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WO1999043356A1 - Conjugues antibiotique-ligand et leur mode d'utilisation - Google Patents

Conjugues antibiotique-ligand et leur mode d'utilisation Download PDF

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Publication number
WO1999043356A1
WO1999043356A1 PCT/CA1998/000817 CA9800817W WO9943356A1 WO 1999043356 A1 WO1999043356 A1 WO 1999043356A1 CA 9800817 W CA9800817 W CA 9800817W WO 9943356 A1 WO9943356 A1 WO 9943356A1
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Prior art keywords
glycolipid
glycomimetic
compound
receptor
moiety
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PCT/CA1998/000817
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English (en)
Inventor
Clifford Lingwood
Murugesapillai Mylvaganam
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Hsc Research Development Limited Partnership
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/CA1998/000142 external-priority patent/WO1998037915A1/fr
Application filed by Hsc Research Development Limited Partnership filed Critical Hsc Research Development Limited Partnership
Priority to AU89679/98A priority Critical patent/AU8967998A/en
Publication of WO1999043356A1 publication Critical patent/WO1999043356A1/fr

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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
    • C07H15/18—Acyclic radicals, substituted by carbocyclic rings
    • 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
    • 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
    • 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
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • Glycolipids have been shown to be involved with the early steps of the infectious process associated with several pathogens. For example, it is believed that oligosaccharide moieties coupled to ceramide lipid bases are used by the infectious agents as anchors or adsorption moieties for invasion of the host cells. Many bacteria and viruses have been found to use extracellular membrane components, such as glycolipids to access host cells.
  • influenza A virus belongs to a family of negative strand RNA viruses called the orthomyxoviridae . They are a major cause of respiratory diseases in humans, of both epidemic and pandemic proportion.
  • Influenza A virus has continued to elude control by vaccines and chemotherapy due to antigenic variation (shift and drift). Antigenic shift is the sudden appearance of a different antigen subtype in a population while antigenic drift arises by mutation in the genes encoding for the antigen.
  • Influenza A has been primarily • contained by preparing effective vaccines based on predictions of future antigenic variation.
  • amantadine hydrochloride has been used in the prophylaxis and treatment of influenza A infections.
  • HA hemagglutinin
  • HA hemagglutinin
  • HA is a non- covalently linked trimer in which each subunit consists of disulfide-linked HA1 and HA2 domains. HA1 is involved in receptor binding while HA2 is a transmembrane anchor. HA binds mainly to cell surface sialic acid (NeuAc) bonded to galactose by ⁇ (2— >6) or ⁇ (2—»3) linkages.
  • NeuAc cell surface sialic acid
  • GalC GalC
  • Another example of a virus binding to a cell through glycolipids is the binding of HIV through gpl20 to GalC, a cell surface carbohydrate.
  • Shiga-like toxins a family of powerful, disease producing toxins, are produced by a common bacteria. Escherichia coli, found in humans and .in animals.
  • the term "SLT” is derived from the cytotoxic nature, structural and functional similarity to Shiga toxin which is a protein cytotoxin produced by Shigella dysenteriae type 1. This Shigella serotype is responsible for the most severe cases of bacillary dysentery. SLTs are also known as verotoxins (VTs) because many of the serotypes that produce this toxin were originally characterized as being vero cell toxinogenic.
  • VTs verotoxins
  • SLT producing E. coli are a heterogeneous group of bacteria that belong to several different O:H:K serotypes; all having the ability to discharge one or more SLTs.
  • SLTs are multimeric proteins composed of an enzymatic (A) subunit and multiple (B) subunits responsible for toxin binding to receptors on host tissues.
  • the binding B oligomers of the SLTs recognize host cell globoseries glycolipid receptors containing at a minimum, the disaccharide unit of ⁇ Gal(l-4) ⁇ Gal at the non-reducing terminus.
  • SLTs Foods of animal origin are a major source of human infection by SLTs. Infants, young children and the elderly are the most susceptible to SLT infection, however, anyone who eats contaminated food is prone to infection. Additionally, infection can be spread by person-to-person transmission which can be especially problematic in day care centers and nursing homes.
  • SLT-producing E coli can also cause edema disease ( ⁇ D) in swine.
  • ⁇ D edema disease
  • Antibiotics have been found to be contraindicated in the treatment of SLT producing E. coli. infection in humans and pigs. Antibiotics often enhance toxin production by the bacteria.
  • Treatment of SLT infection generally relies on management of the physiological complications of the infection, e.g. fluid and electrolyte imbalances. Although certain agents have been used to suppress infection of hosts by pathogens, there are limitations to their use.
  • This invention provides methods and compositions which are useful in the treatment of glycolipid mediated states, such as enteropathogenic and enterohemorrhagic E. coli. (EPEC and EHEC, respectively), e.g., verotoxin producing E. coli. (VT ⁇ C), or viruses e.g. orthomyxoviridae, e.g., influenza A, or HIV.
  • Various pathogens e.g., bacteria or viruses, invade host cells via attachment to or interaction with glycolipids which are associated with the host cell.
  • the present invention serves to inhibit a pathogen from invading a host cell by providing a receptor molecule which has been modified with an active agent; the active agent in combination with the receptor molecule combine with the pathogen, thereby rendering it incapable of invading a host cell, or preferably, eradicating the pathogen.
  • the invention provides methods for treating a glycolipid mediated state in a subject by administering to the subject a therapeutically effective amount of a therapeutic compound, such that the glycolipid mediated state is treated.
  • the therapeutic compound is represented by the structure A-B, in which A is a glycomimetic receptor moiety and B is an active agent.
  • the present invention also provides methods of modulating interaction between a pathogenic microorganism and a glycolipid in a subject by administering to the subject a therapeutically effective amount of a therapeutic compound, such that interaction between a pathogenic microorganism and a glycolipid is modulated.
  • the therapeutic compound is represented by the structure A-B, in which A is a glycomimetic receptor moiety and B is an active agent.
  • the present invention provides methods for treating a state characterized by the presence of a shiga-like toxin in a subject by administering to a subject a therapeutically effective amount of a therapeutic compound, such that a state characterized by the presence of shiga-like toxin is treated.
  • the therapeutic compound is represented by the structure A-B, in which A is a glycomimetic receptor moiety and B is an active agent.
  • the present invention provides methods for treating a state characterized by the presence of a virus in a subject by administering to a subject a therapeutically effective amount of a therapeutic compound, such that a state characterized by the presence of virus is treated.
  • the therapeutic compound is represented by the structure A-B, in which A is a glycomimetic receptor moiety and B is an active agent.
  • the present invention further provides compounds represented by the structure A-B.
  • A is a glycomimetic receptor moiety and B is an active agent.
  • the glycomimetic receptor moiety includes an oligosaccharide moiety coupled to a ceramide lipid base.
  • the glycomimetic receptor moiety is gangliotriaosyl ceramide galNAc ⁇ l-4gal ⁇ l-4glc cer (Gg3) or gangliotetraosyl ceramide gal ⁇ l-4galNAc ⁇ l-4glc cer (Gg4) and derivatives thereof.
  • the glycomimetic receptor moiety includes an oligosaccharide moiety coupled to a serine lipid base.
  • the glycomimetic receptor moiety is glycosyl-N-acyl serine, globotriaosyl-N-acyl serine, or galactosyl-N- acyl serine or derivatives thereof.
  • the glycomimetic receptor moiety includes an oligosaccharide moiety coupled to a sulfogalactosylceramide.
  • Active agents are coupled to the glycomimetic receptor moiety and include antibiotics and carbocyclic compounds. Suitable antibiotics include penicillins, cephams, cephalosporins. Suitable carbocyclic compounds include adamantyl, norbornyl or acridine derivatives.
  • the present invention provides pharmaceutical compositions which include a therapeutically effective amount of a therapeutic compound represented by the structure A-B, in which A is a glycomimetic receptor moiety and B is an active agent and a pharmaceutically acceptable carrier.
  • A is a glycomimetic receptor moiety
  • B is an active agent and a pharmaceutically acceptable carrier.
  • the present invention also provides packaged therapeutic compositions for treating a glycolipid mediated state in a subject.
  • the packaged therapeutic compositions include a container for holding a therapeutically effective amount of a therapeutic compound for treating a glycolipid mediated state in a subject and instructions for using the therapeutic composition for treating the glycolipid mediated state.
  • the therapeutic compound is represented by the structure A-B, in which A is a glycomimetic receptor moiety and B is an active agent.
  • the present invention further provides packaged therapeutic compositions for modulating interaction between a pathogenic microorganism and a glycolipid.
  • the packaged therapeutic composition includes a container for holding a therapeutically effective amount of a therapeutic compound for modulating interaction between a pathogenic microorganism and a glycolipid in a subject and instructions for using the therapeutic composition for modulating interaction between the pathogenic microorganism and the glycolipid.
  • the therapeutic compound is represented by the structure A-B, in which A is a glycomimetic receptor moiety and B is an active agent. - 5 -
  • the present invention further provides an assay for determining gpl20 binding activity, by exposing a gpl20 binding compound to gpl20 such that an intermediate is formed. Unbound gp 120 is removed from the intermediate and the intermediate is exposed to HIV sera. The binding of the HIV sera to the gpl20 is detected thereby determining the gpl20 binding activity of a gpl20 binding compound.
  • the present invention further provides an assay for determining the inhibition between a Shiga-like toxin and its glycolipid receptor by providing a container coated with a glycolipid receptor and an inhibitor. The inhibitor and the glycolipid receptor are then mutually exposed and a Shiga-like toxin is provided. The binding of the Shiga like toxin to the receptor is analyzed thereby, determining the inhibition between a glycolipid receptor and a Shiga-like toxin.
  • Figures 1 A and IB depict deacylation of a ceramide and coupling of an antibiotic to the deacylated ceramide.
  • Figures 2 A and 2B depict oxidation of the sphingosine double bond of glycolipids.
