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WO2002032963A1 - Systèmes immunoadjuvants - Google Patents

Systèmes immunoadjuvants Download PDF

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Publication number
WO2002032963A1
WO2002032963A1 PCT/AU2001/001313 AU0101313W WO0232963A1 WO 2002032963 A1 WO2002032963 A1 WO 2002032963A1 AU 0101313 W AU0101313 W AU 0101313W WO 0232963 A1 WO0232963 A1 WO 0232963A1
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WO
WIPO (PCT)
Prior art keywords
compound
linker
disease
lipophilic
anchor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/AU2001/001313
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English (en)
Inventor
Istvan Toth
Ross Peter Mcgeary
Michael Leo West
Tracie Elizabeth Ramsdale
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Alchemia Pty Ltd
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Alchemia Pty Ltd
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Publication date
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Priority to AU2001295287A priority Critical patent/AU2001295287A1/en
Publication of WO2002032963A1 publication Critical patent/WO2002032963A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • A61K47/54—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 compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H7/00—Compounds containing non-saccharide radicals linked to saccharide radicals by a carbon-to-carbon bond
    • C07H7/02—Acyclic radicals
    • C07H7/033—Uronic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6018—Lipids, e.g. in lipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • A61K2039/645—Dendrimers; Multiple antigen peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support

Definitions

  • This invention relates to delivery systems for pharmaceutically-active agents.
  • the invention relates to compounds comprising a carbohydrate moiety and optionally a lipid moiety, which are useful as delivery agents.
  • synthetic peptides can induce antibodies which are reactive with their cognate sequences in the native proteins, that is, the synthetic peptide and the native protein present the same epitope.
  • Specific antibodies are useful as reagents in a wide variety of investigations.
  • peptide antigens made by peptide synthesis techniques known in the art, are useful for producing immunogens, in immunoprophylaxis and in affinity purification of proteins, antibodies, or other molecules .
  • cognate peptide sequences may not in themselves provide suitable antigens.
  • protein immunogens are glycosylated, and the glycosylation pattern itself may confer higher antigenicity than the cognate peptide sequence.
  • Small peptide molecules or saccharides may not themselves be of sufficient molecular weight to be immunogenic at all or to a sufficient degree, but can be rendered immunogenic or more immunogenic by conjugation to a carrier molecule, for example a protein or a synthetic polymer.
  • a carrier molecule for example a protein or a synthetic polymer.
  • Proteins such as bovine serum albumin (BSA) , keyhole limpet haemocyanin (K H) or diphtheria toxin (DT) , are widely used for this purpose.
  • BSA bovine serum albumin
  • K H keyhole limpet haemocyanin
  • DT diphtheria toxin
  • the conjugated product is antigenic, it comprises a large number of epitopes other than that associated with the synthetic peptide of interest.
  • the MAP comprises a core matrix formed of a low number (n) of sequential levels of dendritically-linked trifunctional amino acids, in practice lysine molecules.
  • the core matrix has 2 n terminal functionalities, in practice amine functionalities, each of which can be conjugated with a synthetic peptide.
  • the MAP was synthesized by forming the core matrix using a conventional stepwise solid phase procedure. Subsequent levels of the core were formed by similar steps. A preformed synthetic peptide antigen molecule was joined to each terminal amine functionality via a triglycyl linker, followed by cleavage from the resin by conventional means .
  • n coupling points When employing polylysine dendrimers for an antigen or pharmaceutically active molecule, 2 n coupling points, where n refers to the number of levels of lysines in the dendrimer, may be generated at each successive branching point.
  • n when using carbohydrates, up to 4 n coupling points, where n refers to the number of monosaccharide units in the carbohydrate, may be generated as each successive monosaccharide is coupled.
  • the higher density using fewer units which is possible when using a carbohydrate core leads to a greater propensity for synergistic effects and cooperative binding.
  • Toth et al have developed a system in which a lipidic adjuvant is chemically conjugated to the antigen of interest.
  • This system combines the MAP system with a lipidic anchor moiety to form a Lipid-Polylysine Core- Peptide (LCP) (Toth et al . , 1993; Toth and Gibbons, British Patent Application No. 9215780.9 (24 July, 1992); Toth and Gibbons, European Patent Application No. 93917902.4).
  • LCP Lipid-Polylysine Core- Peptide
  • LAAs lipid amino acids
  • the carrier, adjuvant and antigen are contained in the same molecule.
  • This molecular entity can be readily synthesised in a single reaction vessel, by step-wise solid phase methods, without requiring the isolation of intermediates.
  • Properties such as molecular weight, charge, lipophilicity, targeting moieties, and radiolabels can readily be varied in the LCP system, by modifying these variables in the LAA.
  • Promising immunological data were obtained in preliminary studies using the LCP system via the parenteral route. For example, the immunogenicity was found to be much greater for synthetic peptides from Group A Streptococci (Pruksakom et al . 1994), foot and mouth disease virus (FMDV) (France et al .
  • S is a carbohydrate core matrix
  • A is a pharmaceutically active moiety
  • L is a linker or covalent bond
  • n is an integer of 2 to 19
  • R' is hydrogen or -X-Z in which Z is a lipophilic anchor and X is a linker or covalent bond.
  • the carbohydrate core matrix S may be a natural or synthetic monosaccharide or an oligosaccharide preferably having 1 to 6 monosaccharide moieties .
