HK1077164A - New immunoeffector compounds - Google Patents

Description

Novel immune effector compounds

Technical Field

The present invention relates generally to immune effector compounds, their use in pharmaceutical compositions, and methods for their production and their use in prophylactic and/or therapeutic vaccination. More particularly, the present invention relates to novel compounds comprising 2-deoxy-2-amino- β -D-glucopyranose (glucosamine) glycosidically linked to a cyclic aminoalkyl (aglycon) group, and their use in pharmaceutical adjuvant systems.

Background

Humoral immunity and cell-mediated immunity are the two major branches of the immune response in mammals. Humoral immunity involves the production of antibodies against foreign antigens. Antibodies are produced by B-lymphocytes. Cell-mediated immunity involves the activation of T-lymphocytes that act on infected cells loaded with foreign antigens or the priming of other cells to act on infected cells. These two branches of the mammalian immune system are important to combat disease. Humoral immunity is the primary line of defense against bacterial pathogens. In the case of viral diseases, the induction of Cytotoxic T Lymphocytes (CTLs) appears to be crucial for protective immunity. Therefore, an effective vaccine preferably stimulates both branches of the immune system to prevent disease.

Vaccines present foreign antigens from pathogenic agents to the host so that the host can mount a protective immune response. Typically, the vaccine antigen is an inactivated or attenuated form of the disease-causing microorganism. The presence of unnecessary components and antigens in these inactivated or attenuated vaccines has prompted increased efforts to improve vaccine components, including the development of well-defined synthetic antigens using chemical and recombinant techniques. Improvements and simplifications in microbial vaccines have resulted in a corresponding loss of potency. Despite the lack of potentially harmful contaminants, low molecular weight synthetic antigens are generally not sufficiently immunogenic themselves. These observations have led researchers to add immune system stimulants called adjuvants to vaccine compositions to boost the activity of vaccine components.

An immunoadjuvant is one that increases the immune response to an antigen, or increases some activity of cells of the immune system, in a subject to whom the antigen is administered, or in an in vitro assay. A number of compounds with varying degrees of adjuvant activity have been prepared and tested (see, e.g., Shimizu et al 1985, Bulusu et al 1992, Ikeda et al 1993, Shimizu et al 1994, Shimizu et al 1995, Miyajima et al 1996). These and other prior adjuvant systems, however, are often toxic, unstable and/or have unacceptably low immunostimulatory effects.

Currently, the only licensed adjuvant for use in humans in the united states is alum, a group of aluminum salts (e.g., aluminum hydroxide, aluminum phosphate) in which vaccine antigens are formulated. Particulate carriers such as alum are reported to promote uptake, processing and presentation of soluble antigens by macrophages. Alum, however, is not without side effects and unfortunately is limited to only humoral (antibody) immunity.

The discovery and development of effective adjuvant systems is essential to improve the efficacy and safety of existing and future vaccines. Therefore, there is a continuing need for new and improved adjuvant systems, especially those capable of stimulating these two effector arms of the immune system, to better facilitate the development of the next generation of synthetic vaccines. The present invention fulfills these and other needs.

Summary of the invention

The compounds of the present invention are immune effector molecules (immunoeffector molecules) capable of enhancing humoral and cell-mediated immune responses to vaccine antigens. The compounds can generally be described as belonging to the class of cyclic AGP compounds, where AGP represents an aminoalkyl glucosaminide phosphate. The term "cyclic AGP" refers to an azacycloalkyl or (azacycloalkyl) alkyl glucosaminide phosphate in which 2-deoxy-2-amino-b-D-glucopyranose (glucosamine) is glycosidically linked to an azacycloalkyl or (azacycloalkyl) alkyl (aglycone) group.

