Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
  • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
  • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
  • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants

Definitions

• the present invention is related to novel polysaccharide derivatives, their preparation and their use in vaccines and immunostimulating compositions.
• the adjuvants are derivatives of polysaccharides recognized by antigen presenting cells (APCs).
• Adjuvants have utility in activating the immune system to increase the efficacy of preventative and therapeutic vaccines.
• Immunoadjuvants have applications in: ( 1 ) the non-specific stimulation of host resistance against infection and cancer, (2) the potentiation of preventative vaccine immunogenicity, and (3) the potentiation of therapeutic vaccine immunogenicity.
• These adjuvants may preferentially enhance cell-mediated immune responses (T cell responses, delayed hypersensitivity), humoral responses (B cell responses, antibody production), or both. Stimulation of humoral immunity is important for prevention of bacterial infections, some viral infections, as well as in therapy of circulating cancers. Cellular immunity is of major importance for solid tumor cancer therapy and some viral diseases.
• the immune system After an initial stimulation by a foreign agent or antigen (such as viruses, bacteria, or parasites), the immune system usually recognizes and reacts to the agent with an accelerated response upon re-exposure. This enhanced response forms the basis for the enormous success of vaccination for disease prevention.
• a foreign agent or antigen such as viruses, bacteria, or parasites
• the immune system After an initial stimulation by a foreign agent or antigen (such as viruses, bacteria, or parasites), the immune system usually recognizes and reacts to the agent with an accelerated response upon re-exposure. This enhanced response forms the basis for the enormous success of vaccination for disease prevention.
• the initial immune response to a foreign antigen requires several days for full response, which is insufficient for protection against infections by highly virulent organisms.
• a way to achieve a faster protective immune response is by vaccination or immunization with a pathogen, which is usually attenuated or dead.
• immunization with killed microorganisms or with pure antigens elicits a poor short term
• this poor immune response can be modified by the addition of adjuvants to the antigen preparation.
• Several polysaccharides (carbohydrate polymers) of mannose (e.g. mannans), ⁇ (l,3) glucose(e.g. glucans), ⁇ (l, 4) acetylated mannose (acemannans), ⁇ (l,4)N-acetyl- glucosamine (chitins), and heteropolysaccharides, such as rhamnogalacturonans
• Antigen presenting cells have specific cell-surface-receptors which recognize and bind the sugar moieties of these and other polysaccharides.
• Antigen presenting cells such as dendritic cells and some macrophages, are responsible for taking up antigens and processing them to small peptides in endolysosomes.
• Processed antigens are expressed on the surface of APCs in conjunction with class II MHC. Specifically, reactive T cells recognize antigen and class II MHC simultaneously, yielding immune responses that are class II MHC restricted.
• B cells are stimulated by processed antigens to produce antibodies.
• APC surface-receptors such as the macrophage mannose receptor and its homologous receptor DEC-205 from dendritic cells
• APC surface-receptors are trans-membrane proteins that mediate endocytosis and apparently play a role in the process of antigen presentation (Stahl, P.D., Current Opinion in Immunology 4:49 (1992); Jiang, W., et al., Nature 575:151 (1995)). Binding of these polysaccharides to such receptors apparently induces immunostimulation, as shown by the increase in phagocytosis, proliferative responses, release of cytokines, and other activities of the immune system. Because of this immunostimulatory activity, these polysaccharides have been proposed as vaccine adjuvants.
• Polysaccharide adj uvants exert an immunomodulating effect by modifying cytokine production, such as upregulating IL-1, and causing a moderate Thl response.
• the immune response produced by the Thl subset of CD4 + T cells induces complement fixing antibodies as well as strong, delayed-type hypersensitivity (DTH) reactions associated with ⁇ -IFN, IL-2 and IL-12.
• DTH delayed-type hypersensitivity
• Polysaccharides' effects on the native protein conformation are moderate, preserving the conformational epitopes necessary to elicit a neutralizing antibody response.
• these adjuvants cannot allow exogenous antigens to be processed via the endogenous pathway, they do not induce a cytotoxic T lymphocyte (CTL) response.
• CTL cytotoxic T lymphocyte
• APCs have cell-surface-receptors specific for certain carbohydrate moieties, the targeting and delivery to these cells of antigens associated with these sugar moieties can be significantly enhanced.
• sugar moieties in the targeting of antigen delivery is not limited to polysaccharide adjuvants.
• the modification of quillaj asaponin carbohydrate side-chains by periodic acid oxidation results in a loss of their adjuvanticity. Presumably, this results because of the loss of their targeting capacity.
• glucans can induce an anti-tumor response in mice, and have a preventive effect on acute sepsis. These effects are dependent on the glucans' molecular weight and their degree of branching.
• Mannans are other polysaccharides with adjuvant activity which presumably exert their effect after binding to the macrophage mannose cell-surface-receptor.
• conjugation of a protein antigen to mannan under oxidizing conditions resulted in a cell-mediated immune response (Apostolopoulos, V. etal , Vaccine 14: 930 (1996)).
• the present invention is directed to chemical conjugates (herein referred to as polysaccharide conjugates) that comprise (i) a polysaccharide capable of binding to the cell surface of Antigen Presenting Cells (APCs), and (ii) one or more molecules having a stable carbonyl group (i.e. an aldehyde or ketone group that is capable of reacting with amino groups to form an imine or Schiff base) wherein molecules (ii) are attached to the polysaccharide (i) through (iii) a direct covalent bond or covalently via a bifunctional linker in a manner that keeps the stable carbonyl group intact.
• APCs Antigen Presenting Cells
• the molecules having an imine-forming carbonyl group can be an aromatic or non-aromatic cyclic, aromatic or non-aromatic heterocyclic or non-cyclic compound.
• aromatic or heteroaromatic ketones and aldehydes are employed as molecules (ii).
• one or more molecules having a stable carbonyl group are covalently attached, either directly or via a bifunctional linking molecule, to non-adjuvant carbohydrate antigens to provide intrinsic adjuvanticity to the non-adjuvant carbohydrate antigens.
• the conjugation of one or more molecules having a stable carbonyl group to said carbohydrate antigens results in a product having increased efficacy of preventive vaccinations compared to the non-conj ugated carbohydrate antigens .
• the present invention is directed to enhancing the potentiation of an immune response in a mammal, comprising administering an effective amount of a polysaccharide conjugate of the present invention to enhance the immune response of a mammal to one or more antigens.
• the present invention is also directed to a method of vaccination, comprising administering one or more antigens, and a polysaccharide conjugate of the present invention.
• the present invention is also directed to pharmaceutical and veterinary compositions comprising one or more of the polysaccharide conjugates of the present invention, and one or more pharmaceutically acceptable diluents, carriers or excipients. These compositions may be employed as immunopotentiators in animals and humans.
• the present invention is also directed to vaccines comprising one or more antigens, and a polysaccharide conjugate of the present invention.
