suitable for reaction with a therapeutic peptide.
  • the resulting linkage can be hydrolytically stable, or alternatively, may be degradable, i.e., hydrolyzable.
  • at least one polymer arm possesses a terminal functional group suitable for reaction with, e.g. , a therapeutic peptide.
  • Branched PEGs such as those represented generally by the formula, R(PEG) y above possess 2 polymer arms to about 300 polymer arms (i.e., n ranges from 2 to about 300).
  • such branched PEGs typically possess from 2 to about 25 polymer arms, such as from 2 to about 20 polymer arms, from 2 to about 15 polymer arms, or from 3 to about 15 polymer arms.
  • Multi-armed polymers include those having 3, 4, 5, 6, 7 or 8 arms.
  • Core molecules in branched PEGs as described above include polyols, which are then further functionalized.
  • polyols include aliphatic polyols having from 1 to 10 carbon atoms and from 1 to 10 hydroxyl groups, including ethylene glycol, alkane diols, alkyl glycols, alkylidene alkyl diols, alkyl cycloalkane diols, 1,5-decalindiol,
  • Cycloaliphatic polyols may also be employed, including straight chained or closed-ring sugars and sugar alcohols, such as mannitol, sorbitol, inositol, xylitol, quebrachitol, threitol, arabitol, erythritol, adonitol, ducitol, facose, ribose, arabinose, xylose, lyxose, rhamnose, galactose, glucose, fructose, sorbose, mannose, pyranose, altrose, talose, tagitose, pyranosides, sucrose, lactose, maltose, and the like.
  • Additional aliphatic polyols include derivatives of glyceraldehyde, glucose, ribose, mannose, galactose, and related stereoisomers.
  • Other core polyomers include derivatives of
  • cyclodextrins include glycerol, pentaerythritol, sorbitol, and trimethylolpropane.
  • the polymer may possess an overall forked structure as described in U.S. Patent No. 6,362,254. This type of polymer is useful for reaction with two therapeutic peptide moieties, where the two therapeutic peptide moieties are positioned a precise or predetermined distance apart.
  • one or more degradable linkages may additionally be contained in the polymer, POLY, to allow generation in vivo of a conjugate having a smaller PEG chain than in the initially administered conjugate.
  • Appropriate physiologically cleavable (i.e., releasable) linkages include but are not limited to ester, carbonate ester, carbamate, sulfate, phosphate, acyloxyalkyl ether, acetal, and ketal. Such linkages when contained in a given polymer segment will often be stable upon storage and upon initial administration.
  • the PEG polymer used to prepare a conjugate may comprise a pendant PEG molecule having reactive groups, such as carboxyl or amino, covalently attached along the length of the PEG rather than at the end of the PEG chain(s).
  • the pendant reactive groups can be attached to the PEG directly or through a spacer moiety, such as an alkylene group.
  • One of ordinary skill in the art can determine the proper molecular size of the water-soluble, non-peptidic polymer. For example, one of ordinary skill in the art, using routine experimentation, can determine a proper molecular size by first preparing a variety of conjugates with different weight-average molecular weights of the polymer and then obtaining the clearance profile for each conjugate by administering the conjugate to a patient and taking periodic blood and/or urine samples. Once a series of clearance profiles has been obtained for each tested conjugate, a conjugate or mixture of conjugates having the desired clearance profile(s) can be determined.
  • polymeric reagent generally refers to an entire molecule, which can comprise a water-soluble polymer segment, as well as additional spacers and functional groups.
  • Each conjugate in the dose of polymer-GLP-1 conjugates contained with the pharmaceutical composition has the water-soluble, non-peptidic polymer covalently attached via an amide-containing linkage to an amino group of the GLP-1 moiety.
  • the linkage will nevertheless contain a amide (i.e., a -NHC(O)- or -C(O)NH-) group.
  • Such factors include, for example, the particular linkage chemistry employed, the particular atoms (if any) surrounding the functional groups effecting the linkage, and so forth.
