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WO1998026662A1 - Composes et methodes pour traiter et prevenir les maladies bacteriennes et virales - Google Patents

Composes et methodes pour traiter et prevenir les maladies bacteriennes et virales Download PDF

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Publication number
WO1998026662A1
WO1998026662A1 PCT/US1997/023374 US9723374W WO9826662A1 WO 1998026662 A1 WO1998026662 A1 WO 1998026662A1 US 9723374 W US9723374 W US 9723374W WO 9826662 A1 WO9826662 A1 WO 9826662A1
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gml
oligo
dendrimer
oligosaccharide
pitc
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PCT/US1997/023374
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WO1998026662A9 (fr
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Cara-Lynne Schengrund
Jeffrey Thompson
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The Penn State Research Foundation
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Priority to AU56101/98A priority Critical patent/AU5610198A/en
Publication of WO1998026662A1 publication Critical patent/WO1998026662A1/fr
Publication of WO1998026662A9 publication Critical patent/WO1998026662A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002—Dendritic macromolecules
    • C08G83/003—Dendrimers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers

Definitions

  • the present invention relates to compounds and methods for treating and preventing bacterial and viral disease, and in particular, carbohydrate derivatives for inhibiting binding to cell receptors.
  • penicillin Like all ⁇ -lactam drugs, it is a compound which selectively inhibits bacterial cell wall synthesis. Because of their relatively high concentration of peptidoglycan, gram-positive organisms tend to be much more susceptible to the effects of penicillin and other ⁇ -lactams than gram-negative organisms. Importantly, because they affect cell wall synthesis, penicillin and the other ⁇ -lactams are only effective against actively growing and dividing cultures. Some organisms are naturally resistant to penicillin. Moreover, following years of use to treat various infections and diseases, penicillin resistance has become increasingly widespread in the microbial populations that were previously susceptible to the action of these drugs. Tetracycline is the drug of choice for some bacteria, such as V. cholerae.
  • gonorrhoeae were found to be penicillin-resistant. This required the use of alternative drugs such as spectinomycin. It can be expected that this trend will continue, with the development of strains that are resistant to sulfonamides, penicillin, spectinomycin, and other antimicrobials. Thus, there remains a need to develop new strategies against bacteria. Ideally; the approach should be effective even against multiple-drug resistant organisms.
  • viruses An ever-increasing number of viruses are being identified as the source of human disease.
  • the better known diseases caused by viruses include, chicken pox, measles, mumps, influenza, hepatitis, poliomyelitis, rabies, and now, of course, HIV.
  • virus-related diseases There are, however, many more virus-related diseases. Indeed, viral infections are estimated to be responsible for more than sixty-percent of human sickness occurring in developing countries. In contrast to the somewhat successful story of antibiotics, efforts to treat viral infection have been largely ineffective. When individuals become infected, modern medicine can do little but ease the symptoms. Viral epidemics have only been avoided by treatment of uninfected individuals with vaccines.
  • a virus is essentially nucleic acid surrounded by a lipid-protein envelope.
  • a virus invades a host cell and uses the host cell's machinery to replicate itself. The latter characteristic makes it especially difficult to find drugs which kill the virus and leave the host unharmed.
  • the present invention relates to compounds and methods for treating and preventing bacterial and viral disease, and in particular, carbohydrate derivatives for inhibiting binding to cell receptors.
  • the present invention contemplates inhibitors to block the binding of microorganisms and/or their toxins to such receptors.
  • the present invention contemplates using oligosaccharide-derivatized dendrimers to 1) inhibit binding of microorganisms (e.g. viruses) to cell surface glycoconjugates, as well as 2) inhibit cells from binding carbohydrate residues present on the surface of the microorganism (e.g. bacteria).
  • the present invention contemplates that such inhibitors can be used for both treatment and prevention of disease (as well as preventing the spread of infection in a subject with nascent disease).
  • the present invention is not limited to in vivo treatment.
  • the present invention contemplates that the inhibitors of the present invention are useful in assays in vitro to assess the susceptibility of microorganisms to inhibition (and to better understand interaction with host cells).
  • glycosylated dendrimers can be used to study the carbohydrate requirements for adherence of a bacteria, virus, or bacterial toxin.
  • the present invention contemplates using such in vitro reactions to obtain information about the optimum spacing between replicate oligosaccharide binding sites, allowing for better inhibitor design.
  • the present invention contemplates that the oligosaccharide derivatives can serve as carriers, i.e. to deliver drugs to microorganisms.
  • the present invention contemplates using (multivalent) oligosaccharide-derivatized dendrimers (i.e. defined clusters) to deliver antibacterial or antiviral agents. In this manner, specificity is achieved (e.g. through the interaction of the oligosaccharide with the specific ligand on the microorganism) and systemic exposure to the antibacterial or antiviral agent (and the consequent toxicity) is avoided.
  • the present invention contemplates methods for synthesizing compounds, comprising: a) providing: i) a source of oligosaccharide; ii) oligosaccharide treatment means; and iii) a polymer; b) treating said source with said treatment means under conditions such that oligosaccharide is released from said source to create free oligosaccharide; and c) mixing said free oligosaccharide with said polymer under conditions such that said free oligosaccharide is covalently attached to said polymer to create a carbohydrate derivative.
  • said source of oligosaccharide is selected from the group consisting of glycoproteins, glycosaminoglycans and glycolipids.
  • glycolipids are used as a source and said glycolipids are selected from the group consisting of neutral glycosphingolipids, gangliosides and sulfatoglycosphingolipids.
  • gangliosides are used as the source and said gangliosides are selected from the group consisting of GM1 (including fucosylated GM1), GM2, GM3, GDla and GDlb.
  • neutral glycosphingolids are used as the source and said neutral glycosphingolipids are selected from the group consisting of glucosylceramide, lactosylceramide, globotriasylceramide, globotetraosylceramide and asialo GM1.
  • treatment means A variety of chemical and enzymatic treatment means are contemplated.
  • said treatment means is triethylamine.
  • said polymer is a branched polymer.
  • said branched polymer is a dendrimer and said carbohydrate derivative is multivalent.
  • the present invention also contemplates a method, comprising: a) providing: i) a source of oligosaccharide selected from the group consisting of glycoproteins, glycosaminoglycans and glycolipids; ii) oligosaccharide treatment means; and iii) a branched polymer; b) treating said source with said treatment means under conditions such that oligosaccharide is released from said source to create free oligosaccharide; and c) mixing said free oligosaccharide with said polymer under conditions such that said free oligosaccharide is covalently attached to said polymer to create a multivalent carbohydrate derivative.