• the present invention is directed to polysaccharide conjugates, comprising:
• Cells APCs
• a stable carbonyl group i.e. an aldehyde or ketone group that is capable of reacting with amino groups to form an imine or
• Schiff base also referred to as an "imine-forming compound”
• polysaccharide (i) is attached (iii) through a direct covalent bond, or covalently via the residue of a bifunctional linker to said one or more molecules (ii).
• the compounds having the imine-forming carbonyl group can be an aromatic or non-aromatic cyclic, aromatic or non-aromatic heterocyclic or non- cyclic compounds.
• aromatic or heteroaromatic ketones and aldehydes are employed as (ii).
• polysaccharide conjugates can be represented by the Formula /:
• P is a polysaccharide that is capable of binding to the cell surface of an
• each L is independently a covalent bond, or the residue of a bifunctional linking molecule
• each I is an imine-forming molecule.
• Preferred imine-forming molecules are residues of aromatic or heteroaromatic compounds having (a) a ketone or aldehyde functionality; and (b) a second functional group that is capable of reacting with a complementary functional group present on said polysaccharide or said bifunctional linking molecule, if present; and x is greater than or equal to one.
• the value of x will be determined by the number of reactive groups that are covalently modified on the polysaccharide. A number of factors and strategies will influence the value of x, as will be more fully detailed herein.
• x will be a function of the number of reactive hydroxyl, terminal end hemiacetal, carboxyl and/or amine groups that are present on the polysaccharide. Because of the diverse molecular weight distribution of useful polysaccharides (P), the degree of modification as expressed by x is expressed as the number of imine-forming groups introduced per hundred glycosyl residues. Using this convention, the value of x can vary from 1 to more than 100, with a preferred range of from 1 to about 50 imine-forming groups per 100 glycosyl residues.
• the ratio of imine-forming molecules varies broadly depending upon the conjugation strategy employed. Control of this ratio is further described herein.
• a free hydroxyl, terminal end hemiacetal, carboxylic acid or amine group of the polysaccharide is employed to covalently link the polysaccharide (P) to either (L) or (I).
• One or more of these reactive groups that are present on the polysaccharide can be first "activated” (as further described herein) to increase the reactivity of these groups, or the polysaccharide can be reacted with an imine- forming compound having an "activated” functional group.
• An additional aspect of the invention is directed to conjugates comprising one or more compounds having a carbonyl group, wherein said compounds are covalently attached, either directly or via the residue of a bifunctional linking molecule, to non-adjuvant carbohydrate antigens to provide intrinsic adjuvanticity to the non-adjuvant carbohydrate antigens.
• the conjugation of an imine-forming molecules to said carbohydrate antigen results in a product having increased efficacy of preventive vaccinations compared to the carbohydrate antigen alone.
• Carbohydrate antigens include polysaccharides, including lipopolysaccharides and peptidoglycans from streptococci, staphylococci, and other bacteria that are used as vaccine antigens. Polysaccharides
• Polysaccharides that can be employed to form conjugates of the present invention include any polysaccharide, natural or chemically modified, that binds to cell surface receptors on APCs.
• useful polysaccharides comprise a minimum of two saccharides, preferably seven or more saccharides, and are unbranched or branched, and can have a molecular weight of from about 1000 to several million Daltons.
• Preferred polysaccharides have a molecular weight of from about 1,000 to about 500,000.
• the polysaccharides may possess chemical modifications as described herein.
• Antigen Presenting Cells or the abbreviation "APCs” for purpose of the present invention mean dendritic cells and macrophages that are responsible for taking up antigens, processing them to small peptides, and expressing them on their surface in conjunction with class II MHC for presentation to T and B cells.
• macrophages and dendritic cells have developed cell surface receptors that recognize the carbohydrate moieties from different microorganisms. These receptors play a critical role in phagocytosis as well as in pinocytosis, two processes that are involved in antigen presentation.
• polysaccharides recognized by these cell-surface-receptors would be suitable for the construction of these adjuvants because such polysaccharides provide an effective mechanism for APC targeting.
• carbohydrate sequences from bacterial, fungal, and animal origins are shared by plant polysaccharides.
• plant polysaccharides can provide a practical source of starting materials in some instances.
• these adjuvants can be prepared with either soluble or insoluble polysaccharides, the soluble forms are preferred.
• the applications of the present disclosure are in no way limited to plant polysaccharides. They can be extended to other carbohydrate-containing compounds from different sources that are recognized by APCs surface receptors. Examples of these other polysaccharides are chitins and dextrans which are of animal and bacterial origin respectively. Examples of suitable carbohydrate- containing compounds are bacterial teichoic acids and their derivatives, bacterial lipopolysaccharides, lipid A, and their derivatives. Finally, conjugation of compounds carrying imine-forming carbonyl groups to non-adjuvant carbohydrate-containing products is contemplated to be useful in providing intrinsic adjuvanticity to these products and to increase the efficacy of preventive immunizations.
• polysaccharides from streptococci, staphylococci, and other bacteria that are used as vaccine antigens.
• preferred polysaccharides that are useful in the present invention are: ⁇ -glucans; mannans; pectins, and 2-acetamido glucans.
• Derivatives, including water-soluble derivatives, of these polysaccharides are also useful. See Provisional Appl. No. 60/083,106, fully incorporated by reference herein. Preferred polysaccharides are more fully described below.
• ⁇ -Glucans have a backbone chain of (l ⁇ 3)-linked ⁇ -D- glucopyranosyl units which has ⁇ -D-glucopyranosyl units attached by (1 ⁇ 6) linkages. They are found in several sources, such as yeast, fungi, algae, and cereals. They have a broad range of molecular weights, i.e. between 5,000 to >500,000, which influence their immunomodulating properties. In general, ⁇ - glucans of high molecular weight that are relatively insoluble in water have higher biological activity. However, this lack of solubility has precluded the systemic administration of glucans.
• Soluble glucans can be prepared by one of the following procedures: i) isolation from yeast extracts
• ⁇ -glucans In ⁇ -glucans the only reducing glucosyl residue (linked at position 3) is located at the terminus of the backbone chain of (1 ⁇ 3)-linked ⁇ -D-glucosyl residues. The glucosyl residues attached by (1 ⁇ 6) linkages to the backbone chain do not have a free reducing group.
• the smallest fragment that binds to the monocyte glucan receptor is a ( 1 ⁇ 3)-linked ⁇ - glucanoheptasaccharide. However, this oligosaccharide does not have immunostimulating activity.
• Mannans are linear or branched polysaccharides formed exclusively of mannose. Mannans are found in plants, mold, bacteria and other organisms. In certain plants, linear mannans consist of ⁇ -(l ⁇ 4) linked mannosyl residues, whereas in some yeasts, the mannosyl residues are linked by ⁇ -( 1 ⁇ 2) and ⁇ -( 1 ->6) linkages.