  • the amide- containing linkage is preferably relatively stable.
  • the nitrogen atom within the amide-containing linkage is contributed by an amine group associated with the GLP-1 moiety.
  • an amine group associated with the GLP-1 moiety contributes the nitrogen for the amide-containing, usually it is the amine acting as a nucleophile for an electrophilically activated polymeric reagent (e.g., a
  • Exemplary electrophilically activated polymeric reagents include
  • an electrophile selected from the group consisting of acetals, esters (such as succinimidyl esters of carboxylic acids) and carbonates.
  • an electrophilically activated polymeric reagent useful in connection with the present invention is encompassed by the following structure:
  • n is from 113 to about 2050 and (GLP-1) is a residue of a GLP-1 moiety.
  • Conjugation of a polymeric reagent to a nitrogen atom within a GLP-1 moiety can be accomplished by a variety of techniques.
  • the GLP-1 moiety is conjugated to a polymeric reagent ranctionalized with an active ester such as a succinimidyl derivative (e.g., an N-hydroxysuccinimide ester).
  • an active ester such as a succinimidyl derivative (e.g., an N-hydroxysuccinimide ester).
  • the polymeric reagent bearing the reactive ester is reacted with the GLP-1 moiety in aqueous media under appropriate pH conditions, e.g. , from pHs ranging from about 3 to about 8, about 3 to about 7, or about 4 to about 6.5.
  • Most polymer active esters can couple to a target peptide such as GLP-1 moiety at physiological pH, e.g., at 7.0. However, less reactive derivatives may require a different pH.
  • activated PEGs can be attached to a peptide such as therapeutic peptide at pHs from about 7.0 to about 10.0 for covalent attachment to an internal lysine.
  • lower pHs are used, e.g., 4 to about 5.75, for preferential covalent attachment to the N-terminus. Conjugation reactions can often be carried out at room temperature, although lower temperatures may also be used.
  • Reaction times are typically on the order of minutes, e.g., 30 minutes, to hours, e.g., from about 1 to about 36 hours), depending upon the pH and temperature of the reaction.
  • Varying ratios of polymeric reagent to the GLP-1 moiety may be employed, e.g., from an equimolar ratio up to a 10-fold molar excess of polymeric reagent. Typically, up to a 5-fold molar excess of polymeric reagent will suffice.
  • reaction can be monitored by withdrawing aliquots from the reaction mixture at various time points and analyzing the reaction mixture by SDS-PAGE or MALDI-TOF mass spectrometry or any other suitable analytical method. Once a plateau is reached with respect to the amount of conjugate formed or the amount of unconjugated polymer remaining, the reaction is assumed to be complete.
  • the resulting product mixture is preferably, but not necessarily purified, to separate out excess reagents, unconjugated reactants (e.g., GLP-1 in unconjugated form) undesired multi-conjugated species, and free or unreacted polymeric reagent.
  • the resulting conjugates can then be further characterized using analytical methods such as MALDI, capillary electrophoresis, gel electrophoresis, and/or chromatography.
  • the nitrogen atom can be associated with the N-terminal amine of the GLP-1 moiety or be associated with the epsilon amine of a lysine residue within the GLP-1 moiety.
  • lysine residues corresponding to positions 26 (i.e., Lys 26 ) and 34 (i.e., Lys 34 ) are preferred, although other nitrogen atom-containing locations (e.g., as may be introduced by aminating the carboxyl terminus or inserting a lysine residue) that may exist in a given GLP-1 moiety are preferred.
  • polymer-GLP-1 conjugates in which it is intended that the N-terminal amine is the most represented location of polymer attachment within a composition it is preferred that at least 60%, more preferably at least 70%, still more preferably at least 80%, and yet still more preferably at least 90% of all polymer-GLP-1 conjugates in the composition have only a single attachment of a water-soluble, non-peptidic polymer attached at the N-terminal amine.