  • the present invention contemplates a method, comprising: a) providing: i) a glycolipid source of oligosaccharide, wherein said glycolipid is selected from the group consisting of neutral glycosphingolipids, gangliosides and sulfatoglycosphingolipids; ii) oligosaccharide treatment means; and iii) a dendrimer; b) treating said glycolipid source with said treatment means under conditions such that oligosaccharide is released from said source to create free oligosaccharide; and c) mixing said free oligosaccharide with said dendrimer under conditions such that said free oligosaccharide is covalently attached to said dendrimer to create a carbohydrate derivative.
  • the present invention also contemplates the resulting compounds.
  • the present invention contemplates a carbohydrate derivative comprising oligosaccharide covalently attached to a non-carbohydrate polymer.
  • said polymer is a branched polymer.
  • said branched polymer is a dendrimer.
  • said oligosaccharide is derived from a glycolipid and said glycolipid is selected from the group consisting of neutral glycosphingolipids, gangliosides and sulfatoglycosphingolipids.
  • the oligosaccharide is derived from a ganglioside and said ganglioside is selected from the group consisting of GM1 (including fucosylated GM1), GM2 and GM3, etc.
  • the present invention specifically contemplates a carbohydrate derivative comprising glycolipid oligosaccharide covalently attached to a dendrimer, wherein said glycolipid is selected from the group consisting of neutral glycosphingolipids, gangliosides and sulfatoglycosphingolipids.
  • the present invention also contemplates methods of treatment (including both acute and preventative treatment).
  • a method of treatment comprising: a) providing: i) a subject having symptoms of (viral or bacterial) disease, and ii) a carbohydrate derivative comprising glycolipid oligosaccharide covalently attached to a dendrimer; b) administering said derivative to said subject under conditions such that said symptoms are reduced.
  • said glycolipid is selected from the group consisting of neutral glycosphingolipids, gangliosides and sulfatoglycosphingolipids
  • said gangliosides are selected from the group consisting of
  • GM1 (including fucosylated GM1), GM2 and GM3, etc.
  • said derivative is multivalent.
  • the present invention contemplates a method of preventative treatment, comprising: a) providing: i) a subject at risk of (viral or bacterial) disease, and ii) a carbohydrate derivative (e.g. multivalent) comprising glycolipid oligosaccharide covalently attached to a dendrimer; b) administering said derivative to said subject.
  • said glycolipid is selected from the group consisting of neutral glycosphingolipids, gangliosides and sulfatoglycosphingolipids.
  • said subject is at risk for bacterial disease and said bacterial disease is cholera.
  • Figure 1 shows the chemical structure of the ganglioside GM1.
  • Figure 2 is a flow chart for the synthesis of the phenylisothiocyanoto derivative of oligo-GMl.
  • Figure 3 is a thin layer chromatograph showing oligo-GMl derivatives.
  • Figure 4 is a flow chart for the synthesis of tetra- and octa(propylene imine) dendrimers.
  • Figure 5 is a thin layer chromatograph showing adherence of horseradish peroxidase conjugated cholera toxin (HRP-CT) to GM1 and oligo-GMl -PITC derivatized dendrimers.
  • HRP-CT horseradish peroxidase conjugated cholera toxin
  • Figure 6 is a graph showing inhibition of adherence of 125 I -labeled choleragenoid to GM1 -coated plastic wells by oligo-GMl -containing ligands.
  • Figure 7 shows predicted lowest-energy conformation of the fully derivatized oligo-GMl -PITC and StarburstTM dendrimers.
  • Figure 8 shows a computer overlay of the crystal structure of choleragenoid with the predicted structure of the tetra(propylene imine)(oligo-GMl-PITC) 4 derivative.
  • Figure 9 shows two graphs depicting adherence of ,25 I-labeled choleragenoid to
  • Figure 9A shows the results when cells were grown in the presence of media containing different amounts of GMl prior to harvest 18 hr later and incubation with 6 nM labeled choleragenoid for 1 hr at 16 ° C.
  • Figure 9B shows the results when cells were grown in media containing 50nM GMl for 18 hrs prior to a 1 hr incubation at 16°C with the indicated concentrations of labeled choleragenoid.
  • Figure 10 shows three graphs depicting adherence of 125 I-labeled choleragenoid (Figure 10A), cholera toxin ( Figure 10B), and the heat labile enterotoxin of E. coli (Figure IOC) to GMl -treated NCTC-2071 cells in the absence or presence of (oligo-GMl - PITC) 7 octa(propylene imine).
  • Figure 11 is a graph showing the tryptophan fluorescence emission spectra for choleragenoid plus oligo-GMl -containing compounds.
  • Figure 12 shows two graphs depicting the tryptophan fluorescence emission spectra for cholera toxin ( Figure 12A) and the heat labile enterotoxin of E. coli ( Figure 12B) plus oligo-GMl -containing compounds.
  • the present invention contemplates oligosaccharides "made from” as well as oligosaccharides "based upon” a variety of sources of carbohydrate.
  • Oligosaccharides "derived from” or “made from” such sources are natural oligosaccharides that are isolated by treatment of such sources (e.g. chemical or enzymatic treatment), while oligosaccharides "based upon” such sources are synthetic and may contain sugar substitutions or deletions, but contain at least three of the same sugars (in the same sequence and linkage) as the natural oligosaccharide.
  • the present invention contemplates isolating oligosaccharides from host cell glycoconjugates or pathogen glycoconjugates, for the preparation of carbohydrate derivatives.
  • oligo-X is meant to indicate the oligosaccharide moiety released (“free") and isolated from the source (“X”); thus, the term “oligo-GMl” is used to indicated the isolated oligosaccharide moiety of GMl (a "glycolipid oligosaccharide”).
  • oligo-GMl is used to indicated the isolated oligosaccharide moiety of GMl (a "glycolipid oligosaccharide”).
  • the present invention contemplates synthetic oligosaccharides based upon host cell glycoconjugates or pathogen glycoconjugates, for the preparation of carbohydrate derivatives.
  • Carbohydrate derivatives are molecules containing natural or synthetic oligosaccharides linked to non-carbohydrate (e.g. protein,protein polymers (such as poly- L-lysine), non-protein polymers, etc.).
  • the present invention contemplates linking oligosaccharides to dendrimers to generate carbohydrate derivatives. It is not intended that the present invention be limited to derivatives wherein only one oligosaccharide is linked. Indeed, in a preferred embodiment, a plurality of oligosaccharides are linked to create a "multivalent” derivative.
  • Carbohydrate conjugates are molecule containing natural or synthetic oligosaccharides linked to carbohydrate (e.g. dextran).
  • Dendrimers are highly branched polymers that originate from a central core. A number of dendrimers are available commercially (e.g. from Aldrich).
  • polymer is intended to indicate all types of polymers (i.e. molecules with repeating units).
  • the present invention contemplates linking oligosaccharides to such polymers as dextran and polyethylene glycol.
  • carrier is intended to indicate that a molecule can carry or deliver a drug or other active ingredient (e.g. oxygen radical) to a target (e.g. host cell, pathogen, etc.). Typically, the carrier is delivered via the bloodstream to the target.