• the mannan In the branched mannans from Saccharomyces cerevisiae (baker's yeast), the mannan consists of a ⁇ -(l ⁇ 6) linked mannopyranosyl backbone structure substituted on the O-2 atoms by side-chains of ⁇ -D-mannopyranosyl, ⁇ - D-mannopyranosyl- ⁇ -( 1 ⁇ 2)- ⁇ -D-mannopyranosyl and ⁇ -D-mannopyranosyl ⁇ -
• S. cerevisiae mannan can also be phosphorylated (Barreto-Bergter and P.A. Gorin, Adv. Carbohydr. Chem. Biochem. 41:61 (1983), Vinogradov, E., et al, Carbohydr. Res. 507:177 (1998)). Although the ability of S. cerevisiae mannans to stimulate cell-mediated immunity is questionable, they enhance the action of lipopolysaccharides in stimulating T-cell responses (Ohta, M., eta , Immunology 60:503 (1987)).
• mannans can exert their immunostimulatory effects by binding to the macrophage mannose-binding cell-surface receptors.
• Pectic polysaccharides are anti- complementary, and they may have different degrees of immunopotentiating activity (Yamada, H., et al, Planta Medica 56:182 (1990)). Oxidation of these polysaccharides with periodic acid results in a loss of anticomplementary activity -l i ⁇
• the polysaccharides showing some immunopotentiating activity and thus, being recognized by cell surface-receptors can be grouped broadly into homogalacturonans, rhamnogalacturonans, arabans, galactans, and arabinogalactans. However, not all of these compounds would have biological activity. In many cases, the activity would be dependent on structure, molecular weight, aggregation state, and other parameters.
• pectic polysaccharides are a group of sugar polymers associated with 1,4-linked ⁇ -D- galactosyluronic acid residues.
• polysaccharides may have several branched oligosaccharides linked to the backbone's galactosyluronic acid residues. From previous studies with saponins and other polysaccharides, branched oligosaccharides appear to be relevant for adjuvanticity.
• 2-Acetamido glucans chitin, murein and their derivatives: Chitinis a linear N- acetyl-D- glucosamine (NAG) polymer linked by ⁇ -( 1 ⁇ 4) linkages that has about
• NAG residues deacetylated 16 percent of its NAG residues deacetylated. It is widely distributed in nature: it has been found in the exoskeleton of arthropods and in the cell walls of fungi. This polysaccharide has chains that form extensive intermolecular hydrogen bonds, making it insoluble in water and in different organic solvents. Removal of chitin's N-acetyl groups by strong alkali treatment yields chitosan, a ⁇ -(l ⁇ 4) poly-D- glucosamine water-soluble polycation. Chitosan with 70% of its N-acetyl groups removed (deacetylated chitin), shows a significant immunostimulating activity (Azuma, I., Vaccine 70:1000 (1992)).
• chitin that may also have immune stimulatory properties.
• Water-soluble alcohol-insoluble chitodextrins composed of heptamers or larger NAG oligosaccharides have been prepared by limited acid hydrolysis (Berger, L. R., et al, Biochim. Biophys. Ada 29:522 (1958)).
• Murein the major component of bacterial cell walls, is a polysaccharide made of ⁇ -(l ⁇ 4) linked NAG, with one of the NAG units substituted at C-3 with an C actic acid group by an ether linkage to yield N-acetyl-D-muramic acid ( ⁇ AM) forming the repeating sequence ⁇ AG- ⁇ AM.
• mureins are water-soluble. In the bacterial cell wall, murein is attached to certain peptides to form a cross-linked peptido-glycan. Because of their structural similarities, chitin and murein are recognized by the enzyme lysozyme, and apparently also by receptors on the macrophage's cell surface. These structural similarities, which are also present in glycol chitin, may explain the immunostimulatory properties of chitin and some of its derivatives.
• the second element of the conjugates of the present invention is one or more molecules having a stable carbonyl group (i.e. an aldehyde or ketone group) that is capable of reacting with amino groups to form an imine or Schiff base.
• the molecules having the imine-forming carbonyl group can be an aromatic or non- aromatic (saturated or partially unsaturated) carbocycle, aromatic or non-aromatic (saturated or partially unsaturated) heterocycle or a non-cyclic, aliphatic compound that may have one or more unsaturated bonds.
• Preferred compounds include mono- and di-substituted C 6 . 10 arylaldehydes and C 6 . 10 aryl(C,. 4 )alkylaldehydes, compounds comprising an aryl group, such as phenyl or naphthyl and include a formyl or formyl(C M )alkyl substituent.
• these compounds further include one or two additional substituents such as halo, hydroxyl, C M alkyl, C,. 4 hydroxyalkyl, C M alkoxy, trifluoromethyl, or benzyloxy.
• Suitable values include benzaldehyde and naphthaldehyde, substituted by one or two of hydroxyl and halo. Examples include 2,3-, 2,4- 2,5-, and 3,4- dihydroxybenzaldehyde, 5-chloro-2-hydroxybenzaldehyde, vanillin, ethyl vanillin, naringenin, 3- and4-hydroxybenzaldehyde, and4-hydroxyphenylacetaldehyde.
• a second preferred group is hydroxyl substituted C, .4 alkyl (C 6 .
• a third preferred group includes heteroaryl aldehydes and heteroaryl ketones.
• Useful heteroaryl groups are thiophene, furan, benzothiophene, benzofuran, pyridine, quinoline, pyridazine, pyrimidine, pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, isoxazole, and oxazole, each having a keto, formyl or formyl(C,.
• furanyl, pyridyl, and indolyl aldehydes and ketones are useful heteroaryl cores.
• useful heteroaryl aldehydes and ketones include pyridoxal, 2-thiophenecarboxaldehyde, and 3-thiophenecarboxaldehyde.
• Another relatively stable group of cyclic compounds that contain imine- forming carbonyl groups are triterpenoids and steroids having a keto, formyl or formylalkyl substitution. Examples include androsterone, formyldienolone, progesterone, prednisolone, and other derivatives.
• Bifunctional linkers are well known in the art for various applications (Hermanson, G.T., Bioconjugate Techniques, Academic Press 1996). A number of bifunctional linkers can be employed to form an attachment between a suitable polysaccharide and a suitable imine-forming compound. "Residue of a bifinctional linker” refers to the structure that links a stable carbonyl compound to the polysaccharide after the terminal ends of the bifunctional linker have covalently bonded to said compound and said polysaccharide.
• Non-limiting examples of linker groups that can be used to link the stable carbonyl containing compound to the polysaccharide are alkylene diamines
• Suitable bifunctional diamine compounds include ethylenediamine, 1,3-propanediamine, 1,4-butanediamine, spermidine, 2,4-diaminobutyric acid, lysine, 3,3'-diaminodipropylamine, diaminopropionic acid, N-(2-aminoethyl)-l,3-propanediamine, 2-(4- aminophenyl)ethylamine, and similar compounds.
• one or more amino acids can be employed as the bifunctional linker molecule.
