WO1996014873A2 - COVALENTLY BOUND β-GLUCAN CONJUGATES IN TARGETED DELIVERY - Google Patents

COVALENTLY BOUND β-GLUCAN CONJUGATES IN TARGETED DELIVERY Download PDF

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WO1996014873A2
WO1996014873A2 PCT/US1995/014800 US9514800W WO9614873A2 WO 1996014873 A2 WO1996014873 A2 WO 1996014873A2 US 9514800 W US9514800 W US 9514800W WO 9614873 A2 WO9614873 A2 WO 9614873A2
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glucan
composition
glucans
agent
cells
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PCT/US1995/014800
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French (fr)
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WO1996014873A3 (en
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Daniel Tuse
Nahid Mohagheghpour
Marcia Dawson
Peter Hobbs
Richard Winant
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Sri International
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to glucan conjugates, particularly ⁇ -1,3-glucan conjugates, for use in targeted delivery of bioactive agents.
  • Glucans have been shown to have properties that are similar to those of endotoxin in increasing nonspecific immunity and resistance to infection.
  • the activities of some glucans (for example, yeast derived glucans) as an immune adjuvant and hemopoietic stimulator compare to those of more complex biological response modifiers (BRMs), such as bacillus Calmette-Guerin (BCG) and Corynebacterium parvum.
  • BRMs complex biological response modifiers
  • the functional activities of yeast glucan are also comparable to those of structurally similar carbohydrate polymers isolated from fungi and plants.
  • These higher molecular weight (1-3)- ⁇ -D-glucans such as schizophyllan, lentinan, krestin, grifolan, and pachyman exhibit similar immunomodulatory activities.
  • the present invention provides a glucan composition containing a ⁇ -1 ,3-glucan covalently attached to a bioactive agent.
  • the ⁇ -1 ,3-glucan is attached to the bioactive agent by means of a hydrolyzable covalent linkage to form the resulting glucan/agent complex.
  • the glucans for use in the present invention may be obtained from various sources, including algae, fungi, plant and bacteria.
  • the glucan contained in the composition is isolated from Euglena.
  • the glucans of the present invention include glucans which are insoluble under normal, physiological conditions. Such glucans may be particulate in nature. The particles typically range in size from about 0.5 to about 10 microns. In a preferred embodiment, the glucan particles are between about 3 and 6 microns in size.
  • the glucans of the present invention may possess a linear ⁇ -1 ,3 configuration.
  • glucans for use in the present invention may possess varying degrees of branching.
  • compositions of the present invention also include glucans which are soluble under physiological conditions.
  • the soluble glucans have at least about 2 repeating monomer units, more preferably, at least about 5 repeating monomer units.
  • the soluble glucans contain polar side chains, such as those containing 2-hydroxyethyl, carboxymethyl, 2-hydroxyethoxyethyl, and 2,3-dihydroxypropyl moieties.
  • the glucan/agent complex of the present invention contains a hydrolyzable bond which covalently links the ⁇ -1 ,3-glucan to the bioactive agent.
  • Such hydrolyzable linkages for use in the present invention include acyl hydrazone, sulfonyl hydrazone, carbamate, carboxy ester, carbonate ester, phosphate, phosphoramidate and amide.
  • the glucan/agent complex may include a variety of bioactive agents such as anti-viral agents, anti-bacterial agents, anti-fungals, anticancer and anti-malarials.
  • the active agent is an anti-mycobacterial agent, such as isoniazid, pyrimethamine, trimethyoprim, trimetrexate, amalcacin, rifampicin and methotrexate.
  • Bioactive agents for use in the invention also include polypeptides, oligonucleotides, and antisense agents (including alternative-backbone antisense agents, such as, phosphoramidates).
  • the invention provides a method for the treatment of a pathogen capable of invading or colonizing phagocytic cells, such as a mycobacteri ⁇ m.
  • administering of a pharmaceutically effective amount of the glucan/agent composition is effective to direct delivery of the bioactive agent to phagocytic cells harboring the pathogen.
  • the agent which is effective for the treatment of the pathogen, is released from the composition by cleavage of the hydrolyzable attachment.
  • the method is used for treatment of a mycobacterium such as M. avium or M. tuberculosis.
  • a mycobacterium such as M. avium or M. tuberculosis.
  • the glucan/agent composition contains a glucan having, for example, 5 or more repeating monomer units.
  • the invention provides a method for delivering an antigen to a phagocytic cell by forming an antigen/glucan complex.
  • the antigen is a polypeptide vaccine, covalently attached to a soluble glucan.
  • Figs. 1A, 1 B illustrate methods of synthesis for ⁇ -glucan/antisense (phosphorothioate) conjugates.
  • Figs. 2A-2D illustrate methods of synthesis for ⁇ -glucan/polypeptide (ribosome inhibitory protein) conjugates.
  • Figs. 3A and 3B show an exemplary synthetic route for forming a glucan/ bioactive agent complex in which herpes simplex virus glycoprotein D HSV gD2 is coupled to a functionalized ⁇ -1 ,3-glucan by means of acyl hydrazone bonds.
  • Figs. 4A, 4B, and 4C are plots illustrating cytokine production by human monocytes following culture of cells in medium containing particulate ⁇ -1 ,3-glucan.
  • Figs. 4A, 4B, and 4C indicate the levels of IL-1 ⁇ , TNF- ⁇ , and IL-6, respectively (in pg/ml, y axis) versus time (hr, x-axis) following stimulation.
  • Cytokine levels for cells exposed to glucan are indicated by closed squares; open circles indicate cytokine levels for non-stimulated control cells maintained in medium alone.
  • Fig. 5 is a plot illustrating the enhanced immunological response of mice immunized with an exemplary gD2-glucan conjugate prepared in accordance with the present invention.
  • Figs. 6A-6E illustrate various methods for preparing functionalized glucans.
  • Fig. 6A outlines the synthesis of 2,3-dihydroxypropylglucan and amine-functionalized glucans.
  • Fig. 6B illustrates a synthetic approach for forming hydrazide-functionalized glucans.
  • Fig. 6C shows the synthesis of tritium-labeled 2-hydroxyethylglucan.
  • Fig. 6D illustrates a preparation of aldehyde-functionalized glucans.
  • Fig. 6E illustrates an alternative method for forming amine-functionalized glucans.
  • Figs. 7A, 7B illustrate methods of synthesis for ⁇ -glucan/polypeptide (CG1-23) conjugates.
  • Figs. 8A-8C illustrate methods of synthesis for ⁇ -glucan/polypeptide (HSV glycoprotein D) conjugates.
  • AG represents ⁇ -glucan.
  • Figs. 9A, 9B illustrate methods of synthesis for ⁇ -glucan/drug (dideoxynucleotide) conjugates. Detailed Description of the Invention
  • a “hydrolyzable bond” is a covalent bond which can be hydrolyzed, typically under acidic conditions in vivo or in vitro.
  • the hydrolyzable bond may be cleaved by acid-catalyzed hydrolysis.
  • the bond may be hydrolyzed by enzymes (e.g., degradative enzymes such as proteinase or phosphatase) or chemical treatment.
  • Acidic conditions may be aqueous or anhydrous, although most typically, acidic conditions signify aqueous environments having Ph values below about 5.
  • the internal phagocyte environment provides the conditions for hydrolyzation, for example, acid-hydrolysis in the lysozyme which typically has an acidic environment generated by means of a proton-pump ATPase.
  • bioactive agent is a compound that possesses biological activity. Upon introduction into the body, a bioactive agent is one which alters the body's function by interactions at the molecular level.
  • Bioactive agents for use in the invention include small molecules, polypeptides, oligonucleotides, antisense agents, and the like. The bioactive agent may be in a prodrug form which is converted to the active agent following administration.
  • Bioactive agents for use in the present invention possess at least one functional group suitable for attachment to a ⁇ -glucan.
  • Glucan refers generically to a variety of naturally occurring homopolysaccharides or polyglucoses, and includes polymers such as cellulose, amylose, glycogen, laminarins, and starch. Glucans are composed of branched and/or unbranched chains of glucose units linked by 1 ,3-, 1 ,4- and 1 ,6-glucosidic bonds that may be of either the a or ⁇ type.
  • glucan or " ⁇ -glucan,” as used herein, refers to a polyglucose macromolecule containing glucopyranose units linked by a series of beta-1 ,3-glucosidic bonds.
  • Particulate glucan refers to a water insoluble glucan, typically from about 0.5-10 ⁇ in size.
  • the present invention provides a ⁇ -glucan composition containing a ⁇ -1 ,3-glucan covalently attached to a bioactive agent by a hydrolyzable bond.
  • the glucan/agent complex of the present invention is useful in the targeted delivery of bioactive agents, particularly to phagocytic cells harboring pathogens.
  • the hydrolyzable bond which acts to temporarily tether the glucan to the bioactive agent, is effective, upon hydrolysis, to release the bioactive agent at its target site, and thereby release the bioactive agent in its active or prodrug form.
  • ⁇ -Glucan is a glucopyranose polysaccharide linked in a ⁇ -1 ,3 configuration.
  • ⁇ -glucans possess a wide range of biological activities, including phagocyte-specific immuno-enhancing activities (Gorecka-Tisera; Scaringi).
  • phagocyte-specific immuno-enhancing activities Gorecka-Tisera; Scaringi.
  • Human and murine monocytes/macrophages express surface cell receptors specific for the ⁇ -(1 ,3)-linked oligosaccharides (Czop).
  • the surface cell receptors mediate the phagocytosis of ⁇ -glucan particles in the absence of opsonic proteins and the production of pro-inflammatory cytokines.
  • ⁇ -glucans for use in the compositions and methods of the present invention are discussed below.
  • Glucans for use in the present invention may be particulate in nature or may be soluble under physiological conditions.
  • the particulate glucans may also provide the starting materials for conversion to soluble ⁇ -glucans.
  • Naturally occurring ⁇ -glucan is typically particulate in nature, and may be derived from a number of sources, including yeast, algae, plant and fungi (Bartnicki-Garcia).
  • the size range of the ⁇ -glucan particles varies widely.
  • a size range typically useful in the practice of the present invention is the range of about 0.5 to about 10 microns.
  • a preferred size range is about 3 to about 6 microns.
  • One common source of ⁇ -glucan is Saccharomyces cerevisiae (Baker's yeast).
  • ⁇ -glucan yields typically range from about 5 to 7 percent of the cellular mass.
  • a preferable source of glucan for use in the invention is algae from the genus E ⁇ g/ena.
  • suitable sources include E. gracilis, E. intermedia, E. piride, and other Euglenoids, such as Astasia longa. Euglena gracilis is particularly preferred.
  • a preferred process for obtaining substantially pure, pyrogen-free ⁇ -glucan from Euglena in relatively high yields is described in co-owned United States Patent No. 5,084,386.
  • Example 2 The cultivation and purification of algal glucan derived from Euglena gracilis is described in Example 1 .
  • the resulting characteristics of the isolated ⁇ -glucan material is summarized in Table 2. This preferred source and method of purification reproducibly provides a nonpyrogenic, linear, high-molecular-weight algal glucan.
  • Particulate algal glucan derived from Euglena gracilis dissolves only at highly alkaline pHs and may be reprecipitated to an amorphous form by lowering the pH to about 6-8. At this point, individual strands typically reanneal by hydrogen bonding to macromolecular aggregates, the viscosity of which is pH dependent.
  • ⁇ -glucans may also be used in the compositions and methods of the invention, such as native branched glucans ( ⁇ -1 ,3- and ⁇ -1 ,6-glucopyranoses) from yeast and filamentous fungi.
  • Bacterial ⁇ -glucans, such as curdlan may also be used. Curdlan is a linear glucan having a molecular weight of about 80 kilodaltons and is insoluble in water.
  • ⁇ -glucan Another source of ⁇ -glucan is pleuran, a fungal glucan having a linear ⁇ -1 ,3-backbone containing branches of ⁇ -1,3-linked oligoglucosides, which are linked to the backbone through ⁇ -1,6-glycosidic bonds (Sandford).
  • glucopyranose polysaccharides which may find use in the present invention include dextran (poly- ⁇ -1 ,6-glucopyranose), methylcellulose (poly- ⁇ -1,4-glucopyranose), and chitopentose (2-acetamido-2-deoxy- ⁇ -1,4-glucopyranose).
  • Dextran may be obtained in a variety of molecular weight ranges and is readily cleared and completely metabolized in humans by dextranases (Jeanes).
  • compositions and methods of the present invention utilize soluble ⁇ -glucans.
  • Soluble glucans are those which are soluble under physiological conditions. Such glucans are particularly preferred for compositions of the present invention intended for therapeutic use. Soluble glucans may be suitable for cellular internalization and clearance through the kidney, since they remain soluble at physiological pH's.
  • Soluble ⁇ -glucans may be either non-functionalized or functionalized. Nonfunctionalized glucans may be obtained by a number of routes.
  • Non-functionalized, water soluble oligomers of ⁇ -glucan from E. gracilis typically contain fewer than 25 monomer units (DP ⁇ 25).
  • Soluble ⁇ -glucans may be obtained by a variety of methods, including the preparation of soluble oligomers by acid-catalyzed cleavage of particulate ⁇ -1 ,3-glucans.
  • One such method of acid-catalyzed cleavage is acetolysis.
  • the glucan is treated with acetic anhydride in concentrated sulfuric acid, followed by alkaline hydrolysis or transesterification of the peracetylated product (Wang).
  • the peracetylated soluble oligomer may also be separated by chromatography or silica gel, and derivatised prior to deacetylation.
  • partial hydrolysis of the glucan may be carried out in 0.5 N hydrochloric acid in dimethyl sulfoxide (Kamo). These methods are somewhat preferable to formic acid hydrolysis (Wang) for large-scale applications.
  • Non-derivatized water soluble oligomers may be fractionated by precipitation from dimethyl sulfoxide with ethanol (Ogawa) or acetone
  • Soluble ⁇ -glucans may also be obtained by derivatization of insoluble glucans with polar side groups, followed by fractionation into varying molecular size ranges.
  • Suitable starting materials for functionalization include particulate glucans as described in Section A above.
  • a variety of soluble glucans may be prepared by varying the stoichiometry of the reagents used for functionalization or by the use of protecting groups to selectively limit the extent of functionalization. For a review of the use of protecting groups in organic synthesis, see Greene.
  • a number of polar side groups may be introduced into the glucan starting material, such as 2-hydroxyethyl, 2,3-dihydroxypropyl, carboxymethyl, depending on the choice of derivatizing agent (Ukai; Seljelid;
  • polar side chains containing hydroxy (Kishida) or carboxy groups (Ohno) are preferred.
  • Representative conversions of particulate glucans to soluble derivatives by functionalization is illustrated in Figs. 6A-E.
  • introduction of glyceryl (2,3-dihydroxypropyl) side chains may be carried out by reacting the glucan starting material 25 in aqueous alkali with epichlorohydrin 49 to form the glucan-epoxypropane intermediate 51 , followed by alkaline hydrolysis, to form the corresponding glyceryl-glucan, 53.
  • This material is referred to as g-glucan.
  • the reactive intermediate epoxy-functionalized glucan, 51 may also be used to introduce other functional groups into the glucan backbone.
  • carboxy groups may be introduced into the glucan molecule to provide soluble glucan derivatives.
  • Carboxymethylation is typically carried out by treating the glucan with chloroacetic acid in aqueous sodium hydroxide, to form water-soluble carboxylated glucan derivatives (59, Fig. 6B).
  • the carboxylated glucan may also be used for drug conjugation through the carboxyl group, to provide glucan/active agents possessing a carbonyl-containing hydrolyzable attachment.
  • Any suitable method may be used to estimate the extent of carboxyl group substitution.
  • One such method is colorimetric analysis using 2,7-dihydroxynaphthalene in sulfuric acid (Ohno).
  • Glucan concentrations in solution may be determined using the anthrone method (Scott).
  • Example 6 An exemplary method for providing soluble derivatives of ⁇ -glucans is provided in Example 6.
  • native algal glucan from Euglena is alkylated with 1-chloro-2-hydroxyethane in aqueous alkali, followed by size-exclusion fractionation and sterilization, to give soluble, pyrogen-free hydroxyethylated glucan of various molecular weight ranges.
  • This methodology dissociates the glucan polysaccharide chains to provide water-soluble material having a lower molecular weight than the native, parent material.
  • Three soluble, derivatized glucan fractions are isolated from the above process for use in the invention.
  • One is a lower molecular weight hydroxyethylated material with a molecular weight less than 5000 daltons.
  • a second medium MW fraction of MW 10,000 to 18,000 daltons is referred to as HE-glucan-SM.
  • the third fractionated glucan material is also a soluble hydroxyethylated material, having a higher molecular weight range from about 20,000 to 100,000 daltons, and is referred to herein as
  • Glucan sources for use in the present invention should be pyrogenfree.
  • One such method for ensuring that the soluble glucan samples are free of pyrogens is to pass the soluble glucan fractions through a "DETO-XI-GEL" pyrogen-chelating column (Pierce, Rockford, IL) to remove possible pyrogens.
  • the glucan fractions are analyzed by the USP (United States Pharmacopia, 1990) rabbit pyrogen test.
  • glucans for use in the present invention are typically functionalized with reactive groups that promote coupling of the glucan backbone to the bioactive agent. Functionalization may be carried out on either particulate or soluble sources of ⁇ -glucan.
  • glucan hydroxyl groups are used to promote coupling.
  • new functional groups are introduced onto the glucan to provide a different chemical site for attachment to the active agent.
  • the choice of functional group introduction depends on a number of factors, including the functional group(s) present in the bioactive agent and the desired covalent attachment.
  • Figs. 6A-E Various synthetic approaches for forming functionalized glucans are shown in Figs. 6A-E.
  • an aldehyde-derivatized glucan, 35 may be conjugated to drugs containing hydrazide groups, or to drugs possessing functionalities which are readily converted to hydrazide.
  • the coupling of an aldehyde-functionalized glucan with a hydrazide-containing drug results in the reversible formation of a glucan/agent complex possessing an acyl hydrazone covalent attachment.
  • Particulate glucan may be similarly functionalized by reaction with periodate after introduction of a low level of 2,3-dihydroxypropyl side chains (or any suitable hydroxy-containing side chain).
  • the dihydroxypropyl side chains introduced into the glucan backbone provide sites for periodate-promoted oxidation.
  • Aldehyde group loading onto a soluble glucan may be estimated using 2,4-dinitrophenylhydrazine (Nathan) or hydroxyjamine hydrochloride (Zhao) to indicate the number of potential sites for drug attachment.
  • Nathan 2,4-dinitrophenylhydrazine
  • Zhao hydroxyjamine hydrochloride
  • optimal release of the bioactive agent from the glucan/agent complex may require a spacer between the glucan backbone and aldehyde groups intended for attachment to the active agent.
  • the introduced aldehyde function separated from the glucan backbone by a spacer, is more accessible to incoming bioactive agents intended for attachment than carbonyl groups contained directly on the glucopyranose rings.
  • An exemplary functionalized glucan containing a spacer between the glucan and the aldehyde sites for drug attachment is shown in Figs. 3A, 3B and Figs. 6D, 6E.
  • the glucan is first activated by reaction with 1 ,1 '-dicarbonyldiimidazole 27 to form the carbonyldiimidazole-activated glucan 29 (Figs. 3A, 3B and 6D).
  • the carbonyldiimidazoleactivated glucan is then treated with an aminoacetal, such as the dimethyl 67 or diethyl 31 acetal of 4-aminobutanal to form the corresponding O-carboxamidobutyraldehyde acetal glucan intermediate 33.
  • the protected glucan is hydrolyzed under mild conditions (0.05 N HCl) prior to coupling with a bioactive agent to form the desired aldehyde-functionalized glucan (e.g., 35 in Figs. 3A, 3B and 6D).
  • a bioactive agent e.g. 35 in Figs. 3A, 3B and 6D.
  • hydrazide-derivatized glucan 61 may be prepared by coupling functionalized carboxymethyl glucan 59 to aqueous hydrazine with a water-soluble carbodiimide (i.e., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EDC) (Hurwitz) or by phase-transfer alkylation (Mann) of glucan with ethyl acrylate followed by treatment of the ester product with hydrazine.
  • EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • Mann phase-transfer alkylation
  • the extent of hydrazide group introduction may be estimated by elemental analysis (Hurwitz) or by reaction with trinitrobenzenesulfonic acid (Miron).
  • a tritium-labeled glucan is shown in Fig. 6C.
  • glyceryl-glucan 53 is oxidized with periodate to the corresponding aldehyde-derivatized glucan intermediate 63, followed by treatment with tritium-labeled sodium borohydride to form 2-hydroxy-2- 3 H-ethylglucan 65.
  • Amine functionalized glucans may be prepared as follows (Fig. 6A). Soluble glucan 25 is first prepared by reaction with epichlorohydrin 49 in aqueous sodium hydroxide to form the epoxy-functionalized glucan derivative, 51. The epoxy group content of epoxy-functionalized glucans may be determined by the method of Sundberg. The epoxy-glucan is then treated with a suitable diamine, such as 1 ,3-diaminopropane, to form the amino-functionalized glucan 57. Unreacted epoxide groups present in the amino-derivatized glucan may be removed by hydrolysis in base.
  • a suitable diamine such as 1 ,3-diaminopropane
  • Particulate glucan may be aminated by derivatization with 4-fluorobenzenesulfonyl chloride (Chang) or with carbonyldiimidazole, followed by treatment with a suitable amine to form aminated glucans, as illustrated in Fig. 6E, 71 and 73.
  • the derivatized glucan, 73 contains a carbamate function linking the terminal amino-containing group to the glucan backbone, and therefore contains fewer basic amino groups than does the amino-functionalized glucan, 71.
  • An appropriate method for determining the extent of -NH 2 loading onto particulate glucans is the trinitrobenzenesulfonic acid difference estimation method (McKelvey).
  • the number of drug attachment sites contained within the functionalized glucan may be altered by chemical blocking methods. For instance, excess amine groups in the amino-functionalized glucan may be blocked by acetylation, using either N-acetylimidazole or N-succinimidyl acetate.
  • Suitable organic solvent-soluble glucan derivatives include the 6-O-trityl ether, 6-O-tosylate, and phenylboronate derivatives. Tritylation by reaction with trityl chloride in pyridine may be used to solubilize the starting glucan by introduction of trityl groups at the 6-position. Selective transformations may then be carried out at the non-protected hydroxy sites within the glucan backbone, such as the 2- and 4-hydroxy functions.
  • oxidation of the reactive hydroxyl groups with acetic anhydride in dimethyl sulfoxide may be used to form the corresponding keto-containing glucan.
  • partial tritylation of the starting glucan may be employed to block some of the reactive hydroxyl groups, resulting in lower levels of functional group introduction into the glucan backbone. The trityl group is removed from the glucan prior to attachment to the bioactive agent by acid hydrolysis.
  • the tosylate of primary hydroxyl groups typically undergoes nucleophilic displacement reactions and may be further derivatized.
  • reaction of tosylated glucan with azide, followed by reduction, for example, by hydrogenation, may be utilized to form the corresponding amino-functionalized glucan.
  • Phenylboronate derivatives may be formed by reaction with phenylboronic acid (de Belder).