  • polymer-GLP-1 conjugates in which it is intended that locations corresponding to lysine residues Lys 26 and Lys 34 [based on a GLP-l (7-36) numbering convention] are the predominant attachment sites, it is preferred that at least 75%, more preferably at least 85%, still more preferably at least 95%, and yet still more preferably at least 99% of all polymer-GLP-1 conjugates in the composition have one attachments at one or both of locations corresponding to lysine residues Lys and Lys . As between, Lys and Lys 34 , it is preferred that the compositions have a majority of conjugates wherein attachment occurs at Lys 26 (e.g., a Lys 26 /Lys 34 ratio of 60/40.
  • the amide- containing linkage that serves to link the GLP-1 moiety to the water-soluble, non-peptidic polymer can include one or more additional atoms in addition to the amide.
  • the one or more additional atoms making up the amide-containing linkage can include one or more of carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms, and combinations thereof.
  • Nonlimiting examples of amide-containing linkages include those selected from the group consisting of -C(0)-NH-, -NH-C(O)-, -NH-C(0)-NH-, -0-C(0)-NH-, -NH-C(0)-0-, -C(0)-NH-CH 2 -, -C(0)-NH-CH 2 -CH 2 -, -CH 2 -C(0)-NH-CH 2 -,
  • amide-containing linkages have the following structures: -C(0)-NH-(CH 2 ) 1-6 -NH-C(0)-, -NH-C(0)-NH-(CH 2 ) 1-6 -NH-C(0)-, and
  • amide-containing linkages may further include an ethylene oxide oligomer chain comprising 1 to 20 ethylene oxide monomer units [i.e., -(CH 2 CH 2 0) 1-2 o]. That is, the ethylene oxide oligomer chain can occur before or after the amide-containing linkage. Also, the oligomer chain would not be considered part of the amide-containing linkage if the oligomer is adjacent to a water-soluble, non-peptidic polymer and merely represents an extension of the polymer.
  • the polymer-GLP-1 conjugates associated with the present invention can be purified to obtain/isolate different conjugate species. Specifically, a product mixture can be purified to obtain the desired numeric isomer. In one embodiment of the invention, the GLP-1 conjugates that make up the dose of polymer-GLP-1 conjugates are mono-conjugates.
  • the strategy for purification of a conjugate reaction mixture will depend upon a number of factors, including, for example, the molecular weight of the polymeric reagent employed, the particular GLP-1 moiety, and the desired characteristics of the product - e.g., monomer, dimer, particular positional isomers, and so forth.
  • conjugates having different molecular weights can be isolated using gel filtration chromatography and/or ion exchange chromatography.
  • chromatography may be used to fractionate different conjugates (e.g., 1-mer, 2-mer, 3-mer, and so forth, wherein “1-mer” indicates one polymer molecule per GLP-1 moeity, "2-mer” indicates two polymers attached to the GLP-1 moiety, and so on) on the basis of their differing molecular weights (where the difference corresponds essentially to the average molecular weight of the water-soluble, non-peptidic polymer). While this approach can be used to separate PEG and other water-soluble, non-peptidic polymer conjugates having different molecular weights, this approach is generally ineffective for separating positional isomers having different polymer attachment sites within the GLP-1 moiety.
  • gel filtration chromatography can be used to separate from each other mixtures of PEG 1-mers, 2- mers, 3-mers, and so forth, although each of the recovered PEG-mer compositions may contain PEGs attached to different reactive amino groups (e.g., lysine residues) or other functional groups of the therapeutic peptide.
  • Separation of positional isomers is typically carried out by reverse phase chromatography using a reverse phase-high performance liquid chromatography (RP-HPLC) CI 8 column (Amersham Biosciences or Vydac) or by ion exchange chromatography using an ion exchange column, e.g., a DEAE- or CM-SepharoseTM ion exchange column available from Amersham Biosciences. Either approach can be used to separate polymer-therapeutic peptide isomers having the same molecular weight (positional isomers).