• an amino acid such as ⁇ -alanine or ⁇ -aminobutyric acid, or an oligopeptide, such as di- or tri- alanine can be employed as a suitable linking molecule.
• Preferred bifunctional linking groups include: -NH-(CH 2 ) r -NH- where r is from 2-5,
• polysaccharide imine-forming compound, linkers and ratios for each may include but are not limited to:
• the present invention is also directed to processes for the preparation of polysaccharide conjugates of the present invention.
• Structure/function studies of adjuvant polysaccharides and saponins have shown that the integrity of the carbohydrate chains' structures are critical for their adjuvanticity.
• the recognition of the carbohydrate moieties by APCs surface-receptors is essential for targeting of the cells as well as to exert their immunostimulatory effects.
• the adjuvant activity of triterpene saponins also requires an aldehyde group in the triterpenoid moiety.
• ⁇ -glucans and ⁇ -mannans are comprised of either glucosyl or mannosyl residues
• the functional groups available for chemical modifications are largely hydroxyl groups (-OH) with limited reactivity.
• the primary hydroxyl groups of glycosyl residues are more reactive than the secondary hydroxyl groups.
• the structure of a particular polysaccharide may impose certain constraints (steric or electronic) on the hydroxyl groups' reactivity. This will create a hierarchy of hydroxyl groups that could favor the production of certain dominant products under limiting reaction conditions.
• the restricted number of terminal reducing sugars in glucans and mannans provides a highly specific site for introduction of new chemical groups. This specificity makes the terminal-end glycosyl hemiacetals a preferred group for commercial production of modified polysaccharide adjuvants of the present invention.
• the chemical modifications described here can be used with soluble or insoluble glucans from different organisms. However, they are provided only as examples, not as limitations of the synthetic procedures available. Because the carbohydrate moieties' role in these new adjuvants is the targeting of APCs, the useful molecular weight range can be very broad, i.e. from about a thousand to several millions. In the present invention, soluble oligo- and polysaccharides of molecular weights ranging from about 1,000 to several hundred thousands are preferred.
• the reducing terminus of oligosaccharides provides a selective and convenient site for the direct covalent attachment of molecules with amino groups, such as bifunctional diamine compounds.
• the reductive amination procedure involves reacting the terminal reducing glycosyl residue(s) in the oligosaccharide (or polysaccharide) with a compound carrying one or more primary amino groups inthe presence of sodium cyanoborohydride (NaCNBH 3 ).
• NaCNBH 3 sodium cyanoborohydride
• the cyanoborohydride anion selectively reduces the imine or Schiff base formed by an aldehyde or ketone and an amine. Since only a small percentage of the time the terminal glucosyl hemiacetals are in their formyl or open form, the reaction may proceed at very low rate. Because the presence of both imine-forming carbonyls and primary amines in a molecule would largely result in the production of undesirable products, the additions are carried on in a two-step procedure, summarized as
• Glucan/mannan oligosaccharides or polysaccharides (1) are suspended in an appropriate solvent, such as aqueous acetonitrile, dimethylformamide (DMF), pyridine, or aqueous buffers containing a tertiary amine buffer pH 9.0, and a suitable diamine compound (2), where n is from about 2 to about 12, preferably, 2 to 4 is added in the same solvent with the pH adjusted to 9.0.
• an appropriate solvent such as aqueous acetonitrile, dimethylformamide (DMF), pyridine, or aqueous buffers containing a tertiary amine buffer pH 9.0, and a suitable diamine compound (2), where n is from about 2 to about 12, preferably, 2 to 4 is added in the same solvent with the pH adjusted to 9.0.
• Suitable bifunctional diamine compounds are ethylenediamine, 1,4- butanediamine, spermidine, 2,4-diaminobutyric acid, lysine, 3,3'- diaminodipropylamine, diaminopropionic acid, N-(2-aminoethyl)-l,3- propanediamine, 2-(4-aminophenyl)ethylamine, and similar compounds].
• the diamine compound that is added should be about 5 to 10-fold excess compared to the molar equivalent of free aldehyde groups in the carbohydrate (i.e. one free aldehyde per carbohydrate polymer chain).
• Aromatic cyclic or heterocyclic compounds with an imine-forming carbonyl group (4), and one or more hydroxyl groups, such as 4- hydroxybenzaldehyde, 2,4-dihydroxybenzaldehyde, vanillin, ethyl vanillin, naringenin, and other similar compounds are preferred for addition to the aminated polysaccharides.
• other compounds having carbonyl groups such as steroid and trite ⁇ enoid derivatives can also be used.
• Step 3 This reaction mixture can be added to the aminated polysaccharides without prior isolation of the intermediate imidazole carbamate.
• the carbamate couples with the modified polysaccharides' amino groups to yield stable carbamate linkages.
• Imidazole carbamate derivatives can be isolated by chromatography, differential extractions, or other procedures.
• the coupling reaction should take place in the presence of equimolar amounts of amino and imidazole carbamate groups.
• the amount of amino groups in the aminated polysaccharide is determined with trinitrobenzenesulfonic acid
• TNBS TNBS
• the aminated glucan or mannan is suspended in a suitable anhydrous organic solvent, such as DMF, dioxane, or pyridine, and the pH is adjusted to about 9.5-10 with triethylamine.
• An aliquot of the carbamate intermediate containing an amount equivalent to the amino groups of the polysaccharide preparation is added, and the reaction is allowed to proceed for 12 to 18 hours at room temperature protected from moisture. About 6-8 volumes of cold ethanol are added to the reaction to precipitate the polysaccharide-aromatic aldehyde conjugate (6), which is collected by filtration.
• the conjugated polysaccharide is redissolved in water, and re-precipitated again with 6-8 volumes of ethanol or other suitable solvent.
• Efficacy of coupling is determined by one of the following methods: i) measuring the residual amino groups in the preparation with TNBS, ii) determining spectrophotometrically the amount of aromatic compound present in the preparation, or iii) by direct estimation of the aldehyde groups either with Schiff reagent or spectrophotometrically with N-methyl benzothiazolone hydrazone (MBTH) (Paz, M.A., et al, Archiv. Biochem. Biophys. 109:548 (1965)).
• MTH N-methyl benzothiazolone hydrazone
• the polysaccharide-aromatic aldehyde conjugate is dissolved in water and is lyophilized. It is also possible to create new aldehyde groups in the polysaccharide chain by mild oxidation with periodic acid. After oxidation, the polysaccharide with the additional aldehyde groups is precipitated with alcohol and subjected to reductive animation as described above.