  • Non limiting examples include, glucose, glucosamine, galactose, mannose, glucuronic acid, iduronic acid, idose, fucose, galactosamine, sucrose, fructose, maltose, lactose, lactosa ine, globotriose, globotetraose, sialyl lewis X, lewis X, lewis Y, lewis b tetrasaccharide, lewis a, sialyl lewis a, chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose, blood group A trisaccharide, blood group b trisaccharide, blood group H dis
  • n will depend on the number of available carbohydrate functional groups on the core matrix S .
  • the available functional groups on S will be free hydroxyl, amino and/or carboxylate groups.
  • S is a monosaccharide
  • n is an integer from 2 to 5 (Ex I)
  • S is a disaccharide
  • n is an integer from 2 to 7 (Ex II)
  • S is a trisaccharide
  • n is an integer from 2 to 10, and so on.
  • the pharmaceutically-active moiety A may be selected from synthetic or natural peptides, proteins, mono- or oligosaccharides , sugar-amino acid conjugates, sugar-peptide conjugates, drugs, pro-drugs or drug like molecules. Also included for moiety A is antibodies or are antigen binding fragments of whole antibody, wherein the fragments retain the binding specificity of the whole antibody molecule.
  • the binding fragments include, for example, Fab, F(ab')2, and Fv fragments. Binding fragments can be obtained using conventional techniques, such as proteolytic digestion of antibody by papsin or pepsin, or through standard genetic engineering techniques that are well known in the art.
  • the pharmaceutically-active moiety may, for example, serve as an antigen or antigenic determinant, toxin, ligand, drug or pro-drug, and may be either antigenic or non-antigenic when taken alone.
  • pharmaceutically active moieties it will be appreciated that they may be the same or different.
  • the pharmaceutically-active moieties A are attached to the core matrix either directly or via the linker L using coupling methods known in the art and compatible with the functionalities on the linker L.
  • the linker L may be alkyl, alkenyl , alkynyl, heteroalkyl, arylalkyl, or heteroarylalkyl of 3 to 12 atoms in length, which may be optionally substituted, branched or linear.
  • linkers L may be attached to the core matrix S by one or more linkage methods known in the art, such as, for example, ether, ester, or amide linkages .
  • the linker X may be: a) a monosaccharide or an oligosaccharide having 1 to 4 monosaccharide moieties, which modifies the physicochemical properties, such as water solubility, targets the compound to specific sites such as mannose, targets an active uptake mechanism such as glucose transport system and/or modifies the immune response; b) a spacer of up to 14 atoms which separates the lipophilic anchor Z from the core matrix S; or c) a peptide or amino acid which modifies the physicochemical properties of the compound, or which provides suitable spacing between the lipophilic anchor Z and the core matrix S .
  • the spacer (b) may be an alkyl, alkenyl, alkynyl, heteroalkyl, arylalkyl or heteroarylalkyl, which may be optionally substituted. Suitable spacers include polyethyleneglycol or polyglycine.
  • the lipophilic anchor Z may be
  • each of R 1 and R 2 are the same or different and selected from: hydrogen or an alkyl or alkenyl having 4 to 24 carbon atoms, which may be optionally substituted with substituents that do not significantly adversely affect the lipophilic nature of the anchor, with the proviso that both R 1 and R 2 are not hydrogen;
  • X is the linker as defined in formula I above, with the proviso that only one of R 1 , R 2 and R 3 can be hydrogen;
  • W is a alkyl or alkenyl group having 4 to 24 carbon atoms which may be optionally substituted with substituents do not significantly adversely affect the lipophilic nature of the anchor
  • (X) is the linking group to the sugar core, with the provisos that at least two of the groups R 4 to R 8 must be OW and only one of the groups may be O(X) or NH(X) . in which R 9 is selected from OW, (X), 0(X), NH-W,
  • R 9 may only be (X), O(X) or NH- (X) if none of R 4 , R 5 , R 6 , R 7 or R 8 are O(X) or NH(X) . additionally, R 4 may be thioalkyl .
  • the compounds of formulae Va, Vb and Vc represent a new class of lipophilic anchors and therefore form another aspect of the invention.
  • Suitable substituents in (a) , (b) , (c) and (d) include halo, hydroxy and thiol . It may be convenient to include substituents so as to confer suitable solvent solubility properties upon the system.
  • the lipoamino acids in (a) may be coupled sequentially, or may be interspersed with up to 4 other amino acid spacers such as serine or arginine which modify the properties such as solubility of the lipophilic anchor.
  • alkyl denotes straight chain, branched or cyclic alkyl, preferably C ⁇ _ 3u alkyl or cycloalkyl.
  • straight chain and branched alkyl examples include methyl, ethyl, propyl , isopropyl, butyl, isobutyl, sec- butyl, tert-butyl, amyl, isoamyl, sec-amyl, 1,2- dimethylpropyl, 1, 1-dimethylpropyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1- dimethylbutyl, 2 , 2-dimethylbutyl , 3 , 3 -dimethylbutyl , 1,2- dimethylbutyl, 1, 3 -dimethylbutyl, 1, 2 , 2-trimethylpropyl , 1, 1, 2-trimethylpropyl, heptyl, 5-methylbexyl , 1- methylhexyl, 2 , 2-dimethypentyl, 3 , 3-dimethylpentyl, 4,4- dimethylpentyl, 1,
  • cyclic alkyl examples include mono- or polycyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and the like.
  • alkenyl denotes groups formed from straight chain, branched or cyclic alkenes including ethylenically mono-, di- or poly-unsaturated alkyl or cycloalkyl groups as defined above, preferably C 2 - 2 ( . alkenyl .