  • Figure 3 depicts coupling of an antibiotic with a deacylated ceramide.
  • Figure 4 depicts a ceramide functionalized with multiple antibiotics.
  • Figure 5 represents functionalization of LysoPE.
  • Figure 6 depicts adamantyl glyconjugates of Gb ⁇ C and LC.
  • Figure 7 depicts TLC analysis of serine and ceramide acids.
  • Figure 8 depicts mass spectra of serine and ceramide acids of behenic analogs of GalC.
  • Figure 9 depicts mass spectra of serine and ceramide acids of palmitic analogs of GalC.
  • Figure 10 depicts fragmentation patterns of serine and ceramide acids.
  • Figures 11A and 1 IB depict ion mass spectra of the serine acid from GM, and its production from GM,.
  • Figure 12 are western blots showing conjugate binding with gpl20.
  • Figure 13A depicts inhibition of HIV coat protein gpl20 binding to GalC and SGC.
  • Figure 13B shows the inhibitor compounds used in the gpl20 inhibition assay.
  • Figure 14 shows glycolipid/lipid binding specificity.
  • Figure 15 shows enhanced inhibitory activity of Gb4-ampicillan compared to ampicillin for uropathogenic E. coli.
  • Figure 16 shows inhibition of VTl binding to Gb3 phospholipid bilayer.
  • Figure 17 shows results of a VTl binding assay with inhibitors.
  • Figure 18 shows a TLC overlay of adamantyl conjugates of Gb3LC and GalC.
  • Figure 19 depicts inhibition by N-adamentylacetyl Gb3 derivatives.
  • Figure 20 depicts inhibtion by Gb3S*NH2 c CONHAda vs. Gb3S» NHAc c CONHAda.
  • Figure 21 shows the titration of the viral antigen.
  • Figure 22 shows the hemagglutination inhibition using SGC-liposomes.
  • Figure 23 shows the determination of the concentration at which SGC- derivatives do not lyse RBC.
  • Figure 24 shows hemagglutination inhibition using SGC derivatives.
  • FIG. 25 shows the TLC mobilities of various SGC derivatives.
  • This invention pertains to methods and compositions which are useful in the treatment of glycolipid mediated states, such as enteropathogenic and enterohemorrhagic E. coli. ( ⁇ P ⁇ C and ⁇ H ⁇ C, respectively), e.g., verotoxin producing E. coli. (VT ⁇ C) or viruses, e.g., HIV or orthomyxoviridae, i.e., influenza A.
  • glycolipid mediated states such as enteropathogenic and enterohemorrhagic E. coli. ( ⁇ P ⁇ C and ⁇ H ⁇ C, respectively), e.g., verotoxin producing E. coli. (VT ⁇ C) or viruses, e.g., HIV or orthomyxoviridae, i.e., influenza A.
  • the present invention pertains to methods for treating a glycolipid mediated state in a subject by administering to the subject a therapeutically effective amount of a therapeutic compound, such that the glycolipid mediated state is treated.
  • the therapeutic compound is represented by the structure A-B, in which A is a glycomimetic receptor moiety and B is an active agent.
  • the methods of the invention can be used therapeutically to treat a subject afflicted by a pathogen or can be used prophylactically in a subject susceptible to pathogens.
  • the methods of the invention are based, at least in part, on inhibiting or preventing interaction between the cell membrane surface and the pathogen.
  • the language "treating a glycolipid mediated state” or “such that the glycolipid mediated state is treated” is intended to include changes in a glycolipid mediated state or condition, as described infra, such that physiological symptoms in a subject can be significantly diminished or minimized.
  • the language also includes control, prevention or inhibition of physiological symptoms or effects associated with a disease state associated with glycolipid mediated states.
  • the control of the glycolipid mediated state or condition is such that the glycolipid mediated state or condition is eradicated.
  • the control is selective such that a particular targeted glycolipid mediated state, e.g., a pathogen, is controlled while other cells and physiological flora which are not detrimental to the subject are allowed to remain substantially uncontrolled or substantially unaffected, e.g., lymphocytes, red blood cells, white blood cells, platelets, growth factors, etc.
  • a particular targeted glycolipid mediated state e.g., a pathogen
  • other cells and physiological flora which are not detrimental to the subject are allowed to remain substantially uncontrolled or substantially unaffected, e.g., lymphocytes, red blood cells, white blood cells, platelets, growth factors, etc.
  • glycolipid as used in "glycolipid mediated state” is art recognized and is intended to include glycolipids, glycoproteins, and glycoamino acids which are associated with or found on the cell or viral surface.
  • pathogen is art recognized and is intended to include disease producing agents, such as organisms capable of causing disease in a subject, e.g., a - 8
  • bacteria e.g., Escherichai coli, Shigella dysenteriae
  • viruses e.g.. HIV, orthomyxoviridae, influenza A. prions and fungi.
  • glycolipid mediated state is intended to include those disease states or conditions caused by or associated with one or more pathogens, e.g., bacteria or viruses. These glycolipid mediated states can include enterotoxins produced by pathogenic bacteria, e.g., Esherichia coli, and are known as shiga-like toxins (SLTs). The term is also intended to include those foreign entities produced by or associated with viruses, e.g., orthomyxoviridae.
  • host cell receptors for adhesion of pathogens have often been found to comprise complex carbohydrates on the host cell surface.
  • carbohydrates have been found to be conjugated to lipid rather than protein, thus host/cell surface glycolipids play an important role as receptors for a variety of bacteria.
  • Binding of influenza A virus to a cell is mediated by hemagglutinin (HA) antigen, which is a glycoprotein protruding from the virion envelope.
  • HA is a non-covalently linked trimer in which each subunit consists of disulfide-linked HA1 and HA2 domains.
  • HA1 is involved in receptor binding while HA2 is a transmembrane anchor.
  • HA binds mainly to cell surface sialic acid (NeuAc) bonded to galactose by ⁇ (2— »6) or ⁇ (2-»3) linkages.
  • the binding of HIV to the cell surface is mediated through an interaction between g ⁇ l20 and GalC.
  • the major species recognized are glycolipids belonging to the ganglio series, globo series, glyco-sphingo series, or sulfatide. Thus, many pathogens have been shown to bind to the lipid-bound carbohydrate.
  • the present invention pertains to ganglio series glycolipid recognition, since SLTs, such as verotoxin producing E.
  • VT ⁇ C demonstrate a high binding affinity for these neutral glycosphingolipids and that this binding is distinct from that of enteropathogenic and commensal E. coli strains.
  • the present invention also pertains to ganglio series glyco-sphingo recognition, since viruses, such as orthomyxoviridae, demonstrate a high binding affinity for these sulfogalactosylceramides.
  • SLT is art recognized and is intended to include cytotoxins similar in structure and function to Shiga toxin.
  • the term is also intended to include verotoxins, based upon structural similarity to shiga toxins by sequencing of relevant genes and are often referred to as SLT1.
  • Known SLTs include SLT-1, SLTII, SLTIII.
  • Variants of SLTII include SLTII; vtx2ha; SLTIIvh; vtx2hb; SLTIIc; SLTIIvp, etc.
  • the term encompasses the presently unknown SLTs or variants thereof that may be discovered in the future, since their characterization as an SLT or variant thereof will be readily determinable by persons skilled in the art.
  • orthomyoxoviridia is art recognized and intended to include those viruses associated with influenza, e.g. Influenza A, B, and C.
  • subject is intended to include mammals having a SLT, including one or more SLT related symptoms, or which are susceptible to pathogens producing SLTs. Examples of such subjects include humans, dogs, cats, pigs, cows, horses, rats and mice.
  • subject is also intended to include mammals having a viral infection, including one or more viral related symptoms, or which are susceptible to viral infection.
  • a therapeutic compound that amount of a therapeutic compound necessary or sufficient to perform its intended function within a subject, e.g., treat a glycolipid mediated state, or a state characterized by the presence of a pathogen, e.g. an SLT or a virus, in a subject.
  • An effective amount of the therapeutic compound can vary according to factors such as the amount of the causative agent already present in the subject, the age, sex. and weight of the subject, and the ability of the therapeutic compounds of the present invention to affect a state in the subject.
  • One of ordinary skill in the art would be able to study the aforementioned factors and make a determination regarding the effective amount of the therapeutic compound without undue experimentation.
  • an in vitro or in vivo assay also can be used to determine an "effective amount" of the therapeutic compounds described infra.
  • the ordinarily skilled artisan would select an appropriate amount of the therapeutic compound for use in the aforementioned assay or as a therapeutic treatment.
  • a therapeutically effective amount preferably diminishes at least one symptom or effect associated with the glycolipid mediated state, SLT, or virus being treated by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects.
  • the therapeutically effective amount diminishes at least one symptom or effect by at least about 90%, more preferably by at least about 95%, and still most preferably 100%.
  • Assays can be designed by one skilled in the art to measure the diminishment of such symptoms and/or effects. Any art recognized assays capable of measuring such parameters are intended to be included as part of this invention. For example, if blood in the stool is treated, then the diminishment of blood in the stool can be measured before and after treatment using an art recognized technique. Likewise, if hypertension is the state being treated, then the - 10 -
  • pressure can be measured before and after treatment for measurement of diminishment of pressure using an art recognized technique.
  • glycomimetic receptor moiety is intended to include those compounds which are glycolipids, glycoproteins, glycoamino acids or derivatives thereof which are recognized by receptors on a cell surface, e.g., cell membrane or cell wall.
  • the interaction between a glycomimetic receptor moiety and the receptor can include adhesion, ionic interactions, charged interactions and the like.
  • glycomimetic receptor moieties include an oligosaccharide moiety which is coupled to a serine or ceramide lipid base.