• Another method to prepare glucan or mannan conjugated to carbonyl carrying compounds is to use the polysaccharides hydroxyl groups for conjugate formation. This method is illustrated by reference to Scheme 2. Because of the number of hydroxyl groups per glycosyl residue, this method allows the preparation of conjugates with higher densities of carbonyl groups. One can activate the polysaccharide's hydroxyl groups and let react with the carbonyl- carrying molecule. Scheme 2 Addition of imine-forming compounds to ⁇ -glucans via -OH groups
• Conjugation of compounds carrying carbonyl groups to the hydroxyl groups of polysaccharides can be made with N,N'-disuccinimidyl carbonate (DSC). Hydroxyl groups that are activated with DSC react almost exclusively with primary amines, avoiding the cross-linking of the polysaccharide chains via their -OH groups. Hydroxyl groups of ⁇ - 1 ,3 glucans are activated with DSC, and subsequently reacted with ethylenediamine to introduce amino groups into the glucan.
• DSC N,N'-disuccinimidyl carbonate
• N- hydroxysuccinimide ( ⁇ HS) esters 3- or 5- formylsalicylic acid-, 4-formylcinnamic acid, and 3- or 4- carboxybenzaldehyde, to form an aromatic aldehyde-glucan conjugate.
• Scleroglucan (7) a ⁇ -1, 3 glucan with single ⁇ -1, 6 linked D-glucose branches on every third glucose unit and with a molecular weight of about 130,000, is dissolved in water (2-5% solution), and lyophilized to yield a powdery product easily suspended in organic solvents.
• the activated scleroglucan dissolved or suspended in 20 ml of water, is slowly added with stirring to 100 ml of 0.2 M K potassium bicarbonate containing 2 ml of ethylenediamine (30 mmoles or lOx excess over the maximum polysaccharides' activated -OH groups) and adjusted to pH 8.5. After 4 hours at room temperature, the reaction is concentrated and dialyzed against water to remove excess of reactants from the aminated scleroglucan derivative (9) and lyophilized. The aminated polysaccharide is then reacted with succinimidyl-3 -formylsalicylic acid ester.
• Succinimidyl-3-formylsalicylic acid ester is prepared as follows: to 0.49 g of 3-formylsalicylic acid (3 mmoles) and 0.42 g of ⁇ HS (3.5 mmoles) dissolved in 10-15 ml of DMF, 1.28 g of CMC or l-cyclohexyl-3-(2-mo ⁇ holinoethyl) carbodiimide/7-toluenesulfonate (3 mmoles) are added and left to react for 5 hours at room temperature protected from moisture.
• the 3-formylsalicylic acid content of the scleroglucan derivative can be determined from its H, C, N, and O elementary composition.
• the content of aromatic aldehyde residues in the scleroglucan derivative can also be determined spectrophotometrically as follows: dissolve 5-8 mg of the scleroglucan derivative in 4 ml of 0.05 M KOH and read the UV spectra between 220 and 320 ran using as a blank a solution of equal concentration of unmodified scleroglucan in 0.05 M KOH. Calculate the 3 -formylsalicylic acid inco ⁇ orated using the extinction coefficient for this compound determined in 0.05 M KOH.
• the aldehyde content can also be determined either colorimetrically with Schiff reagent or spectrophotometrically with MBTH.
• Conjugation of compounds carrying both carbonyl and hydroxyl groups to polysaccharides, such as mannans, can be carried out by activating the polysaccharide hydroxyl groups with ?-toluenesulfonyl (tosyl) chloride (Nilsson, K., et al., A a Chem. Scan. 35: 19 (1981); Nilsson, K., et al, Methods Enzymol 104:56-69 (1984)). See Scheme 3-a. Scheme 3-a Addition of imine-forming compounds to mannan
• the precipitated mannan is collected by filtration, dissolved in water, dialyzed against water to remove salts and lyophilized.
• One g of lyophilized mannan (12) ( ⁇ 6 mmoles mannose), dried by suspension in pyridine and the azeotrope removed, is resuspended in 15 ml of DMF, and mixed with 2.5 g of tosyl chloride (2.15 mmoles) dissolved in 5 ml of DMF.
• To this mixture add 5 ml of pyridine and react with stirring for 18 hours at room temperature, to yield a tosylated mannan (13) having 1 tosyl group for every 20-25 mannosyl residues.
• the degree of tosylation is determined in a sample of the activated mannan that has been precipitated and washed with alcohol as follows. Dissolve 8 mg of the precipitated mannan in 4 ml of 0.1 N KOH, and measure its UV abso ⁇ tion spectra (220 to 360 nm) against a blank of unmodified mannan (8 mg in 4 ml of 0.1 N KOH). Determine the content of tosyl groups by using an extinction coefficient for tosyl of 480 M 'cm "1 at 261 nm.
• tosylated mannan (13) preparation ( ⁇ 0.9 g) resuspended in 20 ml of 0.5 M K bicarbonate, add 2 g of 2-cysteamine HC1 (18 mmoles) and adjust the pH with 1 N KOH to 9.5-10. Let react for 24 hours at 40°C with stirring and under a nitrogen atmosphere to introduce about 1 cysteamine residue per tosylated -OH. Dialyze the reactions against water to remove excess of reactants and salts, and lyophilize the mannan-cysteamine derivative (14). The amount of cysteamine bound to the tosylated mannan can be determined from the derivative's H, C, S, O and N elementary composition.
• the cysteamine inco ⁇ oration can also be estimated by mixing 10 ml of 0.05 M K carbonate buffer pH 9.5 containing 2 mg of the aminated mannan with 1 ml of an aqueous TNBS (7.2 mg/ml) and reacting for 2 hours at 40 °C. Measure the absorbancy at 363 nm against a blank of buffer plus TNBS, and determine the inco ⁇ orated -NH 2 using a molar extinction coefficient of 11,000 M-'cm "1 .
• Scheme 3-b illustrates attachment of an imine-forming compound to mannan-cysteamine.
• the aromatic aldehyde inco ⁇ orated to the mannan can be determined spectrophotometrically as follows. Dissolve 5-8 mg of the mannan derivative in 4 ml of 0.05 M KOH and read the UV spectra between 220 and 320 nm using as a blank a solution of equal concentration of unmodified mannan in 0.05 M KOH. To calculate the p- carboxybenzaldehyde concentration use its extinction coefficient determined in
• the aldehyde content can also be determined either colorimetrically with Schiff reagent or spectrophotometrically with MBTH.
• Carboxylic groups from pectic polysaccharides are reactive groups that can be used to couple the pectins to carbonyl carrying (imine-forming) compounds.
• Carboxylic groups can be coupled specifically to amines by using dicyclohexylcarbodiimide (DCC) and N- hydroxysuccinimide (NHS). See Scheme 4.
• This reaction can be carried out in organic solvents such as dioxane, DMF, pyridine, acetonitrile, or mixtures of the same.
• the coupling reaction can also be carried out in aqueous or semi-aqueous media using one of the water-soluble carbodiimides such as l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or CMC, in conjunction with either N-hydroxysulfosuccinimide (sulfo-NHS) or NHS.