  • Reactive carbamate-functionalized glucans (Bethell 1981 a, 1981 b), such as the corresponding imidazole or 1 ,2,4-triazole-I-carboxylic acid esters, may be produced by reaction with carbonyl diimidazole or ditriazole.
  • the activated carbamates are suitable for further functionalization with amines, either in organic solvents or aqueous buffer, to form the hydrolytically stable N-alkylcarbamate-containing glucans.
  • Conversion of soluble glucan starting materials to amino-functionalized derivatives may also be accomplished by reductive amination, which is specific for the reducing terminus (Gray), to form the corresponding primary amino substituents, or by reductive amination with benzylamine, followed by N-debenzylation and by hydrogenlysis (Yoshida).
  • the derivatized glucans may be coupled to various bioactive agents, depending upon the nature of the terminal reactive groups on the derivatized glucan and the nature of reactive functional groups in the bioactive molecule intended for coupling.
  • Bioactive agents may also be derivatized to introduce functionality appropriate for coupling to the glucan.
  • aldehyde-containing glucans As demonstrated in Figs. 3A and 3B, aldehyde-containing glucans
  • hydrazide-containing molecules such as 45
  • hydrazide-functionalized glucan (as shown in Fig. 6B) may be coupled to aldehyde-containing bioactive agents via hydrazone linkages.
  • Amino-functionalized glucans may be coupled to carboxyl-containing bioactive agents by means of amidelinkages.
  • Glucan hydroxyl-groups may also be utilized as sites of attachment for carbonyl-containing bioactive agents, such as drugs containing either aldehyde or keto-functions. Coupling to the glucan hydroxy groups provides the acetal and ketal-linked glucan-bioactive agent complexes, respectively.
  • Thiolated glucan may be coupled to thiol-containing bioactive agents by means of disulfide bonds.
  • Glucan hydroxy groups may also be used to attach a carboxyl-containing drug via an ester linkage.
  • Preferred glucans for use in the present invention should ideally possess very low toxicities. Acute toxicity is typically evaluated as described in Example 3.
  • glucans intended for use in the invention are administered to healthy mice, usually by ip injection.
  • the dose size is typically varied, and treatments may include one single dose administra tion of glucan over the course of the study, or, alternatively, multiple doses of glucan may be administered over the course of several days.
  • the general health of the mice is observed and the mice are weighed on consecutive days. Some of the visual signs of poor health include an ungroomed coat and lethargy. The resulting characteristics of the glucan-treated mice are compared to a control group, and the general toxicity of the glucan determined.
  • Example 2 show the ability of ⁇ -glucans to protect animals from Listeria challenge.
  • experiments 1 and 2 the mice were challenged with 1 LD 50 of Listeria.
  • the survival of mice treated with various doses of ⁇ -glucans was significantly greater than the survival of saline treated mice.
  • experiment 3 mice were challenged with 6 LD 50 of Listeria to examine the resistance of mice to a large infectious challenge.
  • an additional group of mice was injected with a cellulose preparation which is similar in size to the ⁇ -glucan particles, but is composed of glucose molecules linked in a ⁇ -1,4 configuration.
  • mice treated with three doses of 0.5 mg of ⁇ -glucans prior to challenge had a survival rate of 70%, whereas none of the mice treated with saline survived. Furthermore, all of the animals treated with the cellulose preparation died, indicating that the protection afforded by ⁇ -glucans is not solely related to its particulate nature and that ⁇ -glucans enhanced the ability of mice to resist the Listeria challenge.
  • Enhanced protection may not be due exclusively to the direct effects of ⁇ -glucans on macrophage, because resistance to Listeria requires the development of cell-mediated immunity (CMI), which in turn activates macrophages to kill intracellular parasites (Mackaness; Krahen- buhl and Remington).
  • CMI cell-mediated immunity
  • Mackaness Krahen- buhl and Remington.
  • Example 4 show the ability of ⁇ -glucans to protect animals from tumor challenge.
  • the ability of ⁇ -glucans to modulate the survival of tumor bearing mice was examined in a syngeneic tumor system. Groups of mice were injected with melanoma cells on day 0 and injected iv with various doses of ⁇ -glucans starting on day 1. The survival of mice in each group was compared. Mice treated with five doses of 0.25 mg of ⁇ -glucans had a statistically significant increase in mean survival time compared with mice treated with saline (p ⁇ 0.05). Animals treated with 5 doses of 0.125 mg ⁇ -glucans also demonstrated an increase in survival time. These results indicate that administration of ⁇ -glucans resulted in increased survival of tumor-bearing mice.
  • Example 5 The data presented in Example 5 show that ⁇ -glucans induce colony-stimulating activity in vivo.
  • sera were collected from mice and assayed for bone marrow colony-stimulating activity (CSA).
  • CSA bone marrow colony-stimulating activity
  • LPS lipopolysaccharide
  • CSA was detectable in the sera of mice as early as 2 hours after a single injection of 2.5 or 5 mg of ⁇ -glucans. Elevated serum CSA activity was detectable for at least 24 hours. These results are consistent with the ability of ⁇ -glucans to enhance production of granulocytes and monocytes/macrophages.
  • IL-1 The generation of IL-1 by peritoneal macrophages stimulated with ⁇ -glucans in vitro suggests that intravenous injection of ⁇ -glucans may induce a pyrogenic response.
  • administration of large doses of ⁇ -glucans administered iv to rabbits did not induce immediate febrile response.
  • the potential conflict between the results of the pyrogenicity tests and in vitro IL-1 production by macrophage may be due to difference in the dose of ⁇ -glucans delivered to macrophages in vivo as compared to in vitro. Alternately, the kinetics of IL-1 production induced by ⁇ -glucans in vivo may differ from that observed in vitro.
  • Example 8 demonstrate the ability of ⁇ glucans to stimulate the release of cytokines (including IL-1 ⁇ , TNF- ⁇ and IL-6) from human monocytes.
  • cytokines including IL-1 ⁇ , TNF- ⁇ and IL-6
  • the ability of ⁇ -glucans to induce cytokine production was found to be dose-dependent.
  • the induction of IL-1 release may be beneficial for the use of ⁇ -glucans as vaccine adjuvants since administration of recombinant IL-1 is known to enhance primary and secondary responses to T-dependent antigens and primary responses to a T-independent antigen (Nencioni, et al., 1987).
  • Example 7 Data presented in Example 7 demonstrate that soluble ⁇ -glucans, such as a hydroxyethylated glucan, can bind to and be internalized by both mouse and human macrophages. The binding and uptake of ⁇ -glucans appears to be mediated through a receptor binding mechanism.
  • ⁇ -glucans The ability of ⁇ -glucans to perform as adjuvants in enhancing the immune response of animals was examined as follows.
  • the adjuvant performance of ⁇ -glucans was compared to a low dose of keyhole limpet hemocyanin (KLH), T-cell dependent antigen.
  • KLH keyhole limpet hemocyanin
  • Five mice per group were immunized ip with 1 ⁇ g of KLH delivered in saline, 0.2 ml of CFA, or saline containing various doses of ⁇ -glucans. Animals received a second immunization of 1 ⁇ g of KLH in saline ip on day 21. Serum was collected on various days and assayed for anti-KLH antibodies by ELISA.
  • mice immunized with KLH mixed with ⁇ -glucans demonstrated consistently higher anti-KLH antibody titers than animal immunized with KLH alone.
  • the primary and secondary responses of the KLH/ ⁇ -glucan immunized animals were proportional to the amount of ⁇ -glucan co-injected with the KLH.
  • ⁇ -glucan-N, ⁇ -glucan-D, and ⁇ -glucan-S are used for synthesis of the conjugated antigens.
  • ⁇ -glucan-N and ⁇ -glucan-D are linear, 400,000-molecular-weight ⁇ -glucans that have phosphate groups at approximately every tenth residue.
  • ⁇ -glucan-S is a soluble ⁇ -glucan.
  • phosphate ester groups are employed (Figs. 7A,7B and Figs. 8A-C).
  • This coupling strategy is amenable to (i) high- and low-molecular weight ⁇ -glucans, (ii) particulate and solubilized ⁇ -glucans, and (iii) native and functionalized derivatives of ⁇ -glucans.
  • the phosphate ester groups mimic the natural phosphates on the ⁇ -glucans, and the hydrophobic groups generally should have a weaker antigenic response than hydrophilic groups.
  • the HSV antigens selected for study were HSV-1 glycoprotein D (gD) (Weijer, et al.) and gD peptide fragment 1-23, which is an antigenic determinant capable of inducing HSV-neutralizing antibodies.
  • the HIV antigens are glycoproteins gp1 60 and gp1 20, and peptide fragment 584-609 (SEQ ID NO: 1 , RILAVERYLKDQQLLGLWGCSGKLIC) (Schrier, et al., 1988).
  • conjugation chemistry is useful for linkage to both water-soluble and insoluble ⁇ -glucans.
  • One linkage methodology that can be used to attach peptide gD 1-23 to its tether group is adapted from the method used to couple the synthetic peptide corresponding to residues 1-13 of the gD polypeptide to protein carriers for immunization studies (Cohen, et al., 1984).
  • Peptide CG1-23 (SEQ ID NO:2, CGKYALADASLKMADPNRFRGKDLP) is prepared by the route that was used to prepare peptide CG1-13 (Cohen, et al., 1984).
  • gD peptide 1-23 is extended on its amine terminus by glycine and cysteine residues (peptide CG1-23). Attachment to the protein was effected by the addition of the cysteinyl sulfhydryl group of the modified peptide to the maleimide groups on the protein.
  • the maleimide groups were introduced by reaction of m-maleimido-benzoyl-N-hydroxysuccinimide ester (Klaus and Humphrey) with the e-amino groups of the lysine residues.
  • the introduction of the phosphate esters onto ⁇ -glucan employs reaction of the anhydrous polysaccharides with phosphoramidite reagents such as 2 (Fig. 7A; Coull, et al., 1986), followed by oxidation with iodine and water.
  • the protecting groups are removed and afford the 6-aminohexyl phosphate esters of ⁇ -glucan 5 (Fig. 7A).
  • reaction of ⁇ -glucan with phosphate 3 in the presence of the water-soluble carbodiimide EDC Leclerc and Vogel, 1986; Smrt, 1979
  • removal of the protecting groups also affords 5 (Fig. 7A).
  • FIG. 7A Another strategy for preparing 8 (Fig. 7A, bottom scheme) is to begin with phosphorylation of ⁇ -glucan using polyphosphoric acid, PPA (Suzuki, et al., 1977).
  • PPA polyphosphoric acid
  • the resulting glucan polyphosphate 11 is coupled to the maleimide-alcohol 10 (Fig. 7A) with EDC.
  • Reaction with gD peptide CG1-23 produces 8.
  • An alternative to these loading methods is the derivatization of ⁇ -glucan by carboxymethylation (Winter and Beckmann, 1956), as illustrated in Fig. 7B.
  • the amino group functionality is then introduced by coupling with 1 ,6-diaminohexane (Kasel, et al., 1971 ), giving 14 (Fig. 7B).
  • Reaction with N-hydroxysuccinimide ester 6 and gD peptide CG1-23 affords 15
  • Loading values of a selected antigen onto ⁇ -glucan is established by ultraviolet spectroscopy and elemental analyses (C/N ratios). Phosphate incorporation is determined by the method of Ames (1966). Adducts are purified by gel-filtration and ion-exchange chromatography.
  • ⁇ -glucan 1 is phosphorylated using phosphoramidite chemistry with reagent 16 (Fig. 8A) (Kremsky, et al., 1987). Iodine oxidation followed by hydrolysis give 5-carboxypentyl phosphate 17. Coupling of the carboxyl groups with the amino groups of the lysine residues of gD affords 19 (Fig. 8A).
  • an ether linkage can be used in place of the phosphate diester group to provide adduct 21.
  • Another method of linkage involves modification of the carbohydrate residues on gD, as illustrated in Fig. 8C. Oxidation of gD 18 with periodate introduces aldehyde groups to form oxidized gD intermediate 22
  • Similar synthetic methodology can be employed to prepare conjugates of the HIV antigens to ⁇ -glucan. Because HIV peptide 584-609 has two cysteine residues, the sulfhydryl group of C-603 can be protected as a acetamidomethyl thioether, whereas C-609 can be blocked as the methoxybenzyl thioether. The latter protecting group is preferentially cleaved by treatment with hydrogen fluoride, permitting selective reaction of the sulfhydryl of C-609 with the maleimido adduct formed from reaction of 5 and 6 (Fig. 7A).
  • the acetamidomethyl thioether protecting group on C-603 can then be removed by treatment with Hg(OAc) 2 at pH 4. If the free sulfhydryl group of C-603 proves to be unstable giving peptide dimers that do not produce the desired antigenic effect, it can be replaced by hydroxyl or converted to the methyl thioether. In the event that antigenicity is enhanced by formation of a loop structure caused by disulfide bond formation between C-603 and C-609, a peptide CG584-609 will be prepared where both sulfhydryl groups of C-603 and C-609 are protected by acetamidomethyl groups. Reaction with the maleimido adduct of 5 with 6, followed by removal of the protecting groups, and oxidation affords the desired conjugate bearing a loop structure.
  • HIV gp160- and gp120- ⁇ -glucan adducts can be prepared by the two routes used for gD illustrated in Figs. 8A and 8B; the third route (Fig. 8C) involving Schiff base formation, followed by reduction with hydride may not be desirable because of the presence of critical disulfide bonds in the glycoproteins. Therefore, an alternate strategy can be employed in which the glycoproteins are oxidized with periodate to introduce aldehyde groups onto their sugar functions. These aldehydes form stable hydrazones with the hydrazides prepared from coupling of [ ⁇ -glucan[OP(O)(O -) (CH 2 ) 5 CO 2 H] with excess hydrazine.
  • Peptides gD GC1-23 and HIV 584-609 are prepared by standard solid-phase techniques (Erickson and Merrifield, 1976) on a Beckman
  • CFA Complete Freund's Adjuvant
  • mice are immunized with various antigen and ⁇ -glucan ( ⁇ -glucan-N, ⁇ -glucan-D, and ⁇ -glucan-S) conjugates to determine their effectiveness.
  • ⁇ -glucan-N, ⁇ -glucan-D, and ⁇ -glucan-S conjugates to determine their effectiveness.
  • the dose is based on the loading efficiency of antigen onto the ⁇ -glucan.
  • HSV gD2-glucan conjugate (prepared as in Example 11) exhibited significantly higher anti-gD2 serum antibody titers than did mice immunized with just a mixture of gD2 and glucan. These results are shown in Fig. 5. The results demonstrate the superior properties of the antigen/ ⁇ -glucan conjugates, for example, the ability of ⁇ -glucan/antigen complexes to enhance the immune responses of experimental animals to antigens.
  • mice are injected up to four times, at two-week intervals, with antigen-glucan conjugates.
  • Control animals receive a primary immunization with antigen and CFA (Complete Freund's Adjuvant, Harlow, et al.), and booster immunizations in IFA (Incomplete Freund's Adjuvant, Harlow, et al.). Seven days after each immunization, groups of 3 mice are evaluated for specific immunity to HSV.
  • CFA Complete Freund's Adjuvant, Harlow, et al.
  • Immunogens prepared by covalently linking gD or peptides to particulate and denatured ⁇ -glucan are tested in a similar manner.
  • Antigen- ⁇ -glucan conjugates that elicit significant immunity to HSV are tested in challenge studies to determine the degree of protection that is conferred. Groups of 15 mice are immunized. Control groups consist of untreated mice and mice injected with ⁇ -glucan only, antigen only, or antigen with alum. After completion of the appropriate immunization schedule, induction of HSV-specific immunity is confirmed in 3 to 5 mice from each group. The remaining mice are challenged by ip injection of HSV-1 (Long, et al., 1984).
  • the challenge dose is the minimum lethal dose of HSV as previously determined in untreated mice. Protection is assessed by comparing the survival of immunized mice and non-immunized mice for approximately 50 days. The conjugates conferring complete protection with this challenge dose are re-evaluated for their ability to confer protection against a higher challenge dose.
  • rabbits (two per group) are immunized with the HIV-1 glycoproteins or synthetic peptide 254-274. Animals receive one im injection every two weeks for a total of three injections. Serum is obtained from each rabbit before immunization and seven days after each injection. For comparison, additional animals are immunized with these antigens absorbed onto alum or CFA. Anti-HIV antibody titers from each serum sample are determined by ELISA. Anti-HIV-1-neutralizing antibodies in each serum sample are evaluated on days 7, 14, 21 , and 28, using the procedures described below. Optimized antigen conjugates are identified by this method. These optimized conjugates are then used as "selected" conjugates for further studies.
  • mice are immunized ip with the "selected" HIV-1 antigen/ ⁇ -glucan conjugates.
  • Control animals are immunized with antigen in alum.
  • immune spleen and lymph node cells from groups of 3 mice are evaluated for their CMI response to HIV-1 antigen, using the procedures described below.
  • the effect of multiple immunizations of the conjugate on the cellular immune responses of mice is also evaluated.
  • Serum from immunized mice are assayed for neutralization of HSV-1 infectivity in vitro, using a 50% plaque reduction assay (Weijer, et al.).
  • Two-fold serial dilutions of heat-inactivated serum is incubated with an equal volume of HSV-1 in DMEM containing 5% FCS. After incubation for 2 hours at 37°C, 100 ⁇ l of this mixture is added, in duplicate, to monolayers of Vero cells. After 1 hour of incubation at 37°C, to allow for virus adsorption, an overlay medium containing 0.5% methylcellulose is added.
  • Anti-HIV-1-neutralizing activity of rabbit sera, after heat-inactivation, is estimated by the surviving tissue-culture infectious doses (TCID) employing two assays, quantitative infectivity assay (Gowda, et al., 1989a) and p24 antigen measurement using commercially available ELISA plates.
  • TID tissue-culture infectious doses
  • Two frozen viral stocks are prepared, HIV-1 isolates B-LAV and DV (Institute Pasteur, Paris, France; and Genelabs, Inc., Redwood City, CA, respectively), for use in these experiments.
  • the virus is expanded by de novo infection of VB cells (CD4-positive T lymphoblastic cell line; Chua, et al., 1984).
  • VB cells CD4-positive T lymphoblastic cell line; Chua, et al., 1984.
  • gp120 is present both as an oligomer on virions and as soluble gp120 monomer; the later may cause artifacts in the infectivity assay if it competes significantly with viral associated gp120 for the neutralizing anti- body. Therefore, the soluble gp120 content of the viral stocks is measured using an HIV radioimmune assay (RIA).
  • RIA HIV radioimmune assay
  • VB cells Prior to assay, VB cells are grown at low concentrations to sustain logarithmic growth. On the day of an assay, twofold dilutions of the serum are incubated with the virus stock diluted in culture medium (RPMI).
  • Target cells 1640 with 10% FCS.
  • 0.5 ml of a dilution of the virus-serum mixture is added to "target" VB cells.
  • target cell densities (1 ⁇ 10 6 to 3.13 ⁇ 10 4 /ml) are assayed.
  • Target cells infected in the absence of serum and non-infected cells serve as controls.
  • culture tubes are rotated during the 2-hour infection period.
  • the infected target cells are washed extensively, pelleted and re-suspended in fresh medium.
  • Cell monolayers are prepared by adding 5 ⁇ 10 3 target cells and 5 ⁇ 10 4 "indicator" (i.e., non-infected VB cells) to each well (targetrindicator ratio of 1 :10).
  • a total of eight wells are plated per serum dilution and target cell concentration.
  • the cultures are maintained for seven days and the mean number of syncytia are counted (Gowda, et al., 1989a).
  • the ratio of syncytia induced by the virus inoculum surviving after virus neutralization (Vn) and the number of syncytia induced by the virus inoculum (Vo) are calculated.
  • the virus-surviving fractions (Vn/Vo) from each two-fold serum dilution is plotted versus the reciprocal of the serum dilutions.
  • the neutralizing titer (the reciprocal of the highest dilutions yielding ⁇ 90% virus inhibition) for each experimental group of animals is compared with the neutralizing titer of the control animals.
  • Lymphocyte Proliferation to Viral Antigens Spleen and inguinal lymph node lymphocytes from immunized mice are suspended at 5 ⁇ 10 6 cells/ml in RPMI 1640 containing 5% FCS, 25 mm HEPES, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin. Then 100- ⁇ l aliquots of these cell suspensions are added to microculture wells containing either (i) 100 ⁇ I of various concentrations of the appropriate antigen (HSV or HIV-1 ), or (ii) virus-infected cells. The use of fixed virus-infected cells as an eliciting antigen in vitro has been described (Welling-Wester, et al., 1985).
  • the antigen-specific release of IL-2 by spleen cells from mice immunized with HSV or HIV-1 antigen conjugates is studied as an indicator of the generation of helper T cells.
  • Spleen cells are cultured at 5 ⁇ 10 6 cells/ml in 1 ml of RPMI containing 5 - 10 -5 M 2-mercaptoethanol, 5% FCS, and various concentrations of the appropriate antigens. After 48 hours, the culture supernatants are collected, centrifuged, and assayed for
  • results are be expressed as units of IL-2 per milliliter based on use of a commercially obtained IL-2 standard.
  • CTL cytotoxic T lymphocyte cells
  • NIH3T3 cells are used as target cells because this cell line shares histocompatibility antigens with Balb/c mice (H-2 k ).
  • Cells (2 ⁇ 10 7 ) are infected with HSV at a multiplicity of infection (MOD of 1 for 60 minutes at 37°C.
  • MOD multiplicity of infection
  • the HSV-infected cells are washed once with media, then incubated with 400 ⁇ Ci Na 2 51 CrO 4 for 30 minutes at 37°C.
  • the 51 Cr-labeled target cells are washed three times and resuspended at 1 ⁇ 10 5 cells/ml in RPMI 1640 containing 10% FCS. Aliquots
  • CTL activity against HIV-1 antigens is monitored using a previously described procedure (Takahashi, et al., 1988). Briefly, 3 to 4 weeks after immunization with HIV-1 glycoproteins or synthetic peptides, spleen cells are restimulated in vitro with syngeneic spleen cells infected with a recombinant vaccinia virus expressing HIV env glycoprotein gp120 and gp1 60 (Gowda, et al., 1989b). After culture for 6 days, cytolytic activity of the restimulated cells is measured, using 51 Cr-labeled vaccinia virus-infected syngeneic spleen cells as targets.
  • CTL cytotoxic T lymphocyte cells
  • the present invention describes the use of drug targeting carriers that are specifically internalized by the macrophage.
  • HIV-targeted therapeutics are described.
  • the monocytes/macrophages are an important cellular reservoir for the HIV-1 virus, the causative agent for
  • anti-HIV agents are applicable to other bioactive agents useful for the treatment of other diseases as well, for example, of pathogens (such as viral, bacterial, fungal, and malarial agents) capable of invading or colonizing phagocytic cells.
  • pathogens such as viral, bacterial, fungal, and malarial agents
  • Phagocytic cells include monocytes and macrophages.
  • the ⁇ -glucans of the present invention undergo receptor-mediated endocytosis by the macrophage and, at higher molecular weights, have immunostimulatory effects on the macrophage. Accordingly, these ⁇ -glucans are ideal for cell-specific targeting and, because of their activating properties, for achieving synergistic therapeutic effects.