  • Preferred glycomimetic receptor moieties are Gg3- gangliotriaosyl ceramide, GalNAc ⁇ l-4Gal ⁇ l-4 glucosyl ceramide, Gg4- gangliotetraosyl ceramide-Gal ⁇ l-3GalNAc ⁇ l-4Gal ⁇ l-4 glucosyl ceramide, glycosyl- N-acyl serine, globotriosyl-N-acyl serine, galactosyl-N-acyl serine, and sulfogalactosylceramides (See also, U.S. Patent No.
  • the receptor moiety is not a Gb3 or a Gb4 moiety. In other embodiments, the receptor moiety is not those described in U.S. Patent 5,466,681. In other embodiments, the glycomimetic receptor is a sulfated galactose attached to a sphingosine base.
  • pathogen host cell plasma membrane attachment is an important virulence trait for pathogens.
  • Many specific pathogenic appendages, and adhesion molecules contained within such appendages have been devised to maintain the close apposition of viral or prokaryotic and eukaryotic cell surface membranes.
  • pathogen removal by nonspecific shear forces such interactions can provide the basis for the development of specific biological niches for particular pathogens.
  • Such niches may involve the specific modification of the host cell plasma membrane to better accommodate the requirements of the pathogen.
  • enteropathogenic (EPEC) and enterohemorrhagic E are examples of enteropathogenic (EPEC) and enterohemorrhagic E.
  • coli with eaeA +/- eaeB does not result in the induction of epithelial cell adherence.
  • E ⁇ e mutants still bind to host cells.
  • eae is required for intimate host cell attachment
  • another factor may be required for initial host cell recognition and binding.
  • a homologue of intimin has been identified in VT ⁇ C.
  • the identification of the bundle forming pilus (bfp) in ⁇ P ⁇ C provides the mechanism for the initial host cell attachment of ⁇ P ⁇ C.
  • HA hemagglutinin
  • HA2 structurally resembles to the Fl polypeptide of paramyoxviruses.
  • HA1 is involved in receptor binding while HA2 is a transmembrane anchor.
  • HA binds mainly to cell surface sialic acid (NeuAc) bonded to galactose by ⁇ (2 ⁇ 6) or ⁇ (2-»3) linkages.
  • the NA surface glycoprotein acts as an enzyme and allows the HA to come in to contact with the cellular membrane by acting as a hydrolase and lowering the pH surrounding the cell. This promotes the fusion process and allows the virus to invade the cell (Beers, R.F.(ed) The Role of Immunological Factors in Infectious, Allergic, and Autotoimmune Processes,. (Raven Press: New York, 1978) pp. 455-465.; Outlaw, M.C., Dimmock, N.J., Epidemiol. Infect. (1991), 106:205-220.).
  • the host cell receptors for adhesins of pathogens are believed to comprise complex carbohydrates on the host cell surface.
  • carbohydrates have been found to be conjugated to lipid rather than protein and play an important role as receptors for a variety of pathogens.
  • ganglio series glycolipids primarily Gg3-gangliotriaosyl ceramide, GalNAc ⁇ l-4Gal ⁇ l-4 glucosyl ceramide and Gg4 ⁇ gangliotetraosyl ceramide-Gal ⁇ l-3GalNAc ⁇ l-4Gal ⁇ l-4 glucosyl ceramide
  • ganglio series glycolipids primarily Gg3-gangliotriaosyl ceramide, GalNAc ⁇ l-4Gal ⁇ l-4 glucosyl ceramide and Gg4 ⁇ gangliotetraosyl ceramide-Gal ⁇ l-3GalNAc ⁇ l-4Gal ⁇ l-4 glucosyl ceramide
  • Pathogens which bind to Gg3 and or Gg4 in vitro also bind to the phospholipid, phosphatidyl ethanolamine (PE). Further, binding studies to cells which contain or lack PE, suggest that PE is a significant receptor to mediate pathogen attachment to eukaryotic cells. Not wishing to be bound by theory, it is believed that the binding to eukaryotic cell surface PE allows pathogens to preferentially target apoptotic cells. The loss of plasma membrane phospholipid asymmetry is an early marker of programmed cell death. Thus PE, normally located, for the most part, on the inner leaflet of the plasma membrane bilayer becomes available at the outer leaflet for pathogen binding.
  • PE normally located, for the most part, on the inner leaflet of the plasma membrane bilayer becomes available at the outer leaflet for pathogen binding.
  • apoptotic cells Preferential binding of pathogens to apoptotic cells may allow for the more efficient acquisition of nutrients by the microorganism.
  • Apoptosis has been shown to play a significant role in the turnover of both the respiratory and gastrointestinal epithelia and thus attachment of one pathogens may facilitate that of another.
  • the term "active agent" is intended to include those compounds which inhibit, eliminate, or prevent enterotoxins such as SLTs or viruses from affecting host cells of the subject.
  • the active agent can be an antibiotic known to those skilled in the art.
  • antibiotic is art recognized and is intended to include those substances produced by growing microorganisms and synthetic derivatives thereof, which eliminate or inhibit growth of pathogens and are selectively toxic to the pathogen while producing minimal or no deleterious effects upon the infected host subject.
  • Suitable examples of antibiotics include, but are not limited to, the principle classes of aminoglycosides, cephalosporins, chloramphenicols, fuscidic acids, macrolides, penicillins, polymixins, tetracyclines and streptomycins.
  • the active agents of the invention include penicillins, cephams, cephalosporins and carbocyclic compounds.
  • carbocyclic compound is intended to include carbon cage compounds, such as adamantanes and norbornanes as well as acridines and derivatives thereof. In preferred embodiments, adamantanes and norbornanes can have one or more acetic acid substituents.
  • carbocyclic as used throughout the specification and claims is intended to include both “unsubstituted carbocycles” and “substituted carbocycles”, the latter of which refers to carbocyclic moieties having
  • substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone can include, for example, halogen, hydroxyl. alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl.
  • aryl refers to the radical of aryl groups, including 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl, and the like.
  • aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles", “heteroaryls” or “heteroaromatics”.
  • the aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl.
  • alkenyl and alkynyl refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively. Unless the number of carbons is otherwise specified, "lower alkyl” as used herein means an alkyl group, as defined above, but having from one to ten carbons, - 14 -
  • lower alkenyl and “lower alkynyl” have similar chain lengths.
  • alkoxyalkyl refers to alkyl groups, as described above, which further include oxygen, nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon backbone, e.g., catenary oxygen, nitrogen or sulfur atoms.
  • polycyclyl or “polycyclic radical” refer to the radical of two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings.
  • Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl,
  • heteroatom as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus. It will be noted that the structure of some of the compounds of this invention includes asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis.
  • carbocyclic moieties include substituted or unsubstituted hydrocarbons, e.g., adamantyl; norbornyl; or substituted or unsubstituted aromatic compounds such as naphthyl, quinolyl, acridinyl, tetrahydroacridinyl, anthracenyl, benzopyrenyl, and the like.
  • Large carbocyclic cage moieties such as porphyrins can also be used in the therapeutic compounds and methods of the invention.
  • the carbocyclic moiety preferably has a steric bulk greater than the steric bulk of a - 15 -
  • phenyl group certain compounds in which C is a phenyl group have been found to be ineffective glycolipid mimics.
  • the carbocyclic compounds most preferred are adamantane-3-acetic acid, norbornane, and 1,3-adamantanediacetic acid.
  • acridine and adamantane derivatives as well as those listed in the paragraph supra are not included.
  • the carbocyclic moiety includes a portion which can be coupled to the glycomimetic receptor moiety, e.g., a carboxylic acid, amine or ester. Coupling can be effected by covalent, ionic, charge/charge interactions, etc. for attachment to the glycomimetic receptor moiety.
  • the glycomimetic receptor moiety e.g., a carboxylic acid, amine or ester.
  • Coupling can be effected by covalent, ionic, charge/charge interactions, etc. for attachment to the glycomimetic receptor moiety.
  • aminoadamantanes or aminoacridines can be coupled to the carboxyl group of the oxidized sphigosine moiety.
  • carboxyladamantanes or carboxylacrines e.g., carboxylic acids, can be coupled to the amino group of a deacylated glycolipid.
  • the phrase "associated with a pathogen” is intended to include, but is not limited to, those pathogens, e.g., bacteria, which are pathogenic to the host subject as listed in Table I. It also pertains to viruses and other non-bacterial pathogens, such as. for example, Influenza A.
  • the invention pertains to methods of modulating interaction between a pathogen and a glycolipid in a subject by administering to the subject a therapeutically effective amount of a therapeutic compound, such that interaction between a pathogen and a glycolipid is modulated.
  • the therapeutic compound is represented by the structure A-B, in which A is a glycomimetic receptor moiety and B is an active agent as discussed supra.
  • modulate is intended to include preventing, eradicating, or inhibiting interaction between a pathogen and a glycolipid, e.g., in the context of the therapeutic methods of the invention.
  • modulate includes effects on SLTs, e.g., verotoxin, that diminishes the activity or production of the toxins(s).
  • the therapeutic compound can interact with the toxin(s) to inhibit proteolytic activity.
  • the present invention provides methods for treating a state characterized by the presence of a shiga-like toxin (SLT) in a subject by administering to a subject a therapeutically effective amount of a therapeutic compound, such that a state characterized by the presence of shiga-like toxin is treated.
  • SLT shiga-like toxin
  • the therapeutic compound is represented by the structure A-B, in which A is a glycomimetic receptor moiety and B is an active agent.
  • the language "state characterized by the presence of a SLT” is intended to include those diseases, disorders or conditions which have been associated with a toxin, e.g., an enterotoxin, produced by a pathogen, e.g., bacteria, in that the pathogen is directly or indirectly a causative agent of the disease, disorder or condition.
  • the pathogen does not have to be the sole causative agent of the disease, disorder or condition but be merely responsible for causing some of the symptoms typically associated with the disease, disorder, or condition being treated.
  • the pathogen can be the causative agent alone or at least one other agent can be involved in the state being treated.