  • RP-HPLC reverse phase-high performance liquid chromatography
  • ion exchange column e.g., a DEAE- or CM-SepharoseTM ion exchange column available from Amersham Biosciences.
  • compositions are preferably substantially free of the non-conjugated GLP-1 moiety.
  • compositions preferably are substantially free of all other non-covalently attached water-soluble, non-peptidic polymers.
  • compositions are preferably substantially free of albumin.
  • the pharmaceutical composition will typically satisfy one or more of the following characteristics: at least about 85% of the conjugates in the composition will have one polymer attached to the GLP-1 moiety; at least about 95% of the conjugates in the composition will one polymer attached to the GLP-1 moiety; and at least about 99% of the conjugates in the composition will have one polymer attached to the GLP-1 moiety.
  • the pharmaceutical composition of the invention may contain only one pharmaceutical excipient or the pharmaceutical composition may contain more than one pharmaceutical excipient.
  • the specific pharmaceutical excipient(s) included in the composition can vary and is influenced by the particular needs of the formulation and route of administration.
  • compositions of the invention encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted as well as liquids, as well as for inhalation.
  • suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic endotoxin-free water for injection, dextrose 5% in water, phosphate-buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.
  • suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic endotoxin-free water for injection, dextrose 5% in water, phosphate-buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.
  • solutions and suspensions are envisioned.
  • carbohydrates inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.
  • Representative carbohydrates for use in the compositions of the present invention include sugars, derivatized sugars such as alditols, aldonic acids, esterified sugars, and sugar polymers.
  • Exemplary carbohydrate excipients suitable for use in the present invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), pyranosyl sorbitol, myoinositol and the
  • non-reducing sugars are non-reducing sugars, sugars that can form a substantially dry amorphous or glassy phase when combined with the composition of the present invention, and sugars possessing relatively high glass transition temperatures, or Tgs (e.g., Tgs greater than 40°C, or greater than 50°C, or greater than 60°C, or greater than 70°C, or having Tgs of 80°C and above).
  • Tgs glass transition temperatures
  • Such excipients may be considered glass-forming excipients.
  • Exemplary protein excipients include albumins such as human serum albumin
  • compositions may also include a buffer or a pH-adjusting agent, typically but not necessarily a salt prepared from an organic acid or base.
  • buffers include organic acid salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid.
  • Other suitable buffers include Tris, tromethamine hydrochloride, borate, glycerol phosphate, and phosphate. Amino acids such as glycine are also suitable.
  • compositions of the present invention may also include one or more additional polymeric excipients/additives, e.g., polyvinylpyrrolidones, derivatized celluloses such as hydroxymethylcellulose, hydroxyethylcellulose, and
  • hydroxypropylmethylcellulose FICOLLs (a polymeric sugar), hydroxyethylstarch (HES), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-P-cyclodextrin and sulfobutylether- ⁇ - cyclodextrin), polyethylene glycols, and pectin.
  • FICOLLs a polymeric sugar
  • HES hydroxyethylstarch
  • dextrates e.g., cyclodextrins, such as 2-hydroxypropyl-P-cyclodextrin and sulfobutylether- ⁇ - cyclodextrin
  • polyethylene glycols polyethylene glycols
  • pectin pectin
  • compositions may further include flavoring agents, taste- masking agents, inorganic salts (e.g., sodium chloride), antimicrobial agents (e.g., sodium chloride), antimicrobial agents (e.g., sodium chloride), antimicrobial agents (e.g., sodium chloride), antimicrobial agents (e.g., sodium chloride), antimicrobial agents (e.g., sodium chloride), antimicrobial agents (e.g., sodium chloride), antimicrobial agents, e.g.