  • ⁇ -glucan uptake occurs by specific receptormediated endocytosis ⁇ a process that provides an efficient delivery pathway.
  • the ⁇ -glucans are potent macrophage activators and administration of the ⁇ -glucans appears to enhance cell-mediated immune responses. This enhancement likely occurs by cytokine-mediated cascades initiated by activation. Activation appears to be molecular-weight dependent. Glucans above a specific size (usually above 2.5 glucose residues in length) activate macrophages, a condition that may enhance drug antiviral activity.
  • activated, infected macrophages synthesize virus, but other processes, including endocytosis, are also enhanced, so that cells may be concomitantly more susceptible to antiviral agents.
  • Selection of the appropriate molecular size of the carriers can be evaluated by the methods described below for each ⁇ -glucan/agent conjugate.
  • ⁇ -glucan/agent conjugates are encapsulated during uptake by monocytes/macrophage.
  • the encapsulate is then likely fused with lysosomal vesicles, thereby becoming exposed to the degradative enzymes therein and the more acidic environment.
  • Appropriately designed tether groups for example, hydrolyzable groups, are then cleaved to release the agent from the ⁇ -glucan/agent complex.
  • Phosphate, phosphoramidate hydrazide, and disulfide linkages are exemplary useful tethers.
  • Phosphatases present in lysosomes can cleave the phosphate diester bonds.
  • a phosphate ester linkage at the 5 '-position of 2,3-dideoxycitidine (DDC) can release a pro-drug species of the active DDC triphosphate (Dawson, et al., 1989).
  • the lysosomal pH is sufficiently acidic (pH 5) to effect the release of a tethered agent, where the linkage is, for example, a carbonyl hydrazone group.
  • Disulfide exchange releases toxins from immunoconjugates.
  • Example 6 Preparation of low- (MW ⁇ 5000) and intermediate-molecularweight (MO 10,000 to 18,000) and high-molecular weight (20,000 to 100,000) fractions of soluble ⁇ -glucans is described in Example 6. Briefly, the method consists of alkylation of native ⁇ -glucans with ClCH 2 CH 2 OH in aqueous alkali, followed by size-exclusion fractionation and sterilization, to give soluble, pyrogen-free hydroxyethylated ⁇ -glucans of various molecular weights.
  • Fractions of molecular weight less than 5,000 and 5,000 to 150,000 were fluoresceinated (deBelder and Granoth, 1973) with 5'-[(4,6-dichlorotriazin-2-yl)amino]fluorescein and evaluated against commercially available fluoresceinated dextrans (fl-dextran) (MW 4,000 and 70,000) for uptake in adherent mouse peritoneal macrophages (Example 7).
  • FI-dextran exhibited diffuse homogenous fluorescence in exposed cells, whereas FI- ⁇ -glucan showed a large number of fluorescent aggregates, suggesting different association mechanisms for the two polysaccharides.
  • FI- ⁇ -glucan associated with mouse macrophages more strongly than did FI-dextran, especially in the presence of NaN 3 , which permits receptor binding but prevents internalization.
  • F-I ⁇ -glucan was prevented from binding to its macrophage receptor by excess laminaribiose, a ⁇ -1,3-glucopyranose receptor competitor (Williams, et al., 1988) confirming the identity of this receptor.
  • Both adherent (activated) mouse macrophages and suspended (non-activated) human pro-monocytic cells were incubated with FI- ⁇ -glucan overnight to permit binding and uptake and then exposed to unlabeled ⁇ -glucan to permit displacement of label. There was no displacement of label, indicating that FI- ⁇ -glucan was internalized.
  • Experiments performed with freshly isolated human macrophages yielded similar results. These results demonstrate the specific targeting properties of ⁇ -glucans.
  • DDN dideoxynucleosides
  • DDC dideoxycytosine
  • AZT dideoxycytosine
  • DAI 2',3'-dideoxyinosine
  • Tether functionality for the DDN family is typically introduced onto the 5'-position of the DDNs using a 5'-phosphate ester, prior to glucan coupling.
  • Phosphoramidite chemistry may be used to introduce tether groups on dideoxycytosine, which requires protection of N 4 as the benzoyi group. Dideoxyinosine may be similarly treated, using gentle conditions to avoid depurination.
  • AZT is first converted to the 5'-phosphate (Yoshikawa, and Kato, 1969; Goldman, 1988a; Goldman, 1988b) which is then coupled to the appropriate alcohol using a carbodiimide (Ho, et al., 1981 ).
  • Alkyl groups such as pentamethylene or hexamethylene, can be used as a spacer bridge on the tether. If cleavage using this type of spacer proceeds too slowly, bond lability can be enhanced by incorporation of a hydroxyl group on the ⁇ -carbon adjacent to the phosphate (e.g., -OP(O)(O-)CH 2 CH(OH)-) (Pallesen, et al. , 1987). Hydrolytic susceptibility in the acidic environment of the lysosome may also be enhanced by replacement of the phosphate oxygen by nitrogen [-OP(O)(O-)NHCH 2 -] (Chu, et al., 1983).
  • phosphate e.g., -OP(O)(O-)CH 2 CH(OH)-
  • Hydrolytic susceptibility in the acidic environment of the lysosome may also be enhanced by replacement of the phosphate oxygen by nitrogen [-OP(O)(O-)NHCH 2 -] (Chu
  • Figs. 9A and 9B present exemplary tether syntheses and introduction of tethers onto dideoxynucleosides, respectively.
  • the preparation of reactive tether precursors T1 , T2, and T3 is shown in Fig. 9A.
  • Synthetic approaches for attaching various tether groups e.g., exemplary tethers T1 , T2, T3, and T4 to N4-protected dideoxycytosine is illustrated in Fig. 9B.
  • Each of the tethers possesses a reactive terminal group for glucan coupling.
  • amide e.g.. (PS)-NHCO(CH 2 ) 5 OP(O)(O-)-5'-O-DDN
  • Amide-linked DDN-tether/glucan conjugates can be formed by coupling the amino groups of an amino-derivatized ⁇ -glucan with the carboxyl groups of a tethered DDN using either a water-soluble carbodiimide or, more efficiently, the activated N-hydroxysuccinimide ester of the carboxyl group. Cleavage of the amide bonds may then be effected in vivo by the action of proteases.
  • Hydrazone bonds can be formed by reaction of the formylmethyl or ring ketone groups on the ⁇ -glucan with the carbonyl hydrazide on the tether. This group is readily hydrolyzed in the pH 5 environment of the lysosomes, but relatively stable at physiological pH (Lauzza, et al., 1989).
  • Carbamate-linked DDN-tether/glucan conjugates may. for example, be formed by reaction of the carbonyl imidazolide groups of the ⁇ -glucans with the amines on the tethered DDNs. This group is stable at pH 5-7 (Bethell, et al., 1981 ). Loading of the DDNs on the ⁇ -glucans is established by UV spectroscopy.
  • Anti-Sense Compounds are potentially useful in a variety of applications, for example, as anti-virals. Anti-sense oligodeoxynucleotides and their more nuclease-resistant methylphosphonate and phosphorothioate analogs are reported to inhibit HIV infection in cells (Matsukura, et al., 1989, 1987; Agrawal, et al., 1988; Sarin, et al., 1988). Therapy using anti-sense compounds is limited in one respect by the cost of the anti-sense compounds. Targeting may enhance cellular absorption, thereby reducing the therapeutic concentration required.
  • Phosphorothioate oligonucleotides are more lipophilic than their phosphate analogs and may require coupling to the carrier in organic-aqueous medium rather than aqueous medium. Derivatization of ⁇ -glucans may require partial 6-O-tritylation to achieve sufficient solubility in the reaction medium.
  • the phosphorothioates can be linked to the carriers using disulfide and hydrazone bonds as illustrated in Fig. 1 B). The former has been used to link oligonucleotides and momorcharin to antibodies.
  • phosphorothiate anti-sense oligonucleotides having, for example, an anti-REV HIV sequence can be prepared by automated synthesis using phosphoramidite chemistry and automated introduction of sulfur using the sulfur-transfer reagent 3 H-1,2-benzothiol-3-one-1 ,1-dioxide 75 (Beaucage, et al., 1989). Using this strategy, commercially available, protected phosphoramidite tethers can be employed.
  • a thiol terminus is added by reaction of an amine-terminated S- ⁇ -rev with
  • SPDP N-3-(2-pyridyldithio)propionyloxysuccinimide
  • ⁇ -glucan/polypeptide conjugates Syntheses of exemplary ⁇ -glucan/polypeptide conjugates follow here and is illustrated in Figs. 2A- 2D.
  • Trichosanthin and momorcharin U.S. Patent No. 4, 869,903 may be tethered to ⁇ -glucans using either carbonyl hydrazone and/or disulfide bonds (Figs. 2D, 2B, respectively) to ensure release of the protein within the target cell.
  • Trichosanthin which is not glycosylated, is functionalized with 2-iminothiolane 77 rather than SPDP to maximize protein activity (Lambert, et al., 1985), as shown in Fig. 2A.
  • the glycosylated protein momorcharin is converted to a reactive aldehyde by periodate oxidation and then functionalized by a hydrazone linkage (Fig. 2A). Loading is established using fluoresceinated ⁇ -glucans. (d) Biological Evaluations of ⁇ -Glucan/Agent
  • Conjugates Each conjugate is evaluated for its ability to bind to the surface of the target cell, be transported inside, and release the therapeutic moiety.
  • the ability of the conjugates to control pathogen production, such as HIV production, is compared with that of unbound drug.
  • the characteristic of the conjugates are evaluated as follow: (i) binding of candidates to the macrophage surface, (ii) efficiency of internalization of targeting moieties into macrophage cytoplasm, (iii) effects of ⁇ -glucans on cytokine production and HIV-1 infection (or development of other intracellular pathogen), (iv) anti-HIV activity in de novo infection, and (v) anti-HIV activity of the bioactive agent in chronic infection.
  • Binding of ⁇ -Glucans and Conjugates to Macrophages Binding studies are conducted using the human promonocytic cell line U937, which is maintained in culture and which can be infected with HIV. The studies will be extended to fresh human macro phages. Fluoresceinated ⁇ -glucan conjugates of various molecular sizes are added to cell suspensions and to adherent fractions of human cells. The cells are examined by fluorescence microscopy to determine preliminary binding affinity (Lifson,, et al. , 1986).
  • ⁇ -Glucans and Conjugate Internalization bv Macroohaoes Studies are first conducted using transformed human macrophage cell line U937 and are then extended to normal human macrophages to confirm internalization behavior. Optimum ⁇ -glucan sizes to effect macrophage binding and targeting are determined. ⁇ -glucans can be tested that consist of two or greater repeating monomer units, and typically, five or more repeating monomer units.
  • the DDN conjugates are assayed for the ability to inhibit de novo infection in monocyte/macrophage, whereas the trichosanthin and momorcharin conjugates are evaluated for their ability to inhibit viral production in chronic infection.
  • the antisense phosphorothioates are tested in both assays. Controls consist of free drug, a mixture of drug and ⁇ -glucans, and ⁇ -glucans alone.
  • Anti-HIV-1 activity of the conjugates is determined against HIV-1 isolates (i) HTLV-III Ba-L , a dual tropic isolate recovered from primary cultures of lung tissue from a child having AIDS (Gartner, et al., 1986;
  • Monocyte-enriched populations are prepared from peripheral blood mononuclear leukocytes (PBL) of either HIV-1-seronegative healthy donors (for de novo in vitro-induced chronic infection studies) or donors known to be infected with HIV-1 (for in vivo chronic infection studies) (Ganelig-Meyling, et al., 1980).
  • CD4-positive lymphocytes are separated from PBL of HIV-1 seronegative individuals using a previously described procedure (Gowda, et al. , 1989).
  • CD4-positive cells are activated with PHA (Gowda, et al., 1989).
  • the uninfected CD4-positive lymphoblastoid T-cell line VB (Stein, et al., 1987) can also be employed in the syncytia formation assay.
  • HIV-1 production is assessed by: (1 ) measurement of HIV-1 p24 antigen by ELISA, (2) TCID 50 , and/or (3) syncytia formation.
  • Conjugates demonstrating anti-HIV-1 activity in the above assays are also screened for their ability to inhibit HIV reverse transcriptase (RT) (Smith, et al. , 1987). The presence of HIV-1-specific antigen, viral infectivity, and/or syncytia formation is evidence of infection. RT activity confirms the ability of the virus to replicate.
  • the ability of the conjugates to induce cytokine production in monocyte/macrophage is evaluated as follows. Monocyte/macrophage derived from HIV-1 seronegative individuals are cultured with various concentrations of low- and intermediate-molecular-weight conjugates. Untreated cells, as well as cells treated with lipopolysaccharide (5 ⁇ g/ml), serve as controls. After 24 and 48 hours of stimulation, the levels of GM-CSF, as well as TNF- ⁇ and IL-1 in these cultures is determined by
  • GM-CSGF, M-CSF, and TNF- ⁇ are added to cultures.
  • Monocyte/macrophages isolated from the peripheral blood of HIV-1 seronegative healthy donors are pre-exposed to various concentrations of the DDN or anti-sense conjugates for a minimum of 30 min. Drug-treated cells are then exposed to HIV-1 without washing out the conjugates.
  • Cells are infected with a dose of virus that generally yielded maximum infection of monocyte/macrophages in the experiments described above. At 2 h after incubation with viral inoculum, cells are washed to remove excess virus and cultured in the absence, or continuous presence, of conjugate.
  • Appropriate controls include (1 ) HIV-1-infected cells without con jugate pretreatment, (2) HIV-1-infected cells pre-treated with carrier only, (3) HIV-1-infected cells pre-treated with the unconjugated drug, (4) HIV-1 -infected cells treated with a mixture of carrier and drug, and (5) uninfected cells (cells mock-infected with a cell-free supernatant from uninfected cultures).
  • Cultures are monitored for 28 days by examining (1 ) supernatants for levels of p24 by ELISA, and for infectious virus by RT activity and TCID 50 , (2) cell lysates for cell-associated p24 antigen, (3) monocyte/macrophages for formation of syncytia with VB cells, and (4) immunofluorescence analysis for the proportion of infected cells.
  • (1 ) supernatants for levels of p24 by ELISA, and for infectious virus by RT activity and TCID 50 (2) cell lysates for cell-associated p24 antigen, (3) monocyte/macrophages for formation of syncytia with VB cells, and (4) immunofluorescence analysis for the proportion of infected cells.
  • cell viability is evaluated at various intervals in conjugate-exposed HIV-1-uninfected monocyte/macrophages cultures.
  • Monocyte/macrophages isolated from HIV-1 seropositive patients are treated with the anti-sense or RIP-conjugate, free antisense or RIP, ⁇ -glucans, or a combination of ⁇ -glucans and drug, as described above. Sham-treated cultures serve as controls in these studies. HIV-1 replication in these cultures is measured as described in the previous section.
  • the antiviral activity of the conjugates on in vitro HIV-1-infected monocyte/macrophage is examined.
  • Monocyte/macrophages isolated from PBL of HIV-1 seronegative healthy donors is infected with different HIV-1 isolates, using the acute infection protocol.
  • the efficacy of selected conjugates can be determined using in vitro assays. Simple in vivo studies of the pharmacokinetics and the toxicity of the conjugates demonstrating significant activity are performed in mice. The distribution of radiolabeled conjugates ⁇ 125 l-labeled protein and 14 C-labeled ⁇ -glucans, drug, or ⁇ -glucans ⁇ is determined in Balb/c mice.
  • Pharmacokinetic studies involve determining the clearance rate of ⁇ -glucans or conjugate from blood, as well as distribution volume. Metabolic studies focus on determining stability following in vitro intravenous administration. HPLC analysis indicates whether the labeled drug is found in the size fraction corresponding to the intact conjugate or whether significant quantities of free drug are released into the circulation.
  • mice typically receive an ip injection of conjugate, drug, or ⁇ -glucans in saline, while the control receives saline alone.
  • Animals are monitored visually for toxicity and weighed daily for ten days.
  • conjugates of the present invention can be formulated into the conjugates of the present invention and the therapeutic efficacy of such conjugates tested using appropriate assays.
  • Candidate conjugates useful for therapeutic applications e.g. , in humans or animals
  • Exemplary targets for conjugate drug therapies include anti-viral agents, anti-bacterial agents (including anti-mycobacterial agents), anti-fungal agents, anti-parasite agents, anti-malaria agents and anti-cancer agents.
  • conjugates of the present invention can be used for the treatment of mycobacterial infection, including infection by Mycobacterim avium or Mycobacterium tuberculosis (Example 14).
  • Mycobacterim avium or Mycobacterium tuberculosis
  • One preferred class of compounds for treating mycobacterial infection using the glucan conjugates of the present invention are the diaminoquinazolines.
  • Some drugs useful to generate conjugates for the treatment of mycobacterial infection include the following: isoniazid (Example 13), pyrimethamine, trimethyoprim, trimetrexate, methotrexate, amalcacin and rifampicin.
  • the conjugates of the present invention may have one of three favorable activity profiles leading to an improved therapeutic index: (1 ) higher potency and comparable toxicity, (2) comparable potency and lower toxicity, or (3) both higher potency and lower toxicity. Decreasing toxicity while maintaining potency may permit more extended therapeutic regimens than now possible.
  • mice C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME) or Charles River Laboratories (Willmington, MA). Female BASL/c mice were obtained from the same sources. Euglena gracilis strain z was obtained from the American Type Culture Collection, No. E12716
  • Standard methods for preparing drug conjugates may be found in i) Lindstrom ii) Apelgren; and iii) Kaneko. Standard methodologies for performing various immunological assays, such as ELISA, may be found in Harlow, et al.
  • a 100-ml inoculum of Euglena gracilus strain z was prepared from a culture grown in the dark at 25-27°C, pH 3.5-4.0, with mixing
  • a growth medium having the following composition was employed.
  • the 100-ml inoculum was added to 1 liter of medium in a flask fitted with a stirrer and an aerator.
  • the culture was maintained in the dark at 25-27°C for 72-96 hr.
  • the pH was maintained between 3.0-4.5 and adjusted with sodium hydroxide as necessary.
  • Stirring was maintained at about 175 rpm (120-200 rpm range).
  • the culture was aerated with air and/or pure oxygen at an average flow rate of 1 L/min (0.5-2.3 L/min range) to provide a dissolved oxygen concentration between 8-40 ppm.
  • the filtered cell mass was transferred to a flask and an equal volume of methanol and twice this volume of chloroform were added. This mixture was stirred at ambient temperature for 30-60 min and then vacuum filtered as above. The filtered mass was then taken up in a sufficient volume of 1 N HCl to provide a relatively dilute suspension. This mixture was boiled until no solvent odor was detectable, followed by heating for an additional two hours. The solids were then centrifuged and washed repeatedly with water to provide substantially pure ⁇ -1,3-glucan in the form of a white crystalline solid. This material was stored at 4°C pending use.
  • mice Groups of 10 C57BL/6 mice were injected intravenously (iv) with various doses of pyrogen-free algal glucan from E. gracilis suspended in pyrogen-free saline on Days -3, -2, and -1 .
  • On Day 0 all mice were challenged intraperitoneally (ip) with 1-6 LD 50 of viable Listeria monocytogenes and mortality was monitored for 40 days.
  • the mice were challenged with 1 LD 50 of Listeria.
  • mice were challenged with 6 LD 50 to examine the resistance of mice to large infectious challenge.
  • mice To control for the particulate nature of algal glucan, one additional group of mice was injected with a cellulose preparation ("AVICEL", FMC Corporation, Rockland, MD), similar in size to algal glucan particles but composed of glucose molecules linked in a ⁇ -1 ,4 configuration. The results are shown in Table 3.
  • AVICEL FMC Corporation, Rockland, MD
  • mice C57B1 /6 male mice (Jackson Laboratories or Charles River Laboratories) received a single ip injection of varying doses of the glucan. Control mice received an ip injection of saline. Following glucan injection, all animals were visually monitored for signs of poor health (ungroomed coat, lethargy) and weighed on 10 consecutive days. Injections of up to 2.5 g/kg of algal glucan (approximately 50 mg per mouse) did not adversely impact the health of the animals. The subject mice gained weight at a rate equal to that of control animals and were as physically active as control mice.
  • mice In a second study, suspensions of the ⁇ -1 ,3-glucan were administered intraperitoneally to male mice (Jackson or Charles River Laboratories) in single doses of either 50, 500, or 5000 mg/kg or in doses of 15, 150, or 1500 mg/kg on 8 consecutive days. All mice survived and showed no significant signs of ill health. On day 10, necropsy was performed to determine the effects of the ⁇ -1 ,3-glucan. Minor visceral adhesion was observed macroscopically.
  • ⁇ -1 ,3-glucan from E. gracilis was examined in a syngeneic tumor system using short-term and long-term treatment schedules.
  • mice were injected subcutaneously on the rear flank on day 0 with 1 ⁇ 10 5 B16BL6 melanoma cells. Each group received its respective ⁇ -1 ,3-glucan (in 0.5 ml pyrogen-free saline) or saline treatment on days 1 , 4, 7, 10 and 13 by intravenous injection.
  • mice To evalutate long term treatment ("B" in col 2, Table 4 below) with ⁇ -1 ,3-glucan, groups of ten C57BL/6 mice were injected subcutaneously on the rear flank on day 0 with 1 ⁇ 10 5 B16 melanoma cells. Each group then received its respective ⁇ -1 ,3-glucan or saline treatment on days 1 , 4, 7, 10, 13, 16, 19, 22, 25 and 28 by intravenous injection in 0.5 ml pyrogen-free saline.
  • mice In Vivo Groups of five male mice (Jackson or Charles River Laboratories) were injected intravenously with either 5 mg or 10 mg of ⁇ -1,3 glucan from E. gracilus. One control group of five mice was injected with saline. A second group of mice was injected with lipopolysaccharide (LPS) 25 ⁇ g i.v. to provide a positive control. At various times after injection (2h, 6h, and 24 h), sera were collected from the mice and assayed for bone marrow colony-stimilating activity (CSA).
  • LPS lipopolysaccharide
  • Colony-stimulating activity was detected in the sera of mice as early as 2 hours after a single injection of 2.5 or 5.0 mg of the ⁇ -1,3-glucan. Elevated serum CSA activity was still detectable 24 hours after glucan administration and indicates the enhancement of colony-stimulating activity by administration of glucan.
  • Glycerated glucan (g-glucan) was prepared by treatment of glucan particles with epichlorohydrin and concentrated sodium hydroxide, followed by addition of concentrated sodium hydroxide until the epoxide level was no longer detectable by the thiocyanate method. After neutralization to pH 7 with hydrochloric acid, the salts were removed by dialysis and the g-glucan dried by lyophilization.
  • the reaction mixure was purified by chromatographic passage through a G-25 Sephadex column, using phosphate-buffered saline as the eluent. The void volume was collected and concentrated though a 30,000 molecular weight cutoff membrane. The concentrate was then re-chromatographed and concentrated to a mass of 1 .12 grams. The isolated material was filtered through a 0.45- ⁇ filter and washed with phosphate-buffered saline. The aqueous product solution (1 .36 g) was determined to be 3.39 ⁇ 10 -4 M in fluorescein by ultraviolet spectroscopy (493 nm) in 25 mM sodium borate buffer, pH 9.