• EDC l-ethyl-3-(3-dimethylaminopropyl)carbodiimide
• CMC N-hydroxysulfosuccinimide
• NHS N-hydroxysulfosuccinimide
• the number of amine groups per glycosyl residue can be selected by use of a limiting amount of CMC in the presence of an excess of diamine ligand. This approach is illustrated in Scheme 4. In one example, to
• aminated polysaccharide (17) Separation of the aminated polysaccharide (17) from the other reactants is accomplished by precipitating it with 6-8 volumes of ethanol.
• the ethanol- washed, precipitated, aminated pectin is dissolved in water, dialyzed and lyophilized to yield a powdery product easily dissolved in water, and its amino group content determined with TNBS.
• the aminated pectin is then reacted with succinimidyl-3-formylsalicylic acid ester prepared as follows.
• the 3 -formylsalicylic acid can be replaced by an equimolar amount of 3-carboxybenzaldehyde, 4-formyl-phenoxyacetic acid or 4,5-dioxoheptanoic acid).
• MOBS 4-[N- mo ⁇ holinojbutanesulfonic acid
• the 3-formylsalicylic acid-pectin derivative (18) is precipitated with 6-8 volumes of ethanol, and then collected and washed with ethanol to remove excess of reactants.
• the precipitated pectin derivative is dissolved in a minimal volume of water, dialyzed against water, and lyophilized.
• the aminated pectin can also be reacted with a succinimidyl carbonate intermediate of a hydroxylated, carbonyl-containing compound, such as 4-hydroxyphenylacetaldehyde, 6-hydroxy-2-naphthoquinone,
• aminated pectin (17) can be reacted with a compound containing both a free and a protected aldehyde group, like terephthaldehyde monodiethylacetal.
• the aminated pectin is first reacted with the free aldehyde group to form a stable bond, and subsequently the protected aldehyde is released by mild acid hydrolysis, as described here.
• the terephthaldehyde monodiethylacetal pectin derivative is dissolved in a minimum volume of 0.05 MHC1 and heated at 100°C for 15-20 minutes to convert the acetal to the aldehyde form. After hydrolysis the solution of phenylacetaldehyde pectin derivative (20) is brought to neutrality with NaOH dialyzed against water and lyophilized.
• the aromatic aldehyde content of the pectin derivatives is determined spectrophotometrically as follows: dissolve 5- 8 mg of the conjugated pectin in 3 ml of 0.05 N NaOH and scan the UV spectra between 220 and 320 nm against a blank solution containing the same amount of unmodified pectin.
• the aromatic aldehyde concentration of the pectin derivative is calculated using the extinction coefficient of the free aromatic aldehyde as determined in 0.05 M NaOH.
• the aldehyde content can also be determined spectrophotometrically with MBTH or colorimetrically with Schiff reagent.
• carbonyl containing compounds to pectic polysaccharides can be carried out in organic solvents.
• organic solvents In one example, CMC,
• NHS and a 10-fold excess of a diamine with respect to CMC are added to anhydrous lyophilized pectin (H + form) suspended in DMF-pyridine (6:4, v/v) and the mixture is left to react with stirring at room temperature overnight.
• the number of amine groups per glycosyl residue can be selected by using a limiting amount of CMC in the presence of an excess of diamine ligand. Separation of the aminated polysaccharide from the other reactants is accomplished by precipitating it with 6-8 volumes of ethanol. The ethanol- washed, precipitated, aminated pectin is dissolved in water and lyophilized to yield a powdery product easy to resuspend in DMF-pyridine.
• the aminated pectin is reacted in DMF-pyridine with either (i) a succinimidyl ester of a carboxylated carbonyl-containing compound, such as 4,5- dioxoheptanoic acid, 3- or 5- formylsalicylic acid, 4-formylcinnamic acid, or 3- or 4-carboxybenzaldehyde, or (ii) the succinimidyl carbonate intermediates of a hydroxylated, carbonyl-containing compound, such as 2,4-dihydroxybenzaldehyde, 4-hydroxybenzaldehyde, 4-hydroxyphenylacetaldehyde, 4 ' -hydroxyacetophenone, 6-hydroxy-2-naphthoquinone, formyldienolone, progesterone, androsterone, prednisolone, pyridoxal.
• a succinimidyl ester of a carboxylated carbonyl-containing compound such as 4,5- dioxo
• the aldehyde-containing pectic polysaccharide derivative is then recovered by filtration after mixing the reaction with 6 volumes of ethanol, dissolved in water, dialyzed against water and lyophilized.
• glycol chitin is prepared as follows. To 5 g of finely ground chitin (22) (equal to 21.25 mmoles of N-acetyl glucosamine and 3.75 mmoles glucosamine) suspended in 30 ml of 42% NaOH for 2 hours at room temperature, add with stirring 70 ml of an ice-water mixture to yield a highly viscous dispersion of alkaline-chitin. To this dispersion, 5 g of ethylene oxide (114 mmoles) are added by bubbling into the solution while stirring for 1-2 hours at 30-35 °C.
• glycol chitin (23) is then precipitated with 6 volumes of 80% ethanol, washed on glass filter paper with 80% ethanol, dissolved in water, dialyzed against water, lyophilized and the content of free amine groups determined colorimetrically with TNBS.
• lyophilized gycol chitin about 1.5 mmoles of glucosamine
• pyridine containing 1.60 g of terephthaldehyde monodiethylacetal (7.5 mmoles), 0.4 g sodium cyanoborohydride (6 mmoles) and let react for 4 hours at room temperature.
• glycol chitin (24) Precipitate the terephthaldehyde monodiethylacetal derivative of glycol chitin (24) with 4-6 volumes of ethanol, resuspend the precipitate and wash it with 80% ethanol to remove any excess of reactants. Dissolve the glycol chitin derivative (24) in 40-50 ml of 0.05 M HC1 and heat at 100°C for 20-30 minutes to release the aldehyde groups to yield the benzaldehyde derivative of glycol chitin (25).
• the benzaldehyde content of the glycol chitin derivative is determined spectrophotometrically as follows: dissolve 5-8 mg of the glycol chitin derivative in 4 ml of 0.05 M KOH and read the UV spectra between 220 and 320 nm using as a blank a solution of equal concentration of glycol chitin in 0.05 M KOH. Calculate the inco ⁇ orated benzaldehyde using the extinction coefficient for this compound determined in 0.05 M KOH.
• the aldehyde content can also be determined either spectrophotometrically with MBTH or colorimetrically with Schiff reagent.
• glycol chitin (23), formed as described above, is reacted with succinimidyl-5-formylsalicylic acid ester (10), which is prepared as follows. To 0.5 g of 3 -formylsalicylic acid (3 mmoles) and
• the 5- formylsalicylic acid can be replaced by an equimolar amount of another carboxylated carbonyl-containing compound, such as 4,5-dioxoheptanoic acid, 4- formylcinnamic acid, 3-carboxybenzaldehyde or 4-formylphenoxyacetic acid).