  • alkenyl examples include vinyl, allyl, 1-methylvinyl , butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3- hexenyl, cyclohexenyl, 1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1, 3-butadienyl, 1, 4-pentadienyl, 1,3- cyclopentadienyl, 1, 3-hexadienyl, 1 , 4-hexadienyl , 1,3- cyclohexadienyl, 1, 4-cyclohexadienyl, 1, 3-cycloheptadienyl , 1, 3 , 5-cycloh
  • alkynyl denotes groups formed from straight chain, branched, or mono- or poly- or cyclic alkynes .
  • alkynyl include ethynyl, 1-propynyl, 1- and 2-butynyl, 2-methyl-2-propynyl, 2-pentynyl, 3- pentynyl, 4-pentynyl, 2-hexynyl, 3-hexylnyl, 4-hexynyl, 5- hexynyl , 10-undecynyl, 4-ethyl-l-octyn-3-yl, 7-dodecynyl , 9- dodecynyl, 10-dodecynyl, 3-methyl-l-dodecyn-3-yl, 2- tridecynyl, 11-tridecynyl, 3-tetradecynyl , 7-hexadecynyl, 2- tride
  • aryl used either alone or in compound words such as "heteroaryl” denotes single, polynuclear, conjugated and fused residues of aromatic hydrocarbons or aromatic heterocyclic ring systems.
  • aryl include phenyl, biphenyl, terphenyl, quaterphenyl , phenoxyphenyl , naphthyl, tetrahydronaphthyl , anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenzanthracenyl, phenanthrenyl , fluorenyl, pyrenyl, indenyl , azulenyl, chrysenyl , pyridyl , 4-phenylpyridyl , 3 -phenylpyridyl , thienyl, furyl, pyrryl, pyrrolyl, furanyl, imadazolyl, pyrrol
  • optionally substituted means that a group may or may not be further substituted with one or more groups selected from alkyl, alkenyl, alkynyl, aryl, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, carboxy, benzyloxy, haloalkoxy, haloalkenyloxy, haloaryloxy, nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl , azido, amino, alkylamino, alkenylamino, alkynylamino , arylamino, benzylamino, acylamino, acyl , alkenylacyl, alkynylacyl, arylacyl, acylamino, acyloxy,
  • the compound of formula I is of the formula la
  • S is a mono- or disaccharide example, such as, glucose, galactose, lactose, glucosamine, or glucuronic acid; and n is an integer from 2 to 7; for example in the case of a monosaccharide core matrix n is an integer from 2 to 5, and in the case of a disaccharide core matrix, n is an integer from 2 to 7.
  • X and A are as defined for formula I
  • Z is 2 to 4 lipoamino acids of general formula II as defined above, in which R 1 and R 2 are either H or a linear alkyl or alkenyl group having 6 to 20 carbon atoms;
  • S is a mono- or disaccharide such as glucose, galactose, lactose, glucosamine or glucuronic acid;L is attached to S by an ether or amide bond, and each L is an optionally substituted alkyl or optionally substituted heteroalkyl group of 3 to 12 atoms in length; and n is an integer from 2 to 7; for example in the case of a monosaccharide core matrix n is an integer from 2 to 5 and in the case of a disaccharide core matrix, n is an integer from 2 to 7.
  • Z is a compound of general formula Va, Vb, Vc as defined above .
  • the compound of formula I is of the formula lb
  • the core matrix S is a mono or disaccharide
  • the linkers L are the same or different and selected from linear alkyl or heteroalkyl groups of 3 to 12 atoms in length which may be optionally substituted and are attached to the core matrix S by an ether or amide bond
  • the pharmaceutically active moieties A are the same and are selected from the group consisting of synthetic or natural peptides, proteins, mono or oligosaccharides, sugar-amino acid conjugates, sugar- peptide conjugates, drugs, pro-drugs, and drug-like molecules .
  • the invention also provides a method for the preparation of a compound of formula I, comprising the steps of
  • the person skilled in the art will be aware of suitable methods of performance all of steps (a) to (c) .
  • the method may be performed in solution, on a solid support or by a combination of solution phase and solid supported steps .
  • the linker L is attached to the matrix core S by the formation of an ether bond in solution.
  • the matrix core S - linker L conjugate may then be immobilised onto a solid support or reacted in solution with a pharmaceutically active moiety A to form a covalent linkage between the pharmaceutically active moiety A and the linker L.
  • This covalent bond may be an amide, disulfide, ester, urea or other suitable covalent bond known in the art.
  • step (c) (i) and (ii) a variety of methods known in the art may be used.
  • the linkage may form a urea or thiourea bond between the lipophilic anchor or linker and the core matrix, or a linker such as glutaric acid is coupled to form peptide bonds to both the core matrix and the lipophilic anchor.
  • the formation of the linkage between the matrix core and the lipophilic anchor, with or without a spacer is carried out on a solid support.
  • the compound of formula I is built up by a step-wise procedure from the respective components on a solid support.
  • the invention provides a composition comprising a compound according to the first or the second aspect of the invention, together with a pharmaceutically-acceptable carrier .
  • the invention provides a method of treating or preventing a disease comprising the step of administering to a subject in need thereof a therapeutically-effective amount of a compound according to the first or the second aspect of the invention, together with a pharmaceutically-acceptable carrier.
  • a pharmaceutically-acceptable carrier for the purposes of this specification it will be clearly understood that the word “comprising” means “including but not limited to”, and that the word “comprises” has a corresponding meaning.