  • Particulate glucan derived from Euglena (as in Example 1 ) was dissolved in 5 N sodium hydroxide, followed by treatment with chloroethanol to form soluble derivatives of the linear glucan starting material.
  • the resulting solution was neutralized with dilute HCl, followed by exhaustive dialysis against filtered water using a 3,000- to 5,000-D cutoff tubing.
  • the soluble derivatized glucan fractions having varying molecular weight ranges were separated by column gel permeation chromatography (GPC). The molecular weights of each of the collected fractions were determined by HPLC GPC.
  • glucan-SH soluble, high molecular weight, ⁇ 1 50,000 D
  • glucan-SM soluble, medium weight, ⁇ 18,000 D
  • glucan-SL soluble, low weight material, 3,000 D.
  • Each fraction was passed through a Pierce Detoxi-Gel pyrogen-chelating column prior to use to ensure that the samples remained pyrogen-free.
  • D Preparation of fluoresceinated he-glucan
  • Fluoresceinated hydroxyethylated glucan (fl-he glucan) was prepared in an analogous fashion to Example 6B above.
  • native particulate glucan binds to the surface of macrophage cells. Nearly 80 percent of the mouse macrophage cells contained at least two-surface associated glucan particles.
  • Uptake of hydroxyethylated glucan was determined by fluorescence microscopy. Hydroxyethylated glucan fractions with molecular weights less than 5000 and between 5,000-150,000 were fluoresceinated with 5'-[(4,6-dichloro-triazin-2-yl)aminolfluorescein (fl-he glucan) as in Example 6D above. The fluoresceinated glucan fractions were evaluated against commercially available fluoresceinated dextrans (fl-dextran, MW 4,000 and 70,000) for uptake in adherent mouse peritoneal macrophages. Exposed cells were observed by fluorescence microscopy.
  • FI-dextran exhibited diffuse homogeneous fluorescence in exposed cells, whereas fl-HE glucan showed numerous fluorescent aggregates.
  • FI-he glucan also bound to the surface of a nonactivated human promonocytic cell line U937 in the presence of azide.
  • fl-he glucan was observed to associate more strongly with mouse macrophages than fl-dextran, particularly in the presence of sodium azide
  • ⁇ -1,3-glucan The ability of ⁇ -1,3-glucan to stimulate the release of cytokines from human monocytes was examined.
  • a four-step discontinuous "PERCOLL” gradient was used (Marcowicz), followed by plastic adhesion to obtain monocytes from peripheral blood of healthy donors.
  • Monocytes (1 ⁇ 10 6 ), suspended in 1 ml of RPMI (Roswell Park Memorial Institute, Roswell Park) 1640 medium containing 1 % "NUTRIDOMA” HU (HANA Biologies, Inc., Alameda, CA) and 1 % fetal calf serum were cultured for 4 to 24 hr in the presence of ⁇ -1 ,3-glucan (50 ⁇ g/ml). Control cells were kept in medium alone for the duration of culture. The levels of TNF ⁇ , IL-1 ⁇ , and IL-6 in the culture supernatants were measured by ELISA (R&D System, Minneapolis, MN).
  • the glucan preparation contained 2 ⁇ 10 -5 ⁇ g endotoxin/100 ⁇ g, as determined by the Limulus amebocyte lysate test (BioWhittaker, Walkersville, MD). In these studies, LPS concentrations less than 1 ⁇ 10 -4 ⁇ g/culture failed to increase cytokine production over background levels.
  • TNF ⁇ and IL-1 ⁇ activity were detected in monocyte supernatants as early as 4 hr after stimulation with particulate ⁇ -1 ,3-glucan and peaked by 8 hr.
  • the ability of the glucan to induce cytokine production was found to be dose dependent.
  • HSV-1 gD2 herpes simplex virus glycoprotein
  • Control mice received HSV gD2 alone.
  • Anti-gD2 antibody titers were measured by ELISA 7 days after the third immunization.
  • the glucan preparation used contained 0.158 Endotoxin Units/100 ⁇ g, as determined by the chromogenic Lim ⁇ l ⁇ s Amebocyte Lysate assay (BioWhittaker, Walkersville, MD) (non-pyrogenic concentrations; USP rabbit test).
  • Anti-gD2 antibody titer results are shown in Table 9.
  • the optimum dose of particulate ⁇ -1 ,3-glucan required to enhance the antibody responsiveness of BALB/c mice to gD2 was determined to be 100 ⁇ g. This optimum dosage was utilized in subsequent experiments.
  • BALB/c mice were immunized with three injections, 14 days apart, of 5 ⁇ g HSV gD2 alone, mixed with: (1 ) 100 ⁇ g of particulate ⁇ -1 ,3-glucan (as above), (2) 1000 ⁇ g of soluble high molecular weight glucan, (3) 1000 ⁇ g of soluble medium molecular weight glucan, or (4) adsorbed onto 200 ⁇ g alum. The mice were boosted twice with an equivalent dose of the antigen formulations.
  • Anti-gD2 titers were measured by ELISA 14 days after the first and second injections and 7 days after the third immunization. Anti-gD2 antibody titer results are shown in Table 10.
  • Particulate glucan was as effective as alum in inducing a strong anamnestic response to recombinant gD2. Further, the anti-gD2 antibody titer was 100-1000 fold greater in approximately half of the animals immunized by coadministration of gD2 with soluble glucan compared to that in mice immunized with gD2 alone.
  • Pasteurella-free, New Zealand white, female rabbits each weighing about 2 kg and between 8-9 weeks of age, (Western Oregon Rabbit Company, Philomath, OR) were immunized with 1 mg of an HIV-1 envelope-encoded synthetic peptide (amino acid residues 254-274, having a sequence homologous to the conserved domain of HIV-1 , gp 120) (Ho, et al.) conjugated to bovine serum albumin, BSA, (0.4 mg peptide/0.6 mg BSA) mixed with 5 mg of particulate ⁇ -1,3-glucan per kilogram body weight on days 0, 14, and 28. Control rabbits were injected with HIV-1 peptide/BSA conjugate alone.
  • HIV-1 envelope-encoded synthetic peptide amino acid residues 254-274, having a sequence homologous to the conserved domain of HIV-1 , gp 120
  • BSA bovine serum albumin
  • Control rabbits were injected with HIV-1 peptide/BSA conjugate alone.
  • Serum samples were collected 2 weeks after the last injection and the antibody titers were determined by ELISA using HIV-1 peptide conjugated to KLH as immobilized antigen. Reciprocal of serum dilution: 1250 (HIV-1 synthetic peptide/BSA + glucan); 250 (HIV-1 synthetic peptide/BSA control).
  • ⁇ -1 ,3-glucan was found to enhance the humoral response in rabbits as well as in mice.
  • Rabbits immunized by coadministration of glucan and an HIV-1 envelope-encoded synthetic peptide conjugated to BSA produced specific antibodies that were higher in titers than the response exhibited by rabbits injected with the immunogen alone.
  • Laun. ⁇ -1 ,3-glucan from E. gracilus (200 mg) 25 was activated with 1 ,1 -carbonyldiimidazole 27 (200 mg) in dioxane (10 ml) by stirring for 30 minutes to form the correspondponding imidazolyl carbamate derivative 29.
  • the dioxane-washed, activated glucan 29 was then treated with a 1 .3 M 4-aminobutyraldehye diethyl acetal solution 31 (0.96 ml in 0.7 M aqueous Na 2 CO 3 ) and allowed to stir overnight at pH 10.2.
  • gD2 Functionalization of gD2 to form the hydrazide derivative was carried out as follows.
  • Herpes simplex virus glycoprotein 37 (gD2, 3.8 mg) was thiolated by treatment with 4 mM iminothiolane 39 in 1.9 ml of HEPES buffer for 2 h under argon. The buffer was replaced by 100 mM
  • the gD2 hydrazide 45 was coupled to the O-carboxamidobutyraldehyde derivatized glucan 35 as follows.
  • the gD2 hydrazide 45 (2.8 mg) was added to a suspension containing the derivatized glucan 35 (10 mg, in 1.4 ml of sodium acetate buffer, pH 5.5) and the reaction stirred at ambient temperature. Conjugate formation was then monitored. Aliquots of the reaction mixture were removed, the particles were washed with PBS (pH 7.2) to remove derivatized protein, and the protein and glucan content of the particles determined by the bicinchoninic acid method
  • glucan conjugate particles 47 were harvested by exhaustive washing in PBS at pH 7.2, until protein was no longer detected in the washes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. Protein loadings were determined and ranged from 2.5-8.4 ⁇ g gD2/400 ⁇ g glucan. The above synthetic scheme is outlined in Figs. 3A and 3B.
  • the gD2-glucan conjugate prepared as above was determined to be stable in vitro. During a 14 day storage period at -80°C, less than 0.1 % of the glycoprotein was released, as determined by SDS-PAGE and silver staining techniques.
  • the gD2-glucan conjugate was incubated for 7 h at pH 5.0 and 7.2. Loss of protein was determined by SDS-PAGE and silver staining techniques. Hydrolysis of gD2 from the conjugate was nearly five-fold greater at pH 5.0 than at pH 7.2.
  • the gD2-glucan conjugate adsorbed comparable levels (greater than 90%) of anti-gD2 antibody activity from sera of mice immunized either by coadministration of gD2 and glucan or with the gD2-glucan conjugate. As seen from these experiments, the covalent linking of gD2 to the glucan to form the conjugate did not alter the antigenicity of gD2.
  • mice Female C3H/HeJ mice, 5 per group, were immunized by intraperitoneal injection of 8.4 ⁇ g of the gD2-glucan conjugate (prepared as in Example 11) on days 0 and 14. Control mice received an identical dose of either gD2 (8.4 ⁇ g/dose) alone or mixed with particulate ⁇ -1 ,3-glucan from E. gracilus (100 ⁇ g). The placebo group received 0.2 ml of pyrogen-free phosphate buffered saline. Serum anti-gD2 antibody titers were measured 14 days after the final injection.
  • mice immunized with the gD2-glucan conjugate exhibited significantly higher anti-gD2 serum antibody titers than did mice immunized with a mixture of gD2 and glucan. These results are shown in Fig. 5.
  • a glucan-isoniazid conjugate is prepared as described in Ex. 11.
  • the glucan is activated by treatment with 1 ,1 '-carbonyldiimidazole, followed by reaction with 4-aminobutyraldehye diethyl acetal to form the O-carboxamidobutyraldehyde diethyl acetal-derivatized glucan.
  • isoniazid (4-pyridine carboxylic acid hydrazide)
  • the acetal protecting groups are hydrolyzed to produce the active aldehydecontaining glucan.
  • Treatment of the reactive glucan with isoniazid produces the corresponding glucan-isoniazid complex, containing labile acyl hydrazone linkages which couple the antimycobacterial agent to the glucan.
  • the extent of drug conjugation is determined by Nitrogen Kjeldahl determination and/or acid hydrolysis, followed by HPLC determination of the released isoniazid.
  • mice Ten C57BL/6 mice per group are injected
  • mice intraveneously with three different doses of the glucan-isoniazid complex (prepared as described above) in pyrogen-free saline on days -3, -2, and - 1 .
  • One group of control mice is injected with saline.
  • all mice are challenged intraperitoneally with M. tuberculosis cells (1-6 LD 50 ).
  • the mortality of the mice is monitored for 40 days to examine the ability of mice injected with the glucan-isoniazid complex to resist the M. tuberc ⁇ losis challenge.

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Abstract

Disclosed herein is a glucan composition containing a β-1,3-glucan covalently attached to a bioactive agent. The β-1,3-glucan is attached to the bioactive agent by means of a hydrolyzable covalent linkage to form a glucan/agent complex. Also disclosed are methods relating to the complex of the invention, including a method for the treatment of a pathogen capable of invading or colonizing phagocytic cells, and a method for delivering an antigen to a phagocytic cell.

Description

COVALENTLY BOUND β-GLUCAN CONJUGATES
IN TARGETED DELIVERY
Field of the Invention
The present invention relates to glucan conjugates, particularly β-1,3-glucan conjugates, for use in targeted delivery of bioactive agents.
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Background of the Invention
Glucans have been shown to have properties that are similar to those of endotoxin in increasing nonspecific immunity and resistance to infection. The activities of some glucans (for example, yeast derived glucans) as an immune adjuvant and hemopoietic stimulator compare to those of more complex biological response modifiers (BRMs), such as bacillus Calmette-Guerin (BCG) and Corynebacterium parvum. The functional activities of yeast glucan are also comparable to those of structurally similar carbohydrate polymers isolated from fungi and plants. These higher molecular weight (1-3)-β-D-glucans such as schizophyllan, lentinan, krestin, grifolan, and pachyman exhibit similar immunomodulatory activities.
Various preparations of both particulate and soluble glucans have been tested in animal models to evaluate biological activities. The administration of glucan was shown to be beneficial in animal models of trauma, wound healing and tumorigenesis. However, various insoluble and soluble preparations of β-glucan differ significantly in biological specificity and potency, with effective dosages varying from 25 to 500 mg/kg intravenously or intraperitoneally (ip) in models for protection against infection and for hemopoiesis. Summary of the Invention
The present invention provides a glucan composition containing a β-1 ,3-glucan covalently attached to a bioactive agent. The β-1 ,3-glucan is attached to the bioactive agent by means of a hydrolyzable covalent linkage to form the resulting glucan/agent complex. The glucans for use in the present invention may be obtained from various sources, including algae, fungi, plant and bacteria. In a preferred embodiment, the glucan contained in the composition is isolated from Euglena.
The glucans of the present invention include glucans which are insoluble under normal, physiological conditions. Such glucans may be particulate in nature. The particles typically range in size from about 0.5 to about 10 microns. In a preferred embodiment, the glucan particles are between about 3 and 6 microns in size.
The glucans of the present invention may possess a linear β-1 ,3 configuration. Alternatively, glucans for use in the present invention may possess varying degrees of branching.
The compositions of the present invention also include glucans which are soluble under physiological conditions. In one embodiment, the soluble glucans have at least about 2 repeating monomer units, more preferably, at least about 5 repeating monomer units. In an alternate embodiment, the soluble glucans contain polar side chains, such as those containing 2-hydroxyethyl, carboxymethyl, 2-hydroxyethoxyethyl, and 2,3-dihydroxypropyl moieties.
The glucan/agent complex of the present invention contains a hydrolyzable bond which covalently links the β-1 ,3-glucan to the bioactive agent. Such hydrolyzable linkages for use in the present invention include acyl hydrazone, sulfonyl hydrazone, carbamate, carboxy ester, carbonate ester, phosphate, phosphoramidate and amide.
The glucan/agent complex may include a variety of bioactive agents such as anti-viral agents, anti-bacterial agents, anti-fungals, anticancer and anti-malarials. In one embodiment of the invention, the active agent is an anti-mycobacterial agent, such as isoniazid, pyrimethamine, trimethyoprim, trimetrexate, amalcacin, rifampicin and methotrexate. Bioactive agents for use in the invention also include polypeptides, oligonucleotides, and antisense agents (including alternative-backbone antisense agents, such as, phosphoramidates).
In another aspect, the invention provides a method for the treatment of a pathogen capable of invading or colonizing phagocytic cells, such as a mycobacteriυm. In the method of the invention, administering of a pharmaceutically effective amount of the glucan/agent composition is effective to direct delivery of the bioactive agent to phagocytic cells harboring the pathogen. After delivery to the phagocytes, the agent, which is effective for the treatment of the pathogen, is released from the composition by cleavage of the hydrolyzable attachment.
In one embodiment of the invention, the method is used for treatment of a mycobacterium such as M. avium or M. tuberculosis. In providing a method for treating M. tuberculosis, the glucan/agent composition contains a glucan having, for example, 5 or more repeating monomer units.
In another aspect, the invention provides a method for delivering an antigen to a phagocytic cell by forming an antigen/glucan complex. In one embodiment of the above method, the antigen is a polypeptide vaccine, covalently attached to a soluble glucan.
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples. Brief Description of the Figures
Figs. 1A, 1 B illustrate methods of synthesis for β-glucan/antisense (phosphorothioate) conjugates.
Figs. 2A-2D illustrate methods of synthesis for β-glucan/polypeptide (ribosome inhibitory protein) conjugates.
Figs. 3A and 3B show an exemplary synthetic route for forming a glucan/ bioactive agent complex in which herpes simplex virus glycoprotein D HSV gD2 is coupled to a functionalized β-1 ,3-glucan by means of acyl hydrazone bonds.
Figs. 4A, 4B, and 4C are plots illustrating cytokine production by human monocytes following culture of cells in medium containing particulate β-1 ,3-glucan. Figs. 4A, 4B, and 4C indicate the levels of IL-1β, TNF-α, and IL-6, respectively (in pg/ml, y axis) versus time (hr, x-axis) following stimulation. Cytokine levels for cells exposed to glucan are indicated by closed squares; open circles indicate cytokine levels for non-stimulated control cells maintained in medium alone.
Fig. 5 is a plot illustrating the enhanced immunological response of mice immunized with an exemplary gD2-glucan conjugate prepared in accordance with the present invention.
Figs. 6A-6E illustrate various methods for preparing functionalized glucans. Fig. 6A outlines the synthesis of 2,3-dihydroxypropylglucan and amine-functionalized glucans. Fig. 6B illustrates a synthetic approach for forming hydrazide-functionalized glucans. Fig. 6C shows the synthesis of tritium-labeled 2-hydroxyethylglucan. Fig. 6D illustrates a preparation of aldehyde-functionalized glucans. Fig. 6E illustrates an alternative method for forming amine-functionalized glucans.
Figs. 7A, 7B illustrate methods of synthesis for β-glucan/polypeptide (CG1-23) conjugates.
Figs. 8A-8C illustrate methods of synthesis for β-glucan/polypeptide (HSV glycoprotein D) conjugates. In these figures, AG represents β-glucan.
Figs. 9A, 9B illustrate methods of synthesis for β-glucan/drug (dideoxynucleotide) conjugates. Detailed Description of the Invention
I. Definitions
The following terms, as used herein, have the meanings as indicated:
A "hydrolyzable bond" is a covalent bond which can be hydrolyzed, typically under acidic conditions in vivo or in vitro. The hydrolyzable bond may be cleaved by acid-catalyzed hydrolysis. Alternately, the bond may be hydrolyzed by enzymes (e.g., degradative enzymes such as proteinase or phosphatase) or chemical treatment. Acidic conditions may be aqueous or anhydrous, although most typically, acidic conditions signify aqueous environments having Ph values below about 5. In some embodiments of the invention, the internal phagocyte environment provides the conditions for hydrolyzation, for example, acid-hydrolysis in the lysozyme which typically has an acidic environment generated by means of a proton-pump ATPase.
As used herein, a "bioactive agent" is a compound that possesses biological activity. Upon introduction into the body, a bioactive agent is one which alters the body's function by interactions at the molecular level. Bioactive agents for use in the invention include small molecules, polypeptides, oligonucleotides, antisense agents, and the like. The bioactive agent may be in a prodrug form which is converted to the active agent following administration. Bioactive agents for use in the present invention possess at least one functional group suitable for attachment to a β-glucan.
"Glucan" refers generically to a variety of naturally occurring homopolysaccharides or polyglucoses, and includes polymers such as cellulose, amylose, glycogen, laminarins, and starch. Glucans are composed of branched and/or unbranched chains of glucose units linked by 1 ,3-, 1 ,4- and 1 ,6-glucosidic bonds that may be of either the a or β type.
Unless specifically stated otherwise "glucan" or "β-glucan," as used herein, refers to a polyglucose macromolecule containing glucopyranose units linked by a series of beta-1 ,3-glucosidic bonds. "Particulate glucan" refers to a water insoluble glucan, typically from about 0.5-10 μ in size. II. Glucans for Use in the Present Invention
The present invention provides a β-glucan composition containing a β-1 ,3-glucan covalently attached to a bioactive agent by a hydrolyzable bond. The glucan/agent complex of the present invention is useful in the targeted delivery of bioactive agents, particularly to phagocytic cells harboring pathogens. The hydrolyzable bond, which acts to temporarily tether the glucan to the bioactive agent, is effective, upon hydrolysis, to release the bioactive agent at its target site, and thereby release the bioactive agent in its active or prodrug form.
β-Glucan is a glucopyranose polysaccharide linked in a β-1 ,3 configuration. β-glucans possess a wide range of biological activities, including phagocyte-specific immuno-enhancing activities (Gorecka-Tisera; Scaringi). Experiments performed in support of the present invention demonstrate that polysaccharides, such as β-glucans, are useful carriers for such cell-specific targeting.
Human and murine monocytes/macrophages express surface cell receptors specific for the β-(1 ,3)-linked oligosaccharides (Czop). The surface cell receptors mediate the phagocytosis of β-glucan particles in the absence of opsonic proteins and the production of pro-inflammatory cytokines.
Exemplary β-glucans for use in the compositions and methods of the present invention are discussed below. A. Preparation and Isolation of Particulate β-Glucans
Glucans for use in the present invention may be particulate in nature or may be soluble under physiological conditions. The particulate glucans may also provide the starting materials for conversion to soluble β-glucans.
Naturally occurring β-glucan is typically particulate in nature, and may be derived from a number of sources, including yeast, algae, plant and fungi (Bartnicki-Garcia). The size range of the β-glucan particles varies widely. A size range typically useful in the practice of the present invention is the range of about 0.5 to about 10 microns. A preferred size range is about 3 to about 6 microns. One common source of β-glucan is Saccharomyces cerevisiae (Baker's yeast). In isolation methods where the glucan is extracted from the cell wall, β-glucan yields typically range from about 5 to 7 percent of the cellular mass.
A preferable source of glucan for use in the invention is algae from the genus Eυg/ena. Examples of suitable sources include E. gracilis, E. intermedia, E. piride, and other Euglenoids, such as Astasia longa. Euglena gracilis is particularly preferred. A preferred process for obtaining substantially pure, pyrogen-free β-glucan from Euglena in relatively high yields is described in co-owned United States Patent No. 5,084,386.
The cultivation and purification of algal glucan derived from Euglena gracilis is described in Example 1 . The resulting characteristics of the isolated β-glucan material is summarized in Table 2. This preferred source and method of purification reproducibly provides a nonpyrogenic, linear, high-molecular-weight algal glucan.
Particulate algal glucan derived from Euglena gracilis dissolves only at highly alkaline pHs and may be reprecipitated to an amorphous form by lowering the pH to about 6-8. At this point, individual strands typically reanneal by hydrogen bonding to macromolecular aggregates, the viscosity of which is pH dependent.
Other β-glucans may also be used in the compositions and methods of the invention, such as native branched glucans (β-1 ,3- and β-1 ,6-glucopyranoses) from yeast and filamentous fungi. Bacterial β-glucans, such as curdlan, may also be used. Curdlan is a linear glucan having a molecular weight of about 80 kilodaltons and is insoluble in water.
Another source of β-glucan is pleuran, a fungal glucan having a linear β-1 ,3-backbone containing branches of β-1,3-linked oligoglucosides, which are linked to the backbone through β-1,6-glycosidic bonds (Sandford).