  • benzalkonium chloride sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as 'TWEEN 20" and 'TWEEN 80," and pluronics such as F68 and F88, available from BASF), sorbitan esters, lipids (e.g., phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, although preferably not in liposomal form), fatty acids and fatty esters, steroids (e.g., cholesterol), and chelating agents (e.g., zinc and other such suitable cations).
  • lipids e.g., phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines, although preferably not in liposomal form
  • fatty acids and fatty esters steroids (e.g., cholesterol), and chelating agents (e.g., zinc and other such suitable cations).
  • compositions according to the present invention are listed in “Remington: The Science & Practice of Pharmacy,” 21 st ed., Williams & Williams, (2005), and in the “Physician's Desk Reference,” 60th ed., Medical Economics, Montvale, N.J. (2006).
  • a pharmaceutical preparation if in solution form, can be housed in a syringe.
  • the amount of any individual excipient in the composition will vary depending on the activity of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects.
  • the excipient or excipients will be present in the composition in an amount of about 1% to about 99% by weight, from about 5% to about 98% by weight, from about 15 to about 95% by weight of the excipient, or with concentrations less than 30% by weight. In general, a high concentration of the therapeutic peptide is desired in the final pharmaceutical formulation.
  • compositions described herein can be administered by any of a number of routes including without limitation, oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal), intrathecal, and pulmonary.
  • routes including without limitation, oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal), intrathecal, and pulmonary.
  • Preferred forms of administration include parenteral and pulmonary.
  • Suitable formulation types for parenteral administration include ready- for-injection solutions, dry powders for combination with a solvent prior to use, suspensions ready for injection, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration, among others.
  • a method comprising delivering a conjugate to a patient, the method comprising the step of administering to the patient a pharmaceutical composition as provided herein.
  • Administration can be effected by any of the routes herein described.
  • the method may be used to treat a mammal suffering from diabetes (e.g., Type II diabetes).
  • the actual dose of the conjugate to be administered will vary depending upon the age, weight, and general condition of the subject as well as the severity of the condition being treated, the judgment of the health care professional, and conjugate being administered.
  • a therapeutically effective dosage amount of a therapeutic peptide conjugate as described herein will range from about 0.001 mg per day to about 1000 mg per day for an adult.
  • dosages may range from about 0.1 mg per day to about 100 mg per day, or from about 1.0 mg per day to about 10 mg/day.
  • corresponding doses based on international units of activity can be calculated by one of ordinary skill in the art.
  • the unit dosage of any given conjugate (again, such as provided as part of a pharmaceutical composition) can be administered in a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth.
  • the specific dosing schedule will be known by those of ordinary skill in the art or can be determined
  • Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Once the clinical endpoint has been achieved, dosing of the composition is halted.
  • GLP-1 conjugates Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Bio-Rad system (Mini-PROTEAN III Precast Gel Electrophoresis System), and Invitrogen system (XCell SureLock Mini-Cell). Samples were mixed with sample buffer. Then, the prepared samples were loaded onto a gel and run for approximately thirty minutes.
  • RP-HPLC reversed phase high-performance liquid chromatography
  • the protein and PEG-protein conjugates were eluted with a linear gradient 25%-65% over 30 minutes, and were visualized a Diode Array detector at 220, 280 and 320 nm.
  • Molecular weights GLP-1 compounds were analyzed with MALDI-TOF spectrophotometer, and sites of PEGylation were confirmed with peptide mapping using Trypsin/Glu-C. The digestion product was analyzed by LC/MALDI-TOF.
  • dimers identified through RP-HPLC indicate protein dimer aggregates (and lack any polymeric component).
  • HiTrap SP Sepharose HP cation exchange column (Amersham Biosciences) was used with the AKTA Explorer 100 system (GE Bioscience) to purify the PEG-GLP-1 conjugates.