• another carboxylated carbonyl-containing compound such as 4,5-dioxoheptanoic acid, 4- formylcinnamic acid, 3-carboxybenzaldehyde or 4-formylphenoxyacetic acid.
• the 3 -formylsalicylic acid derivative of glycol chitin (26) is precipitated with 6 volumes of ethanol and washed with 80% ethanol to remove excess reactants.
• the precipitated glycol chitin derivative is dissolved in 50-60 ml of water, dialyzed against water, and lyophilized.
• the 3 -formylsalicylic acid content of the glycol chitin derivative is determined spectrophotometrically as follows: dissolve 5-8 mg of the glycol chitin derivative in 4 ml of 0.05 M KOH and read the UV spectra between 220 and 320 nm using as a blank a solution of equal concentration of unmodified glycol chitin in 0.05 M KOH.
• APCs surface receptors include chitins and dextrans which are of animal and bacterial origin respectively.
• suitable carbohydrate-containing compounds are bacterial teichoic acids and their derivatives, bacterial lipopolysaccharides, lipid A, and their derivatives.
• Conjugation of compounds carrying imine-forming carbonyl groups to non-adjuvant carbohydrate-containing products is contemplated to be useful in providing intrinsic adjuvanticity to these products and to increase the efficacy of preventive immunizations.
• Examples of these products are polysaccharides from streptococci, staphylococci, and other bacteria that are used as vaccine antigens.
• a conjugate of the present invention for example a compound of Formula / or a physiologically acceptable salt thereof, may be used for the treatment of diseases where there is a defect in the immune system and/or an ineffective host defense mechanism, or to enhance activity to the immune system above normal levels.
• a compound of the invention or a physiologically acceptable salts thereof may administered for the treatment or prophylaxis of immunodeficient mammals alone or combination with other therapeutic agents, for example, with other antiviral agents, or with other anticancer agents.
• a conjugate of the present invention for example a compound of
• Formula/ and physiologically acceptable salts thereof may be administered for the treatment or prophylaxis of immunodeficient mammals alone or in combination with other therapeutic agents, for example, with other antiviral agents, or with other anticancer agents.
• Immune adjuvants are compounds which, when administered to an individual or tested in vitro, increase the immune response to an antigen in a subject or in a test system to which the antigen is administered.
• an “effective amount” is meant an amount of a conjugate of the present invention that will restore immune function to substantially normal levels, or increase immune function above normal levels in order to eliminate infection.
• Immune function is defined as the development and expression of humoral (antibody-mediated) immunity, cellular (T-cell-mediated) immunity, or macrophage and granulocyte mediated resistance.
• immunodeficient patient is employed to describe patients with a deficient or defective immune system.
• An immunodeficient patient can be characterized by means of a T-lymphocyte proliferation assay. Using this assay immunodeficient patients are characterized by a reduced ability of the T-cells to respond to stimulation by mitogens.
• An example of a mitogen commonly used in this assay is phytohaemagglutinin (PHA).
• T-cells that orchestrate the immune response.
• T-cells require two signals in order to initiate an effective immune response:
• T-cells In the absence of signal (ii), T-cells fail to respond and may also become chronically paralyzed or anergic. Persistent viral and bacterial infections and neoplastic disease can produce T-cell hyporesponsiveness by interfering in various ways with the delivery of secondary signals and in this way evade the immune response.
• the conjugates of the present invention appear to work by substituting or otherwise compensating for deficient costimulatory signals to T-cells.
• Recent studies (Rhodes, J, Immunology Today 17:436 (1996)) have shown that exogenous Schiff-base-forming compounds can substitute for natural donors of carbonyl groups and provide a costimulatory signal to CD4 T helper (Th) cells.
• Treatment of APCs with galactose oxidase to form new aldehyde groups resulted in an adjuvant effect when administered with an antigen to mice.
• immune system deficiency is common in immature or premature infants (neonates). It may also result from suppression by certain drugs which may be deliberate e.g. as a side-effect of cancer chemotherapy. Disordered growth of one or more constituent parts of the immune system, e.g. as in certain forms of cancer, may also result in immunodeficiency. Immune deficiency can also be caused by viral infections, including human immunodeficiency virus (HIV).
• HIV human immunodeficiency virus
• a further aspect of the present invention provides a method of treating immunodeficient patients, which comprises administering to a mammal an effective amount of a conjugate of the present invention, for example a compound of Formula /, or a physiologically acceptable salt thereof.
• a further aspect of the present invention provides for the use of a conjugate of the present invention, for example a compound of Formula / or a physiologically acceptable salt thereof for the treatment and/or prophylaxis of acute and chronic viral infections.
• Examples of acute viruses against which immunopotentiatory therapy with a conjugate of the present invention are: he ⁇ es viruses, influenza viruses, parainfluenza viruses, adenoviruses, coxsakie viruses, picorna viruses, rotaviruses, hepatitis A virus, mumps virus, rubella virus, measles virus, pox viruses, respiratory syncytial viruses, papilloma viruses, and enteroviruses, arenavirus, rhinoviruses, poliovirus, Newcastle disease virus, rabies virus, and arboviruses.
• chronic viral infections against which immunopotentiatory therapy with conjugates of the present invention may be used are persistent he ⁇ es virus infections, Epstein Barr virus infection, persistent rubella infections, papillomavirus infections, hepatitis virus infections and human immunodeficiency virus infections.
• conjugates of the invention can be employed alone or in combination with other therapeutic agents for the treatment of the above infections or conditions.
• Combination therapies according to the present invention comprise the administration of at least one conjugate of the present invention, for example a compound of the Formula / or a physiologically acceptable salt thereof and at least one other pharmaceutically active ingredient.
• the pharmaceutically active ingredient(s) and compounds of the present invention may be administered together or separately and, when administered separately this may occur simultaneously or sequentially in any order.
• the amounts of the pharmaceutically active ingredient(s) and compounds of the present invention and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect.
• the combination therapy involves the administration of one compound of the present invention and one of the agents mentioned herein below.
• agents that are effective for the treatment of HIV infections or associated conditions such as 3 '-azido-3 '-deoxythymidine (zidovudine), other 2',3 '-dideoxynucleosides such as
• interferons such as ⁇ -interferon, renal excretion inhibitors such as probenecid, nucleoside transport inhibitors such as dipyridamole; pentoxifylline, N-acetylcysteine, precession, ⁇ -trichosanthin, phosphonoformic acid, as well as immunomodulators such as interleukin II, granulocyte macrophage colony stimulating factors, erythropoetin, soluble CD4 and genetically-engineered derivatives thereof.
• HB V infections examples include carbovir, oxathiolan nucleoside analogs such as cis- 1 -(2-hydroxymethyl)- l ,3 -oxathiolan-5 -yl)cytosine or cis- ⁇ -(2 -(hydroxymethyl)-
• the present invention provides the use of a conjugate of the present invention, for example a compound of Formula /or a physiologically acceptable salt thereof, for the manufacture of a medicament for the treatment and/or prophylaxis of Pneumocystis carinii infections in mammals.