  • Figure 1 is a schematic representation of a representative lipid-sugar immunogen construct according to the invention, in which A is an antigenic epitope, L is a linking arm, and n refers to the length of the lipid chain.
  • the present invention relates to a new antigen delivery system, the lipid-sugar- immunogen, wherein lipid anchors comprised of lipid- aminoacids (LAA) , liposaccharides, lipoglycerols, or similar structural motifs, are coupled via a linker, which may be peptidic, polyethyleneglycolic, aryl or otherwise, to a carbohydrate core structure.
  • the carbohydrate core structure is composed of a mono or oligosaccharide, in which some, but not necessarily all, carbohydrate functional groups are coupled, either directly or via a linking or spacer arm, to a pharmaceutically active molecule of interest.
  • the structure of the lipid-sugar- immunogen is schematically illustrated in Figure 1. The entire system may be synthesised while coupled to a solid support .
  • the unique fixed structural features of carbohydrates allow for multiple presentation of a pharmacologically interesting molecule in a three- dimensional space. This provides the opportunity for a more concerted approach to antigen delivery, whereby optimal distances between antigenic epitopes can be established for the highest activity, even to the extent of employing two different epitopes in concert. For instance it may be advantageous to provide a molecule where improved immunoprophylaxis is achieved by raising antibodies to more than one epitope.
  • the compounds, compositions and methods of the invention are useful for immunoprophylaxis, toxin binding or drug delivery.
  • the compound of formula I is useful as a deliver system for pharmaceutically-active agents .
  • subject refers to any animal having a disease or condition which requires treatment with a pharmaceutically-active agent.
  • the subject may be a human, or may be a domestic or companion animal. While it is particularly contemplated that the compounds of the invention are suitable for use in medical treatment of humans, it is also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as felids, canids , bovids, and ungulates .
  • the methods of this invention involve in one embodiment, (1) the administration of compound of formula I, prior to, together with, or subsequent to the administration of a pharmaceutically-active agent; or (2) the administration of a combination of compound of formula I and a pharmaceutically-active agent.
  • terapéuticaally effective amount is meant an amount of a compound of the present invention effective to yield a desired therapeutic response. For example to prevent or treat a disease which is effected by administration of a pharmaceutically-active agent .
  • a “pharmaceutical carrier” is a pharmaceutically acceptable solvent, suspending agent or vehicle for delivering the compound of formula I and/or pharmaceutically-active agent to the subject.
  • the carrier may be liquid or solid and is selected with the planned manner of administration in mind.
  • the compound of formula I and/or pharmaceutically-active agent may be administered orally, topically, or parenterally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.
  • parenteral as used herein includes subcutaneous injections, aerosol, intravenous, intramuscular, intrathecal, intracranial, injection or infusion techniques.
  • the present invention also provides suitable topical, oral, and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention.
  • the compounds of the present invention may be administered orally as tablets, aqueous or oily suspensions, lozenges, troches, powders, granules, emulsions, capsules, syrups or elixirs.
  • the composition for oral use may contain one or more agents selected from the group of sweetening agents, flavouring agents, colouring agents and preserving agents in order to produce pharmaceutically elegant and palatable preparations.
  • the tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be, for example, (1) inert diluents, such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintegrating agents, such as corn starch or alginic acid;
  • binding agents such as starch, gelatin or acacia
  • lubricating agents such as magnesium stearate, stearic acid or talc.
  • These tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Coating may also be performed using techniques described in the U. S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotic therapeutic tablets for control release.
  • the compound of formula I as well as the pharmaceutically-active agent useful in the method of the invention can be administered, for in vivo application, parenterally by injection or by gradual perfusion over time independently or together. Administration may be intravenously, intra-arterial, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally . For in vi tro studies the agents may be added or dissolved in an appropriate biologically acceptable buffer and added to a cell or tissue. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, anti-microbials, anti-oxidants, chelating agents, growth factors and inert gases and the like.
  • the invention can be used to prevent or treat any disease associated with the use of pharmaceutically-active agents, including, for example, neoplasms, cancers (eg., cancers of the breast, lung, prostate, kidney, skin, neural, ovary, uterus, liver, pancreas, epithelial, gastric, intestinal, exocrine, endocrine, lymphatic, haematopoietic system or head and neck tissue), fibrotic disorders and the like.
  • neoplasms eg., cancers of the breast, lung, prostate, kidney, skin, neural, ovary, uterus, liver, pancreas, epithelial, gastric, intestinal, exocrine, endocrine, lymphatic, haematopoietic system or head and neck tissue
  • cancers eg., cancers of the breast, lung, prostate, kidney, skin, neural, ovary, uterus, liver, pancreas, epithelial, gastric, intestinal, exocrine,
  • disorders of the central nervous system including Alzheimer's disease (AD) and other forms of dementia and memory loss, motor neurone diseases, disorders of cardiovascular system including cardiac hypertrophy, congestive heart failure, hypertension, hormonal imbalance, atherosclerosis, disorders of development and growth including, disorders of glucose and fat metabolism and the like.
  • AD Alzheimer's disease
  • cardiovascular system including cardiac hypertrophy, congestive heart failure, hypertension, hormonal imbalance, atherosclerosis, disorders of development and growth including, disorders of glucose and fat metabolism and the like.
  • the terms “treating”, “treatment” and the like are used herein to mean affecting a subject, tissue or cell to obtain a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure of a disease.