  • the conjugate solution was loaded on a column that was pre-equilibrated in 20 mM NaOAc buffer, pH 4.0 (buffer A) and then washed with ten column volumes of buffer A to remove any unreacted PEG reagent. Subsequently, a linear gradient of buffer A and buffer B (lOmM NaOAc with 1.0 M NaCl buffer, pH 4.0) was applied over 20 column volumes. The eluent was monitored by UV detector at 220 nm, 280 nm and 320 nm. The purity of individuals fractions of GLP-1 conjugates was determined by RP-HPLC and SDS-PAGE.
  • Nter-GLP-1 is provided in FIG. 1.
  • the PEG 2 -ru-40K-Nter-GLP-l and unreacted PEG are indicated and the lines correspond to absorbance at various wavelengths (e.g., 280 nm and 225 nm).
  • Purity analysis of PEG 2 -ru-40 -Nter-GLP-l by reverse phase HPLC determined that the purity of the purified conjugate was determined to be 100% at 280 nm. See FIG. 2.
  • 40K-NHS 40K-NHS
  • 200 mG/niL stock solutions of were prepared in 2 mM HC1, and a molar ratio of 2.5: 1 (PEG/GLP-1) was added to GLP-1 solution to reach a final GLP-1 concentration of 1.5 mG/mL (0.45 mM).
  • MES buffer 0.5 M, pH 6.0 was added to the PEG-GLP-1 mixture to a final concentration of 20 mM, and the PEGylation was allowed to react for three hours.
  • a solution containing 1 M Hydroxyamine and 1 M glycine (pH 6.0) was added to the reaction mixture to final concentrations of 100 mM Hydroxyamine and 100 mM glycine to stop the PEGylation reaction and remove undesired imidazole PEG side product.
  • the reaction was allowed to continue for one hour, and was diluted with H 2 0 to the conductivity below 0.5 mS/cm (25 °C). pH was then adjusted to 4.0 using glacial acetic acid prior to column chromatography purification.
  • Lys26/34-GLP-l is provided in FIG. 3.
  • the PEG 2 -ru-40K-Lys26/34-GLP-l and unreacted PEG are indicated and the lines correspond to absorbance at various wavelengths (e.g., 280 nm and 225 nm).
  • Purity analysis of PEG 2 -ru-40K-Lys26/34-GLP-l by reverse phase HPLC determined that the purity of the purified conjugate was determined to be 99.6% at 280 nm.
  • the peaks at 18.7 minutes may represent the di-PEGylated GLP-1 conjugate. See FIG. 4.
  • mPEG-ButyrALD 30kDa, stored at -80 °C under argon, was warmed to ambient temperature under nitrogen purging.
  • 60 mG/mL stock solutions of were prepared in 2 mM HC1, and a molar ratio of 3: 1 (PEG/GLP-1) was added to GLP-1 solution to reach a final GLP-1 concentration of 1.5 mG/mL (0.45 mM).
  • NaoAc buffer (1 M, pH 4.0
  • Butyr ALD-GLP- 1 is provided in FIG. 5.
  • the PEG-30K-Nter-ButyrALD-GLP-l and unreacted PEG are indicated and the lines correspond to absorbance at various wavelengths (e.g., 280 nm and 225 nm).
  • Purity analysis of PEG-30K-Nter-ButyrALD-GLP-l by reverse phase HPLC determined that the purity of the purified conjugate was determined to be 97% at 280 nm with a retention time at 17 minutes. The peak at 15.2 minutes represent GLP-1 in unconjugated form. See FIG. 6.
  • PEG 2 -ru-40K-Nter-GLP- 1 and PEG 2 -ru-40K-Lys26/34-GLP-l were shown to be stable in not only fresh murine and rodent plasma, but also in fresh murine and rodent blood.
  • the two conjugates remained stable, as evidenced by no detectable appearance of PEG or GLP-1 from either conjugate at 28 hours when tested via LC/MS system containing Agilentl200 and Qtrap400 TripleQual Spectrophotometer.