• the present invention provides for the use of a conjugate of the present invention, for example a compound of Formula / or a physiologically acceptable salt thereof to treat conditions resulting from relative T-cell deficiencies such as DiGeorge Syndrome, fungal infections, mycoplasma infections, tuberculosis, leprosy, and systemic lupus erythemotosus.
• a conjugate of the present invention for example a compound of Formula / or a physiologically acceptable salt thereof for the manufacture of a medicament for the treatment and/or prophylaxis of cancer in mammals.
• cancers particularly suitable for treatment with compounds the present invention are: melanoma, breast cancer, colon cancer, cancer of the head and neck, gastric cancer, renal cancer, laryngeal cancer, rectal cancer, and non-Hodgkins lymphoma. Cancers that express tumor specific antigens or antigens rarely expressed or expressed at very low density on normal cells, are likely therapeutic targets. Cancers which contain tumor specific cytotoxic T-cells which are anergic or otherwise ineffective are likely targets for therapy. Surgically resected tumors where there is a high risk of recurrence are also suitable for therapy with compounds of the present invention.
• a further aspect of the present invention provides for the use, as a vaccine adjuvant, of a conjugate of the present invention, for example a compound of Formula / or a physiologically acceptable salt thereof.
• a vaccine may therefore be prepared by formulating an antigenic component with a conjugate of the present invention.
• Compounds of the present invention may be administered to a human recipient by a route selected from oral, parenteral (including subcutaneous, intradermal, intramuscular and intravenous), rectal and inhalation.
• a route selected from oral, parenteral (including subcutaneous, intradermal, intramuscular and intravenous), rectal and inhalation The size of an effective dose of a compound will depend upon a number of factors including the identity of the recipient, the type of immunopotentiation involved, the severity of the condition to be treated and the route of administration, and will ultimately be at the discretion of the attendant physician.
• the effective dose will generally be in the range of 0.03 to 250 mg per individual, and most preferably between about 0.05 to about 100 mg per dose.
• Immune stimulators are preferably administered only once or twice a week, and in some cases, less frequently. Frequency and length of treatment vary among species and individuals.
• the formulations of the present invention comprise a compound of the present invention, together with one or more acceptable carriers therefor and optionally other therapeutic ingredients.
• the carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
• Immune adjuvants are compounds which, when administered to an individual or tested in vitro, increase the immune response to an antigen in a subj ect or in a test system to which the antigen is administered. Some antigens are weakly immunogenic when administered alone or are toxic to a subject at concentrations that evoke useful immune responses in a subject. An immune adjuvant can enhance the immune response of the subject to the antigen by making the antigen more strongly immunogenic. The adjuvant effect can also result in the ability to administer a lower dose of antigen to achieve a useful immune response in a subject.
• the immunogen-inducing activity of compounds and compositions of the present invention can be determined by a number of known methods.
• the increase in titer of antibody against a particular antigen upon administration of a composition of the present invention can be used to measure immunogenic activity. (Dalsgaard, K. Acta Veterinia Scandinavica 69:1-40 (1978)).
• One method requires injecting CD-I mice intradermally with a test composition that includes one or more exogenous antigens. Sera is harvested from mice two weeks later and tested by ELISA for anti-immunogen antibody.
• compositions of the invention are useful as vaccines to induce active immunity towards antigens in subjects. Any animal that may experience the beneficial effects of the compositions of the present invention within the scope of subjects that may be treated.
• the subjects are preferably mammals, and more preferably humans.
• Conjugates of the present invention can be employed as a sole adjuvant, or alternatively, can be administered together with other adjuvants.
• adjuvants useful with the present invention include oil adjuvants (for example, Freund's Complete and Incomplete), saponins, modified saponins, liposomes, mineral salts (for example, AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH 4 (SO 4 ), silica, alum,
• polynucleotides for example, poly IC and poly AU acids
• certain natural substances for example, wax D from Mycobacterium tuberculosis, as well as substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella
• bovine serum albumin diphtheria toxoid, tetanus toxoid, edestin, keyhole
• the non-saponin adjuvant may comprise a protein fragment comprising at least the immunogenic portion of the molecule.
• immunogenic macromolecules which can be used in the practice of the invention include, but are not limited to, polysaccharides, tRNA, non- metabolizable synthetic polymers such as poly vinylamine, polymethacrylic acid, polyvinylpyrrolidone, mixed polycondensates (with relatively high molecular weight) of 4',4-diaminodiphenyl-methane-3,3'-dicarboxylic acid and 4-nitro-2- aminobenzoic acid (See Sela, M., Science 765:1365-1374 (1969)) or glycolipids, lipids or carbohydrates.
• the conjugates of the present invention exhibit adjuvant effects when administered over a wide range of dosages and a wide range of ratios to one or more particular antigens being administered.
• the conjugates can be administered either individually or admixed with other substantially pure adjuvants to achieve an enhancement of immune response to an antigen.
• the conjugates of the present invention can be utilized to enhance the immune response to one or more antigens.
• Typical antigens suitable for the immune-response provoking compositions of the present invention include antigens derived from any of the following: viruses, such as influenza, feline leukemia virus, feline immunodeficiency virus, HIV-1 , HIV-2, rabies, measles, hepatitis B, or hoof and mouth disease; bacteria, such as anthrax, diphtheria, Lyme disease, or tuberculosis; or protozoans, such as Babeosis bovis or Plasmodium.
• the antigen can be proteins, peptides, polysaccharides, or mixtures thereof. The proteins and peptides may be purified from a natural source, synthesized by means of solid phase synthesis, or may be obtained means of recombinant genetics.
• the conjugates of the present invention can also be administered alone to potentiate the immune system for treatment of chronic infectious diseases, especially in immune compromised patients.
• infectious diseases for which conjugates of the present invention can be employed for therapeutic or prophylactic treatment are described in U.S. Patent No. 5, 508,310.
• Potentiation of the immune system by saponin conjugates can also be useful as a preventative measure to limit the risks of nosocomial and/or post-surgery infections.
• Administration of the compounds useful in the method of present invention may be by parenteral, intravenous, intramuscular, subcutaneous, intranasal, or any other suitable means.
• the dosage administered may be dependent upon the age, weight, kind of concurrent treatment, if any, and nature of the antigen administered.
• the saponin/antigen conjugates may be administered over a wide range of dosages and a wide range of ratios to the antigen being administered.
• the initial dose may be followed up with a booster dosage after a period of about four weeks to enhance the immunogenic response. Further booster dosages may also be administered.
• the conjugates of the present invention may be employed in such forms as capsules, liquid solutions, emulsions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions, emulsions or suspensions.
• any inert carrier is preferably used, such as saline, or phosphate-buffered saline, or any such carrier in which the compounds used in the method of the present invention have suitable solubility properties for use in the methods of the present invention.

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