  • Treating covers any treatment of, or prevention of disease in a vertebrate, a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject that may be predisposed to the disease, but has not yet been diagnosed as having it; (b) inhibiting the disease, ie., arresting its development; or (c) relieving or ameliorating the effects of the disease, ie., cause regression of the effects of the disease.
  • the invention includes various pharmaceutical compositions useful for ameliorating disease.
  • the pharmaceutical compositions according to one embodiment of the invention are prepared by bringing a compound of formula I, analogue, derivatives or salts thereof and one or more pharmaceutically-active agents or combinations of compound of formula I and one or more c pharmaceutically- active agents into a form suitable for administration to a subject using carriers, excipients and additives or auxiliaries.
  • carriers or auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial, anti- oxidants, chelating agents and inert gases.
  • Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington's Pharmaceutical Sciences, 15th ed. Easton: Mack Publishing Co. , 1405-1412,1461-1487 (1975) and The National Formulary XIV., 14th ed. Washington: American Pharmaceutical Association (1975) , the contents of which are hereby incorporated by reference.
  • the pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman's The Pharmacological Basis for Therapeutics (7th ed.).
  • the pharmaceutical compositions are preferably prepared and administered in dose units.
  • Solid dose units are tablets, capsules and suppositories.
  • different daily doses can be used for treatment of a subject. Under certain circumstances, however, higher or lower daily doses may be appropriate.
  • the administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.
  • the pharmaceutical compositions according to the invention may be administered locally or systemically in a therapeutically effective dose. Amounts effective for this use will, of course, depend on the severity of the disease and the weight and general state of the subject.
  • dosages used in vi tro may provide useful guidance in the amounts useful for in si tu administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of the cytotoxic side effects.
  • animal models may be used to determine effective dosages for treatment of the cytotoxic side effects.
  • Formulations for oral use may be in the form of hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin. They may also be in the form of soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions normally contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspension.
  • excipients may be (1) suspending agent such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; (2) dispersing or wetting agents which may be (a) naturally occurring phosphatide such as lecithin; (b) a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate; (c) a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethylenoxycetanol; (d) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol such as polyoxyethylene sorbitol monooleate, or (e) a condensation product of ethylene oxide with a
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension.
  • This suspension may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1 , 3-butanediol .
  • the acceptable vehicles and solvents that may be employed are water,
  • Liposomes can be formed from a variety of phospholipids , such as cholesterol, stearylamine, or phosphatidylcholines .
  • Dosage levels of the compound of formula I of the present invention are of the order of about 0.5mg to about 20mg per kilogram body weight, with a preferred dosage range between about 0.5mg to about lOmg per kilogram body weight per day (from about 0.5gms to about 3gms per patient per day) .
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage will vary depending upon the host treated and the particular mode of administration.
  • a formulation intended for oral administration to humans may contain about 5mg to lg of an active compound with an appropriate and convenient amount of carrier material which may vary from about 5 to 95 percent of the total composition.
  • Dosage unit forms will generally contain between from about 5mg to 500mg of active ingredient.
  • the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
  • solvates may form solvates with water or common organic solvents .
  • Such solvates are encompassed within the scope of the invention.
  • the compounds of the present invention may additionally be combined with other compounds to provide an operative combination. It is intended to include any chemically compatible combination of pharmaceutically- active agents, as long as the combination does not eliminate the activity of the compound of formula I of this invention.
  • the invention will now be further described by way of reference only to the following non-limiting examples. It should be understood, however, that the examples following are illustrative only, and should not be taken in any way as a restriction on the generality of the invention described above. Furthermore, it will be clearly understood that although the invention is illustrated particularly with reference to delivery of antigens, the invention is not restricted solely to the delivery of antigens, but may be employed in the delivery of any pharmaceutically active moiety.
  • the lipidic anchor may be absent or present; when the anchor is absent, the carbohydrate scaffold becomes a useful tool for the ultiple presentation of pharmaceutically active molecules of interest, displaying the unique structural features described above which allow for a fixed presentation of multiple copies of a pharmaceutically active moiety in three-dimensional space. This may be of particular benefit where multivalency plays an important binding.
  • the lipoophilic anchor may be used to target particular cell membranes, to form liposomes or to induce aggregation, by exploiting the particulate forming properties of the anchor [10,11].
  • the invention provides a novel vesicular drug delivery system in which both drug and particulate carrier are present in the same molecule.
  • the protected amino acids 2- (lH-benzotriazole-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate (HBTU) activating reagent and the auxiliary nucleophile hydroxybenzotriazole (HOBt) were used in equimolar quantities.
  • N,N-diisopropylethylamine (DIEA) was used to both neutralise the resin for coupling and for in si tu activation of the amino acids.
  • DIEA diisopropylethylamine
  • a second coupling was performed, using a symmetrical anhydride of the N ⁇ -Boc amino acids in dichloromethane (20ml) and N-methylpyrrolidinone (5ml).
  • the protecting groups used for the synthesis of peptides were Boc groups for the ⁇ -amino-termini, Acm for Cys, 2-Cl-Z for Lys , Bzl for Thr and 2-Br-Z for Tyr. In all the couplings the coupling efficiency was more than 99.8 % as indicated by quantitative ninhydrin testing.
  • deprotection of the N-terminus was performed using TFA (10ml, 2 x 1 min) .
  • the deprotected resin-peptide was neutralised with 10% N,N-diisopropylethylamine in dichloromethane.