  • Examples include uncomplicated diarrhea, bloody diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS), fluid electrolyte imbalances, anemia, renal failure and/or hypertension manifested by the presence of symptomatic responses, such as gastritis, (Salmonella typh ⁇ ), food poisoning (E. coli O157:H7), bascillary dysentery (Shigella dysenteria), pneumonia ((Psuedomonas aerugenosa) and cholera (Vivrio cholerae).
  • symptomatic responses such as gastritis, (Salmonella typh ⁇ ), food poisoning (E. coli O157:H7), bascillary dysentery (Shigella dysenteria), pneumonia ((Psuedomonas aerugenosa) and cholera (Vivrio cholerae).
  • Preferred examples include those symptoms associated with E. coli. - 18 -
  • HUS Hemolytic uremic syndrome
  • VTEC verotoxin producing E. coli.
  • verotoxin targets the endothelial cells within the microvasculature of the gastrointestinal tract and the pediatric renal glomerulus.
  • VTEC are not believed to be invasive and thus the clinical pathology is the result of translocation of verotoxin across the gastrointestinal barrier to the systemic circulation.
  • Structural studies indicate that the verotoxin receptor glycolipid (globotriaosyl ceramide-Gb3) is not present on the gastrointestinal epithelial cell surface and therefore the mechanism by which the toxin translocates from the GI tract is essentially unknown.
  • Studies in vitro and in animal models however indicate that the attachment of the verotoxin producing E. coli organism to the host epithelial cell membrane may be intimately involved in the mechanism by which the toxin translocates.
  • attachment of the organism to the gastrointestinal host cell plasma membrane is an important virulence trait in the induction of diarrhea.
  • Verotoxins comprise a family of subunit toxins which target the glycolipid globotriaosyl ceramide (Gb3) expressed on the surface of sensitive cells.
  • Gb3 glycolipid globotriaosyl ceramide
  • the language "treating or treatment of the state characterized by the presence of an SLT" is intended to include the alleviation of or diminishment of at least one symptom typically associated with the state.
  • the treatment also includes alleviation or diminishment of more than one symptom.
  • the treatment cures, e.g., substantially eliminates, the symptoms associated with the state.
  • the present invention pertains to compounds represented by the structure A-B, in which A is a glycomimetic receptor moiety and B is an active agent.
  • Synthesis of the compounds represented by the structure A-B can be accomplished by various approaches detailed as follows (For example, see also Sakac, D. et al. "Purification of the Testicular Galactolipid 3' Phosphoadenosine 5' Phosphosulfate Sulfotransferase" J. Biol. Chem. 267:1655-1659 (1992); Lingwood CA. "The Production of Glycolipid Affinity Matrices by Use of Heterobifunctional Crosslinking Reagents" J. Lipid Res. 25: 1010-1012 (1984); Lingwood CA. and Taylor T. "Synthesis and Use of Galactolipid Sulfotransferase Substrate-analogue Affinity - 19 -
  • Gg3/Gg4 or PE allows for the accumulation of a lipid receptor-analog/antibiotic conjugate at the bacterial cell surface.
  • Antibiotics which are active at the pathogenic surface are coupled to derivatives of either Gg3/Gg4,SGCor PE.
  • These "receptocides" are bound by the bacterium and this accumulation results in the more efficient inhibition of pathogen membrane assembly.
  • these receptocides function as anti- adherents to prevent the attachment of the pathogen to host cells.
  • any development of resistance due to the loss of such adhesin species can be avoided since loss of the adhesin, in order to avoid binding of the receptocide, can also result in the loss of ability to bind to host cells.
  • the lipid binding specificity is restricted to pathogens and thus would spare the beneficial commensal E. coli strains , in contrast to broad spectrum antibiotics.
  • Gg4 can be prepared from GM1 and Gg3 from GM2 ganglioside (both commercially available) by mild acetic acid hydrolysis to remove the sialic acid.
  • Gg4 is first treated with aqueous base when the aminosugar is preferentially deacylated (since the lipid moiety is sequestered in micelles) and the free amine is alkylated, e.g., dimethylated.
  • the ceramide of the dimethyl Gg4 is then deacylated with alcoholic base and the free amine of the sphingosine base is coupled, for example, to the carboxyl group of an antibiotic, for example N-acetyl penicillin, as shown in Figures 1A and IB (Schemes 1A and IB).
  • an antibiotic for example N-acetyl penicillin
  • oxidative cleavage of the double bond in the sphingosine of glycosphingolipids affords a carboxylic acid ("glycosphingosinic" acid) derivative for coupling to amino containing antibiotics. Oxidation ofsphingosine double bond of glycolipids by ozonolysis has been previously described.
  • R represents groups such as hydrogen, alkyl ketones, e.g., methyl ketone, aryl ketones, e.g., phenyl ketone. alkyl or aryl esters, e.g., t-butyl ester and other acyl groups.
  • the oxidation procedure is carried out in a neutral tertiary butyl alcohol solution and utilizes catalytic amounts of KMn ⁇ 4(plus a regeneration system to prevent Mn ⁇ 2 precipitation).
  • This method provides the advantages that i) tertiary butyl alcohol is not liable to KMn ⁇ 4 oxidation, ii) lack of precipitation prevents product loss by adsorption.
  • the procedure affords high yields (40-80%) of the ceramide oligiosaccharide as the single product.
  • the allylic alcoholic function as a whole undergoes oxidation as depicted in Scheme B.
  • This oxidation procedure utilizes catalytic amounts of KMn ⁇ 4 (plus a regeneration system to prevent Mn ⁇ 2 precipitation) in a basic solution of tertiary butyl alcohol This procedure affords high yields (40-80%) of the serine oligiosaccharide.
  • Rl H limited amount RT O
  • the glycolipid is first deacylated to remove the fatty acid and the free amine is alkylated, e.g., methylated resulting in the dimethylation of the aminosugar in Gg3.
  • the sugar residues are then acetylated prior to oxidation of the sphingosine double bond as in Figures 2A and 2B (Schemes 2A and 2B) and Scheme B.
  • the carboxylic acid can be activated using procedures known in the art, for example, N-OH succinimide and coupled, using dicyclohexylcarbodiimide, to the amino function of an antibiotic as shown in Figure 3 (Scheme 3, shown for Gg3) and Scheme C EDAC is l-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
  • the hydroxyl groups can be regenerated by deacetylation using triethylamine base.
  • R2 H. CH3CO, PhCO, COOtBu NH
  • Preferred embodiments include monoalkylated, dialkylated, monoarylated or diarylated deacylated glycolipids described by the above procedures.
  • the resultant amino functionality of the deacylated glycolipid can be treated with alkylating or arylating agents known in the art.
  • the amine is dialkylated or diarylated with lower alkyl groups, e.g., methyl, ethyl, propyl, or aryl groups whose steric bulk do not interfere with the bioreactivity of the resultant conjugate, e.g., benzyl, benzoyl, aryl.
  • the glycomimetic receptor moiety, A can be a sulfated saccharide.
  • the sphingosine moiety of a SGC can be left intact or oxidized to yield an amine.
  • the amine can then be coupled to a rigid hydrophobic moiety, such as a norbornyl or adamantyl group.
  • the sphingosine chain can be further oxidized yielding either a ceramide or serine derivative (Scheme D).
  • therapeutic compounds depicted below can be prepared by known coupling reactions, e.g., etherification reactions, where X, Y and R represent soluble Gg3 or Gg4 mimics.
  • the structures of the receptocides made can be determined by FAB mass spectrometry, proton NMR as well as those techniques known to persons of skill in the art. - 27 -
  • adhesin targeted antibiotics should allow the administration of pathogencidal doses which represent significantly lower antibiotic doses when considered on a molar basis.
  • the binding of the receptocides by the SLTs, e.g., VTEC, result in the concentration of the antibiotic at the pathogen surface for more efficient inhibition of membrane assembly.
  • Judicious selection of antibiotics with activity against SLTs may result in the generation of new potent treatments for the effective and selective elimination of SLTs from the subject, e.g., human, GI tract.
  • the topology of the adhesins on the SLTs, e.g., VTEC, surface may not optimally correspond to the surface location of the antibiotic binding proteins
  • Suitable spacer groups are known in the art and can include anhydrides, haloalkylamines and the like.
  • the present invention pertains to pharmaceutical compositions which include a therapeutically effective amount of a therapeutic compound represented by the structure A-B, in which A is a glycomimetic receptor moiety and B is an active agent, described supra, and a pharmaceutically acceptable carrier.
  • A is a glycomimetic receptor moiety
  • B is an active agent, described supra.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it can performs its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol;
  • polyols such as glycerin, sorbitol. mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar: buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.
  • buffering agents such as magnesium hydroxide and aluminum hydroxide
  • certain embodiments of the present compounds can contain a basic functional group, such as amino or alkylamino. and are, thus, capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids.
  • pharmaceutically acceptable salts refers to the relatively non- toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See. e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).
  • the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases.
  • pharmaceutically acceptable salts refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine.
  • Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like.
  • Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.
  • esters refers to the relatively non-toxic, esterified products of the compounds of the present invention. These esters can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a
  • esterifying agent Carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst. Hydroxyl containing derivatives can be converted into esters via treatment with an esterifying agent such as alkanoyl halides.
  • esterifying agent such as alkanoyl halides.
  • the term is further intended to include lower hydrocarbon groups capable of being solvated under physiological conditions, e.g., alkyl esters, methyl, ethyl and propyl esters. (See, for example, Berge et al., supra.)
  • the invention further contemplates the use of prodrugs which are converted in vivo to the therapeutic compounds of the invention (see, e.g., R.B. Silverman, 1992, “The Organic Chemistry of Drug Design and Drug Action", Academic Press, Chp. 8).
  • prodrugs can be used to alter the biodistribution (e.g., to allow compounds which would not typically enter the reactive site of the protease) or the pharmacokinetics of the therapeutic compound.