Additional glucopyranose polysaccharides which may find use in the present invention include dextran (poly-α-1 ,6-glucopyranose), methylcellulose (poly- β-1,4-glucopyranose), and chitopentose (2-acetamido-2-deoxy-β-1,4-glucopyranose). Dextran may be obtained in a variety of molecular weight ranges and is readily cleared and completely metabolized in humans by dextranases (Jeanes). B. Preparation and Isolation of Soluble β-Glucans
In some embodiments, the compositions and methods of the present invention utilize soluble β-glucans. Soluble glucans, according to the present invention, are those which are soluble under physiological conditions. Such glucans are particularly preferred for compositions of the present invention intended for therapeutic use. Soluble glucans may be suitable for cellular internalization and clearance through the kidney, since they remain soluble at physiological pH's.
Soluble β-glucans may be either non-functionalized or functionalized. Nonfunctionalized glucans may be obtained by a number of routes.
Non-functionalized, water soluble oligomers of β-glucan from E. gracilis typically contain fewer than 25 monomer units (DP< 25).
Soluble β-glucans may be obtained by a variety of methods, including the preparation of soluble oligomers by acid-catalyzed cleavage of particulate β-1 ,3-glucans. One such method of acid-catalyzed cleavage is acetolysis. In acetolysis, the glucan is treated with acetic anhydride in concentrated sulfuric acid, followed by alkaline hydrolysis or transesterification of the peracetylated product (Wang). The peracetylated soluble oligomer may also be separated by chromatography or silica gel, and derivatised prior to deacetylation. Alternatively, partial hydrolysis of the glucan may be carried out in 0.5 N hydrochloric acid in dimethyl sulfoxide (Kamo). These methods are somewhat preferable to formic acid hydrolysis (Wang) for large-scale applications.
Non-derivatized water soluble oligomers may be fractionated by precipitation from dimethyl sulfoxide with ethanol (Ogawa) or acetone
(Kamo), by ultrafiltration, or by gel filtration (Janusz).
Soluble β-glucans may also be obtained by derivatization of insoluble glucans with polar side groups, followed by fractionation into varying molecular size ranges. Suitable starting materials for functionalization include particulate glucans as described in Section A above. A variety of soluble glucans may be prepared by varying the stoichiometry of the reagents used for functionalization or by the use of protecting groups to selectively limit the extent of functionalization. For a review of the use of protecting groups in organic synthesis, see Greene. A number of polar side groups may be introduced into the glucan starting material, such as 2-hydroxyethyl, 2,3-dihydroxypropyl, carboxymethyl, depending on the choice of derivatizing agent (Ukai; Seljelid;
Williams). Typically, polar side chains containing hydroxy (Kishida) or carboxy groups (Ohno) are preferred. Representative conversions of particulate glucans to soluble derivatives by functionalization is illustrated in Figs. 6A-E.
As shown in Fig. 6A, introduction of glyceryl (2,3-dihydroxypropyl) side chains may be carried out by reacting the glucan starting material 25 in aqueous alkali with epichlorohydrin 49 to form the glucan-epoxypropane intermediate 51 , followed by alkaline hydrolysis, to form the corresponding glyceryl-glucan, 53. This material is referred to as g-glucan. The reactive intermediate epoxy-functionalized glucan, 51 , may also be used to introduce other functional groups into the glucan backbone.
Alternatively, carboxy groups may be introduced into the glucan molecule to provide soluble glucan derivatives. Carboxymethylation is typically carried out by treating the glucan with chloroacetic acid in aqueous sodium hydroxide, to form water-soluble carboxylated glucan derivatives (59, Fig. 6B). Additionally, the carboxylated glucan may also be used for drug conjugation through the carboxyl group, to provide glucan/active agents possessing a carbonyl-containing hydrolyzable attachment.
Any suitable method may be used to estimate the extent of carboxyl group substitution. One such method is colorimetric analysis using 2,7-dihydroxynaphthalene in sulfuric acid (Ohno). Glucan concentrations in solution may be determined using the anthrone method (Scott).
An exemplary method for providing soluble derivatives of β-glucans is provided in Example 6. In this approach, native algal glucan from Euglena is alkylated with 1-chloro-2-hydroxyethane in aqueous alkali, followed by size-exclusion fractionation and sterilization, to give soluble, pyrogen-free hydroxyethylated glucan of various molecular weight ranges. This methodology dissociates the glucan polysaccharide chains to provide water-soluble material having a lower molecular weight than the native, parent material. Three soluble, derivatized glucan fractions are isolated from the above process for use in the invention. One is a lower molecular weight hydroxyethylated material with a molecular weight less than 5000 daltons. A second medium MW fraction of MW 10,000 to 18,000 daltons is referred to as HE-glucan-SM. The third fractionated glucan material is also a soluble hydroxyethylated material, having a higher molecular weight range from about 20,000 to 100,000 daltons, and is referred to herein as
HE-glucan-SH.
Glucan sources for use in the present invention should be pyrogenfree. One such method for ensuring that the soluble glucan samples are free of pyrogens is to pass the soluble glucan fractions through a "DETO-XI-GEL" pyrogen-chelating column (Pierce, Rockford, IL) to remove possible pyrogens. In an alternate approach, the glucan fractions are analyzed by the USP (United States Pharmacopia, 1990) rabbit pyrogen test.
C. Functionalization of Glucans
Prior to coupling to the bioactive agent, glucans for use in the present invention are typically functionalized with reactive groups that promote coupling of the glucan backbone to the bioactive agent. Functionalization may be carried out on either particulate or soluble sources of β-glucan.
In some instances, functionalization is used to activate the glucan hydroxyl groups to promote coupling. In other instances, new functional groups are introduced onto the glucan to provide a different chemical site for attachment to the active agent. The choice of functional group introduction depends on a number of factors, including the functional group(s) present in the bioactive agent and the desired covalent attachment. Various synthetic approaches for forming functionalized glucans are shown in Figs. 6A-E.
As described in Example 11 and in Figs. 3A and 3B, an aldehyde-derivatized glucan, 35 (Bogwald) may be conjugated to drugs containing hydrazide groups, or to drugs possessing functionalities which are readily converted to hydrazide. The coupling of an aldehyde-functionalized glucan with a hydrazide-containing drug results in the reversible formation of a glucan/agent complex possessing an acyl hydrazone covalent attachment. Since underivatized β-1 ,3-glucans possess periodate-cleavable glycol functions only at the reducing and nonreducing termini, periodatepromoted cleavage cannot be used to generate a sufficient number of aldehyde groups for coupling to a bioactive agent. However, this reagent can be used effectively to transform functionalized glucans, such as glyceryl-glucan, to aldehyde-containing materials. Preparation of glyceryl-glucan is described in Example 6A. Glyceryl-glucan, which has a 1 ,2-glycol side chain, is suitable for oxidation by periodate. The level of substitution is controlled by adjusting the stoichiometry of the reagents and the reaction time.
Particulate glucan may be similarly functionalized by reaction with periodate after introduction of a low level of 2,3-dihydroxypropyl side chains (or any suitable hydroxy-containing side chain). The dihydroxypropyl side chains introduced into the glucan backbone provide sites for periodate-promoted oxidation.
Aldehyde group loading onto a soluble glucan may be estimated using 2,4-dinitrophenylhydrazine (Nathan) or hydroxyjamine hydrochloride (Zhao) to indicate the number of potential sites for drug attachment.
In some instances, optimal release of the bioactive agent from the glucan/agent complex may require a spacer between the glucan backbone and aldehyde groups intended for attachment to the active agent. The introduced aldehyde function, separated from the glucan backbone by a spacer, is more accessible to incoming bioactive agents intended for attachment than carbonyl groups contained directly on the glucopyranose rings. An exemplary functionalized glucan containing a spacer between the glucan and the aldehyde sites for drug attachment is shown in Figs. 3A, 3B and Figs. 6D, 6E.
In an exemplary synthetic approach, the glucan is first activated by reaction with 1 ,1 '-dicarbonyldiimidazole 27 to form the carbonyldiimidazole-activated glucan 29 (Figs. 3A, 3B and 6D). The carbonyldiimidazoleactivated glucan is then treated with an aminoacetal, such as the dimethyl 67 or diethyl 31 acetal of 4-aminobutanal to form the corresponding O-carboxamidobutyraldehyde acetal glucan intermediate 33. The protected glucan is hydrolyzed under mild conditions (0.05 N HCl) prior to coupling with a bioactive agent to form the desired aldehyde-functionalized glucan (e.g., 35 in Figs. 3A, 3B and 6D).
Alternatively, reactive hydrazide functions may be introduced onto the glucan backbone. As seen in Fig. 6B, hydrazide-derivatized glucan 61 may be prepared by coupling functionalized carboxymethyl glucan 59 to aqueous hydrazine with a water-soluble carbodiimide (i.e., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EDC) (Hurwitz) or by phase-transfer alkylation (Mann) of glucan with ethyl acrylate followed by treatment of the ester product with hydrazine. The extent of hydrazide group introduction may be estimated by elemental analysis (Hurwitz) or by reaction with trinitrobenzenesulfonic acid (Miron).
Preparation of a tritium-labeled glucan is shown in Fig. 6C. In the exemplary synthesis shown, glyceryl-glucan 53 is oxidized with periodate to the corresponding aldehyde-derivatized glucan intermediate 63, followed by treatment with tritium-labeled sodium borohydride to form 2-hydroxy-2-3H-ethylglucan 65.
Amine functionalized glucans may be prepared as follows (Fig. 6A). Soluble glucan 25 is first prepared by reaction with epichlorohydrin 49 in aqueous sodium hydroxide to form the epoxy-functionalized glucan derivative, 51. The epoxy group content of epoxy-functionalized glucans may be determined by the method of Sundberg. The epoxy-glucan is then treated with a suitable diamine, such as 1 ,3-diaminopropane, to form the amino-functionalized glucan 57. Unreacted epoxide groups present in the amino-derivatized glucan may be removed by hydrolysis in base. Substitution of ammonium hydroxide (Mazid) in place of the diamine reactant forms the corresponding 3-amino-2-hydroxypropyl glucan (Fig. 6A, 55). The primary amine content of the amine-functionalized glucan may be determined by quantitative ninhydrin assay.
Particulate glucan may be aminated by derivatization with 4-fluorobenzenesulfonyl chloride (Chang) or with carbonyldiimidazole, followed by treatment with a suitable amine to form aminated glucans, as illustrated in Fig. 6E, 71 and 73. The derivatized glucan, 73, contains a carbamate function linking the terminal amino-containing group to the glucan backbone, and therefore contains fewer basic amino groups than does the amino-functionalized glucan, 71. An appropriate method for determining the extent of -NH2 loading onto particulate glucans is the trinitrobenzenesulfonic acid difference estimation method (McKelvey).
The number of drug attachment sites contained within the functionalized glucan may be altered by chemical blocking methods. For instance, excess amine groups in the amino-functionalized glucan may be blocked by acetylation, using either N-acetylimidazole or N-succinimidyl acetate.
In many cases, conversion of particulate glucan to soluble glucan starting materials is desired. Additionally, the prudent selection of functional group introduction into the glucan may provide enhanced selectivity in subsequent functionalizations by the use of known protecting groups.
Suitable organic solvent-soluble glucan derivatives include the 6-O-trityl ether, 6-O-tosylate, and phenylboronate derivatives. Tritylation by reaction with trityl chloride in pyridine may be used to solubilize the starting glucan by introduction of trityl groups at the 6-position. Selective transformations may then be carried out at the non-protected hydroxy sites within the glucan backbone, such as the 2- and 4-hydroxy functions.
For instance, oxidation of the reactive hydroxyl groups with acetic anhydride in dimethyl sulfoxide may be used to form the corresponding keto-containing glucan. Alternatively, partial tritylation of the starting glucan may be employed to block some of the reactive hydroxyl groups, resulting in lower levels of functional group introduction into the glucan backbone. The trityl group is removed from the glucan prior to attachment to the bioactive agent by acid hydrolysis.
For tosyl-containing glucans, the tosylate of primary hydroxyl groups typically undergoes nucleophilic displacement reactions and may be further derivatized. In one exemplary approach, reaction of tosylated glucan with azide, followed by reduction, for example, by hydrogenation, may be utilized to form the corresponding amino-functionalized glucan.
Additional transformations of soluble, hydroxyethylated glucan to form functionalized glucans for attachment to bioactive agents include the following. Phenylboronate derivatives may be formed by reaction with phenylboronic acid (de Belder). Reactive carbamate-functionalized glucans (Bethell 1981 a, 1981 b), such as the corresponding imidazole or 1 ,2,4-triazole-I-carboxylic acid esters, may be produced by reaction with carbonyl diimidazole or ditriazole. The activated carbamates are suitable for further functionalization with amines, either in organic solvents or aqueous buffer, to form the hydrolytically stable N-alkylcarbamate-containing glucans.
Conversion of soluble glucan starting materials to amino-functionalized derivatives may also be accomplished by reductive amination, which is specific for the reducing terminus (Gray), to form the corresponding primary amino substituents, or by reductive amination with benzylamine, followed by N-debenzylation and by hydrogenlysis (Yoshida).
The derivatized glucans may be coupled to various bioactive agents, depending upon the nature of the terminal reactive groups on the derivatized glucan and the nature of reactive functional groups in the bioactive molecule intended for coupling. Bioactive agents may also be derivatized to introduce functionality appropriate for coupling to the glucan.
As demonstrated in Figs. 3A and 3B, aldehyde-containing glucans
35 may be coupled to hydrazide-containing molecules, such as 45, by means of hydrazone linkages. Conversely, hydrazide-functionalized glucan (as shown in Fig. 6B) may be coupled to aldehyde-containing bioactive agents via hydrazone linkages. Amino-functionalized glucans may be coupled to carboxyl-containing bioactive agents by means of amidelinkages. Glucan hydroxyl-groups may also be utilized as sites of attachment for carbonyl-containing bioactive agents, such as drugs containing either aldehyde or keto-functions. Coupling to the glucan hydroxy groups provides the acetal and ketal-linked glucan-bioactive agent complexes, respectively. Thiolated glucan may be coupled to thiol-containing bioactive agents by means of disulfide bonds. Glucan hydroxy groups may also be used to attach a carboxyl-containing drug via an ester linkage.
D. Toxicoloαical Properties of Glucans
Preferred glucans for use in the present invention should ideally possess very low toxicities. Acute toxicity is typically evaluated as described in Example 3.
In a typical toxicity study, glucans intended for use in the invention are administered to healthy mice, usually by ip injection. The dose size is typically varied, and treatments may include one single dose administra tion of glucan over the course of the study, or, alternatively, multiple doses of glucan may be administered over the course of several days. Following glucan administration, the general health of the mice is observed and the mice are weighed on consecutive days. Some of the visual signs of poor health include an ungroomed coat and lethargy. The resulting characteristics of the glucan-treated mice are compared to a control group, and the general toxicity of the glucan determined.
III. β-Glucan/Agent Complexes: Targeted Delivery, Immunological
Properties and Applications
Experiments performed in support of the present invention demonstrate the immunopotentiating effects of β-glucans. The following studies demonstrate the ability of β-glucans to enhance resistance to bacteria and tumor challenges, stimulate the release of colony-stimulating factor in vivo and IL-1 in vitro, and enhance humoral immunity to protein antigens.
The data presented in Example 2 show the ability of β-glucans to protect animals from Listeria challenge. In experiments 1 and 2 (Example 2), the mice were challenged with 1 LD50 of Listeria. The survival of mice treated with various doses of β-glucans was significantly greater than the survival of saline treated mice. In experiment 3 (Example 2), mice were challenged with 6 LD50 of Listeria to examine the resistance of mice to a large infectious challenge. To control for the particulate nature of β-glucans, an additional group of mice was injected with a cellulose preparation which is similar in size to the β-glucan particles, but is composed of glucose molecules linked in a β-1,4 configuration.
In experiment 3, animals treated with three doses of 0.5 mg of β-glucans prior to challenge had a survival rate of 70%, whereas none of the mice treated with saline survived. Furthermore, all of the animals treated with the cellulose preparation died, indicating that the protection afforded by β-glucans is not solely related to its particulate nature and that β-glucans enhanced the ability of mice to resist the Listeria challenge.
Enhanced protection may not be due exclusively to the direct effects of β-glucans on macrophage, because resistance to Listeria requires the development of cell-mediated immunity (CMI), which in turn activates macrophages to kill intracellular parasites (Mackaness; Krahen- buhl and Remington). Experiments performed in support of the present invention indicate that macrophages elicited in vivo or cultured in vitro (48 hours) with β-glucans do not demonstrate enhanced killing of Listeria in vitro, suggesting that glucans may amplify the anti-Listeria CMI response.
The data presented in Example 4 show the ability of β-glucans to protect animals from tumor challenge. The ability of β-glucans to modulate the survival of tumor bearing mice was examined in a syngeneic tumor system. Groups of mice were injected with melanoma cells on day 0 and injected iv with various doses of β-glucans starting on day 1. The survival of mice in each group was compared. Mice treated with five doses of 0.25 mg of β-glucans had a statistically significant increase in mean survival time compared with mice treated with saline (p < 0.05). Animals treated with 5 doses of 0.125 mg β-glucans also demonstrated an increase in survival time. These results indicate that administration of β-glucans resulted in increased survival of tumor-bearing mice.
The data presented in Example 5 show that β-glucans induce colony-stimulating activity in vivo. At various times after iv injection of β-glucans, sera were collected from mice and assayed for bone marrow colony-stimulating activity (CSA). As a positive control, additional groups of mice were injected with lipopolysaccharide (LPS). Results show that
CSA was detectable in the sera of mice as early as 2 hours after a single injection of 2.5 or 5 mg of β-glucans. Elevated serum CSA activity was detectable for at least 24 hours. These results are consistent with the ability of β-glucans to enhance production of granulocytes and monocytes/macrophages.
Experiments performed in support of the present invention demonstrate the ability of β-glucans to stimulate cytokine production by macrophages and monocytes. The ability of β-glucans to stimulate the release of IL-1 from peritoneal macrophages in vitro was demonstrated as follows. Peritoneal exudate cells were collected from mice injected 3 days earlier with 1.5 ml of sterile 10% proteose peptone. The cells were plated into 24-well culture plates. After 2 hours, non-adherent cells were removed by vigorous washing and the adherent cells were cultured with various concentrations of β-glucans. Forty-eight hours later, the culture supernatants were collected, centrifuged, and tested for IL-1 activity using the standard thymocyte proliferation assay. A dose dependent increase in IL-1 activity was observed in the supernatants from macrophage incubated with β-glucans.
The generation of IL-1 by peritoneal macrophages stimulated with β-glucans in vitro suggests that intravenous injection of β-glucans may induce a pyrogenic response. However, administration of large doses of β-glucans administered iv to rabbits did not induce immediate febrile response. The potential conflict between the results of the pyrogenicity tests and in vitro IL-1 production by macrophage may be due to difference in the dose of β-glucans delivered to macrophages in vivo as compared to in vitro. Alternately, the kinetics of IL-1 production induced by β-glucans in vivo may differ from that observed in vitro.
The data presented in Example 8 demonstrate the ability of βglucans to stimulate the release of cytokines (including IL-1β, TNF-α and IL-6) from human monocytes. As shown above for mouse macrophages, the ability of β-glucans to induce cytokine production was found to be dose-dependent. The induction of IL-1 release may be beneficial for the use of β-glucans as vaccine adjuvants since administration of recombinant IL-1 is known to enhance primary and secondary responses to T-dependent antigens and primary responses to a T-independent antigen (Nencioni, et al., 1987).
Data presented in Example 7 demonstrate that soluble β-glucans, such as a hydroxyethylated glucan, can bind to and be internalized by both mouse and human macrophages. The binding and uptake of β-glucans appears to be mediated through a receptor binding mechanism.
The ability of β-glucans to perform as adjuvants in enhancing the immune response of animals was examined as follows. In preliminary experiments performed in support of the present invention, the adjuvant performance of β-glucans was compared to a low dose of keyhole limpet hemocyanin (KLH), T-cell dependent antigen. Five mice per group were immunized ip with 1 μg of KLH delivered in saline, 0.2 ml of CFA, or saline containing various doses of β-glucans. Animals received a second immunization of 1 μg of KLH in saline ip on day 21. Serum was collected on various days and assayed for anti-KLH antibodies by ELISA. Mice immunized with KLH mixed with β-glucans demonstrated consistently higher anti-KLH antibody titers than animal immunized with KLH alone. The primary and secondary responses of the KLH/β-glucan immunized animals were proportional to the amount of β-glucan co-injected with the KLH.
Further experiments demonstrated that co-administration of β-glucans with a Herpes Simplex Virus I glycoprotein D2 antigen enhanced animal immunogenic responsiveness to the antigen (Example 9) relative to administration of the antigen alone. Similar results were obtained with a protein from Human Immunodeficiency Virus (HIV) envelope (Example 10).
Experiments performed with this HIV antigen (gp120) demonstrated that β-glucan was found to enhance the humoral response in rabbits as well as in mice. A. β-Glucan Antigen Conjugation and Applications of Conjugates
Further experiments performed in support of the present invention demonstrated that administration of β-glucans conjugated to antigens (i.e., covalently bound) lead to an improved antigenic response in animals relative to both antigen alone and antigen mixed with β-glucans.
β-glucan-N, β-glucan-D, and β-glucan-S are used for synthesis of the conjugated antigens. β-glucan-N and β-glucan-D are linear, 400,000-molecular-weight β-glucans that have phosphate groups at approximately every tenth residue. β-glucan-S is a soluble β-glucan. To minimize the antigenicity of the tether groups that link the β-glucans to the antigen, phosphate ester groups are employed (Figs. 7A,7B and Figs. 8A-C). This coupling strategy is amenable to (i) high- and low-molecular weight β-glucans, (ii) particulate and solubilized β-glucans, and (iii) native and functionalized derivatives of β-glucans. The phosphate ester groups mimic the natural phosphates on the β-glucans, and the hydrophobic groups generally should have a weaker antigenic response than hydrophilic groups.
The HSV antigens selected for study were HSV-1 glycoprotein D (gD) (Weijer, et al.) and gD peptide fragment 1-23, which is an antigenic determinant capable of inducing HSV-neutralizing antibodies. The HIV antigens are glycoproteins gp1 60 and gp1 20, and peptide fragment 584-609 (SEQ ID NO: 1 , RILAVERYLKDQQLLGLWGCSGKLIC) (Schrier, et al., 1988).
The following conjugation chemistry is useful for linkage to both water-soluble and insoluble β-glucans. One linkage methodology that can be used to attach peptide gD 1-23 to its tether group is adapted from the method used to couple the synthetic peptide corresponding to residues 1-13 of the gD polypeptide to protein carriers for immunization studies (Cohen, et al., 1984). Peptide CG1-23 (SEQ ID NO:2, CGKYALADASLKMADPNRFRGKDLP) is prepared by the route that was used to prepare peptide CG1-13 (Cohen, et al., 1984). To facilitate purification it may be necessary to replace methionine-11 of the peptide with norleucine (Coull, et al., 1986). In the method, gD peptide 1-23 is extended on its amine terminus by glycine and cysteine residues (peptide CG1-23). Attachment to the protein was effected by the addition of the cysteinyl sulfhydryl group of the modified peptide to the maleimide groups on the protein. The maleimide groups were introduced by reaction of m-maleimido-benzoyl-N-hydroxysuccinimide ester (Klaus and Humphrey) with the e-amino groups of the lysine residues.