  • a drug or other active ingredient e.g. oxygen radical
  • the present invention contemplates using the carbohydrate derivatives of the present invention as carriers.
  • a subject with symptoms of (bacterial or viral) disease is meant to indicate a human or animal (whether cow, horse, sheep, etc.) having detectable symptoms (i.e. detectable by observation or diagnostic testing of fluid and/or tissue samples) known to those skilled in the art to be associated with disease.
  • detectable symptoms i.e. detectable by observation or diagnostic testing of fluid and/or tissue samples
  • HIV infection is associated with immune deficiencies that are readily detectable; on the other hand, HIV can also be associated with readily detectable retroviral nucleic acid in serum or plasma of the patient.
  • bacterial disease is typically associated with fever; on the other hand, specific diseases have diarrhea as a characteristic symptom.
  • the present invention is not limited to any one symptom for any one disease.
  • Symptoms are "reduced" when there is a detectable quantitative reduction.
  • fever and/or respiration rate can be quantitatively detected and measured and thus a reduction in fever and/or respiration can be readily detected and quantitated.
  • fluid loss is detectable and can be measured. It is not intended that the present invention be limited to precise levels or reductions of a particular magnitude. Most importantly, the present invention is not limited to “cures" or complete elimination of each and every symptom. It is sufficient that there is a reduction in one or more symptoms, regardless of the magnitude of the reduction. For example, in the case of cholera, a reduction in fluid loss can help to stabilize the patient, even though other symptoms of disease are present.
  • the present invention contemplates both acute treatment and preventative treatment.
  • preventative treatment subjects "at risk for (bacterial or viral) disease” are treated.
  • Subjects are “at risk” where disease is prevalent in the area (e.g. V. cholera in East Africa, Bangladesh and parts of India) or detected in members of the immediate population (e.g. within a city for humans; within a farm or herd for animals).
  • the present invention also contemplates that immune deficient patients and hospitalized patients (regardless of their immune state) are, by definition, at risk. However, immune competent individuals are not at risk unless the above-discussed criteria are satisfied.
  • the present invention relates to compounds and methods for treating and preventing bacterial and viral disease, and in particular, carbohydrate derivatives for inhibiting binding to cell receptors. While it is not intended that the present invention be limited to a precise mechanisms by which a benefit is achieved, it is contemplated that, in one embodiment, the carbohydrate derivatives of the present invention mimic the carbohydrate portion of the cell receptor and thereby provide an alternative target for the microorganism and/or a microbial toxin.
  • Oligosaccharides can differ from one another in composition and sequence in a manner analogous to polypeptides.
  • Oligosaccharides with the identical composition and sequence can, moreover, differ in sugar linkages. Added to this level of diversity is the fact that a given carbohydrate chain can be found on either glycoproteins, glycosaminoglycans or glycolipids, each with their own spatial orientation in the cell membrane. It is not intended that the present invention be limited to a particular source of carbohydrate. Derivatives made from carbohydrate chains of glycoproteins, glycosaminoglycans or glycolipids are contemplated. Within glycolipids, carbohydrate derivatives made from (or based upon) oligosaccharides of glycosphingolipids are, in particular, contemplated.
  • Glycosphingolipids have three major structural features: a long-chain base, a fatty acid moiety, and a carbohydrate chain. Heterogeneity of the carbohydrate chain, as mentioned above for carbohydrates in general, can be found in glycosphingolipids with carbohydrate chains of different length (as small as one sugar to as large as twenty sugars), different sugar composition (glucose, galactose, glucosamine, galactosamine, fucose, and neuraminic acid are the most common), different sugar linkages (the glycosidic linkage can involve a number of different carbon atoms and can be found in two spatial orientations), and different sugar sequences.
  • Glycosphingolipids are usually broadly divided into two classes: those that contain only neutral sugars (neutral glycosphingolipids) and those that contain sialic acid (acidic glycosphingolipids, or "gangliosides").
  • the present invention contemplates oligosaccharides made from (or based upon) all three classes.
  • the sphingosine base and the fatty acid moiety (together called ceramide) comprise the hydrophobic region of the molecule (see Figure 1 ) whereas the carbohydrate chain makes up the hydrophilic region of the molecule. This amphiphilicity of glycosphingolipids makes them well-suited to a position in the cell membrane such that the ceramide portion is imbedded in the lipid bilayer and the carbohydrate portion extends outward from the membrane.
  • a number of bacterial toxins, bacteria and viruses have been found to recognize and adhere to the oligosaccharide portion of glycosphingolipid receptors.
  • the present invention contemplates inhibitors to block the binding of such organisms and/or their toxins to such receptors.
  • the present invention contemplates inhibiting bacterial toxin binding to cell receptors.
  • a variety of toxins are known to bind carbohydrate. While not limited to particular toxins, the present invention contemplates, in particular, inhibiting cholera toxin, and the heat labile enterotoxins of E. coli and Campylobacter jejuni, all three of which recognize the oligosaccharide portion of ganglioside GMl as a cell surface receptor.
  • Antigenic cross-reactivity has been observed between cholera toxin and both heat labile enterotoxins.
  • the structures of cholera toxin and the heat labile enterotoxin of E. coli are quite similar. Both have five binding sites which are spaced at similar distances and are on one side of the protein. Cholera is a severe problem for third world countries and travelers' diarrhea is a problem for many people all over the world. At the moment there is no preventative for these toxin-induced diseases. 2. Binding Of Bacteria
  • the present invention contemplates inhibiting binding of bacteria (or portions thereof) to cell receptors.
  • bacteria A variety of bacteria are known to bind carbohydrate.
  • Pseudomonas aeruginosa pili mediate adherence to asialo-GMl [(See Lee et al., Molecular Microbiol. 11 :705 (1994)] and Helicobacter pylori can adhere to gangliotetraosylceramide, gangliotriaosylceramide, and phosphatidylethanolamine [(See Lingwood et al., Infection and Immunity 61 :2474 (1993)].
  • the present invention specifically contemplates using asialo-GMl, as well as gangliotetraosylceramide and gangliotriaosylceramide, as sources of oligosaccharide for the preparation of carbohydrate derivatives.
  • Pneumocystis carinii which causes pneumonia in patients with impaired immunity adheres to cells that have mannose receptors.
  • the bacteria has a mannose-rich surface component (glycoprotein A) that is recognized by mannose receptors present on macrophage within the lung (O'Riordan et al, Infection and Immunity 63: 779- 784, 1995).
  • the derivatized dendrimer e.g. containing mannose or an oligosaccharide comprising mannose
  • the present invention is contemplated by the present invention to keep the cell from binding carbohydrate residues present on the surface of the bacteria.