  • a carboxylic acid group can be esterified. e.g., with a methyl group or an ethyl group to yield an ester.
  • the ester When the ester is administered to a subject, the ester is cleaved, enzymatically or non- enzymatically, reductively or hydrolytically, to reveal the anionic group.
  • An anionic group can be esterified with moieties (e.g., acyloxymethyl esters) which are cleaved to reveal an intermediate compound which subsequently decomposes to yield the active compound.
  • the prodrug is a reduced form of a sulfate or sulfonate, e.g.. a thiol, which is oxidized in vivo to the therapeutic compound.
  • an anionic moiety can be esterified to a group which is actively transported in vivo, or which is selectively taken up by target organs.
  • the ester can be selected to allow specific targeting of the therapeutic moieties to particular reactive sites, as described below for carrier moieties.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulf ⁇ te, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, rectal, vaginal and/or parenteral
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
  • Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • a compound of the present invention may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxyme hylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol
  • compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liposomes are microscopic spherical membrane-enclosed vesicles or sacks (20-30 ⁇ m in diameter) made artificially in the laboratory using a variety of methods. Within the scope of the present invention, the liposomes should be non-toxic to living cells and they should deliver the contents, in this case a compound of the invention to the infected area.
  • the liposomes according to the present invention may be of various sizes and may comprise either one or several membrane layers separating the internal and external compartments.
  • An important element of the present invention is that a sufficient amount of compound be sequestered so that a relatively low concentration of compound is required for delivery to the infected area and further that
  • Liposome structures according to the present invention includes small unilamellar vesicles (less than 250 angstroms in diameter), large unilamellar vesicles (greater than 500 angstroms in diameter) and multilamellar vesicles depending upon the quantity of compound required to be encapsulated.
  • small unilamellar vesicles are preferred since the compound, according to the present invention is only required in low concentrations.
  • the liposomes may be made from natural and synthetic phospholipids, and glycolipids and other lipids and lipid congeners; cholesterol, cholesterol derivatives and other cholesterol congeners; charged species which impart a net charge to the membrane; reactive species which can react after liposome formation to link additional molecules to the lysome membrane; and other lipid soluble compounds which have chemical or biological activities.
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable
  • nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body.
  • dosage forms can be made by dissolving or dispersing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel.
  • compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use. which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol. polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride
  • the absorption of the drug in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenteral ly-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular. subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex. weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that
  • a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • intravenous and subcutaneous doses of the compounds of this invention for a patient when used for the indicated analgesic effects, will range from about 0.0001 to about 200 mg per kilogram of body weight per day, more preferably from about 0.01 to about 150 mg per kg per day, and still more preferably from about 0.2 to about 140 mg per kg per day.
  • the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • a compound of the present invention While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical composition.
  • the present invention pertains to packaged therapeutic compositions for treating a glycolipid mediated state in a subject.
  • the packaged therapeutic compositions include a container for holding a therapeutically effective amount of a therapeutic compound for treating a glycolipid mediated state in a subject and instructions for using the therapeutic composition for treating the glycolipid mediated state.
  • the therapeutic compound is represented by the structure A-B, in which A is a glycomimetic receptor moiety and B is an active agent.
  • the present invention pertains to packaged therapeutic compositions for modulating interaction between a pathogen and a glycolipid.
  • the packaged therapeutic composition includes a container for holding a therapeutically effective amount of a therapeutic compound for modulating interaction between a pathogen and a glycolipid in a subject and instructions for using the therapeutic composition for modulating interaction between the pathogen and the glycolipid.
  • the therapeutic compound is represented by the structure A-B, in which A is a glycomimetic receptor moiety and B is an active agent.
  • Advantages of the invention include i) early diagnosis of SLTs, so that receptocide therapy can be initiated prior to the production of significant levels of SLTs, e.g., verotoxin, within the GI tract and ii) methods to target antibiotics to the pathogenic microorganism and not commensal GI organisms.
  • SLTs e.g., verotoxin
  • Reagents were purchased from the following suppliers: Caledon - trifluoroacetic anhydride, K2CO3, sodium cyanoborohydride (NaB ⁇ CN). triethylamine (Et ⁇ N); Aldrich - 37% aqueous formalin solution, 0.5N H2SO4 solution, trichloroacetic anhydride, acetic anhydride, diphenyl succinimidyl phosphate (PNHS); BDH (Toronto, Ontario) - ANALAR KMn ⁇ 4 « ANALAR NaHSOs, 30% H2O2; Sigma (St.
  • Solvents were dried by storing over activated (-120° C for 16 hrs) molecular sieves. Solvent systems are given in volume ratios. Crown ether (10 g) was recrystallized from a hexane (4 to 5 mL) solution at -20° C, washed with cold (-20° C) hexanes (1 mL) and dried at 40° C under a stream of N2.
  • BSA (99%, essentially fatty acid free) was purchased from Sigma.
  • Recombinant gpl20 was purchased from Intracell (CA), anti-human IgG horse radish peroxidase conjugate from Bio-Rad and human sera from HIV patients containing anti- gpl20 antibodies was a gift from Dr. S. Read, Division of Infectious Disease, HSC
  • Globotetraosyl ceramide, Gb4»C, Globotriaosyl ceramide, Gb3 # C and Lactosyl ceramide, Lac » C were purified from human kidney (Boyd, B. and Lingwood, C. A. (1989) Nephron 51 , 207-210) and Forssmann, Gb5 » C was purified from sheep - 39 -
  • GMj'C monosialylganglioside
  • bovine brain Yamakawa, T., Irie, R. and Iwanaga, M. (1960) J. Biochem. 48, 490-497) according to previously published procedures.
  • Galactocerebroside, Gal » C was purchased from Sigma.
  • Gangliotetraosyl ceramide, Gg4»C was prepared by acid hydrolysis of GMj 'C with 1 M acetic acid at 80° C for 1 hour (Head, S., Ramotar, K. and Lingwood, C A. (1990) Infect.
  • the yield of methylated product was >90% by TLC. TLC showed that the methylated compound has a reduced mobility compared to the deacyl forms.
  • the Rf values for Gal » S and Gal » SNNMe2 are 0.80 and 0.75 in CHCl 3 :MeOH:H 2 O; 60:35:8 or 0.38 and 0.31 in CHCl 3 :MeOH:H 2 O; 65:25:4 respectively.
  • Gal'SNTfa (GalQl ⁇ spfiin ⁇ osineiVTfa), Ga SNTca (GalQI ⁇ sphingosineNTca)
  • Acetylating reagents N-acetyl imidazole and N-trihaloacetyl imidazole, were prepared by adding a DCM solution of anhydride - for example (CL3CO)2 ⁇ (0.85 g, 2.7 mmol) dissolved in DCM (2mL) and the resulting solution was divided in 3 portions and added at 15 minute intervals, to an imidazole (0.41 g, 6.0 mmol) suspension in DCM (3 mL). The reaction mixture was stirred for 2 hours and was assumed to be approximately a 0.5 M solution of the imidazole derivative.
  • anhydride - for example (CL3CO)2 ⁇ 0.85 g, 2.7 mmol
  • a solution of the imidazole derivative was added to a DCM suspension of GSL » S (1 mg/mL).
  • N-trichloroacetyl imidazole solution (20 ⁇ L. 10 ⁇ mol) was added to a suspension of Gal » S (3 mg in 3 mL of CH2CI2, 6 ⁇ mol), and the reaction was monitored by TLC (CHCl3:MeOH:H2O; 70:30:2). Appearance of many orcinol positive products suggested some degree of acylation of OH groups.
  • the reaction mixture was dried under a stream of N2, redissolved in DCE and loaded on to a silica column (0.5 X 6 cm, in DCE) and eluted with CHC MeOH; 98:2 (batch elution, 25 mL) and then with CHCl3:MeOH:H2 ⁇ ; 80:20:2 (10, 3 mL fractions were collected).
  • the estimated product yield by TLC was >90%.
  • Method A Suitable for natural, NAc and NNMe2 derivatives.
  • a mixture of 1 :2 acetic anhydride and pyridine 1 mg/mL of lyso GSL was added to a dried sample of natural GSL, GSL S or GSL SNNMe2 and stirred at 37° C.
  • the reactions were monitored every 30 minutes by TLC using DCE: lso PrOH; 80: 15 as solvent system, and upon completion, dried under a stream of N2.
  • Method B Suitable for the preparation of NTca(OAc) n and NT/a(OAc) n derivatives.
  • a mixture of 2: 1 trifluoroacetic anhydride and glacial acetic acid (1 mL/mg of glycolipid) was added to a dried sample of NT a or NTca, GSL derivatives and stirred at 25° C.
  • the reactions were monitored every 30 minutes by TLC using DCE: lso PrOH; 80: 15 as solvent system, and upon completion, dried under a stream of N2-
  • the peracetylated crude was dissolved in DCE (1 mL) and loaded on to a silica column (for 3 mg. 0.5 X 5 cm in DCE) and eluted DCM:MeOH; 25 :Y, Y being methanol which was varied from 100 ⁇ L in increments of 100 ⁇ L, where for each case 6, 4 mL fractions were collected. It is noteworthy that the mobility of most of the peracetylated derivates during column chromatography vary significantly with the degree of silica gel activation, and concomitant changes of the solvent ratio of the eluent may be necessary.
  • Reagent A 2:1 mixture of t ⁇ uOH ⁇ O. Solutions of NaIO4 (0.4 M), K2CO3 (0.25 M) and KMn ⁇ 4 (0.05 M). Quenching solution: A5:l mixture of 0.24 M NaHSO3 solution and 0.5 M H2SO4 solution.