The introduction of the phosphate esters onto β-glucan employs reaction of the anhydrous polysaccharides with phosphoramidite reagents such as 2 (Fig. 7A; Coull, et al., 1986), followed by oxidation with iodine and water. The protecting groups are removed and afford the 6-aminohexyl phosphate esters of β-glucan 5 (Fig. 7A). Alternatively, reaction of β-glucan with phosphate 3 in the presence of the water-soluble carbodiimide EDC (Leclerc and Vogel, 1986; Smrt, 1979) and removal of the protecting groups also affords 5 (Fig. 7A). Reaction of the primary amino groups of 5 with 6 introduces the maleimide groups, which are available for reaction with the gD peptide CG1-23 (Butler, et al., 1969) to give glucan-gD conjugate 8 (Fig. 7A).
Another strategy for preparing 8 (Fig. 7A, bottom scheme) is to begin with phosphorylation of β-glucan using polyphosphoric acid, PPA (Suzuki, et al., 1977). The resulting glucan polyphosphate 11 is coupled to the maleimide-alcohol 10 (Fig. 7A) with EDC. Reaction with gD peptide CG1-23 produces 8. An alternative to these loading methods is the derivatization of β-glucan by carboxymethylation (Winter and Beckmann, 1956), as illustrated in Fig. 7B. The amino group functionality is then introduced by coupling with 1 ,6-diaminohexane (Kasel, et al., 1971 ), giving 14 (Fig. 7B). Reaction with N-hydroxysuccinimide ester 6 and gD peptide CG1-23 affords 15
(Fig. 7B).
Loading values of a selected antigen onto β-glucan is established by ultraviolet spectroscopy and elemental analyses (C/N ratios). Phosphate incorporation is determined by the method of Ames (1966). Adducts are purified by gel-filtration and ion-exchange chromatography.
A second strategy for coupling β-glucan to gD is outlined in Fig. 8A. β-glucan 1 is phosphorylated using phosphoramidite chemistry with reagent 16 (Fig. 8A) (Kremsky, et al., 1987). Iodine oxidation followed by hydrolysis give 5-carboxypentyl phosphate 17. Coupling of the carboxyl groups with the amino groups of the lysine residues of gD affords 19 (Fig.
8A). Alternately, as shown in Fig. 8B, an ether linkage can be used in place of the phosphate diester group to provide adduct 21.
Another method of linkage involves modification of the carbohydrate residues on gD, as illustrated in Fig. 8C. Oxidation of gD 18 with periodate introduces aldehyde groups to form oxidized gD intermediate 22
(Chua, M., et al., 1984), which can then form Schiff bases with the amino groups of 5. Reduction with sodium cyanoborohydride affords the stable amino bonds of 23 (Fig. 8C). Purification of the adducts is effected by gel filtration and ion exchange chromatography. Loading is established by spectrophotometric methods.
Loading levels with both classes of conjugates are varied to optimize the antigenic response. It may also be necessary to increase the length of the alkyl tether group to enhance antigenicity (for example, from n = 6 to n = 10 or 12). The optimal length of the tether group can be determined based on experimentation.
Similar synthetic methodology can be employed to prepare conjugates of the HIV antigens to β-glucan. Because HIV peptide 584-609 has two cysteine residues, the sulfhydryl group of C-603 can be protected as a acetamidomethyl thioether, whereas C-609 can be blocked as the methoxybenzyl thioether. The latter protecting group is preferentially cleaved by treatment with hydrogen fluoride, permitting selective reaction of the sulfhydryl of C-609 with the maleimido adduct formed from reaction of 5 and 6 (Fig. 7A).
The acetamidomethyl thioether protecting group on C-603 can then be removed by treatment with Hg(OAc)2 at pH 4. If the free sulfhydryl group of C-603 proves to be unstable giving peptide dimers that do not produce the desired antigenic effect, it can be replaced by hydroxyl or converted to the methyl thioether. In the event that antigenicity is enhanced by formation of a loop structure caused by disulfide bond formation between C-603 and C-609, a peptide CG584-609 will be prepared where both sulfhydryl groups of C-603 and C-609 are protected by acetamidomethyl groups. Reaction with the maleimido adduct of 5 with 6, followed by removal of the protecting groups, and oxidation affords the desired conjugate bearing a loop structure.
HIV gp160- and gp120-β-glucan adducts can be prepared by the two routes used for gD illustrated in Figs. 8A and 8B; the third route (Fig. 8C) involving Schiff base formation, followed by reduction with hydride may not be desirable because of the presence of critical disulfide bonds in the glycoproteins. Therefore, an alternate strategy can be employed in which the glycoproteins are oxidized with periodate to introduce aldehyde groups onto their sugar functions. These aldehydes form stable hydrazones with the hydrazides prepared from coupling of [β-glucan[OP(O)(O -) (CH2)5CO2H] with excess hydrazine.
Peptides gD GC1-23 and HIV 584-609 are prepared by standard solid-phase techniques (Erickson and Merrifield, 1976) on a Beckman
Model 990C automated peptide synthesizer using commercially available t-BOC-amino acid polystyrene resin and t-BOC protected amino acids with suitable side-chain protecting groups.
Preparation of a β-Glucan/HSV gD2 conjugate with a hydrolyzable, acid-labile hydrazone linkage is described in Example 11.
The above described coupling methods are applicable to other polypeptide antigens in addition to those described above. Other useful antigens particularly include vaccine polypeptides. Exemplary targets for vaccine development, using the methods of the present invention, include Malaria, Varicella-Zoster Virus, Cytomegalovirus, Pneumococcus, Cholera, Rotaviruses, Measles, Respiratory Syncytial Virus, Tuberculosis and Hepatitis Viruses.
B. Immunological Studies Using Herpes Simplex Virus Antigens Successful immunizations have been reported in mice injected ip with a total of 6 μg of gD in Complete Freund's Adjuvant (CFA) divided over six biweekly injections (Long, et al., 1984) or guinea pigs that were immunized intradermally (id) in the footpad or intramuscularly (im) in the thigh muscle three times at 3-week intervals with 6.25 μg of recombinant gD in various adjuvants (Sanchez-Pescador, et al., 1988).
Mice are immunized with various antigen and β-glucan (β-glucan-N, β-glucan-D, and β-glucan-S) conjugates to determine their effectiveness. In formulations where the antigen is covalently linked to β-glucan, the dose is based on the loading efficiency of antigen onto the β-glucan.
Female C3H/HeJ mice immunized by intraperitoneal injection of the
HSV gD2-glucan conjugate (prepared as in Example 11) exhibited significantly higher anti-gD2 serum antibody titers than did mice immunized with just a mixture of gD2 and glucan. These results are shown in Fig. 5. The results demonstrate the superior properties of the antigen/β-glucan conjugates, for example, the ability of β-glucan/antigen complexes to enhance the immune responses of experimental animals to antigens.
In addition to the effects of β-glucan complexes on immune responses to a single dose of antigen, the effect of multiple immunizations of β-glucan complex on the humoral and cellular immunity is also evaluated. In these studies, mice are injected up to four times, at two-week intervals, with antigen-glucan conjugates. Control animals receive a primary immunization with antigen and CFA (Complete Freund's Adjuvant, Harlow, et al.), and booster immunizations in IFA (Incomplete Freund's Adjuvant, Harlow, et al.). Seven days after each immunization, groups of 3 mice are evaluated for specific immunity to HSV.
Immunogens prepared by covalently linking gD or peptides to particulate and denatured β-glucan are tested in a similar manner.
Antigen-β-glucan conjugates that elicit significant immunity to HSV are tested in challenge studies to determine the degree of protection that is conferred. Groups of 15 mice are immunized. Control groups consist of untreated mice and mice injected with β-glucan only, antigen only, or antigen with alum. After completion of the appropriate immunization schedule, induction of HSV-specific immunity is confirmed in 3 to 5 mice from each group. The remaining mice are challenged by ip injection of HSV-1 (Long, et al., 1984).
In initial studies the challenge dose is the minimum lethal dose of HSV as previously determined in untreated mice. Protection is assessed by comparing the survival of immunized mice and non-immunized mice for approximately 50 days. The conjugates conferring complete protection with this challenge dose are re-evaluated for their ability to confer protection against a higher challenge dose.
C. Immunological Studies Using Human Immunodeficiency
Virus Antigens
To evaluate the effects of the β-glucan/HIV antigen complexes, rabbits (two per group) are immunized with the HIV-1 glycoproteins or synthetic peptide 254-274. Animals receive one im injection every two weeks for a total of three injections. Serum is obtained from each rabbit before immunization and seven days after each injection. For comparison, additional animals are immunized with these antigens absorbed onto alum or CFA. Anti-HIV antibody titers from each serum sample are determined by ELISA. Anti-HIV-1-neutralizing antibodies in each serum sample are evaluated on days 7, 14, 21 , and 28, using the procedures described below. Optimized antigen conjugates are identified by this method. These optimized conjugates are then used as "selected" conjugates for further studies.
The ability of optimal doses of β-glucan to enhance the response of rabbits to lower concentrations of antigens is also examined. Further, the effect of the conjugates of the present invention on a single immunization with antigen is evaluated. In these studies, rabbits receive a primary immunization with the "selected" antigen conjugate and serum is collected at weekly intervals for 28 days. The evaluation of neutralizing antibodies is carried out as described below.
In addition to the rabbit studies, Balb/c mice are immunized ip with the "selected" HIV-1 antigen/β-glucan conjugates. Control animals are immunized with antigen in alum. Three to four weeks later, immune spleen and lymph node cells from groups of 3 mice (conjugate, alum, and control) are evaluated for their CMI response to HIV-1 antigen, using the procedures described below. In addition to examining the response of mice to a single injection of the conjugate, the effect of multiple immunizations of the conjugate on the cellular immune responses of mice is also evaluated.
D. Immunolooical Assay Procedures
1 . In vitro Virus Neutralization
Serum from immunized mice are assayed for neutralization of HSV-1 infectivity in vitro, using a 50% plaque reduction assay (Weijer, et al.). Two-fold serial dilutions of heat-inactivated serum is incubated with an equal volume of HSV-1 in DMEM containing 5% FCS. After incubation for 2 hours at 37°C, 100 μl of this mixture is added, in duplicate, to monolayers of Vero cells. After 1 hour of incubation at 37°C, to allow for virus adsorption, an overlay medium containing 0.5% methylcellulose is added.
These cultures are incubated for 2 to 3 days, after which the cells are fixed with formalin and stained with Giemsa solution. The number of plaques are counted and the neutralization titer is calculated as the reciprocal of the serum dilution producing a 50% reduction in the number of plaques.
Anti-HIV-1-neutralizing activity of rabbit sera, after heat-inactivation, is estimated by the surviving tissue-culture infectious doses (TCID) employing two assays, quantitative infectivity assay (Gowda, et al., 1989a) and p24 antigen measurement using commercially available ELISA plates. Two frozen viral stocks are prepared, HIV-1 isolates B-LAV and DV (Institute Pasteur, Paris, France; and Genelabs, Inc., Redwood City, CA, respectively), for use in these experiments.
The virus is expanded by de novo infection of VB cells (CD4-positive T lymphoblastic cell line; Chua, et al., 1984). In a typical viral stock, gp120 is present both as an oligomer on virions and as soluble gp120 monomer; the later may cause artifacts in the infectivity assay if it competes significantly with viral associated gp120 for the neutralizing anti- body. Therefore, the soluble gp120 content of the viral stocks is measured using an HIV radioimmune assay (RIA).
Prior to assay, VB cells are grown at low concentrations to sustain logarithmic growth. On the day of an assay, twofold dilutions of the serum are incubated with the virus stock diluted in culture medium (RPMI
1640 with 10% FCS). After a 1-hour incubation at 37°C, 0.5 ml of a dilution of the virus-serum mixture is added to "target" VB cells. For each viral strain, five serum dilutions and six target cell densities (1 × 106 to 3.13 × 104/ml) are assayed. Target cells infected in the absence of serum and non-infected cells serve as controls.
To assure mixing, culture tubes are rotated during the 2-hour infection period. The infected target cells are washed extensively, pelleted and re-suspended in fresh medium. Cell monolayers are prepared by adding 5 × 103 target cells and 5 × 104 "indicator" (i.e., non-infected VB cells) to each well (targetrindicator ratio of 1 :10). A total of eight wells are plated per serum dilution and target cell concentration. The cultures are maintained for seven days and the mean number of syncytia are counted (Gowda, et al., 1989a).
The ratio of syncytia induced by the virus inoculum surviving after virus neutralization (Vn) and the number of syncytia induced by the virus inoculum (Vo) are calculated. The virus-surviving fractions (Vn/Vo) from each two-fold serum dilution is plotted versus the reciprocal of the serum dilutions. The neutralizing titer (the reciprocal of the highest dilutions yielding ≥ 90% virus inhibition) for each experimental group of animals is compared with the neutralizing titer of the control animals.
For confirmation, a sample of target cells cultured alone is tested for cell-associated P24 antigen using commercially available ELISA plates.
2. Lymphocyte Proliferation to Viral Antigens Spleen and inguinal lymph node lymphocytes from immunized mice are suspended at 5 × 106 cells/ml in RPMI 1640 containing 5% FCS, 25 mm HEPES, 100 U/ml penicillin, and 100 μg/ml streptomycin. Then 100-μl aliquots of these cell suspensions are added to microculture wells containing either (i) 100 μI of various concentrations of the appropriate antigen (HSV or HIV-1 ), or (ii) virus-infected cells. The use of fixed virus-infected cells as an eliciting antigen in vitro has been described (Welling-Wester, et al., 1985).
These cultures are incubated for a total of 5 days at 37°C, with 3H-thymidine at 1 μCi/ml added during the last 18 hr. Subsequently, these cultures are harvested onto glass fiber filters and counted with a scintillation cocktail on a Packard TriCarb 2000RA scintillation counter. Results are expressed as mean counts per minute of triplicate wells.
3. IL-2 Production
The antigen-specific release of IL-2 by spleen cells from mice immunized with HSV or HIV-1 antigen conjugates is studied as an indicator of the generation of helper T cells. Spleen cells are cultured at 5 × 106 cells/ml in 1 ml of RPMI containing 5 - 10-5 M 2-mercaptoethanol, 5% FCS, and various concentrations of the appropriate antigens. After 48 hours, the culture supernatants are collected, centrifuged, and assayed for
IL-2 activity.
Two-fold serial dilutions of culture supernatant is added in triplicate to microculture wells containing 5 × 103 HT-2 cells (a murine IL-2-dependent cell line). These cultures are incubated for 24 hours at 37°C, with 3H-thymidine added during the last 4 hours.
Subsequently, the cultures are harvested for measurement of radiolabeled thymidine incorporated into newly synthesized DNA. Results are be expressed as units of IL-2 per milliliter based on use of a commercially obtained IL-2 standard.
4. Cvtotoxic T Lymphocyte Activity
The development of cytotoxic T lymphocyte cells (CTL) specific for HSV-infected cells is measured in a 3-hour chromium release assay as described by Lawman (1980). Spleen and lymph node cells are prepared from immunized animals as described. Different numbers of effector cells are added in triplicate to microculture wells in 0.1 ml of RPMI 1640 containing 10% FCS to evaluate effector-to-target ratios of 100: 1 , 50: 1 , 25:1 , and 10: 1 .
NIH3T3 cells are used as target cells because this cell line shares histocompatibility antigens with Balb/c mice (H-2k). Cells (2 × 107) are infected with HSV at a multiplicity of infection (MOD of 1 for 60 minutes at 37°C. The HSV-infected cells are washed once with media, then incubated with 400 μCi Na2 51CrO4 for 30 minutes at 37°C.
The 51Cr-labeled target cells are washed three times and resuspended at 1 × 105 cells/ml in RPMI 1640 containing 10% FCS. Aliquots
(100 μI) of target cells are added to wells containing effector cells, media (spontaneous release), or 0.5% "TRITON X-100" (maximum release). The plates are centrifuged at 200 x g, for 4 minutes and incubated at 37°C. After 3 hours of incubation, the supernatant of each well is removed and counted on a Packard Cobra 5005 gamma counter. Results of this assay are expressed as percent specific lysis, which is calculated by the formula:
Figure imgf000032_0001
CTL activity against HIV-1 antigens is monitored using a previously described procedure (Takahashi, et al., 1988). Briefly, 3 to 4 weeks after immunization with HIV-1 glycoproteins or synthetic peptides, spleen cells are restimulated in vitro with syngeneic spleen cells infected with a recombinant vaccinia virus expressing HIV env glycoprotein gp120 and gp1 60 (Gowda, et al., 1989b). After culture for 6 days, cytolytic activity of the restimulated cells is measured, using 51Cr-labeled vaccinia virus-infected syngeneic spleen cells as targets.
The development of cytotoxic T lymphocyte cells (CTL) specific for the antigens present in an antigen/β-glucan conjugate indicates that the conjugate is capable of potentiating an immune response.
In view of the above guidance, other antigens of interest can be formulated into the conjugates of the present invention and the immunogenicity of such conjugates tested using appropriate assays. Candidate conjugates useful for vaccine and other applications can be identified by the methods described above. E. β-Glucan Antigen Conjugation and Applications of Conjugalas
The present invention describes the use of drug targeting carriers that are specifically internalized by the macrophage. In one embodiment, HIV-targeted therapeutics are described. The monocytes/macrophages are an important cellular reservoir for the HIV-1 virus, the causative agent for
AIDS. The following conjugation reactions and general embodiments described for anti-HIV agents are applicable to other bioactive agents useful for the treatment of other diseases as well, for example, of pathogens (such as viral, bacterial, fungal, and malarial agents) capable of invading or colonizing phagocytic cells. Phagocytic cells include monocytes and macrophages.
As described herein, the β-glucans of the present invention undergo receptor-mediated endocytosis by the macrophage and, at higher molecular weights, have immunostimulatory effects on the macrophage. Accordingly, these β-glucans are ideal for cell-specific targeting and, because of their activating properties, for achieving synergistic therapeutic effects.
As described above, β-glucan uptake occurs by specific receptormediated endocytosis╌ a process that provides an efficient delivery pathway. Further, the β-glucans are potent macrophage activators and administration of the β-glucans appears to enhance cell-mediated immune responses. This enhancement likely occurs by cytokine-mediated cascades initiated by activation. Activation appears to be molecular-weight dependent. Glucans above a specific size (usually above 2.5 glucose residues in length) activate macrophages, a condition that may enhance drug antiviral activity. Unlike un-activated cells, activated, infected macrophages synthesize virus, but other processes, including endocytosis, are also enhanced, so that cells may be concomitantly more susceptible to antiviral agents. Selection of the appropriate molecular size of the carriers can be evaluated by the methods described below for each β-glucan/agent conjugate.
β-glucan/agent conjugates are encapsulated during uptake by monocytes/macrophage. The encapsulate is then likely fused with lysosomal vesicles, thereby becoming exposed to the degradative enzymes therein and the more acidic environment. Appropriately designed tether groups, for example, hydrolyzable groups, are then cleaved to release the agent from the β-glucan/agent complex.
Phosphate, phosphoramidate hydrazide, and disulfide linkages are exemplary useful tethers. Phosphatases present in lysosomes can cleave the phosphate diester bonds. A phosphate ester linkage at the 5 '-position of 2,3-dideoxycitidine (DDC) can release a pro-drug species of the active DDC triphosphate (Dawson, et al., 1989). The lysosomal pH is sufficiently acidic (pH 5) to effect the release of a tethered agent, where the linkage is, for example, a carbonyl hydrazone group. Disulfide exchange releases toxins from immunoconjugates.
Preparation of low- (MW < 5000) and intermediate-molecularweight (MO 10,000 to 18,000) and high-molecular weight (20,000 to 100,000) fractions of soluble β-glucans is described in Example 6. Briefly, the method consists of alkylation of native β-glucans with ClCH2CH2OH in aqueous alkali, followed by size-exclusion fractionation and sterilization, to give soluble, pyrogen-free hydroxyethylated β-glucans of various molecular weights. Fractions of molecular weight less than 5,000 and 5,000 to 150,000 were fluoresceinated (deBelder and Granoth, 1973) with 5'-[(4,6-dichlorotriazin-2-yl)amino]fluorescein and evaluated against commercially available fluoresceinated dextrans (fl-dextran) (MW 4,000 and 70,000) for uptake in adherent mouse peritoneal macrophages (Example 7).
Exposed cells were observed by fluorescence microscopy. FI-dextran exhibited diffuse homogenous fluorescence in exposed cells, whereas FI-β-glucan showed a large number of fluorescent aggregates, suggesting different association mechanisms for the two polysaccharides. FI-β-glucan associated with mouse macrophages more strongly than did FI-dextran, especially in the presence of NaN3, which permits receptor binding but prevents internalization. These results show that FI-β-glucan was surface bound and internalized by a different mechanism than dextran.
F-Iβ-glucan was prevented from binding to its macrophage receptor by excess laminaribiose, a β-1,3-glucopyranose receptor competitor (Williams, et al., 1988) confirming the identity of this receptor. Both adherent (activated) mouse macrophages and suspended (non-activated) human pro-monocytic cells were incubated with FI-β-glucan overnight to permit binding and uptake and then exposed to unlabeled β-glucan to permit displacement of label. There was no displacement of label, indicating that FI-β-glucan was internalized. Experiments performed with freshly isolated human macrophages yielded similar results. These results demonstrate the specific targeting properties of β-glucans.
Following here are exemplary methods for forming β-glucan/drug conjugates, as well as methods for testing the uptake and therapeutic efficacy of such conjugates. Embodiments of several conjugates are summarized in the Table 11 below.
Figure imgf000035_0001
1 . Synthetic Methods
(a) Anti-viral Drugs. An exemplary class of anti- viral drugs, for delivery to macrophage, are the dideoxynucleosides (DDN), including, dideoxycytosine (DDC), (Yarchoan, et al., 1988a; Yarchoan, et al., 1988b; Yarchoan, et al., 1989) AZT, (Yarchoan, et al., 1988a; Yarchoan, et al., 1988b; Yarchoan, et al., 1989) and 2',3'-dideoxyinosine (DDI) (Ahluwalia, et al., 1987). These drugs inhibit HIV reverse transcriptase (RT) and cause oligodeoxynucleotide chain termination after phosphorylation to the 5'-triphosphate in the cell (Perno, et al., 1988a; Perno, et al., 1988b; Perno, et al., 1989; Ahluwalia, et al., 1987; Kelley, et al.,
1987) and therefore inhibit acute HIV infection. These agents are currently in clinical use. The drugs, however, have undesirable side effects that may be reduced by targeted drug delivery to the macrophage/monocytes.
Synthesis of exemplary β-glucan/drug conjugates follows here and is illustrated in Figs. 9A and 9B, 1A-B, and 2A-D. Tether functionality for the DDN family is typically introduced onto the 5'-position of the DDNs using a 5'-phosphate ester, prior to glucan coupling. Phosphoramidite chemistry may be used to introduce tether groups on dideoxycytosine, which requires protection of N4 as the benzoyi group. Dideoxyinosine may be similarly treated, using gentle conditions to avoid depurination. Due to the susceptibility of the azide group of AZT to reduction by phosphoramidite reagents, AZT is first converted to the 5'-phosphate (Yoshikawa, and Kato, 1969; Goldman, 1988a; Goldman, 1988b) which is then coupled to the appropriate alcohol using a carbodiimide (Ho, et al., 1981 ).