  • HIV- 1 Human immunodeficiency virus type 1
  • the present invention contemplates inhibiting HIV binding using a polyvalent galactosyl ligand (including but not limited to carbohydrate derivatives comprising dendrimers having galactose moieties or dendrimers having oligosaccharide comprising galactose, such as oligosaccharides having terminal galactose residues).
  • a polyvalent galactosyl ligand including but not limited to carbohydrate derivatives comprising dendrimers having galactose moieties or dendrimers having oligosaccharide comprising galactose, such as oligosaccharides having terminal galactose residues.
  • Rotavirus is believed to adhere to carbohydrate receptors on epithelial cells in the small intestine. The binding is thought to be essential for infection and is probably carbohydrate mediated.
  • Neutral lipids reported to function as receptors are gal( ⁇ l- 3)galNAc( ⁇ l-4)glc( ⁇ l-4)glc( ⁇ l-l)cer and pentaosylceramides with terminal galNAc residues. Srnka et al., Virol. 190:794 (1992).
  • the present invention specifically contemplates carbohydrate derivatives wherein in such oligosaccharides are employed.
  • carbohydrate chains are present on a number of classes of molecules.
  • the present invention contemplates isolating the carbohydrate chain free of the rest of the molecule and attaching it to a dendrimer.
  • the present invention be limited to particular methods for isolating carbohydrate.
  • a variety of methods are available. For example, several procedures have been developed for the cleavage and purification of the oligosaccharide portion of glycosphingolipids. See e.g., S. Hakomori, J. Lipid Res., 7: 789 (1966).
  • the initial reaction is the oxidative cleavage of the C-4-C-5 double bond of the sphingosine base.
  • the free, intact oligosaccharide is then released by treatment with alkali.
  • a simpler procedure for the cleavage and isolation of the oligosaccharide portion consists of the selective oxidation (by e.g. 2,3-dichloro-5,6-dicyanobenzoquinone) of the allylic OH-3 of the sphingenine base. Then, the oligosaccharide can be cleaved from the 3-ketosphingolipid intermediate by a base-catalyzed ⁇ -elimination (e.g. by treatment with a treatment means such as triethylamine). See M. Miljkovic and C-L. Schengrund,
  • the above-described oligosaccharides are coupled (covalently or non-covalently) to macromolecules. It is not intended that the present invention be limited to particular coupling chemistries or strategies. A variety of approaches are contemplated.
  • aryl-amine groups are introduced into the terminal reducing end of oligosaccharides by reacting them with 2-(4-aminophenyl)-ethylamine. After subsequent conversion to the corresponding saccharide-phenylisothiocyanato derivatives, saccharides can be covalently linked to macromolecules. For example, saccharides can be linked to free lysylamine groups of proteins.
  • phenylisothiocyanate derivative instead of reductive amination to directly couple the oligosaccharide to the dendrimer
  • the phenylisothiocyanate provides an additional 8.7 A spacer which computer modeling indicates would provide a better "fit” between ligand and toxin, and 2) the reaction is more efficient.
  • reductive amination reactions in aqueous medium are usually slow when molar amounts of aldehyde or amine are insufficient to drive the reaction. Synthesis of the phenylamine derivative by reductive amination is feasible because the molar amount of amine far exceeded the amount of oligo (such as oligo- GMl).
  • the present invention contemplates attaching oligosaccharides (covalently or non-covalently) to dendrimers. It is not intended that the present invention be limited to particular dendrimers.
  • poly(propylene imine) dendrimers having four or eight primary amino groups and a StarburstTM (PAMAM) dendrimer having eight primary amino groups are used as core molecules, to which phenylisothiocyanate derivatized oligosaccharides are covalently attached to yield multivalent oligosaccharides.
  • the oligosaccharides that are attached are isolated from glycolipids, and more particularly glycosphingolipids, and preferably glycosphingolipids involved in pathogen-binding or toxin-binding. D. In Vivo Targeting
  • the present invention contemplates inhibiting of receptor binding both in vitro and in vivo.
  • a variety of strategies are contemplated including but not limited to the use of dendrimers as carriers.
  • Goers et al. U.S. Patent No.4,867,973, hereby incorporated by reference
  • the conjugates of the present invention do not require antibody. Instead, specificity is conferred by the carbohydrate interaction with the corresponding ligand.
  • microorganisms e.g. bacteria, viruses, etc.
  • host cells e.g. virally infected host cells and cancer cells.
  • the carbohydrate derivatives of the present invention may be prepared either as liquid solutions or suspensions, or in solid forms.
  • Oral formulations e.g., for gastrointestinal diseases usually include such normally employed additives such as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and typically contain l%-95% of active ingredient, preferably 2%-70%.
  • the compositions are also prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • formulation containing the carbohydrate derivatives of the present invention may be achieved by a variety of routes, including but not limited to, intravenous injection. However, as noted above, for certain diseases, oral delivery is contemplated.
  • V. cholera toxin B subunit horseradish peroxidase conjugate (CT-B-HRP)
  • V. cholera toxin B subunit (CT-B)
  • goat anti-choleragenoid antibody were obtained from List Biological Laboratories (Campbell, CA); generation
  • Bio-Gel P-2 [fine 45-90 mm (wet)] from Bio-Rad (Hercules, CA); centriconTM-3 and centriconTM-10 filters from Amicon Corp. (Lexington, MA); PD-10 (Sephadex G-25M) columns, Sephadex G-25, and DEAE-Sephadex A-25 from Pharmacia LKB Biotechnology Inc. (Uppsala, Sweden); HPTLC (Silica Gel-60) plates from VWR (Bridgeport, NJ), Immunolon 1 Removawell strips from Dynatech Labs Inc. (Chantilly, VA); AlchemyTM
  • the present invention contemplates isolating oligosaccharides from a variety of sources, including glycosphingolipids.
  • gangliosides such as GMl
  • purification was carried out using bovine brain.
  • the partial acid hydrolysis of disialo-, trisialo-, or mixed gangliosides was carried out. Briefly, 200 mg of gangliosides were dissolved by sonication in 1ml of 0.1N H 2 SO 4 and then incubated at 80°C for 45 min.
  • GMl was isolated from the supernatant by chromatography on a DEAE-Sephadex A-25 column (100 ml bed vol). The column was eluted with a step gradient of methanol/chloroform/water (60/30/8, by vol) to methanol/chloroform/l .OM sodium acetate (60/30/8, by vol). GMl was recovered in the fraction eluted with methanol/chloro form/0.2M sodium acetate (60/30/8, by vol).
  • GMl fraction After the GMl fraction was eluted, polysialylated lipids were recovered using methanol/chloroform/l .OM sodium acetate (60/30/8, by vol). Ganglioside containing fractions were rotoevaporated to remove organic solvents and dialyzed against water to remove salts prior to being dried under vacuum from the frozen state (lyophilized). Purity of each fraction was determined using HPTLC with chloroform/methanol/0.3% CaCl 2 (60/35/8, by vol) or chloroform/ isopropanol/50mM KC1 (2/13.4/4.6, by vol). Sialic acid containing glycosphingo-lipids were visualized with resorcinol spray.