  • Peracetylated glycolipid (0.5 mg; depending on the GSLs this might vary from 1 to 0.3 ⁇ mol) was dissolved in tBuOH/H2O (500 ⁇ L) and solutions of NaIO4 (30 ⁇ L, 10 ⁇ mol), K2CO3 (10 ⁇ L, 2.5 ⁇ mol) and KMnO4 (15 ⁇ L, 0.75 ⁇ mol) were added in the given sequence. The resulting purple, turbid mixture was stirred at 37° C for 2 to 3 hours, depending on the GSL. If purified peracetylated derivatives are employed, the overall color of the reaction mixture should not diminish during the course of the reaction.
  • oxidation was carried out in a more polar medium containing 1 :1 tBuOH:H2 ⁇ .
  • Deprotection of the ceramidic acids or the serine oligosaccharide acids were carried out by treating 0.5 mg of dry ceramidic acid with 1 mL of triethyl amine solution (Et3N:MeOH:H2 ⁇ ; 2:6: 10) at 37° C for 2 to 3 hours.
  • Et3N:MeOH:H2 ⁇ ; 2:6: 10 triethyl amine solution
  • the reaction mixture was then stirred at overnight at 37°C for Gb4 and at the same temperature for three hours for Gg3-
  • the samples were then dried and passed through a 2 cm silica column in a pasteur pipette with a glass wool plug and .5 cm suspension of celite in a 2: 1 solution of chloroform:MeOH.
  • the product was run through the column twice with a solvent of
  • Oxidized Gb4 was coupled to ampicillin using the following procedure. 200 ⁇ l of dry oxidized Gb4 was added to three tubes with 20 mgs of Ampicillin and .5 mis of dry MeOH. In tube 1 , 500 ⁇ l of a 5: 1 solution of dioxane:triethylamine was also added. In tube 2, 500 ⁇ l of a 5:1 solution of DMF:triethylamine was added. In tube 3, 500 ⁇ l of a 5: 1 solution of acetone:triethylamine was added. No difference in the products from the three reactions was found.
  • Oxidation of Gal(OAc)4 » C(OAc) by KMn ⁇ 4 in acetone was carried out according to the published procedure (MacDonald, D. L., L., P. and Hakomori, S. I. (1980) J. Lipid Res. 21, 642-645), except product purification was similar to the isolation of ceramidic acids described in the new method.
  • Lanes 7, 10. 12, 13, 15 and 19 correspond to (OAc) 5 GalC, SGC (upper) and SGG (lower), (OAc) SGC Gbs, GMj , and lyso Gb3 (Gb3»S), respectively. Asterisks indicate intermediates formed during the reaction.
  • the TLC plates show the relative purity of the products formed.
  • Gg4 « C- s COOH Permethylation of ceramidic acid, Gg4 « C- s COOH was performed according to published methods (Fan, J. Q., Huynh, L. H., Reinhold, B. B., Reinhold, V. N., Takegawa, K., Iwahara, S., Kondo, A., Kato, I. and Lee, Y. C. (1996) Glycoconj. J. 13, 643-648).
  • a suspension of NaOH in DMSO 100 ⁇ L of 5% suspension was added and incubated at 25° C for 1 hour.
  • Figure 8 shows the mass spectra of serine and ceramide acids of behenic analogues of GalC.
  • (+) mode shows peaks at 612.6 and 634.6. These peaks represent the molecular weight of the behenic serine analogue of GalC (589) with one and two accompanying sodium atoms (612.6 and 634.6, respectively).
  • the peak at 588.4 represents the molecular weight of the GalC-behenic serine acid analog without one hydrogen ion.
  • GalC-behenic is represented by a single peak at 807.0.
  • peaks at 706.8, 684.8, 664.6, and 642.6 for the positive mode mass spectra.
  • peaks at 528.4 and 550.4 The peaks at 528.5 and 550.4 are the compound plus one sodium ion and the compound plus two sodium ions, respectively.
  • the minor peaks at 558.6 and 580.4 represent incompletely oxidized ceramide acid analogues.
  • the major peak at 504.4 is the molecular weight of the compound with out one hydrogen ion.
  • the GalC palmitic analogue is represented by a single at 722.6.
  • peaks are found at 558.4 and 580.4 in (+) mode, representing the compound plus one and two sodium ions, respectively.
  • GAL(OAc) 4 'C(OAc)- s COOH and GAL(OAc) 4 « S(OAc)-COOH 500 ⁇ g were deprotected with 1 ;1 ;0.2 Et3N/MeOH/H2 ⁇ at 37°C for 16 hours. The reaction mixture was dried. The crude deprotected acids, Gal » C- s COOH and Gal » S-COOH were dissolved (0.5 mL of C:M:W; 90: 10:1) and loaded on to a silica column (0.5 X 2 cm) and eluted, first with C:M:W; 80:20:2 (5 mL) and then with MeOH (6 mL).
  • Gal'C- s COOH and Gal*S-COOH precursors were converted to the corresponding NHS derivatives by treating (dissolved in 5:1 AcCN:Et3N to give a final concentration of 1 mg/mL) with PNHS (Giambattista, M. D., Nyssen, E., Pecher, A. and Cocito, C (199) J. Biol. Chem. 29, 9203-921 1 ; Ogura, H., Nagai, S. and Takeda, K. (1980) Tetrahedron Lett. 21 , 1467-1468) or OxNHS (Kometani, T., Fitz, T. and Watt, D. S. (1986)
  • nitrocellulose membranes were blocked with 5% milk powder, 0.05% tween-20 in lO M TBS for 2 hours. Rinsed 3 times (10 to 15 minutes each) with 0.05% tween-20 in 10 mM TBS and incubated with rgpl20, 1 :1000 dilution in 3% milk powder in 10 mM TBS for 2 hours. Washed as described above and incubated with human HIV serum, 1 :50 dilution in 5% milk powder, 0.05% tween-20 in 10 mM TBS for 2 hours.
  • the blots were incubated with the secondary antibody (anti-human IgG horse radish peroxidase conjugate), 1 : 1000 dilution in 5% milk powder, 0.05% tween-20 in 10 mM TBS for 45 minutes. Finally the blots were rinsed 3 times with 0.05% tween-20 in 10 mM TBS and a fourth rinse with only 10 mM TBS. Binding was visualized according to previously published procedure (Lingwood, C. A., Law, H., Richardson, S., Petric, M., Brunton, J. L., DeGrandis, S. and Karmali, M. (1987) J. Biol. Chem. 262.
  • the galactosyl serine oligosaccharide conjugate (GalS»NAcCOHN) n BSA is not recognized by gpl20, indicating the presence of at least one of the hydrocarbon chains is essential for binding. This is consistent with the lack of binding inhibition by free galactose (Bhat, S., Spitalinik, S. L., Gonzalez-Scarano, F. and Silberberg, D. H. (1991) Proc. Natl. Acad. Sci. USA 88, 7131-7134).
  • Influenza A virus also binds to galactosyl ceramide or sulfatide (Suzuki, T., Sometani, A., Yamazaki, Y., Horiki, G., Mitzutani, Y., Masuda, H., Yamada, M., Tahara, H., Xu, G., Miyamoto, D., Oku, N., Okada, S., Kisio, M., Hasagawa, A., Ito, T., Kawaoka, Y. and Suzuki, Y. (1996) Biochem. J 318, 389-393), and again the lipid moiety is important for binding.
  • the BSA conjugate may therefore also bind this virus.
  • the Western blot analysis method was also used for the GalC «BSA conjugates analyzed in Figure 12a.
  • a western blot analysis of GalC-glycoconjugates (3 to 4 mg each) binding to recombinant gpl20 was done.
  • Lane A was BSA.
  • Lanes B, C, and H were GalC» c CONH)nBSA.
  • Lanes D and E were (GalS»NHOC)nBSA.
  • Lanes F and G were (GalBehenicC* c CONH)nBSA.
  • the conjugates in lanes A,D, and F were derived using di(N-succinimydyl)oxalate.
  • the conjugates in lanes C,E, and g were derived using succimidyl phosphate, while the conjugat in lane H was derived using EDAC and NHS. In all cases a ratio of 1 :1 w/w of BSA:Gb3C»CCOOH was used. This Western blot shows binding to Gb3 occurs for all the compounds tested. The upper bands indicate other degrees of binding. BSA can bind Gb3 in a variety of ways and the smaller bands are indicative of minor forms of binding.
  • FIG. 12b A slot blot analysis was done on lanes B, C, D, and H ( Figure 12b).
  • the slot blot shows dose response for different GalC conjugates. The highest concentrations correspond to 2 to 3 mg.
  • Figures 12c and d are dot blot analyses showing dose response for (Gb3C
  • Figure 12c corresponds to a dot blot with conjugates derived using di(N- succinimydyl)oxalate
  • Figure 12d corresponds to conjugates derived from using EDAC and NHS.
  • Stock solutions (1 mg/ml) of inhibitors were made up in ethanol. Appropriate amounts (i.e., such that at the end of all the manipulations the inhibitor concentrations will be either 100 ⁇ M or 10 ⁇ M) from these stock solutions were transferred into polypropylene tubes and solvent removed under a flow of N 2 .
  • the dried samples were dissolved in 900 ⁇ L of 50 mM TBS by sonication and vortexing. To each tube was added 50 ⁇ L of 0.2% BSA in TBS and 50 ⁇ L of 0.2% gelatin and vortexed well and incubated at room temperature for 2 hours. To these solutions was added 5 ⁇ L gpl20 (l ⁇ g/5 ⁇ L in PBS) stock solution and vortexed (1 min) and shaked at room temperature for 2 hours.
  • Gp 120 was washed (3 times with 5mL of 50 mM TBS) and the plates were incubated with sera from human HIV patients (1 :50; human sera: 50 mM TBS containing 1 % gelatin for 2 hours at room temperature and washed as above.
  • the plates were incubated with goat anti-human IgG horse radish peroxidase conjugate (1 : 1000; GAH:50mM TBS containing 1% gelatin) for 45 minutes and washed as above; developed with 4-chloro-l-naphthol (3 mg/mL freshly prepared solution in methanol mixed) with 5 volumes of 50 mM TBS and 1 :1000 dilution of H 2 O 2 .