Once internalized in the cell and transported to the lysosomal vesicles, the phosphate esters are cleaved by phosphatases to release the drugs. This type of linkage has been successfully employed for the preparation of conjugates of DDC-5'-(6-aminohexyl)phosphate (Dawson, et al.. 1989) and ribavirin-5'-(5-carboxypentyl)phosphate (Dawson, et al., 1989). Hydrolysis studies on ribavirin-5'-succinate having carboxylic ester linkages indicated that this linkage was too labile at physiological pH, whereas the more hindered 2,3-dimethyl-succinate was too stable
(Dawson, et al., 1989). In contrast, the 5'-phosphate ester of DDC was stable to hydrolysis at pH 7.2 but was metabolized intra-cellularly to release drug.
Alkyl groups, such as pentamethylene or hexamethylene, can be used as a spacer bridge on the tether. If cleavage using this type of spacer proceeds too slowly, bond lability can be enhanced by incorporation of a hydroxyl group on the β-carbon adjacent to the phosphate (e.g., -OP(O)(O-)CH2CH(OH)-) (Pallesen, et al. , 1987). Hydrolytic susceptibility in the acidic environment of the lysosome may also be enhanced by replacement of the phosphate oxygen by nitrogen [-OP(O)(O-)NHCH2-] (Chu, et al., 1983).
Figs. 9A and 9B present exemplary tether syntheses and introduction of tethers onto dideoxynucleosides, respectively. The preparation of reactive tether precursors T1 , T2, and T3 is shown in Fig. 9A. Synthetic approaches for attaching various tether groups (e.g., exemplary tethers T1 , T2, T3, and T4) to N4-protected dideoxycytosine is illustrated in Fig. 9B. Each of the tethers possesses a reactive terminal group for glucan coupling.
Linkage of dideoxynucleosides bearing functionalized tether groups to derivatized β-glucans may be effected, for example, by means of (i) amide (e.g.. (PS)-NHCO(CH2)5OP(O)(O-)-5'-O-DDN), (ii) hydrazone (e.g.. (PS)-CR = NNHCO(CH2)5OP(O)(O-)-5'-O-DDN), or (Hi) carbamate bonds (e.g., (PS)-OCONH(CH6)6OP(O)(O-)-5'-O-DDN), as will be described below.
Amide-linked DDN-tether/glucan conjugates can be formed by coupling the amino groups of an amino-derivatized β-glucan with the carboxyl groups of a tethered DDN using either a water-soluble carbodiimide or, more efficiently, the activated N-hydroxysuccinimide ester of the carboxyl group. Cleavage of the amide bonds may then be effected in vivo by the action of proteases.
Hydrazone bonds can be formed by reaction of the formylmethyl or ring ketone groups on the β-glucan with the carbonyl hydrazide on the tether. This group is readily hydrolyzed in the pH 5 environment of the lysosomes, but relatively stable at physiological pH (Lauzza, et al., 1989).
Carbamate-linked DDN-tether/glucan conjugates may. for example, be formed by reaction of the carbonyl imidazolide groups of the β-glucans with the amines on the tethered DDNs. This group is stable at pH 5-7 (Bethell, et al., 1981 ). Loading of the DDNs on the β-glucans is established by UV spectroscopy.
(b) Anti-Sense Compounds. Anti-sense compounds are potentially useful in a variety of applications, for example, as anti-virals. Anti-sense oligodeoxynucleotides and their more nuclease-resistant methylphosphonate and phosphorothioate analogs are reported to inhibit HIV infection in cells (Matsukura, et al., 1989, 1987; Agrawal, et al., 1988; Sarin, et al., 1988). Therapy using anti-sense compounds is limited in one respect by the cost of the anti-sense compounds. Targeting may enhance cellular absorption, thereby reducing the therapeutic concentration required.
Synthesis of an exemplary β-glucan/anti-sense conjugate follows here. Phosphorothioate oligonucleotides are more lipophilic than their phosphate analogs and may require coupling to the carrier in organic-aqueous medium rather than aqueous medium. Derivatization of β-glucans may require partial 6-O-tritylation to achieve sufficient solubility in the reaction medium. The phosphorothioates can be linked to the carriers using disulfide and hydrazone bonds as illustrated in Fig. 1 B). The former has been used to link oligonucleotides and momorcharin to antibodies.
A carbamate linkage is used to evaluate whether a stable conjugate would display antisense activity. As illustrated in Fig. 1 A, phosphorothiate anti-sense oligonucleotides having, for example, an anti-REV HIV sequence (S-α-rev) can be prepared by automated synthesis using phosphoramidite chemistry and automated introduction of sulfur using the sulfur-transfer reagent 3H-1,2-benzothiol-3-one-1 ,1-dioxide 75 (Beaucage, et al., 1989). Using this strategy, commercially available, protected phosphoramidite tethers can be employed.
If a tritylated thiol-terminated tether cannot be successfully deprotected using Ag(l) in the presence of phosphorothioate groups, a thiol terminus is added by reaction of an amine-terminated S-α-rev with
N-3-(2-pyridyldithio)propionyloxysuccinimide (SPDP) (Dillman, et al.,
1988). The free thiol group on the tether of S-α-rev is allowed to react with the 2-pyridyldithiopropionyl groups on the derivatized glucan.
Unreacted 2-pyridyldithiopropionyl groups are removed by reaction with mercaptoethanol.
The inverse reaction using free thiol groups on the glucan is less desirable because cross-linking may occur. Loading of S-α-rev is established by UV analysis. (c) Protein Agents. A number of polypeptide agents have been identified that are useful therapeutic compounds, for example, the ribosome inhibitory proteins (RIPs) trichosanthin and momorcharin (Kubota, et al., 1986). Selectivity of such proteins may be enhanced by cell-type specific targeting. Such targeting may also reduce their systemic side effects.
Syntheses of exemplary β-glucan/polypeptide conjugates follow here and is illustrated in Figs. 2A- 2D. Trichosanthin and momorcharin (U.S. Patent No. 4, 869,903) may be tethered to β-glucans using either carbonyl hydrazone and/or disulfide bonds (Figs. 2D, 2B, respectively) to ensure release of the protein within the target cell.
Trichosanthin, which is not glycosylated, is functionalized with 2-iminothiolane 77 rather than SPDP to maximize protein activity (Lambert, et al., 1985), as shown in Fig. 2A. The glycosylated protein momorcharin is converted to a reactive aldehyde by periodate oxidation and then functionalized by a hydrazone linkage (Fig. 2A). Loading is established using fluoresceinated β-glucans. (d) Biological Evaluations of β-Glucan/Agent
Conjugates. Each conjugate is evaluated for its ability to bind to the surface of the target cell, be transported inside, and release the therapeutic moiety. The ability of the conjugates to control pathogen production, such as HIV production, is compared with that of unbound drug. The characteristic of the conjugates are evaluated as follow: (i) binding of candidates to the macrophage surface, (ii) efficiency of internalization of targeting moieties into macrophage cytoplasm, (iii) effects of β-glucans on cytokine production and HIV-1 infection (or development of other intracellular pathogen), (iv) anti-HIV activity in de novo infection, and (v) anti-HIV activity of the bioactive agent in chronic infection.
(i) Binding of β-Glucans and Conjugates to Macrophages. Binding studies are conducted using the human promonocytic cell line U937, which is maintained in culture and which can be infected with HIV. The studies will be extended to fresh human macro phages. Fluoresceinated β-glucan conjugates of various molecular sizes are added to cell suspensions and to adherent fractions of human cells. The cells are examined by fluorescence microscopy to determine preliminary binding affinity (Lifson,, et al. , 1986).
Exposure (15 min to 24 h) of macrophages to β-glucan conjugates at 4°C in the presence of NaN3 is used to establish binding, which is quantitated by flow cytometric analysis. Applying this procedure to the cell line and then normal human macrophages allows the optimization binding affinity for various conjugates (including the same agent conjugated to β-glucans of differing molecular weight). This allows identification of molecular weight ranges that allow selection of optimum molecular size for targeting conjugates to human cells.
(ii) β-Glucans and Conjugate Internalization bv Macroohaoes. Studies are first conducted using transformed human macrophage cell line U937 and are then extended to normal human macrophages to confirm internalization behavior. Optimum β-glucan sizes to effect macrophage binding and targeting are determined. β-glucans can be tested that consist of two or greater repeating monomer units, and typically, five or more repeating monomer units.
Internalization of fluoresceinated β-glucan conjugates is followed by using an ACAS 570 laser scanning fluorescence microscope. After binding of the conjugates to the cells, at saturating concentrations of β-glucan conjugates that exhibit the highest binding affinity at 4°C, exposed cells are incubated in the absence of azide at 37°C to allow internalization.
Internalization is monitored from 1 5 minutes to 24 hours, after adding 100-fold excess of unlabeled β-glucan to displace surface label. The internalized fluoresceinated β-glucan is measured with the ACAS 570. The results of these screening studies establishes optimum size ranges for uptake of β-glucan conjugates. Because uptake by virally-infected cells may be altered, these results are confirmed in HIV-1 infected monocyte/macrophage. (iii) Effects of β-glucan Conjugates on Cytokine Production and HIV-1 Infection. In vitro evaluation of the conjugates is performed using HI V-1-infected monocyte/macrophage. The DDN conjugates are assayed for the ability to inhibit de novo infection in monocyte/macrophage, whereas the trichosanthin and momorcharin conjugates are evaluated for their ability to inhibit viral production in chronic infection. The antisense phosphorothioates are tested in both assays. Controls consist of free drug, a mixture of drug and β-glucans, and β-glucans alone.
Anti-HIV-1 activity of the conjugates is determined against HIV-1 isolates (i) HTLV-IIIBa-L, a dual tropic isolate recovered from primary cultures of lung tissue from a child having AIDS (Gartner, et al., 1986;
provided by Primate Research Institute, New Mexico State University), and (ii) isolate DV (Genelabs, Inc., isolate). These viral isolates are grown in normal human monocyte/macrophage or phytohemagglutinin (PHA) activated CD4-positive T cells.
Monocyte-enriched populations are prepared from peripheral blood mononuclear leukocytes (PBL) of either HIV-1-seronegative healthy donors (for de novo in vitro-induced chronic infection studies) or donors known to be infected with HIV-1 (for in vivo chronic infection studies) (Ganelig-Meyling, et al., 1980). CD4-positive lymphocytes are separated from PBL of HIV-1 seronegative individuals using a previously described procedure (Gowda, et al. , 1989). At 72 hours prior to infection with HIV-1 , or at co-cultivation with infected monocyte/macrophage in the syncytia assay, CD4-positive cells are activated with PHA (Gowda, et al., 1989). The uninfected CD4-positive lymphoblastoid T-cell line VB (Stein, et al., 1987) can also be employed in the syncytia formation assay.
To evaluate HIV-1 infection and for viral detection, monocyte/macrophage are infected with HIV-1 immediately after separation or after 5-14 days in culture. Initially, HIV-1 production is assessed by: (1 ) measurement of HIV-1 p24 antigen by ELISA, (2) TCID50, and/or (3) syncytia formation. Conjugates demonstrating anti-HIV-1 activity in the above assays are also screened for their ability to inhibit HIV reverse transcriptase (RT) (Smith, et al. , 1987). The presence of HIV-1-specific antigen, viral infectivity, and/or syncytia formation is evidence of infection. RT activity confirms the ability of the virus to replicate. These bioassays allow the monitoring of effectiveness and mode of action of the conjugates.
The ability of the conjugates to induce cytokine production in monocyte/macrophage is evaluated as follows. Monocyte/macrophage derived from HIV-1 seronegative individuals are cultured with various concentrations of low- and intermediate-molecular-weight conjugates. Untreated cells, as well as cells treated with lipopolysaccharide (5 μg/ml), serve as controls. After 24 and 48 hours of stimulation, the levels of GM-CSF, as well as TNF-α and IL-1 in these cultures is determined by
ELISA (Genzyme, and Research and Diagnostic Systems, respectively).
Next, the effects of β-glucans alone on cytokine and viral production in HIV-1 infected monocyte/macrophage is examined using these procedures. To determine whether alteration of HIV-1 production is mediated by a given cytokine, polyclonal neutralizing antibodies to human
GM-CSGF, M-CSF, and TNF-α (Genzyme) are added to cultures.
(iv) Effects of Conjugates on Primary In Vitro HIV-1 Infection. The kinetics of HIV-1 production in freshly isolated and cultured monocyte/macrophage is established after exposure of the cells to various concentrations of HIV-1 isolates using the above described procedures. Viral replication in HIV-1-exposed cultures is followed for 28 days by examining cell and supernatant samples collected weekly for TCID50, RT activity, and/or p24 content. In addition, HIV-1-infected monocyte/macrophage, as well as co-cultures of monocyte/macrophage and VB cells, are monitored for syncytia formation.
Monocyte/macrophages isolated from the peripheral blood of HIV-1 seronegative healthy donors are pre-exposed to various concentrations of the DDN or anti-sense conjugates for a minimum of 30 min. Drug-treated cells are then exposed to HIV-1 without washing out the conjugates.
Cells are infected with a dose of virus that generally yielded maximum infection of monocyte/macrophages in the experiments described above. At 2 h after incubation with viral inoculum, cells are washed to remove excess virus and cultured in the absence, or continuous presence, of conjugate. Appropriate controls include (1 ) HIV-1-infected cells without con jugate pretreatment, (2) HIV-1-infected cells pre-treated with carrier only, (3) HIV-1-infected cells pre-treated with the unconjugated drug, (4) HIV-1 -infected cells treated with a mixture of carrier and drug, and (5) uninfected cells (cells mock-infected with a cell-free supernatant from uninfected cultures).
Cultures are monitored for 28 days by examining (1 ) supernatants for levels of p24 by ELISA, and for infectious virus by RT activity and TCID50, (2) cell lysates for cell-associated p24 antigen, (3) monocyte/macrophages for formation of syncytia with VB cells, and (4) immunofluorescence analysis for the proportion of infected cells. To establish that the antiviral effect of the conjugates is not an artifact of drug toxicity, cell viability is evaluated at various intervals in conjugate-exposed HIV-1-uninfected monocyte/macrophages cultures.
Because monocyte/macrophage from different donors may vary as to permissiveness for HIV-1 replication, and because different HIV-1 isolates may exhibit different levels of susceptibility to the drugs, the results obtained above are confirmed by screening monocyte/macrophage from a number of blood donors as well as DV isolate. (v) Effect of Conjugates on Chronic HIV-1
Infection. Monocyte/macrophages isolated from HIV-1 seropositive patients are treated with the anti-sense or RIP-conjugate, free antisense or RIP, β-glucans, or a combination of β-glucans and drug, as described above. Sham-treated cultures serve as controls in these studies. HIV-1 replication in these cultures is measured as described in the previous section.
Alternatively, the antiviral activity of the conjugates on in vitro HIV-1-infected monocyte/macrophage is examined. Monocyte/macrophages isolated from PBL of HIV-1 seronegative healthy donors is infected with different HIV-1 isolates, using the acute infection protocol.
After the extent of infection is established, cells are exposed to the conjugates as described above.
The efficacy of selected conjugates can be determined using in vitro assays. Simple in vivo studies of the pharmacokinetics and the toxicity of the conjugates demonstrating significant activity are performed in mice. The distribution of radiolabeled conjugates╌ 125l-labeled protein and 14C-labeled β-glucans, drug, or β-glucans╌ is determined in Balb/c mice.
Pharmacokinetic studies involve determining the clearance rate of β-glucans or conjugate from blood, as well as distribution volume. Metabolic studies focus on determining stability following in vitro intravenous administration. HPLC analysis indicates whether the labeled drug is found in the size fraction corresponding to the intact conjugate or whether significant quantities of free drug are released into the circulation.
The acute toxicity study can also be conducted in adult mice (five of each sex per dose). Mice typically receive an ip injection of conjugate, drug, or β-glucans in saline, while the control receives saline alone.
Animals are monitored visually for toxicity and weighed daily for ten days.
The above teachings allow the development of new chemical strategies for selective drug targeting to the macrophage. In view of the above guidance, other drugs of interest can be formulated into the conjugates of the present invention and the therapeutic efficacy of such conjugates tested using appropriate assays. Candidate conjugates useful for therapeutic applications (e.g. , in humans or animals) can be identified by the methods described above. Exemplary targets for conjugate drug therapies, using the methods of the present invention, include anti-viral agents, anti-bacterial agents (including anti-mycobacterial agents), anti-fungal agents, anti-parasite agents, anti-malaria agents and anti-cancer agents.
In one embodiment, conjugates of the present invention can be used for the treatment of mycobacterial infection, including infection by Mycobacterim avium or Mycobacterium tuberculosis (Example 14). One preferred class of compounds for treating mycobacterial infection using the glucan conjugates of the present invention are the diaminoquinazolines. Some drugs useful to generate conjugates for the treatment of mycobacterial infection include the following: isoniazid (Example 13), pyrimethamine, trimethyoprim, trimetrexate, methotrexate, amalcacin and rifampicin.
The conjugates of the present invention may have one of three favorable activity profiles leading to an improved therapeutic index: (1 ) higher potency and comparable toxicity, (2) comparable potency and lower toxicity, or (3) both higher potency and lower toxicity. Decreasing toxicity while maintaining potency may permit more extended therapeutic regimens than now possible.
Materials and Methods
C57BL/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME) or Charles River Laboratories (Willmington, MA). Female BASL/c mice were obtained from the same sources. Euglena gracilis strain z was obtained from the American Type Culture Collection, No. E12716
(Rockville, ME). Chemical reagents were typically obtained from Aldrich Chemical Co. (Milwaukee, WI) or Sigma Chemical Co. (St. Louis, MO). Methods for introducing reactive groups onto glucan molecules may be found in the following: i) Kishida, ii) Bogwald, iii) Miron, iv) Nathan, v) Hurwitz, vi) Mazid, vii) Chang, viii) Bethell (a), (b), and ix) McKelvy.
Standard methods for preparing drug conjugates may be found in i) Lindstrom ii) Apelgren; and iii) Kaneko. Standard methodologies for performing various immunological assays, such as ELISA, may be found in Harlow, et al.
Example 1
Purification of Glucan
A. Cultivation of Euglena gracilus
A 100-ml inoculum of Euglena gracilus strain z was prepared from a culture grown in the dark at 25-27°C, pH 3.5-4.0, with mixing
(120-200 rpm) for 72 hours. A growth medium having the following composition was employed.
Figure imgf000046_0001
The 100-ml inoculum was added to 1 liter of medium in a flask fitted with a stirrer and an aerator. The culture was maintained in the dark at 25-27°C for 72-96 hr. The pH was maintained between 3.0-4.5 and adjusted with sodium hydroxide as necessary. Stirring was maintained at about 175 rpm (120-200 rpm range). The culture was aerated with air and/or pure oxygen at an average flow rate of 1 L/min (0.5-2.3 L/min range) to provide a dissolved oxygen concentration between 8-40 ppm.
B. Purification
All purification procedures were conducted under aseptic conditions. Cells were harvested by centrifuging the culture at 3000-4000 rpm for 5-10 min. The cell mass was then washed with pyrogen-free water and recentrifuged. Optionally, the cells may be sonicated to disrupt the cell wall. The water-washed solids were then placed in a flask equipped with a stirrer and an equal volume of methanol was added. The mixture was heated at 65°C for 5-10 minutes with stirring and allowed to cool. Two volumes of chloroform were then added and the mixture was stirred for an additional 20-30 min at ambient temperature. The cell mass was then vacuum-filtered and washed with 2-3 additional volumes of chloroform. The filtered cell mass was transferred to a flask and an equal volume of methanol and twice this volume of chloroform were added. This mixture was stirred at ambient temperature for 30-60 min and then vacuum filtered as above. The filtered mass was then taken up in a sufficient volume of 1 N HCl to provide a relatively dilute suspension. This mixture was boiled until no solvent odor was detectable, followed by heating for an additional two hours. The solids were then centrifuged and washed repeatedly with water to provide substantially pure β-1,3-glucan in the form of a white crystalline solid. This material was stored at 4°C pending use.
Analysis of the elemental composition of several batches of β-1 ,3-glucan illustrated that the purification procedure results in preparations that are consistent in composition and possess the properties described in Table 2.
Figure imgf000047_0001
Example 2
Survival of Algal Glucan-Treated Mice
Challenged with L. monocytogenes
Groups of 10 C57BL/6 mice were injected intravenously (iv) with various doses of pyrogen-free algal glucan from E. gracilis suspended in pyrogen-free saline on Days -3, -2, and -1 . On Day 0, all mice were challenged intraperitoneally (ip) with 1-6 LD50 of viable Listeria monocytogenes and mortality was monitored for 40 days. In experiments 1 and 2, the mice were challenged with 1 LD50 of Listeria. In experiment 3, mice were challenged with 6 LD50 to examine the resistance of mice to large infectious challenge. To control for the particulate nature of algal glucan, one additional group of mice was injected with a cellulose preparation ("AVICEL", FMC Corporation, Rockland, MD), similar in size to algal glucan particles but composed of glucose molecules linked in a β-1 ,4 configuration. The results are shown in Table 3.
Figure imgf000048_0001
Animals treated with β-1 ,3-glucan prior to the challenge exhibited a survivial rate of 70%. In contrast, none of the mice treated with saline survived. The high mortality rate (100%) of the animals pre-treated with "AVICEL" indicated that the action of the β-1 ,3-glucan was not solely due to its particulate nature and suggests that administration of the glucan enhanced the ability of mice to resist the Listeria challenge.
Example 3
β-1 ,3-Glucan Toxicity
Acute toxicity experiments were performed to evaluate the impact of large intraperitoneally-administered doses of β-1 ,3-glucan from E.
gracilis on the general health of normal mice. In a first study, C57B1 /6 male mice (Jackson Laboratories or Charles River Laboratories) received a single ip injection of varying doses of the glucan. Control mice received an ip injection of saline. Following glucan injection, all animals were visually monitored for signs of poor health (ungroomed coat, lethargy) and weighed on 10 consecutive days. Injections of up to 2.5 g/kg of algal glucan (approximately 50 mg per mouse) did not adversely impact the health of the animals. The subject mice gained weight at a rate equal to that of control animals and were as physically active as control mice.
In a second study, suspensions of the β-1 ,3-glucan were administered intraperitoneally to male mice (Jackson or Charles River Laboratories) in single doses of either 50, 500, or 5000 mg/kg or in doses of 15, 150, or 1500 mg/kg on 8 consecutive days. All mice survived and showed no significant signs of ill health. On day 10, necropsy was performed to determine the effects of the β-1 ,3-glucan. Minor visceral adhesion was observed macroscopically.
The lack of significant toxicity of β-1 ,3-glucan demonstrates a broad range of useful β-1 ,3-glucan concentrations for therapeutic use.
Example 4
Effect of β-1 ,3-Glucan Treatments on the Survival of C57BL/6 Mice Inoculated with B16 Melanoma Cells
The effect of β-1 ,3-glucan from E. gracilis on the survival time of tumor-bearing mice was examined in a syngeneic tumor system using short-term and long-term treatment schedules.