  • GMl was identified by its co-mobility with standard GMl and its ability to function as a ligand for the binding subunit of cholera toxin. Recovered polysialylated gangliosides were rehydrolyzed and the procedure to isolate GMl repeated.
  • GMl used for the isolation of oligo-GMl contained both N-acetyl- and N-glycolylneuraminic acid
  • the GMl was hydrolyzed and the released carbohydrate was analyzed by anion exchange chromatography using pulsed amperometric detection (Dionex, technical note 20, 1989).
  • oligo-GMl oligo-GMl
  • oligo-GMl oligosaccharide moiety of GMl
  • Figure 2 is a flow chart showing the synthesis scheme.
  • Compound 1 was converted to 2, the aminophenyl derivative, by reductive amination.
  • Compound 3 the phenylisothiocyanate derivative of oligoGMl was produced by reacting compound 2 with thiophosgene.
  • the R in this particular case indicates the gal ⁇ l- 3 galNAc ⁇ 1-4 [sialic acid ⁇ 2-3]gal ⁇ l-4 portion of the oligosaccharide (although the reaction can be extended to other compounds such that R can comprise a variety of different sugars, linkages and sequences).
  • 1 ImM oligo-GMl was reductively aminated at 37°C for 90 ruin 122mM 2-(4-aminophenyl)-ethylamine in 200mM borate buffer, pH 8.0, containing 32mM sodium cyanoborohydride.
  • the aminophenyl derivative of oligo-GMl was separated from unreacted oligo-GMl by chromatography on a Bio-Gel P2 column.
  • a thin layer chromatograph of GMl and the oligo-GMl derivatives is shown in Figure 3 (the origin is indicated by "O").
  • the compounds in each lane were as follows: a) GMl, B) oligo-GMl, C) the aminophenyl derivative of oligo-GMl, and D) the phenylisothiocyanate derivative of oligo-GMl.
  • the plate was developed in methanol/n-butanol/water (2/1/1, by vol) and the sialic acid containing compounds visualized using resorcinol spray.
  • Rf values were 0.84, 0.76, 0.48, and 0.80, for GMl, oligo-GMl, oligo-GMl phenylamine, and oligo-GMl -PITC, respectively. Average percent yields were 31% for oligo-GMl, and 80% for the aminophenyl derivative. Purity of the derivatives as determined by densitometric scanning of bands on a thin layer chromatograph (Stratagene Eagle Eye scanner and NIH Image software) was essentially 100% for oligo-GMl and 90% for the oligo-GMl aminophenyl derivative.
  • Synthesis of 1, an 0.5 generation poly(propylene imine) dendrimer was accomplished by reacting 1 ,4-diaminobutane with acrylonitrile. Borane mthyl sulfide was used to reduce compound 1 to produce the tetra(proplylene imine) dendrimer with four terminal primary amino groups, compound 2. Repetition of the addition and reduction reactions yields 3, the octa(propylene imine) dendrimer, with eight terminal primary amino groups. Compound 3 is drawn in Figure 4 in its computer-modeled, lowest energy conformation. The reduction was carried out as follows.
  • Methyl borate was removed by resuspending the residue in 50 ml of methanol and drying it by rotoevaporation three times. Unreduced dendrimer was removed from the first generation product by dissolving the residue in 100ml of water and extracting it three times with 100 ml of chloroform. The extracted aqueous phase was dried, yielding the dendrimer-(NH 2 HC1) 4 (generation 1.0).
  • a second generation dendrimer was obtained by converting the dendrimer-(NH2 HC1) 4 to dendrimer-(CN) 8 and then reducing it to obtain dendrimer-(NH 2 HCl) g (generation 2.0) using the procedures described above except that the molar ratio of borane methylsulfide to dendrimer-(CN) 8 was 8.5 to one ( Figure 4).
  • IR was routinely used to monitor the presence of nitriles and primary amines during the stepwise synthesis of the tetra- and octa(propylene imine) dendrimers.
  • Generation 0.5 and 1.5 nitrile containing dendrimers, had a characteristic peak at ⁇ 2260 cm-1 (data not shown). This peak was absent in generation 1.0 and 2.0 dendrimers which had four and eight primary amine groups, respectively, and absorbed in the frequency range indicative of primary amines, 3350-3500 cm-1.
  • Generation 0.5 and 1.5 which lacked primary amines and moved from the origin upon HPTLC, were ninhydrin negative.
  • the mass of the first generation dendrimer as determined by low resolution -Fab mass spectroscopy was 317(+
  • This example describes the synthesis of Oligo-GMl Dendrimers. Coupling of the oligo-GMl -PITC to each of the dendrimer cores was accomplished by combining oligo-
  • GM1-PITC with the dendrimer in 0.2M borate buffer, pH 9.0.
  • a 5:1 molar ratio of oligosaccharide-PITC to dendrimer was used for dendrimers with four primary amino groups, and a 9: 1 molar ratio for dendrimers having eight primary amino groups.
  • the mixture was stirred for 15 hr at 37°C.
  • Oligo-GMl dendrimers were isolated from lower molecular weight components by size exclusion centrifugation, using the appropriate centriconTM filter. Retentates were rinsed three times by addition of water followed by reconcentration prior to drying under vacuum from the frozen state.
  • This example describes the synthesis of Oligo-GMl Dendrimers using the commercially available StarburstTM dendrimers.
  • Coupling of the oligo-GMl -PITC was accomplished by combining oligo-GMl -PITC with the dendrimer in 0.2M borate buffer, pH 9.0. A 20: 1 molar ratio of oligosaccharide-PITC to dendrimer was used and the mixture was stirred for 30 hr at 37°C. Separation and analysis was carried out as described above for the synthesized dendrimers.
  • the oligo-GMl -PITC-StarburstTM dendrimer was both resorcinol and ninhydrin positive, even though more rigorous conditions were used for its synthesis compared to those used for the linkage of oligo ⁇
  • CT-B cholera toxin B subunit
  • Chemiluminescent detection was used to monitor adherence of the toxin to A) asialo GMl, B) GMl, C) tetra (propylene imine)(oligo-GMl-PITC) 4 , D) octa(propylene imine)(oligo-GMl-PITC) 7 , and E) polyclonal anti cholera toxin binding subunit antibody (Figure 5).
  • Lanes A' - E' show the same compounds as in A - E overlaid with HRP-CT that was preincubated with GMl . Plates were developed in methanol/ glacial acetic acid/water (15:7.5:0.25, by vol.). The origin is indicated by the O.