  • the resin-peptide was carefully washed with DMF after each deprotection and neutralisation step. Conjugation of lipoamino acids. Lipoamino acids were incorporated into the matrix core (S) - linker (X) chain by as N- Boc protected residues using standard
  • HBTU/HOBt/DIEA mediated coupling strategies When lipoamino acids were incorporated at the C-terminus of the linker a spacer (Gly and/or GABA) was used to overcome the difficulty in attaching a lipoamino acid to resin directly. -The 2- ( te-rt-butoxycarbonylamino) -alkanoic acids were synthesized from 1-bromoderivatives (Gibbons et al . , 1990). The linker-conjugate was removed from the resin support with high HF method (1.5ml p-cresol, 15 ml HF) to yield the crude construct, which was precipitated with ether and redissolved in 95 % acetic acid.
  • high HF method 1.5ml p-cresol, 15 ml HF
  • Analytical HPLC separation was carried out on a Vydac C ⁇ 8 5 RAC column (4.6 x 250 mm at a constant flow rate of 1.2 ml min "1 .
  • Mobile phases employed were: (A) 0.06 % TFA (aq ) and (B) 0.06 % TFA in 90 % acetonitrile (aq) .
  • HPLC grade acetonitrile (Aldrich) and water were filtered through a 23 ⁇ m membrane filter and degassed with helium flow prior to use.
  • Analytical PR-HPLC was performed using a Waters 616/600S twin pump system and 486 tunable absorbance detector controlled via a Millennium software package.
  • Post-column eluent was monitored by UV absorbance at 214 nm.
  • the gradient employed for separation was 0 - 100 % (B) in 20 min linearly (Retention times in Table 4).
  • For preparative separation a TSK-GEL preparative Ci 8 column was used (30 x 250 mm) .
  • the gradient employed for separation was 0 - 70 % (B) in 180 min linearly, staying at this concentration for 60 min and decreasing steadily to 0% (B) for 30 min at a constant flow of 7 ml min "1 .
  • the gradient was effected by two microprocessor- controlled Gilson 302 single piston pumps. Compounds were detected with a Holochrome UV-VIS detector at 230 nm.
  • Mass spectra were run on a Fisons VG-TOF spectrometer using matrix assisted laser desorption ionisation (MALDI) or a VG Analytical ZAB- SE instrument, using fast atom bombardment (FAB) ionisation or a Finnigan MassLab Navigator Quadrupole Mass Spectrometer using electrospray ionisation.
  • MALDI matrix assisted laser desorption ionisation
  • FAB fast atom bombardment
  • N 2 flow 300 1/hr, temp. 180°C, cone voltage 49 V.
  • the carrier proteins is conjugated to the peptides.
  • Either KLH or BSA may be used as the immunogen carrier in the ELISA experiments.
  • the glutaraldehyde method is typically used for conjugation, but other conjugating agents such as HOBT, water-soluble carbodiimide etc. may be used if necessary.
  • the unused coupling agent is removed by passing through Sephadex G-25 column. Conjugated samples are lyophilized and kept at -186°C.
  • the system is injected directly. Rabbits are used to produce polyclonal antibodies using the immunization protocol 1-21-35-60, i.e. immunisation on days 1, 21 35 and 60.
  • Methyl 2, 3 , 4 6-tetra-O-allyl - -D-glucopyranoside (2) .
  • Methyl 2 , 3 , 4, 6-Tetra-O- (3 ' - (2 ' ' - aminoethylthio) propyl) - ⁇ -D-galactopyranoside (4) is dissolved in DMF and to this solution is added a solution of a pharmaceutically active agent containing a free carboxylate function, HOBT and diisopropylethylamine .
  • the mixture is stirred at room temperature for 30 minutes to form an amide linkage by reaction of the activated carboxylate with the free amine.
  • the azido sugar 8 (0.38 g, 0.4 mmol) dissolved in ethyl acetate (10 mL) was hydrogenated using Pd (10% on charcoal, 90 mg, 10%) catalyst for 2 days at room temperature.
  • the catalyst was filtered off and washed with ethyl acetate (40 mL) and the filtrate was evaporated.
  • the residue was purified with ethyl acetate-ether (9:1) eluent containing 0.5% triethylamine .
  • the amino sugar 9 (140 mg, 0.15 mmol) was refluxed with succinic anhydride (17 mg, 1.7 mmol) in dry CH 2 C1 2 (5 mL) overnight. The solvent was evaporated and the residue was purified by chromatography with CHCl -MeOH
  • the azido sugar 16 (110 mg, 0.1 mmol) dissolved in ethyl acetate (10 mL) was hydrogenated using Pd catalyst (10% on charcoal, 60 mg) for 2 days at room temperature.
  • the amino sugar 17 (45 mg, 0.04 mmol) was refluxed with the succinic anhydride (5.4 mg, 0.05 mmol) in dry CH 2 C1 2 (5 mL) overnight. The solvent was evaporated and the residue was purified by chromatography with CHCl 3 -MeOH (95:5).
  • Resin-bound glycosides 10 and 18 were treated with hydrazine hydrate (67 ⁇ L, 1.38 mmol) in ethanol (5 mL) at 60°C for 1 day.
  • hydrazine hydrate 67 ⁇ L, 1.38 mmol
  • ethanol 5 mL
  • the glycoside was cleaved from the resin by stirring the resin with TFA- water-TIS (95/2.5/2.5) for 2 h.
  • the resin was filtered off and washed with TFA.