To evaluate short term treatment ("A" in col 2 of Table 4, below), ten C57BL/6 mice per group were injected subcutaneously on the rear flank on day 0 with 1 × 105 B16BL6 melanoma cells. Each group received its respective β-1 ,3-glucan (in 0.5 ml pyrogen-free saline) or saline treatment on days 1 , 4, 7, 10 and 13 by intravenous injection.
To evalutate long term treatment ("B" in col 2, Table 4 below) with β-1 ,3-glucan, groups of ten C57BL/6 mice were injected subcutaneously on the rear flank on day 0 with 1 × 105 B16 melanoma cells. Each group then received its respective β-1 ,3-glucan or saline treatment on days 1 , 4, 7, 10, 13, 16, 19, 22, 25 and 28 by intravenous injection in 0.5 ml pyrogen-free saline.
Figure imgf000050_0001
A consistently significant increase in survival time was observed for animals treated with 0.5 mg of particulate β-1 ,3-glucan. More variable responses were seen with lower doses of particulate β-1 ,3-glucan. Example 5
Induction of Colony-Stimulating Activity In Vivo Groups of five male mice (Jackson or Charles River Laboratories) were injected intravenously with either 5 mg or 10 mg of β-1,3 glucan from E. gracilus. One control group of five mice was injected with saline. A second group of mice was injected with lipopolysaccharide (LPS) 25 μg i.v. to provide a positive control. At various times after injection (2h, 6h, and 24 h), sera were collected from the mice and assayed for bone marrow colony-stimilating activity (CSA).
Figure imgf000050_0002
Colony-stimulating activity was detected in the sera of mice as early as 2 hours after a single injection of 2.5 or 5.0 mg of the β-1,3-glucan. Elevated serum CSA activity was still detectable 24 hours after glucan administration and indicates the enhancement of colony-stimulating activity by administration of glucan.
Example 6
Preparation of Soluble Glucan Derivatives
Lower molecular weight glucan materials, soluble under physiological conditions, were prepared as follows.
A. Preparation of olvcerated glucan (o-glucan)
Glycerated glucan (g-glucan) was prepared by treatment of glucan particles with epichlorohydrin and concentrated sodium hydroxide, followed by addition of concentrated sodium hydroxide until the epoxide level was no longer detectable by the thiocyanate method. After neutralization to pH 7 with hydrochloric acid, the salts were removed by dialysis and the g-glucan dried by lyophilization. B. Preparation of fluoresceinated g-glucan
The pH of a stirred, aqueous solution of glycerated glucan (24.4 mg) in 2 ml of H2O was raised to pH 10-11 by addition of a 0.02 M solution of potassium hydroxide, to which was added in a portionwise fashion, 5'-[(4,6-dichlorotriazin-2-yl)amino]fluorescein (fl) (5 mg), over a three hour period. Over the course of the addition, the pH was maintained between 10-11 by addition of 0.02 M potassium hydroxide at 30 minute intervals. Upon completion of the addition of the fluorescein reagent, the reaction mixture was stirred for an additional hour, followed by stirring at 4° C overnight. The reaction mixure was purified by chromatographic passage through a G-25 Sephadex column, using phosphate-buffered saline as the eluent. The void volume was collected and concentrated though a 30,000 molecular weight cutoff membrane. The concentrate was then re-chromatographed and concentrated to a mass of 1 .12 grams. The isolated material was filtered through a 0.45-μ filter and washed with phosphate-buffered saline. The aqueous product solution (1 .36 g) was determined to be 3.39 × 10-4 M in fluorescein by ultraviolet spectroscopy (493 nm) in 25 mM sodium borate buffer, pH 9.
C. Preparation of hydroxyethylated glucan (he-glucan)
Particulate glucan derived from Euglena (as in Example 1 ) was dissolved in 5 N sodium hydroxide, followed by treatment with chloroethanol to form soluble derivatives of the linear glucan starting material. The resulting solution was neutralized with dilute HCl, followed by exhaustive dialysis against filtered water using a 3,000- to 5,000-D cutoff tubing. The soluble derivatized glucan fractions having varying molecular weight ranges were separated by column gel permeation chromatography (GPC). The molecular weights of each of the collected fractions were determined by HPLC GPC. Three differing molecular weight populations were collected: glucan-SH (soluble, high molecular weight, ~ 1 50,000 D), glucan-SM (soluble, medium weight, ~ 18,000 D), and glucan-SL (soluble, low weight material, 3,000 D). Each fraction was passed through a Pierce Detoxi-Gel pyrogen-chelating column prior to use to ensure that the samples remained pyrogen-free. D. Preparation of fluoresceinated he-glucan
Fluoresceinated hydroxyethylated glucan (fl-he glucan) was prepared in an analogous fashion to Example 6B above.
Example 7
Specificity of β-1 ,3-Glucan in Targeting
to Murine Macrophages
A. Binding Specificity of Particulate Glucan
Mouse peritoneal macrophages were plated onto coverslips and treated for 10 min at 25°C with various soluble polysaccharide binding inhibitor solutions, as shown in column 1 , Table 6. Particulate native β- 1 ,3-glucan was then added to each of the polysaccharide-treated macrophage plates and incubation was continued for 15 minutes. The plates were then washed vigorously. The percentage of cells on duplicate coverslips having at least two surface-associated native β-1,3-glucan particles was determined microscopically and the corresponding binding inhibition values determined relative to non-preincubated macrophage cells. The results are presented in Table 6.
Figure imgf000053_0001
As illustrated above, native particulate glucan binds to the surface of macrophage cells. Nearly 80 percent of the mouse macrophage cells contained at least two-surface associated glucan particles. The binding inhibition studies indicate the binding of particulate glucan to the macrophage surface at a specific β-glucopyranose receptor. Binding was inhibited in a dose-dependent manner by the soluble yeast glucan, HE-glucan (IC50= 10μg/ml). However, concentrations over one log unit higher of the putative binding inhibitors, dextran, methylcellulose, and chitopentose, only partially inhibited binding. The inhibitors contain glucopyranose linkages different from those of β-1 ,3-glucan, and the partial inhibition by these compounds suggests the receptor specificity of particulate glucan binding to the macrophage surface.
B. Uptake of Soluble HE-Glucan in Adherent Mouse Peritoneal Macrophages
Uptake of hydroxyethylated glucan (he-glucan; Example 6) was determined by fluorescence microscopy. Hydroxyethylated glucan fractions with molecular weights less than 5000 and between 5,000-150,000 were fluoresceinated with 5'-[(4,6-dichloro-triazin-2-yl)aminolfluorescein (fl-he glucan) as in Example 6D above. The fluoresceinated glucan fractions were evaluated against commercially available fluoresceinated dextrans (fl-dextran, MW 4,000 and 70,000) for uptake in adherent mouse peritoneal macrophages. Exposed cells were observed by fluorescence microscopy. FI-dextran exhibited diffuse homogeneous fluorescence in exposed cells, whereas fl-HE glucan showed numerous fluorescent aggregates. In other experiments, FI-he glucan also bound to the surface of a nonactivated human promonocytic cell line U937 in the presence of azide.
Figure imgf000054_0001
fl-he glucan was observed to associate more strongly with mouse macrophages than fl-dextran, particularly in the presence of sodium azide
(sodium azide permits receptor binding but prevents internalization). The difference in visual appearance of fl-dextran versus fl-he glucan, especially in the presence of azide, suggests a difference association mechanism for the two polysaccharides.
C. Label Displacement From fl-HE Glucan
The label was displaced from the cell surface by excess laminaribiose, a β-1 ,3-glucopyranose receptor competitor, confirming the identity of this receptor. Both adherent (activated) mouse macrophages and suspended (nonactivated) human promonocytic cells (cell line 4937, from
ATCC) were incubated with fl-he glucan overnight to permit binding and uptake and were then exposed to unlabeled HE glucan for 2 hr at 37°C to permit displacement of label. Labeled fl-he glucan was not displaced, indicating that fl-he glucan was internalized. A study with freshly isolated human macrophages yielded similar results. The above data suggest that soluble derivatives of β-1,3-glucan, such as hydroxyethylated glucan, can bind to and be internalized by both mouse and human macrophages by receptor-mediated mechanisms. Example 8
Induction of Cytokine Production by β-1 ,3-Glucan from E. gracilus
The ability of β-1,3-glucan to stimulate the release of cytokines from human monocytes was examined. A four-step discontinuous "PERCOLL" gradient was used (Marcowicz), followed by plastic adhesion to obtain monocytes from peripheral blood of healthy donors. Monocytes (1 × 106), suspended in 1 ml of RPMI (Roswell Park Memorial Institute, Roswell Park) 1640 medium containing 1 % "NUTRIDOMA" HU (HANA Biologies, Inc., Alameda, CA) and 1 % fetal calf serum were cultured for 4 to 24 hr in the presence of β-1 ,3-glucan (50 μg/ml). Control cells were kept in medium alone for the duration of culture. The levels of TNFα, IL-1β, and IL-6 in the culture supernatants were measured by ELISA (R&D System, Minneapolis, MN).
The glucan preparation contained 2 × 10-5 μg endotoxin/100μg, as determined by the Limulus amebocyte lysate test (BioWhittaker, Walkersville, MD). In these studies, LPS concentrations less than 1 × 10-4 μg/culture failed to increase cytokine production over background levels.
Figure imgf000055_0001
As illustrated in Table 8 and in Fig. 4, TNFα and IL-1β activity were detected in monocyte supernatants as early as 4 hr after stimulation with particulate β-1 ,3-glucan and peaked by 8 hr. The cultures of monocytes in medium alone led to the secretion of cytokines, but at considerably lower levels. The ability of the glucan to induce cytokine production was found to be dose dependent.
Example 9
Anti-oD2 Antibody Response for
β-1 ,3-Glucan Formulations
Three groups of female BALB/c mice (n = 3), 5 weeks of age, were immunized by intraperitoneal injection of 5 μg of herpes simplex virus glycoprotein, HSV-1 gD2, (provided by Chiron Corp., Emeryville, CA) mixed with 10, 100, or 500 μg of particulate β- 1 ,3-glucan from E. gracilus. Control mice received HSV gD2 alone. Anti-gD2 antibody titers were measured by ELISA 7 days after the third immunization. The glucan preparation used contained 0.158 Endotoxin Units/100 μg, as determined by the chromogenic Limυlυs Amebocyte Lysate assay (BioWhittaker, Walkersville, MD) (non-pyrogenic concentrations; USP rabbit test). Anti-gD2 antibody titer results are shown in Table 9.
Figure imgf000056_0001
Based on the above, the optimum dose of particulate β-1 ,3-glucan required to enhance the antibody responsiveness of BALB/c mice to gD2 was determined to be 100 μg. This optimum dosage was utilized in subsequent experiments. In a related study, BALB/c mice were immunized with three injections, 14 days apart, of 5 μg HSV gD2 alone, mixed with: (1 ) 100 μg of particulate β-1 ,3-glucan (as above), (2) 1000 μg of soluble high molecular weight glucan, (3) 1000μg of soluble medium molecular weight glucan, or (4) adsorbed onto 200 μg alum. The mice were boosted twice with an equivalent dose of the antigen formulations. Anti-gD2 titers were measured by ELISA 14 days after the first and second injections and 7 days after the third immunization. Anti-gD2 antibody titer results are shown in Table 10.
Figure imgf000057_0001
Particulate glucan was as effective as alum in inducing a strong anamnestic response to recombinant gD2. Further, the anti-gD2 antibody titer was 100-1000 fold greater in approximately half of the animals immunized by coadministration of gD2 with soluble glucan compared to that in mice immunized with gD2 alone. Example 10
Antibody Response for Rabbits Immunized with HIV-1 Envelope-Encoded Synthetic Peotide
Mixed with Particulate β-1 ,3-Glucan
from E. gracilus
Pasteurella-free, New Zealand white, female rabbits, each weighing about 2 kg and between 8-9 weeks of age, (Western Oregon Rabbit Company, Philomath, OR) were immunized with 1 mg of an HIV-1 envelope-encoded synthetic peptide (amino acid residues 254-274, having a sequence homologous to the conserved domain of HIV-1 , gp 120) (Ho, et al.) conjugated to bovine serum albumin, BSA, (0.4 mg peptide/0.6 mg BSA) mixed with 5 mg of particulate β-1,3-glucan per kilogram body weight on days 0, 14, and 28. Control rabbits were injected with HIV-1 peptide/BSA conjugate alone. Serum samples were collected 2 weeks after the last injection and the antibody titers were determined by ELISA using HIV-1 peptide conjugated to KLH as immobilized antigen. Reciprocal of serum dilution: 1250 (HIV-1 synthetic peptide/BSA + glucan); 250 (HIV-1 synthetic peptide/BSA control).
β-1 ,3-glucan was found to enhance the humoral response in rabbits as well as in mice. Rabbits immunized by coadministration of glucan and an HIV-1 envelope-encoded synthetic peptide conjugated to BSA produced specific antibodies that were higher in titers than the response exhibited by rabbits injected with the immunogen alone. Example 11
Synthesis of β-1 ,3-Glucan Conjugates
A. Preparation of a β-1 ,3-Glucan/Herpes Simplex Virus Glucoprotein D HSV gD2 Conjugate with an Acid-Labile Hydrazone Linkage
For a description of aldehyde functionalization of glucans, see
Laun. β-1 ,3-glucan from E. gracilus (200 mg) 25 was activated with 1 ,1 -carbonyldiimidazole 27 (200 mg) in dioxane (10 ml) by stirring for 30 minutes to form the correspondponding imidazolyl carbamate derivative 29. The dioxane-washed, activated glucan 29 was then treated with a 1 .3 M 4-aminobutyraldehye diethyl acetal solution 31 (0.96 ml in 0.7 M aqueous Na2CO3) and allowed to stir overnight at pH 10.2. A 2.1 M ethanolamine solution (8 ml of ethanolamine in 0.8 M Na2CO3, pH 10.2) was then added to the reaction flask and the resulting mixture was stirred for an additional one hour at room temperature. The ethanolamine treatment was then repeated. The desired O-carboxamidobutyraldehyde diethyl acetal glucan 33 was recovered and washed with six 10-ml volumes of water. The acetal protecting groups were hydrolyzed just prior to coupling the glucan to the reactive gD2 reagent by heating with 1 M HCl for five minutes to form the desired O-carboxamido-butyraldehyde derivatized glucan 35. The derivatized glucan was determined to contain about 0.12 aldehyde groups per mole of glucose, as determined by titration with hydroxylamine hydrochloride (Zhao).
Functionalization of gD2 to form the hydrazide derivative was carried out as follows. Herpes simplex virus glycoprotein 37 (gD2, 3.8 mg) was thiolated by treatment with 4 mM iminothiolane 39 in 1.9 ml of HEPES buffer for 2 h under argon. The buffer was replaced by 100 mM
Tris, pH 6.7, by gel filtration over Sephadex G-25. Ellman's test (Stewart) indicated 2 moles of thiol per mole of gD2 in the intermediate 41. This solution was concentrated to 2 mg protein per ml prior to the addition of 4-{N-maleimidomethyl)cyclohexane-1-carbonyl hydrazide 43 in 0.16 ml of DMSO, with stirring. After 2 hr, excess reagent was removed and the buffer was changed to 100 mM sodium acetate, pH 5.5, by gel filtration on Sephadex G-25. The solution was concentrated to 2 mg protein/ml.
The gD2 hydrazide 45 was coupled to the O-carboxamidobutyraldehyde derivatized glucan 35 as follows. The gD2 hydrazide 45 (2.8 mg) was added to a suspension containing the derivatized glucan 35 (10 mg, in 1.4 ml of sodium acetate buffer, pH 5.5) and the reaction stirred at ambient temperature. Conjugate formation was then monitored. Aliquots of the reaction mixture were removed, the particles were washed with PBS (pH 7.2) to remove derivatized protein, and the protein and glucan content of the particles determined by the bicinchoninic acid method
(Pierce Chemical Co., Protocol 23225x, 1991) and the anthrone reaction (Scott), respectively. At sufficient protein loading levels, the glucan conjugate particles 47 were harvested by exhaustive washing in PBS at pH 7.2, until protein was no longer detected in the washes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. Protein loadings were determined and ranged from 2.5-8.4 μg gD2/400 μg glucan. The above synthetic scheme is outlined in Figs. 3A and 3B.
B. In Vitro Stability
The gD2-glucan conjugate prepared as above was determined to be stable in vitro. During a 14 day storage period at -80°C, less than 0.1 % of the glycoprotein was released, as determined by SDS-PAGE and silver staining techniques.
C. Acid Lability of the Hydrazone Linkage
The gD2-glucan conjugate was incubated for 7 h at pH 5.0 and 7.2. Loss of protein was determined by SDS-PAGE and silver staining techniques. Hydrolysis of gD2 from the conjugate was nearly five-fold greater at pH 5.0 than at pH 7.2.
D. Antioenicitv of the gD2-Glucan Conjugate
Pooled serum (1 :20 dilution) from 3 C3H/HeJ mice immunized twice with 5 μg gD2 mixed with 100μ g of particulate β-1 ,3-glucan from E. gracilus was incubated at ambient temperature with increasing quantities of either gD2-glucan conjugate (prepared as described above, 8.4 μg gD2 per 100 μg glucan) or O-carboxamidobutyraldehyde glucan (derivatized glucan prior to coupling to activated gD2). After a 2 hour incubation period, the serum was removed by centrifugation and tested for anti-gD2 activity against gD2 by ELISA.
The gD2-glucan conjugate adsorbed comparable levels (greater than 90%) of anti-gD2 antibody activity from sera of mice immunized either by coadministration of gD2 and glucan or with the gD2-glucan conjugate. As seen from these experiments, the covalent linking of gD2 to the glucan to form the conjugate did not alter the antigenicity of gD2.
Example 12
Enhanced Adjuvant Activity of the oD2-Glucan Conjugate
Female C3H/HeJ mice, 5 per group, were immunized by intraperitoneal injection of 8.4 μg of the gD2-glucan conjugate (prepared as in Example 11) on days 0 and 14. Control mice received an identical dose of either gD2 (8.4 μg/dose) alone or mixed with particulate β-1 ,3-glucan from E. gracilus (100 μg). The placebo group received 0.2 ml of pyrogen-free phosphate buffered saline. Serum anti-gD2 antibody titers were measured 14 days after the final injection.
Mice immunized with the gD2-glucan conjugate exhibited significantly higher anti-gD2 serum antibody titers than did mice immunized with a mixture of gD2 and glucan. These results are shown in Fig. 5.
Example 13
Preparation of a Glucan-lsoniazid Conjugate
A glucan-isoniazid conjugate is prepared as described in Ex. 11. The glucan is activated by treatment with 1 ,1 '-carbonyldiimidazole, followed by reaction with 4-aminobutyraldehye diethyl acetal to form the O-carboxamidobutyraldehyde diethyl acetal-derivatized glucan. Just prior to treatment with isoniazid (4-pyridine carboxylic acid hydrazide), the acetal protecting groups are hydrolyzed to produce the active aldehydecontaining glucan. Treatment of the reactive glucan with isoniazid produces the corresponding glucan-isoniazid complex, containing labile acyl hydrazone linkages which couple the antimycobacterial agent to the glucan. The extent of drug conjugation is determined by Nitrogen Kjeldahl determination and/or acid hydrolysis, followed by HPLC determination of the released isoniazid.
Example 14
Effect of β-1 ,3-Glucan/lsoniazid Conjugate
Treatments on the Survival of C57BL/6 Mice
Inoculated with M. Tuberculosis
Ten C57BL/6 mice per group are injected
intraveneously with three different doses of the glucan-isoniazid complex (prepared as described above) in pyrogen-free saline on days -3, -2, and - 1 . One group of control mice is injected with saline. On day 0, all mice are challenged intraperitoneally with M. tuberculosis cells (1-6 LD50). The mortality of the mice is monitored for 40 days to examine the ability of mice injected with the glucan-isoniazid complex to resist the M. tubercυlosis challenge.
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001

Claims

IT IS CLAIMED: 1 . A glucan composition comprising:
a β-1 ,3-glucan covalently attached to a bioactive agent to form a glucan/agent complex, wherein said covalent attachment is hydrolyzable.
2. The composition of claim 1 , wherein said glucan is insoluble under normal physiological conditions.
3. The composition of claim 2, where said glucan is particulate in nature.
4. The composition of claim 3, where said particulate glucan is in the size range of between about 0.5 to 10 microns in size.
5. The composition of claim 4, where said particulate glucan is in the size range of between about 3 to 6 microns.
6. The composition of claim 1 , where said glucan has a linear β-1 ,3 configuration.
7. The composition of claim 1 , where said glucan is obtained from a source selected from the group consisting of algae, fungi, plant and bacteria.
8. The composition of claim 7, where said glucan is isolated from Euglena.
9. The composition of claim 1 , where said glucan is soluble under normal physiological conditions.
10. The composition of claim 9, where said glucan has at least about 5 monomer repeating units.
11. The composition of claim 9, where said glucan contains polar side chains selected from the group consisting of hydroxyethyl, carboxymethyl, 2-hydroxyethoxyethyl and dihydroxypropyl.
12. The composition of claim 1 , where said covalent attachment is selected from the group consisting of acyl hydrazone, phosphate, phosphoramidate and amide.
13. The composition of claim 12, where said covalent attachment is a hydrazone bond.
14. The composition of claim 1 , where said bioactive agent is selected from the group consisting of anti-viral agents, anti-bacterial agents, anti-cancer, anti-fungal agents, and anti-malarial agents.
15. The composition of claim 1 , where said bioactive agent is an anti-mycobacteriai agent.
16. The composition of claim 15, where said agent is a diaminoquinazoline.
17. The composition of claim 15, where said agent is selected from the group consisting of isoniazid, pyrimethamine, trimethyoprim, trimetrexate, amalcacin, rifampicin and methotrexate.
18. The composition of claim 1 , where said bioactive agent is a polypeptide.
19. The composition of claim 18, where said polypeptide is a polypeptide vaccine.
20. The composition of claim 1 , where said bioactive agent is an oligonucieotide.
21. The composition of claim 1 , where said bioactive agent is an antisense agent.
22. A method for the treatment of a pathogen capable of invading or colonizing phagocytic cells, comprising:
administering in a pharmaceutically effective amount, the composition of any of claims 1 to 21 , wherein said composition is effective to direct delivery of the bioactive agent to phagocytic cells harboring the pathogen, and
after delivery of the agent to said phagocytic cells, releasing the agent from said composition by cleavage of the hydrolyzable attachment, where said bioactive agent is effective for the treatment of said pathogen.
23. The method of claim 22, where said pathogen is a mycobacterium.
24. The method of claim 22, where said pathogen is Mycobacterium avium or Mycobacterium tuberculosis.
25. A method of delivering an antigen to a phagocytic cell, comprising:
contacting the composition of any of claims 1-15, 18 or 19, where said bioactive agent is an antigen and said complex is a glucan/antigen complex, with phagocytic cells, where said cells internalize the complex, and whereby said internalization is effective to free said antigen from the glucan/antigen complex by cleavage of the hydrolyzable attachment.
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