  • well-binding assays were done to determine the effectiveness of the oligo-GMl dendrimers at inhibiting the adherence of cholera toxin B subunit to GM1- coated wells. Briefly, GMl in methanol was added to plastic microtiter wells and allowed to dry. Potential nonspecific binding sites were blocked by incubating the wells with phosphate buffered saline containing 0.1% BSA for 1 hr at 37°C. After 1 hr, the buffer was removed and the wells used for the binding assay. Wells lacking GMl, but blocked in the same way, were used to determine nonspecific binding.
  • Labelled cholera binding subunit ( ⁇ 6nM) was preincubated for 1 hr at 37°C in the presence or absence of inhibitor in phosphate buffered saline (pH 7.2) containing 0.1% BSA and then added to GM1- coated plastic wells. After incubating for 1 hr at 37°C, the toxin was removed, the wells washed seven times with PBS, and bound 125 I-labeled toxin determined by counting in a gamma counter. The results are shown in Figure 6. Each point is the average of quadruplicate samples.
  • the closed circles indicate octa(propylene imine) (oligo-GM 1-PITC) 7 ; the closed squares indicate tetra(propylene imine) (oligo-GM 1-PITC) 4 ; the open circles indicate StarburstTM (oligo-GMl -PITC) 6 ; the closed triangles indicate GMl; and open triangles indicate oligo-GMl. From the results it is clear that all oligo-GMl dendrimers were more effective at inhibiting the adherence of 125 I-labeled cholera toxin B subunit to GMl -coated wells than was GMl.
  • the concentration of poly(propylene imine) oligo-GMl -PITC dendrimer needed to inhibit adherence of the binding subunit to GMl by 50% was 3nM for the octa(propylene imine)(oligo-GMl-PITC) 7 dendrimer and 7-8nM for both the tetra(propylene imine)(oligo-GM 1 -PITC) 4 and StarburstTM (oligo-GM 1 -PITC) 6 dendrimers compared to an average of 40nM for GMl .
  • the IC50 for oligo-GMl was lO ⁇ M. Comparable concentrations of underivatized or acetylated dendrimers did not inhibit adherence of the binding subunit to GMl -coated wells. Replicate experiments gave similar results.
  • This example describes molecular modeling to predict the lowest energy conformation and size of each of the fully derivatized oligo-GMl -PITC dendrimers.
  • the software used was AlchemyTM III.
  • Each of the oligo-GMl -PITC dendrimers was predicted to be large enough to span the diameter of the cholera toxin B subunit.
  • average molecular distances between the oligo-GMl moieties of the oligo-GMl -PITC dendrimers were compared to the geometry of the GMl binding sites predicted by x-ray crystallographic structure determination of the toxin B subunit.
  • Figure 7 shows the predicited lowest-energy conformation of the fully derivatized oligo-GMl -PITC and StarburstTM dendrimers.
  • the tetra(propylene imine) oligo-GMl - PITC dendrimer is on the left
  • the octa(propylene imine) oligo-GMl-PITC dendrimer is in the center
  • the StarburstTM oligo-GMl-PITC dendrimer is on the right.
  • An oligo- GMl-PITC moiety can be seen at the end of each dendrimer arm. Structures shown are uniformly scaled to show their relative sizes.
  • the octa(propylene imine) oligo-GM 1 -PITC-dendrimer which if completely derivatized was predicted to have its oligo-GMl-PITC moieties spaced approximately the same distance apart as the binding sites on the B subunit, was the best inhibitor.
  • the StarburstTM oligo-GMl -PITC-dendrimer which, if fully derivatized, was predicted to have its oligo-GMl-PITC moieties too far apart for optimal interaction with the binding sites on the B subunit, was a less effective inhibitor.
  • poly(propylene imine) dendrimer would be more flexible than the StarburstTM.
  • the poly(propylene imine) dendrimer core is linear in nature, whereas the StarburstTM dendrimer core contains amide bonds. The presence of the amide bond restricts the freedom of rotation at that site thereby decreasing its molecular flexibility.
  • the three dimensional structure of the core molecules predicted more "intramolecular room" around each of the arms of the poly(propylene imine) dendrimers compared to those of the StarburstTM dendrimer.
  • the oligo-GMl-PITC moieties on the tetra(propylene imine) dendrimer may not exactly fit the binding sites on the B subunit, it is possible that the molecular flexibility of the dendrimer arms would allow the oligosaccharides to move into appropriate positions.
  • oligo-GMl-PITC-dendrimer In order to effectively interact with the cholera toxin B subunit, the oligo-GMl-PITC moieties of the dendrimer need to be in appropriate alignment with the binding sites, which are all on one side of the binding subunit.
  • Figure 8 shows an overlay of the crystal structure of the choleragenoid with the AlchemyTM III predicted structure of the tetra(propylene imine)(oligo-GMl-PITC) 4 derivative.
  • the relative sizes of the toxin ( green), and the tetra(propylene imine) oligo- GM 1 -PITC dendrimer (multi-color) are shown.
  • the structures indicate that the potential exists for the choleragenoid (GMl -binding sites are located near the "points" of the toxin pentamer) to adhere to more than one of the oligo-GMl-PITC moieties at the end of each arm of the dendrimer.
  • adherence of the choleragenoid to three oligo-GMl-PITC moieties would result in blockage of the central pore through which the
  • This example describes the ability of oligo-GMl-PITC derivatized dendrimers to inhibit adherence of choleragenoid, cholera holotoxin, and the heat labile enterotoxin of E. coli to GMl on the surface of viable NCTC-2071 cells (chemically transformed murine fibroblasts) was investigated. Since each binding subunit of the toxins has a single tryptophan residue, the effect that adherence of the toxin to either GMl, oligo-GMl, or derivatized dendrimer had on their tryptophan fluorescence emission spectra was determined to ascertain whether each ligand induced a comparable change in the tryptophan microenvironment.
  • NCTC-2071 cells Chemically transformed, GMl -deficient, mouse fibroblast NCTC-2071 cells were cultured, asceptically, in defined NCTC-135 media (Sigma Chemical Co.) with L- glutamine and 0.22% sodium bicarbonate. Cultures were grown at 37°C in 95%air/5% > CO2 and 90%) humidity. Confluent cells were harvested by first exposing them for one minute at 37°C to 0.05%) trypsin versene containing 0.1 % glucose and then rapping the flask sharply to dislodge the cells.
  • NCTC-135 medium supplemented with 3% fetal bovine serum (FBS) and the cells pelleted by centrifugation at 200 X g for five minutes. The supernatant was discarded and the cells resuspended in fresh, unsupplemented, NCTC-135 media. Number of viable cells was determined by counting trypan blue negative cells in a hemocytometer. Aliquots containing ⁇ 5 X 106 cells were then seeded into 75 cm 2 flasks in a total volume of 10 ml of fresh NCTC-135 media.