  • the gradient settings for GLP-1 conjugates and PEG quantification were as follows: mobile phase A - 0.1 % fomic acid/H 2 0; and mobile phase B - 0.1 % fomic acid/acetonitrile, wherein the parameters for each step were as follows: step 0, total time - 0.00, flow rate 500 ⁇ /min, 80%A 20%B; step 1 , total time - 1.00, flow rate 500 ⁇ /min, 80%A/20%B; step 2, total time - 6.00, flow rate 500 ⁇ /min, 55%A/45%B; step 3, total time - 1 1.00, flow rate 500 ⁇ /min, 35%A/65%B; step 4, total time - 1 1.30, flow rate 500 ⁇ /min, 10%A/90%B; step 5, total time - 13.00, flow rate 500 ⁇ /min, 10%A/90%B; and step 6, total time - 13.01, flow rate 750 ⁇ /min, 80%A/20%B.
  • An Intrada reverse phase column WP-
  • the gradient settings for GLP-1 (7-36) and GLP-1 (9-36) quantification were as follows: mobile phase A - 10 mM ammonium acetate/H 2 0; and mobile phase B - 10 mM ammonium acetate/acetonitrile, wherein the parameters for each step were as follows: step 0, total time - 0.00, flow rate 500 ⁇ /min, 75%A/25%B; step 1, total time - 1.00, flow rate 500 ⁇ /min, 75%A/25%B; step 2, total time - 8.00, flow rate 500 ⁇ /min, 55%A/45%B; step 3, total time - 8.30, flow rate 750 ⁇ /min, 5%A/95%B; step 4, total time - 10.30, flow rate 750 ⁇ /min, 5%A/95%B; step 5, total time - 10.31, flow rate 750 ⁇ /min, 75%A/25%B; and step 6, total time - 12.30, flow rate 750 ⁇ /min, 75%A
  • the plasma incubation and extraction procedure involved a 5 mL aliquot of freshly collected, heparin treated, EDTA-free pooled s/d rat plasma, or
  • IXfreeze/thawed rat plasma into a 15 mL corning centrifuge tube. To this was added 1.2 mL of 5X PBS solution to 5 mL plasma, which was then mixed by inverting the tube. The final concentration of PBS in rat plasma was 1 X PBS. The tube was centrifuged and kept on ice until used. Immediately before plasma incubation, the plasma was pre- warmed to 37 °C for five minutes. Plasma incubation time points were set as follows: T 0, 0.25 hour, 0.5 hour, 1 hour, 2 hour, 3 hour, 4 hour, 6 hour, 8 hour, 12 hour, and 24 hour. For each time point, duplicate samples were prepared by labeling 2 1.5 mL Eppendorf tubes. Protease inhibitor mix (25 uL of 10X HALT) prepared according to conventional methods, was added to the tubes and tubes were kept on ice until use.
  • Protease inhibitor mix 25 uL of 10X HALT
  • protease inhibitor mix 100 uL of
  • 10X Halt solution or 1/10 volume of the blood to be collected, was placed into pre-chilled, heparin coated tubes. Approximately 1 mL of blood was added to the tube and mixed gently by inverting the tube five times. Following collection, tubes containing samples were centrifuged at approximately 2500rpm for five minutes (at 2-6°C). Thereafter, two aliquots of resulting plasma (approximately 200 uL each) were transferred to pre-labeled tubes. The tubes were frozen on dry ice and stored at -80°C pending analysis.
  • Quantification of GLP-1 analytes with LC/MS Quantification of GLP-1 analytes with LC/MS. Source ions and daughter ions were listed in individual windows, and the quantification of individual analytes were performed with Multiple Reaction Monitoring (MRM). The Limit of Quantification (LOQ) and Quantification range were 10 nG/mL and 10-1000 nG/mL, respectively, for active GLP-1 (7-36), inactive GLP-1 (9-36), PEG-GLP-1, and free PEG.