  • the solvent was evaporated and the residue was neutralized, and triturated with ether.
  • the precipitate was analyzed by MS (FAB) and elemental analysis. Cleavage of resin bound, deprotected 10 yielded 19, while cleavage of resin bound, deprotected 18 yielded 20.
  • Tetra ( tetrahydropyranyloxy-ethoxy) ethyl 1-S- methyl galactoside 21 (60 mg) dissolved in chloroform - methanol (1/1; 20 ml) was treated with 3N HCl (30 ⁇ l) at 50°C for 4 hours. The solvent was evaporated and the product was dried by repeated evaporation with toluene. The product 22 (36 mg, 97%) is a thick syrup.
  • H6 B 4.00, 4H, br t(J " 5.1), 2x OCH 2 ; 3.79, 1H, d(J 2.9), H4; 3.61, 3H, s, OMe; 3.60, 1H, t(J " 9.5), H5 ; 3.50, 1H, t(J 8.9), H2; 3.34, 1H, dd(J " 2.8, 9.5), H3.
  • N- (butoxycarbonyl) ethylenediamine [Muller, D. , Zeltser, I., Bitan, G. and Gilon, C, J. Org. Chem . , 1997, 62, 411-416] (3.10 g, 19.4 mmol), DIEA (4.50 ml, 3.33 g, 25.8 mmol) and N,N-dimethylformamide (40 ml) .
  • HBTU 4.90 g, 12.9 mmol
  • Azido 2, 3 , 6, 2 X 3 ' , 4 ' , 6 ' -Hepta-O- (2-cyanoethyl ) - lactopyranoside 32 The lactose azide 31 (2.46 g, 6.3 mmol) was suspended in CH 3 CN (50 mL) and acrylonitrile (12.8 mL, 10.4 g, 194 mmol) and DBU (1.2 mL, 1.2 g, 7.8 mmol) were added.
  • a second and third lipoaminoacid is then added and deprotected using the same protocol. If the coupling is less than 99% complete, the resin is drained and resubmitted to the coupling step for a further 30 minutes. If the coupling is still less than 99% after a second coupling, the resin is drained and treated with 10% acetic anhydride in methanol for 5 minutes, drained, washed and the Boc group cleaved. When the construct has the desired number of lipoaminoacids (in this case 3), the terminal Boc is cleaved and a sugar linker conjugate may be added.
  • the desired number of lipoaminoacids in this case 3
  • compound 10 or 18 (4 eq) with HOBT (4 eq) and DIPEA (6 eq) is added and the resin shaken for 30 minutes.
  • Quantitative ninhydrin assay is again used to asses the completion of the reaction and if necessary a second coupling and or capping with acetic anhydride performed.
  • the phthaloyl groups are then removed from the amino side chains by treatment with hydrazine hydrate as detailed above ad the resin is washed with DMF.
  • a pharmaceutically active agent with a free carboxyl (10 eq) , HOBT (10 eq) and DIPEA (16 eq) is then added and the mixture shaken for 2 hours.
  • Quantitative ninhydrin is again used to determine the extent of coupling and if required a second coupling or capping with acetic anhydride is performed.
  • the resin is washed with DMF, 50% methanol/DCM and finally DCM then air dried before submitting to cleavage using the Hi/Low HF procedure [2].
  • Methyl 2-azido-2-deoxy-l-thio- ⁇ -D-glucopyranoside 35 (1.0 g, 4.255 mmol) was dissolved in anhydrous DMF (50 mL) .
  • NaH (0.68 g (60 % oil dispersion), 17.02 mmol) was added to the mixture and stirred for 10 minutes, followed by 1-bromooctane (5.15 mL, 29.78 mmol). Additional NaH (0.3 g (60% oil dispersion), 7.5 mmol) and 1-bromooctane (2.5 mL, 14.47 mmol) were added after three hours and the reaction left overnight.
  • Methyl 2-azido-2-deoxy-3 , 4, 6-tri-O-octyl-l-thio- ⁇ -D-glucopyranoside 36 200 mg, 0.349 mmol
  • DTT 110 mg, 0.699 mmol
  • dry triethylamine 97 ⁇ L, 0.699 mmol
  • Nitrogen was bubbled in to the reaction mixture for 30 minutes, followed by overnight stirring at room temperature. Additional DTT (110 mg, 0.699 mmol) and dry triethylamine (97 ⁇ L, 0.699 mmol) were added and stirring continued for 24 hours.
  • the solution was diluted with CHC1 3 (7 mL) and washed with 10 % citric acid solution (2 x 20 mL) , saturated NaHC0 solution (2 x 20 mL) and saturated brine solution (2 x 20 mL) .
  • the aqueous layers were back- extracted with CHC1 (20 mL) and the organic layers combined, dried over MgS0 4 , filtered and evaporated to dryness.
  • the crude residue was purified by silica gel chromatography, using CHC1 3 to CHCl 3 -MeOH (10-1) as the mobile phase.
  • compounds 40, 41 and 42 can be prepared by reaction of the lipidic anchor 37 with matrix-core - linker components 10, 18 and 30 respectively under the same conditions .
  • the solution was diluted with DCM (5 mL) and washed with water (5 mL) , 5% citric acid solution (5 mL) , saturated NaHC0 3 solution (5 mL) and saturated brine solution (5 mL) .
  • the aqueous layers were back-extracted with DCM (5 mL) and the organic layers combined, dried over MgS0 , filtered and evaporated to dryness.