  • FBS fetal bovine serum
  • NCTC-2071 cells contain little cell surface GMl, it was necessary to determine how much GMl had to be added to the cells to provide binding sites for the toxins, and how much labeled toxin would be used in the assays. Therefore, NCTC-2071 cells were grown for 18 hrs prior to harvest in media supplemented with increasing amounts of GMl (0 to 500nM). After harvesting cells by scraping them into PBS, they were recovered by low speed centrifugation and washed three times with PBS. After the third rinse cells were resuspended in a small volume of NCTC-135 media and the number of viable cells determined by counting trypan blue negative cells in a hemocytometer.
  • Binding to added GMl was obtained by subtracting the counts associated with cells that were grown in media alone from those associated with cells grown in media containing GMl .
  • Figure 9A shows the results where cells were grown in the presence of media containing different amounts of GMl prior to harvest 18 hr later and incubation with 6nM 125 I-labeled choleragenoid for 1 hr at 16 ° C.
  • Figure 9B shows the results cells were grown in media containing 50nM GMl for 18 hrs prior to a 1 hr incubation at 16°C with the indicated concentrations of labeled choleragenoid.
  • Cell-associated labeled choleragenoid was determined by counting in a gamma counter.
  • the effectiveness of (oligo-GM 1-PITC) 7 octa(propylene imine) at inhibiting the adherence of labeled choleragenoid or toxin to GMl -treated cells was determined as follows. Two nM labeled protein was preincubated for 1 hr at 37°C with different concentrations of (oligo-GMl -PITC)7 octa(propylene imine) prior to its addition to GM1- treated or control (grown in media alone) cells. The concentration of ligand used was based on the amount found previously to inhibit adherence of labeled choleragenoid or holotoxin to GMl -coated plastic wells by 50% (IC50s).
  • Specific binding was defined as the amount of label bound to GMl -treated cells minus that bound to the same number of control cells. Values for each experimental point are the average of quadruplicate samples and each experiment was usually done twice. Experiments were also carried out in which the preincubation of toxin with dendrimer was omitted. In these experiments, inhibitor was added to the cells just prior to the addition of labeled toxin: the rest of the procedure was unchanged. In some of the assays done at 37°C, lOO ⁇ g of bacitracin was added/ml of incubation media to reduce the possibility of receptor-mediated endocytosis during the incubation period.
  • Figure 10 shows the adherence of 125 I-labeled choleragenoid (Figure 10A), cholera toxin ( Figure 10B), and the heat labile enterotoxin of E. coli (Figure IOC) to GMl -treated NCTC-2071 cells in the absence or presence of (oligo-GM 1 -PITC) 7 octa(propy lene imine) .
  • Average cell -associated counts are indicated by striped bars for experiments in which labeled choleragenoid was preincubated with (oligo-GM 1-PITC) 7 octa(propylene imine) for 1 hr at 37°C prior to incubation with cells for 1 hr at 16"C, by open bars for experiments in which labeled choleragenoid or toxin was preincubated with derivatized dendrimer for 1 hr at 37°C prior to incubation with cells for 1 hr at 37°C, by solid bars for experiments in which dendrimer and choleragenoid or toxin were added directly to the cells which were then incubated for 1 hr at 37 ° C, and by dotted bars for experiments in which toxin and dendrimer were added directly to cells which were then incubated for 1 hr at 37°C in media containing lOO ⁇ g/ml of bacitracin.
  • Cell-associated label was determined by counting in a
  • the results provide a quantitative assessment of the efficacy of (oligo-GMl -PITC) 7 octa(propylene imine) as an inhibitor.
  • a significant reduction in adherence of 125 I-labeled choleragenoid, cholera holotoxin, or heat-labile enterotoxin of E. coli to GMl -treated cells was seen when (oligo-GM 1-PITC) 7 octa(propylene imine) was present ( Figure 10A- C).
  • Inhibition of choleragenoid binding to GMl -treated cells by (oligo-GMl -PITC) 7 octa(propylene imine) depended upon the concentration of derivatized dendrimer used.
  • Counts per min shown in the graphs were obtained by subtracting label associated with control cells incubated with choleragenoid or holotoxin alone. This was done because the addition of 4 or 16 nM (oligo-GMl -PITC) 7 octa(propylene imine) plus labeled choleragenoid or holotoxin directly to control cells resulted in the adherence of as much label to the control cells as was found in association with GMl -treated cells. Interestingly, this binding was always significantly less than the amount of label associated with GMl treated cells incubated with choleragenoid or toxin alone.
  • 6nM choleragenoid to GMl -treated and control cells was also monitored using immunofluorescence.
  • Cells (3 X 10 5 ) were seeded and grown in each of eight separate chambers on a microscope slide. Cells in half of the chambers were treated with GMl as above, the others with media alone.
  • Choleragenoid (6nM) was pre-incubated at 37°C for 1 hr in the presence or absence of 30nM (oligo-GM 1-PITC) 7 octa(propylene imine) or
  • EXAMPLE 10 This example describes experiments to determine the effect of (oligo-GMl -PITC) 7 octa(propylene imine) on NCTC-2071 cell viability. Cconfluent cells were grown in media containing the dendrimer at a concentration of 500nM. This concentration was selected because it was ten times the highest concentration of derivatized dendrimer used in most studies. After 18 hrs, media was removed, the cells harvested into PBS by scraping, and the number of trypan blue negative and positive cells counted using a hemocytometer.
  • EXAMPLE 11 This example describes tyrptophan fluorescence analyses.
  • the effect of potential ligands on the intrinsic tryptophan fluorescence of choleragenoid and the holotoxins was monitored using an Aminco-Bowman® series 2 luminescence spectrometer and associated software from SLM-Aminco® (Rochester, NY).
  • Figure 11 shows the tryptophan fluorescence emission spectra for choleragenoid plus oligo-GMl -containing compounds.
  • Choleragenoid was incubated at 37°C for 1 hr in the absence (solid circles), or presence of a five-fold molar excess of GMl (open cirlces), oligo-GMl (solid squares), (oligo-GM 1-PITC) 7 octa(propylene imine) (open squares) , or (oligo-GMl -PITC) 4 tetra(propylene imine) (open triangles).
  • Choleragenoid excited by ultraviolet light with a wavelength of 282 nm, had a tryptophan emission spectra with a maximum at approximately 350 nm. Similar emission spectra were observed when the choleragenoid was incubated with a five-fold molar excess of either of three gangliosides, GDla, GTlb, or asialo-GMl, that fail to function as ligands for the binding subunit.
  • derivatized dendrimers such as the oligo- GMl-PITC-derivatized dendrimers
  • oligosaccharide in a clustered arrangement, are as effective or better ligands than the natural source (e.g. GMl) for binding pathogens and/or their toxins (e.g. cholera toxin).
  • an oligo-GM 1 -PITC-derivatized dendrimer was a better inhibitor of the adherence of the binding subunit than was native GMl is important because it may provide a model for developing compounds that can inhibit the adherence of pathogens or toxins that recognize glycosphingolipids as a ligand.