  • Figure 13 shows inhibition of HIV coat protein gpl20 binding to Galactocerebroside (GalC) and Sulfatide (SGC). The results are also summarized in Table 2 below. Table 2 shows that unoxidized compounds are more efficient inhibitors of GalC and that none of the compounds were noticeably effective against GalC under the given conditions.
  • Inhibition is estimated by comparing the intensity of gpl20 binding for each inhibition assay and the control. The control has O% inhibition. Assay for all the compounds were done at the same time. N/C: Not conclusive N/I: No inhibition
  • VTEC strains Figure 14
  • binding of commensal and non-pathogenic laboratory E. coli strains to these lipid structures in vitro appears to correlate, with bacterial pathogenicity.
  • E. coli adherence is defined by a two stage process; firstly as a loose attachment to host cells, and secondly, a more consolidated tight attachment involving the intimin protein.
  • this initial loose attachment is mediated by the bundle forming pilus.
  • VTEC the mechanism of such initial host cell interaction is unknown. It is believed that the selective binding of Gg4 and PE by VTEC may function in place of the bfp-mediated attachment.
  • Dimethyl deacyl Gg3 was coupled to N-acetyl penicillin (as indicated for Gg4 in Figure IB) and tested the efficacy of the conjugate ('receptocide') on the growth of Hemophilus influenzae (which has been shown to bind Gg3 in vitro).
  • N-acetyl penicillin is considered a poor antibiotic for Hemophilus influenzae and was found by agar gel diffusion to be virtually ineffective against this organism under the conditions used.
  • the "receptocide” was found to be almost as effective to inhibit H. influenzae as penicillin.
  • targeting the antibiotic by coupling to Gg3 had a major effect (several orders of magnitude) to increase antimicrobial efficacy.
  • globotetraosyl ceramide (Gb4) was coupled to ampicillin via oxidation of the glycolipid as shown in Figure 3 for Gg3- Uropathogenic E. coli express P pili to mediate binding to globoseries glycolipids.
  • Figure 15 it can be seen that the Gb4-ampicillin conjugate was effective to inhibit the growth of an uropathogenic E. coli (more than ampicillin itself) but was not effective for a VTEC strain (which does not bind Gb4). It was observed that the VTEC was less sensitive to Gb4-ampicillin than to ampicillin, suggesting that such receptocides may be even more specific than expected. The uncoupled glycolipid demonstrated no inhibitory effect.
  • the concept includes that the molecule contains a truncated glycolipid (glycolipid acid) in which both the fatty acid has been removed and the sphingosine double bond cleaved (i.e., combination of i and ii above), with a rigid hydrophobic group which can mimic the effect of the lipid moiety of sugar conformation without allowing the lateral lipid packing that results in the formation of lamellar and micellar glycolipid structures in aqueous buffers.
  • Two such soluble Gb3 analogs were produced using the oxidative hydrolysis procedure described above: an adamantyl and an acridine conjugate to the glycolipid acid. The adducts were filtered prior to use to ensure solubility.
  • micromolar range This is the first description of any effective soluble competitive inhibitor of verotoxin receptor glycolipid binding. In this assay, the free globotriaose is totally ineffective.
  • the wells were incubated for two hours at room temperature with varying amounts of VTl (10 to 50 ⁇ g in 0.2% BSA in PBS) where the final incubation volume in each well was constant (60 ⁇ L) and washed as described above.
  • the samples were then incubated with PHI (60 ⁇ L, 1 mg/mL in 0.2% BSA in PBS) for 1 hour at room temperature, washed as described above, incubated with GAM (sigma, 1 :2000 dilution in 0.2% BSA in PBS) for 1 hour at room temperature, washed as described above, and then washed with PBS.
  • the samples were then developed with 100 ⁇ L of substrate solution ( 0.5 mg/mL of ABTS (2,2'-azino- bis(3-ethylbenzthiozoline-6-sulfonic acid) diammonium salt) in citrate buffer (0.08 M citric acid and 0.1 M NaH 2 PO ) and recorded after 10 to 15 minutes at 415 nM.
  • substrate solution 0.5 mg/mL of ABTS (2,2'-azino- bis(3-ethylbenzthiozoline-6-sulfonic acid) diammonium salt
  • citrate buffer 0.08 M citric acid and 0.1 M NaH 2 PO
  • the results from the assay are shown in Figure 17.
  • the typical binding assay with VTl shows that the adamantine derivative from ceramide acid has a very high affinity and that the derivative has an affinity between that of natural Gb3 and ceramide-adamantine derivatives.
  • LC derivatives showed no binding under these conditions. This suggested that the Gb3 compounds have
  • the TLC overlay of adamantyl conjugates of Gb3 LC and GalC is shown in Figure 18.
  • the TLC plate shows the relative migration of adamantyl glyconjugates of Gb3 LC and GalC and natural GSLs.
  • Lane A shows three bands: GalC, Gb3 and Gb4C (1 ⁇ g each).
  • Lane B shows the same compounds (2 ⁇ g each).
  • Lane C is Gb3C* s CONHAda.
  • Lane D is Gb 3 C» c CONHAda.
  • Lane E is LC « s CONHAda.
  • Lane F is LC « c CONHAda.
  • Lane G is GalC» s CONHAda
  • Lane H is GalC « c CONHAda.
  • the plate was developed with orcinol spray.
  • a second TLC plate was run having the same amount of lipids and run under identical conditions. It was analyzed by using TLC overlay with verotoxin. In this assay, single bands in Lanes A, B, C, and D are visible corresponding to the mobility of Gb3- This assay shows that verotoxin binds to Gb3 but not the other compounds present in these lanes. In Lane F, verotoxin is shown to bind to LC» c CONHAda.
  • Verotoxin was preincubated with the inhibitor for 2 hours and this mixture was then added to the Gb 3 coated wells.
  • this mixture was then added to the Gb 3 coated wells.
  • 22 ⁇ L was transferred into a glass tube, dried added 220 ⁇ L of PBS (2.5 mM of inhibitor) and from this solution a serial 2 fold dilutions were carried out with PBS where the final 1 10 ⁇ L aliquot was discarded.
  • To each dilution added 100 ⁇ L of VT solution (160 ng/mL in PBS) and incubated for 2 hours at room temperature. Then 60 ⁇ L aliquots were transferred into wells (3 wells) containing Gb 3 and incubated for 2 hours are room temperature.
  • FIG 19 shows the inhibition by N-adamantylacetyl Gb3 derivatives using the above mentioned assay.
  • PHI is a natural monoclonal antibody against VTl .
  • the results of the assays shows that Gb3S «NHCOCH2Ada (A) was the most efficient inhibitor against VTl binding. At concentrations around 10 ⁇ 2 mM of inhibitor, almost none of the VTl is bound. The hydrophobic tail is believed to be important to the binding. It was noted that without the tail, the inhibition was reduced.
  • Figure 20 shows that Gb3S «NHCOCH Ada and Gb3S «NHAc c CONHAda are both apparently successful inhibitors of VTl binding at concentrations lO' ⁇ mM of inhibitor. Gb3S» NH2 c CONHAda was not found to block the binding of VTl.
  • SGC Sulfogalactosylceramide
  • the resulting protected compound was then oxidized using a 2:1 solution of tBuOH: water and NaIO4 and KMn ⁇ 4 at 37°C for sixteen hours.
  • the resulting compound was then deprotected using triethylamine, water, and methanol at 37°C for sixteen hours.
  • hemagglutinin When hemagglutinin (HA) is mixed with RBC they cause hemagglutination, a process by which the RBC's are linked together in a mesh-like network. Hemagglutination can be visualized in v-shaped wells, where agglutinated RBC settle out of suspension to form a diffused red layer at the bottom of the well ("button").
  • the RBC is not the type of host cell which the virus normally infects, but expresses on its surface NeuAc containing receptors similar to those found on mucous cells of the respiratory tract.
  • Figure 21 shows an assay for the determination of 1HA unit.
  • 1 HA unit the following procedure was used. To a two fold dilutions of viral antigens in PBS (50 ⁇ L) in a V-shaped 96-well plate, 50 ⁇ L of PBS and 150 ⁇ L of RBC (0.5% in PBS , 6xl0 7 cells) were added and the reaction mixture was then incubated for 1 hour at 4 °C The lowest antigen concentration that gave a positive hemagglutination was taken 1 HA unit and the next highest as 2HA and so on. The HA used was
  • Inhibition was carried out using 8 HAU and 10 A 7 RBC.
  • a positive control of the assay was done using V-shaped wells charged with the following reactants 150 ⁇ l of 0.5% RBC (6 x 10 cells) in PBS. 50 ⁇ l of viral antigen in PBS, and 50 ⁇ l of PBS. The reaction was then incubated at 4°C for one hour. This resulted in no inhibition and, hence, no button. A negative control for hemagglutination was done without viral antigen.
  • SGC liposomes were used in the following procedure. 150 ⁇ l of 0.5% RBC (6 x 10 cells) in PBS, 50 ⁇ l of viral antigen in PBS, and 50 ⁇ l of soluble SGC inhibitor dissolved in PBS. It was then incubated for an hour at 4°C and - 56 -
  • Each well contained RBC-SGC-liposomes at varying concentrations. Hemagglutinin was inhibited at 12.5 mM indicating that the viral antigen was neutralized by SGC-liposomes ( Figure 22). This was confirmed by the viral antigen binding to SGC. Inhibition was carried out using 8 HAU and 10 A 7 RBC Liposome ratio was 1 :6:3 - SGCPCChol. It was determined that to completely inhibit 1 HAU, 1.56 mM of SGC-liposomes are necessary.
  • each well contained RBC and soluble SGC derivatives in varying concentrations ( Figure 23).
  • the wells to the left of the button showed lysis of RBC. an in vitro effect.
  • the hemagglutination inhibition assays were carried out at inhibitor concentrations which would not lyse the RBC.