  • the crude residue was purified by silica gel chromatography, using CHC1 3 to CHCl 3 -MeOH (10-1) as the mobile phase.
  • the product was isolated a white film (8 mg, 23%) .
  • compounds 44, 45 and 46 can be prepared by reaction of the lipidic anchor 34 with matrix-core - linker components 10, 18 and 30 respectively under the same conditions .
  • Gal- ⁇ -l,3-Gal- ⁇ -l,4-Glc and Gal- ⁇ -1 , 3-Gal- ⁇ -l, 4- GlcNAc are carbohydrate epitopes present on the surface of non-human cells and are responsible for the recognition of xenobiotic invasion. Constructs containing these epitopes may be pharmacologically useful in for example sequestering antibodies, eliciting an antibody response or binding other agents which are reactive with these epitopes, for example clostridium difficile derived toxin a and toxin b.
  • N, N-Diisopropylethylamine (48 ⁇ l, 35 mg, 0.27 mmol) was added to a stirred suspension of the Glucosyl- linker-carboxylic acid 30 (128 mg, 0.136 mmol), HBTU (57 mg, 0.15 mmol) and ammonium chloride (15 mg, 0.27 mmol) in tetrahydrofuran (2.0 ml). After stirring overnight, the mixture was diluted with ethyl acetate (100 ml) and washed with 5% hydrochloric acid (2 x 10 ml), saturated NaHC0 3 (2 x 10 ml) , water (10 ml) and brine (10 ml) , then dried
  • the protected glucosyl-linker-amide above (6.9 mg, 7.4 ⁇ mol) was dissolved in CH 2 C1 2 (1.0 ml). Trifluoroacetic acid (1.0 ml) was added, and the solution was allowed to stand for 5 min before being evaporated, then coevaporated with several portions of CH 2 C1 2 . The residue was then lyophilised three times from 1:1 acetonitrile water.
  • compound 48 was prepared by substituting (Gala ( 1 ⁇ 3 ) Gal ⁇ ( 1 ⁇ 4 ) Glc-NHCONH (CH 2 ) 5 C0 2 H) for (Gala ( 1 ⁇ 3 ) Gal ⁇ ( 1 ⁇ 4 ) GlcNAc-NHCONH (CH 2 ) 5 C0 2 H) .
  • Boc - Glycine was coupled to MBHA resin using standard peptide coupling procedures outlined above. Deprotection of the BOC group and subsequent coupling of three units of 2-aminododecanoic acid through standard BOC/ HBTU chemistry outlined above, yielded the lipidic anchor on resin. Compound 30 was then coupled after hydrogenolysis of the benzyl ester, to yield the linker -core matrix - linker -lipidic anchor construct. The Boc groups were removed with TFA and the peptide sequence synthesized on this construct by sequential HBTU couplings and BOC deprotections. Finally the construct was cleaved from the resin by HF-cleavage as outlined above, and after workup compound 49 was isolated.
  • the construct was characterized by mass spectroscopy m/z 14300 M+H reconstruct. Serum IgG antibody responses to this construct were measured in groups of mice. Antibody titres were compared with those observed for the free peptide and the LCP system (lipoaminoacid - polylysine system). In all cases, the antigen was injected with complete freunds adjuvant. Mice were dosed at day 1 with subsequent boosts at days 7 and 14. Antibody titres were determined after 21 days .
  • Raw data indicates that the free peptide/complete freunds adjuvant system elicited a titre of approximately 10 4 antibodies, the peptide-LCP/complete freunds adjuvant system elicited a titre of approximately 10 6 antibodies, and compound 49/complete freunds adjuvant elicited a titre of 3 x 10 6 antibodies.
  • these experiments indicate that compound 49 elicits an increased immunological response over the free peptide. Further immunological work is ongoing with this and other constructs .

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Abstract

La présente invention concerne des composés représentés par la formule générale (I). dans cette formule, S est la matrice d'un noyau glucide. A est un fragment pharmaceutiquement actif. L est un lien ou une liaison covalente. 'n' est un entier valant de 2 à 19, et R' est hydrogène, thioalkyle ou -X-Z dans lequel Z est un ancrage lipophile et X est un lien ou une liaison covalente. L'invention concerne également les formules Va, Vb et Vc, dans lesquelles R4 à R9 sont tels que définis dans les spécifications. L'invention concerne enfin des systèmes d'apport pour des agents pharmaceutiquement actifs.
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US7737287B2 (en) 2002-03-28 2010-06-15 Alchemia Limited Anomeric derivatives of monosaccharides
WO2003093286A1 (fr) * 2002-05-03 2003-11-13 Alchemia Limited Disaccharides pour la decouverte de medicaments
US7875707B2 (en) 2002-05-03 2011-01-25 Alchemia Limited Disaccharides for drug discovery
US7700577B2 (en) 2002-10-17 2010-04-20 Alchemia Limited Carbohydrate based anti-bacterials
EP1917041A4 (fr) * 2005-08-24 2010-09-29 Cedars Sinai Medical Center Utilisation de traitements a base de fructose pour lutter contre le cancer
US8241607B2 (en) 2005-08-24 2012-08-14 Cedars-Sinai Medical Center Use of fructose-based compounds for the diagnosis of cancer
WO2012060718A1 (fr) * 2010-11-05 2012-05-10 Sally Diana Poppitt Prévention et traitement du syndrome métabolique par l'administration d'un acide gras trans, ou d'un sel, d'un ester ou d'un précurseur de celui-ci, ou d'acide sialique sous forme libre ou liée

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