  • the lipid per se can not be used because it has been shown that exogenous glycosphingolipids can become functional components of a cell's plasma membrane. As a result, a previously nonsusceptible cell can be made susceptible to the toxic agent.
  • multivalent oligosaccharides that function as efficient ligands for specific pathogens can be designed and synthesized. This approach may be useful for preventing or treating illnesses in which the binding of a pathogenic agent to a clustered carbohydrate ligand is an essential step in the etiology of the disease.
  • a possible reason for the efficacy of the oligosaccharide derivatized dendrimers is that the dendrimer core promotes the radial distribution of the added oligosaccharide moieties, effectively clustering them into an "artificial" micelle.
  • the use of dendrimers for the synthesis of multivalent oligosaccharides has the advantage of providing more defined products than are obtained using protein, peptide, or polyacrylamide cores.

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Abstract

L'invention concerne des méthodes permettant de synthétiser des composés utiles pour traiter et prévenir les maladies bactériennes et virales. Ces composés contiennent un oligosaccharide lié à des polymères et, plus spécifiquement, à des dendrimères.
PCT/US1997/023374 1996-12-19 1997-12-19 Composes et methodes pour traiter et prevenir les maladies bacteriennes et virales WO1998026662A1 (fr)

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WO2000015240A1 (fr) * 1998-09-14 2000-03-23 Starpharma Limited Compositions antimicrobiennes et antiparasitaires a base de dendrimere anionique ou cationique
WO2000008467A3 (fr) * 1998-08-07 2000-07-06 Univ Alberta Traitement d'infections bacteriennes
WO2000029556A3 (fr) * 1998-11-17 2000-11-23 Us Health Identification des glycosphingolipides qui favorisent la penetration du vih-1 dans les cellules
US6440405B1 (en) 1999-06-07 2002-08-27 University Of Delaware Quaternary ammonium functionalized dendrimers and methods of use therefor
FR2830017A1 (fr) * 2001-09-27 2003-03-28 Centre Nat Rech Scient Materiau compose d'au moins un polymere biodegradable et de cyclodextrines
US6579906B2 (en) 2000-06-09 2003-06-17 University Of Delaware Dendrimer biocide-silver nanocomposites: their preparation and applications as potent antimicrobials
WO2004041310A1 (fr) * 2002-11-08 2004-05-21 Danmarks Fødevareforskning Preparation de conjugues de dendrimere et de glucide chimiquement bien definis
WO2008091246A1 (fr) * 2007-01-22 2008-07-31 Allexcel, Inc. Polymères amphiphiles à auto-assemblage en tant qu'agents antiviraux
WO2009005353A1 (fr) * 2007-07-02 2009-01-08 Erasmus University Medical Center Rotterdam Composés d'oligosaccharide de liaison élevée, compositions et leurs utilisations
US7572459B2 (en) 1998-09-14 2009-08-11 Starpharma Pty Ltd. Anionic or cationic dendrimer antimicrobial or antiparasitic compositions
CN115073321A (zh) * 2022-07-20 2022-09-20 山东新华制药股份有限公司 一种1,4-双[双(2-氰基乙基)氨基]丁烷的制备方法

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US5470843A (en) * 1992-12-11 1995-11-28 Hoechst Aktiengesellschaft Carbohydrate-containing polymers, their preparation and use

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US5346696A (en) * 1991-06-19 1994-09-13 Korea Green Cross Corporation Asialoglycoprotein - conjugated medicinal agent
US5470843A (en) * 1992-12-11 1995-11-28 Hoechst Aktiengesellschaft Carbohydrate-containing polymers, their preparation and use

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000008467A3 (fr) * 1998-08-07 2000-07-06 Univ Alberta Traitement d'infections bacteriennes
US6310043B1 (en) 1998-08-07 2001-10-30 Governors Of The University Of Alberta Treatment of bacterial infections
AU754331B2 (en) * 1998-08-07 2002-11-14 Governors Of The University Of Alberta, The Treatment of bacterial infections
US7572459B2 (en) 1998-09-14 2009-08-11 Starpharma Pty Ltd. Anionic or cationic dendrimer antimicrobial or antiparasitic compositions
US6464971B1 (en) 1998-09-14 2002-10-15 Starpharma Limited Anionic or cationic dendrimer antimicrobial or autiprotozoan compositions
WO2000015240A1 (fr) * 1998-09-14 2000-03-23 Starpharma Limited Compositions antimicrobiennes et antiparasitaires a base de dendrimere anionique ou cationique
WO2000029556A3 (fr) * 1998-11-17 2000-11-23 Us Health Identification des glycosphingolipides qui favorisent la penetration du vih-1 dans les cellules
US6440405B1 (en) 1999-06-07 2002-08-27 University Of Delaware Quaternary ammonium functionalized dendrimers and methods of use therefor
US6579906B2 (en) 2000-06-09 2003-06-17 University Of Delaware Dendrimer biocide-silver nanocomposites: their preparation and applications as potent antimicrobials
FR2830017A1 (fr) * 2001-09-27 2003-03-28 Centre Nat Rech Scient Materiau compose d'au moins un polymere biodegradable et de cyclodextrines
WO2003027169A1 (fr) * 2001-09-27 2003-04-03 Centre National De La Recherche Scientifique (C.N.R.S) Materiau compose d'au moins un polymere biodegradable et de cyclodextrines
WO2004041310A1 (fr) * 2002-11-08 2004-05-21 Danmarks Fødevareforskning Preparation de conjugues de dendrimere et de glucide chimiquement bien definis
WO2008091246A1 (fr) * 2007-01-22 2008-07-31 Allexcel, Inc. Polymères amphiphiles à auto-assemblage en tant qu'agents antiviraux
JP2010516673A (ja) * 2007-01-22 2010-05-20 オールエクセル，インコーポレイティド 抗ウイルス薬としての自己集合性の両親媒性高分子
AP2523A (en) * 2007-01-22 2012-11-30 Allexcel Inc Self-assembling amphiphilic polymers as antiviral agents
KR101405764B1 (ko) 2007-01-22 2014-06-10 올엑셀, 인크. 항바이러스성 제제로서 자기 조립성 양친매성 폴리머
EA026125B1 (ru) * 2007-01-22 2017-03-31 Элексел, Инк. Самособирающиеся амфифильные полимеры в качестве противовирусных средств
WO2009005353A1 (fr) * 2007-07-02 2009-01-08 Erasmus University Medical Center Rotterdam Composés d'oligosaccharide de liaison élevée, compositions et leurs utilisations
CN115073321A (zh) * 2022-07-20 2022-09-20 山东新华制药股份有限公司 一种1,4-双[双(2-氰基乙基)氨基]丁烷的制备方法
CN115073321B (zh) * 2022-07-20 2023-10-13 山东新华制药股份有限公司 一种1,4-双[双(2-氰基乙基)氨基]丁烷的制备方法

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