  • SGC-adamantane, SGC-norbornane and SGC-adamantane-3 -acetic acid were found to lyse RBC at concentrations above 0.75 ⁇ M, 1.5 ⁇ M and 1.5 ⁇ M respectively.
  • Oxidized STC-adamantane has no lysing effect on the RBC, because the hydrophobic "tail" of GC derivatives was crucial to lyse the RBC ( Figure 23).
  • SGC liposomes were prepared according to the published procedure (Arab. S. and Lingwood, CA. Glycoconj. J. 13:159-166 (1996). Multilamellar vesicles containing PC/Chol/SGCs were used in order to avoid the high surface curvature of small unilamellar liposomes and the possible exclusion of the GSL during preparation of the large unilamellar liposomes by extrusion or solvent injection of the lipid mixtures.
  • a chloroform:methanol (2: 1 v/v) solution of the lipid mixtures in weight ratio of 1 :5:2.5 of SGCPCChol (as used in the microtitre plate binding assay) were added to a screw capped glass tube and after evaporation of the solvent under a stream of nitrogen, was further dried under a vacuum for 1 hour. Then the lipids were dispersed in 60 mM Tris - 57 -
  • TBS buffered saline
  • the suspension was dispersed by alternate vigorous vortexing and heating for 30_s at 90-95°C (5-10 °C above the lipid transition temperature) for a period of 10 minutes. Background binding to control liposomes lacking SGC , routinely >6% was subtracted. Measurements were made in triplicate.
  • HAU hemagluggution unit
  • SGC-norbornane and SGC-adamantane-3-acetic acid were found to inhibit 2 HAU at concentrations of 1.5 ⁇ M and 0.75 ⁇ M respectively (see Figure 24).
  • Each V-shaped well contained a RBC.SGC derivative at varying concentrations and a viral antigen.
  • SGC-norbornane was found to inhibit hemagglutination at 1.5 ⁇ M and 0.75 ⁇ M.
  • Non-sulfated GC-norbornane was ineffective in inhibiting hemagglutination, as was the case with the oxidized SGC-adamantane derivative. Inhibition was carried out with 2 HAU and 6 x 10 A 7 RBC.
  • Oxidized-SGC-adamantane did not inhibit hemagglutination even at concentrations as high as 50 ⁇ M. It was inferred that the hydrocarbon "tail" ensures the proper presentation of the sugar, to bind to hemagglutinin.

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Abstract

L'invention concerne des méthodes de traitement d'un état induit par des glycolipides chez un sujet. Une dose efficace d'au moins un composé thérapeutique représenté par la structure A-B, dans laquelle A est une fraction récepteur glycomimétique et B est un agent actif, est administrée à un sujet, de manière que le traitement de l'état induit par glycolipide se produise. Des méthodes consistent également à administrer une dose efficace d'au moins un composé thérapeutique, ou d'un sel pharmaceutiquement acceptable de celui-ci, à un sujet de manière à traiter un état pathologique associé à une toxine de type Shiga (SLT). L'invention concerne également des compositions pharmaceutiques conditionnées de traitement des SLT. Le conditionnement comprend un récipient destiné à contenir une dose efficace d'une composition pharmaceutique et des instructions d'utilisation de la composition pharmaceutique destinée au traitement des SLT. La composition pharmaceutique comprend au moins un composé thérapeutique destiné à moduler une SLT chez un sujet.
PCT/CA1998/000817 1998-02-25 1998-08-26 Conjugues antibiotique-ligand et leur mode d'utilisation WO1999043356A1 (fr)

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Cited By (23)

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WO2004058304A1 (fr) * 2002-12-20 2004-07-15 Glycomimetics, Inc. Oligosaccharides et conjugues pour le traitement d'infections par bacterie pseudomonale
US7060685B2 (en) 2002-05-16 2006-06-13 Glycomimetics, Inc. Compounds and methods for inhibiting selectin-mediated function
US7361644B2 (en) 2003-11-19 2008-04-22 Glycomimetics, Inc. Specific antagonist for both E- and P-selectins
WO2008133652A3 (fr) * 2006-11-09 2009-03-05 Sudhir Paul Anticorps reconnaissant des épitopes binaires et stimulants immunitaires des superantigènes des lymphocytes b
US7517980B2 (en) 2005-08-09 2009-04-14 Glycomimetics, Inc. Glycomimetric inhibitors of the PA-IL lectin, PA-IIL lectin or both the lectins from Pseudomonas
WO2010027479A3 (fr) * 2008-09-08 2010-10-07 Children's Medical Center Corporation Administration mucosale de molécules, protéines ou particules thérapeutiques couplées à des lipides céramiques
USRE44778E1 (en) 2005-09-02 2014-02-25 Glycomimetics, Inc. Heterobifunctional pan-selectin inhibitors
US8895510B2 (en) 2008-04-08 2014-11-25 Glycomimetics, Inc. Pan-selectin inhibitor with enhanced pharmacokinetic activity
US8921328B2 (en) 2010-09-14 2014-12-30 Glycomimetics, Inc. E-selectin antagonists
US9109002B2 (en) 2011-12-22 2015-08-18 Glycomimetics, Inc. E-selectin antagonist compounds, compositions, and methods of use
US9867841B2 (en) 2012-12-07 2018-01-16 Glycomimetics, Inc. Compounds, compositions and methods using E-selectin antagonists for mobilization of hematopoietic cells
US10519181B2 (en) 2014-12-03 2019-12-31 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectins and CXCR4 chemokine receptors
US11045485B2 (en) 2016-01-22 2021-06-29 Glycomimetics, Inc. Glycomimetic inhibitors of PA-IL and PA-IIL lectins
US11072625B2 (en) 2016-10-07 2021-07-27 Glycomimetics, Inc. Highly potent multimeric e-selectin antagonists
EP3700549A4 (fr) * 2017-10-27 2021-08-18 Children's Medical Center Corporation Lipides à base de céramides à chaîne courte et leurs utilisations
US11197877B2 (en) 2017-03-15 2021-12-14 Glycomimetics. Inc. Galactopyranosyl-cyclohexyl derivauves as E-selectin antagonists
US11291678B2 (en) 2016-03-02 2022-04-05 Glycomimetics, Inc Methods for the treatment and/or prevention of cardiovascular disease by inhibition of E-selectin
EP3773564A4 (fr) * 2018-04-12 2022-04-20 Children's Medical Center Corporation Véhicules d'administration à base d'un lipide de type céramide et leurs utilisations
US11433086B2 (en) 2016-08-08 2022-09-06 Glycomimetics, Inc. Combination of T-cell checkpoint inhibitors with inhibitors of e-selectin or CXCR4, or with heterobifunctional inhibitors of both E-selectin and CXCR4
US11548908B2 (en) 2017-12-29 2023-01-10 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectin and galectin-3
US11707474B2 (en) 2018-03-05 2023-07-25 Glycomimetics, Inc. Methods for treating acute myeloid leukemia and related conditions
US11712446B2 (en) 2017-11-30 2023-08-01 Glycomimetics, Inc. Methods of mobilizing marrow infiltrating lymphocytes and uses thereof
US11845771B2 (en) 2018-12-27 2023-12-19 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectin and galectin-3

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WO2004058304A1 (fr) * 2002-12-20 2004-07-15 Glycomimetics, Inc. Oligosaccharides et conjugues pour le traitement d'infections par bacterie pseudomonale
US7361644B2 (en) 2003-11-19 2008-04-22 Glycomimetics, Inc. Specific antagonist for both E- and P-selectins
US7517980B2 (en) 2005-08-09 2009-04-14 Glycomimetics, Inc. Glycomimetric inhibitors of the PA-IL lectin, PA-IIL lectin or both the lectins from Pseudomonas
USRE44778E1 (en) 2005-09-02 2014-02-25 Glycomimetics, Inc. Heterobifunctional pan-selectin inhibitors
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US8895510B2 (en) 2008-04-08 2014-11-25 Glycomimetics, Inc. Pan-selectin inhibitor with enhanced pharmacokinetic activity
US9534009B2 (en) 2008-04-08 2017-01-03 Glycomimetics, Inc. Pan-selectin inhibitor with enhanced pharmacokinetic activity
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US9796745B2 (en) 2011-12-22 2017-10-24 Glycomimetics, Inc. E-selectin antagonist compounds, compositions, and methods of use
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US11987598B2 (en) 2011-12-22 2024-05-21 Glycomimetics, Inc. E-selectin antagonist compounds, compositions, and methods of use
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US10519181B2 (en) 2014-12-03 2019-12-31 Glycomimetics, Inc. Heterobifunctional inhibitors of E-selectins and CXCR4 chemokine receptors
US11045485B2 (en) 2016-01-22 2021-06-29 Glycomimetics, Inc. Glycomimetic inhibitors of PA-IL and PA-IIL lectins
US11291678B2 (en) 2016-03-02 2022-04-05 Glycomimetics, Inc Methods for the treatment and/or prevention of cardiovascular disease by inhibition of E-selectin
US11433086B2 (en) 2016-08-08 2022-09-06 Glycomimetics, Inc. Combination of T-cell checkpoint inhibitors with inhibitors of e-selectin or CXCR4, or with heterobifunctional inhibitors of both E-selectin and CXCR4
US11780873B2 (en) 2016-10-07 2023-10-10 Glycomimetics, Inc. Highly potent multimeric e-selectin antagonists
US11072625B2 (en) 2016-10-07 2021-07-27 Glycomimetics, Inc. Highly potent multimeric e-selectin antagonists
US11197877B2 (en) 2017-03-15 2021-12-14 Glycomimetics. Inc. Galactopyranosyl-cyclohexyl derivauves as E-selectin antagonists
US11878026B2 (en) 2017-03-15 2024-01-23 Glycomimetics, Inc. Galactopyranosyl-cyclohexyl derivatives as e-selectin antagonists
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