  • MRM Multiple Reaction Monitoring
  • GLP-1 (7-36) and GLP-1 (9-36) were each spiked in fresh rat plasma at the final concentrations of 1000 nG/mL. The plasma was maintained under 37 °C incubation, and fractions of the plasma were taken at pre-set time points for quantification. The stabilities of the two peptides, as revealed by Tl/2 values, obtained from First order, single exponential decay fitting, were 23 minutes and 2.1 hours, respectively.
  • PEG 2 -ru-40K-Nter-GLP-l (7-36) was spiked in fresh rat plasma.
  • the plasma was maintained was under 37 °C incubation, and fractions of the plasma were taken at pre-set time points for quantification.
  • the conjugate was associated with no detection of free PEG or GLP-1 peptides and underwent no obvious changes (less than 10 % variation, which was within experimental deviation).
  • Receptor binding assay RINm5F cells (rat insulinoma stably expressing GLP-
  • cAMP stimulation assay Rat insulinoma (RINm5F) cells in passages 12-15 were seeded at 30,000 cells/well in 96-well plates and grown overnight. The cells were washed twice with Dulbecco's phosphate-buffered saline (D-PBS), then pre-incubated for twenty minutes in D-PBS containing 0.1% BSA, 500 ⁇ 3-isobutyl-methylxanthine (IBMX), and 100 uM RO 20-1724 at room temperature.
  • D-PBS Dulbecco's phosphate-buffered saline
  • IBMX 3-isobutyl-methylxanthine
  • PEG 2 -ru-40K-Lys 26/34 -GLP-l exhibited minimal or no in vitro biological activity. Binding and activation (cAMP stimulation) studies with RINm5F cells, a rat insulinoma cell line expressing the GLP-1 receptor, indicated that PEG 2 -ru-40 -N ter -GLP-l exhibited no binding activity and 0.2% of the activation activity relative to GLP-1. PEG 2 -ru-40K-Lys 26/34 -GLP-l exhibited 0.9% and 0.1% of the binding and activation activity relative to GLP-1,
  • 40K-Lys 26/34 -GLP-l are expected as conjugation of the N-terminal amine group and lysines within GLP-1 with large polymers (> 10 kDa) are often known to reduce activity.
  • the binding and cAMP stimulation activities of PEG-30 -N ter -ButyrALD-GLP- 1 were not determined but the activities are expected to be comparable to the activities of the two conjugates containing the amide linkage.
  • mice (BKS.Cg- Lepr db /Lepr db /OlaHsd) were obtained from Harlan
  • the two conjugates containing the amide linkage may bind to the GLP-1 receptor with a slow on-rate but also a very slow off-rate.
  • the prolonged presence of the conjugates on the GLP-1 receptor may result in a protracted activation of the receptor and an increased biological response.
  • the extended half-life of activity of PEG2-ru-40K-N ter -GLP-l and PEG 2 -ru-40K-Lys 26/34 -GLP-l are expected as PEGylation of GLP-1 with a 40 kD PEG is expected to increase the peptide's circulation half-life.

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Abstract

L'invention concerne des peptides qui sont chimiquement modifiés par une fixation covalente d'un oligomère soluble dans l'eau. Un conjugué de l'invention, lorsqu'il est administré par l'une des nombreuses voies d'administration, présente des caractéristiques qui sont différentes des caractéristiques du peptide non fixé à l'oligomère soluble dans l'eau.
PCT/US2011/057267 2010-10-22 2011-10-21 Conjugués polymère-glp 1 pharmacologiquement actifs WO2012054822A1 (fr)

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Cited By (2)

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US20160017016A1 (en) * 2013-03-14 2016-01-21 Medimmune Limited Pegylated glucagon and glp-1 co-agonists for the treatment of obesity
EP3858866A4 (fr) * 2018-09-26 2021-12-01 Jiangsu Gensciences Inc. Protéine de fusion glp1-fc et conjugué associé

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