The compounds of the present invention comprise 2-deoxy-2-amino- β -D-glucopyranose (glucosamine) glycosidically linked to a cyclic aminoalkyl (aglycon) group. The compounds are phosphorylated at the 4 or 6-position of the glucosamine ring and acylated at the aglycone nitrogen and at the 2 and 3-positions of the glucosamine ring with alkanoyloxytetradecanoyl residues. The compounds of the present invention are generally represented by structural formula (I):

and pharmaceutically acceptable salts thereof, wherein X is-O-or-NH-and Y is-O-or-S-; r¹，R²And R³Are each independently (C)₉-C₁₄) Acyl groups, including saturated, unsaturated and branched acyl groups; r⁴is-H or-PO₃R⁷R⁸Wherein R is⁷And R⁸Are each independently H or (C)₁-C₄) An aliphatic group; r⁵is-H, -CH₃or-PO₃R⁹R¹⁰Wherein R is⁹And R¹⁰Are each independently selected from-H and (C)₁-C₄) An aliphatic group; r⁶Independently selected from H, OH, (C)₁-C₄) Oxyaliphatic radical, -PO₃R¹¹R¹²，-OPO₃R¹¹R¹²，-SO₃R¹¹，-OSO₃R¹¹，-NR¹¹R¹²，-SR¹¹，-CN，-NO₂，-CHO，-CO₂R¹¹and-CONR¹¹R¹²Wherein R is¹¹And R¹²Are each independently selected from H and (C)₁-C₄) An aliphatic group; provided that R is⁴And R⁵One is a phosphorus-containing group and if R is⁴is-PO₃R⁷R⁸Then R is₅Is not-PO₃R⁹R¹⁰Wherein^*1-3"and^**"denotes a chiral center;

wherein n, m, p and q are each independently integers of 0 to 6, provided that the sum of p and m is 0 to 6.

In certain embodiments of the compounds of the present invention, X and Y are each oxygen, R⁴Is PO₃R⁷R⁸，R⁵And R⁶Is H, and n, m, p, and q are integers from 0 to 3. In a more preferred embodiment, R⁷And R⁸is-H. In an even more preferred embodiment, n is 1, m is 2, and subscripts p and q are 0. In an even more preferred embodiment, R¹，R²And R³Is C₉-C₁₃Acyl group, most preferably C₁₀-C₁₂An acyl group. In a still more preferred embodiment of the present invention,^*1-3is in the R configuration, Y is in the equatorial position, and^**is in the S configuration. Particularly preferred is (N- [ (R) -3-tetradecanoyloxytetradecanoyl group]- (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-phosphono-2- [ (R) -3-tetradecanoyloxytetradecanoylamino]-3-O- [ (R) -3-tetradecanoyloxytetradecanoyl group]-beta-D-glucopyranoside, formula (II),

(N- [ (R) -3-dodecanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-dodecanoyloxytetradecanoylamino ] -3-O- [ (R) -3-dodecanoyloxytetradecanoyl ] - β -D-glucopyranoside, structural formula (III),

and (N- [ (R) -3-decanoyloxytetradecanoyl ] - (S) -2-pyrrolidinomethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-decanoyloxytetradecanoylamino ] -3-O- [ (R) -3-decanoyloxytetradecanoyl ] -beta-D-glucopyranoside, formula (IV),

and pharmaceutically acceptable salts thereof.

The invention also provides a pharmaceutical composition comprising a compound having the above general formula and specific structural formula. The pharmaceutical composition may be combined with various antigens in various formulations known to those skilled in the art.

The compounds of the invention may also be used in methods of inducing an immune response in a subject. The method entails administering to the subject a therapeutically effective amount of one or more compounds of the invention, preferably a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

The invention also includes methods of treating a mammal having or susceptible to a pathogenic infection, cancer or an autoimmune disease. The method entails administering to the mammal a therapeutically effective amount of one or more compounds of the present invention, preferably a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

Still further, the present invention includes a method for treating a disease or condition ameliorated by the production of nitric oxide in a subject. The method entails contacting the subject with an effective amount of one or more compounds of the invention, or with an effective amount of a composition comprising one or more compounds of the invention and a pharmaceutically acceptable carrier. In some embodiments, the compounds of the present invention may be administered 48 hours prior to ischemia, no more than ischemia, and during ischemia.

Detailed description of the invention

Definition of

The term "acyl" refers to those groups derived from organic acids by removal of the hydroxyl portion of the acid. Thus, acyl is meant to include, for example, acetyl, propionyl, butyryl, decanoyl, and pivaloyl. For example, "(C)₉-C₁₄) Acyl "refers to an acyl group having 9 to 14 carbons.

Unless otherwise specified, the term "aliphatic" by itself or as part of another substituent means a straight or branched chain, or cyclic, hydrocarbon moiety, including moieties containing both cyclic and chain members, which may be fully saturated or mono-or polyunsaturated, having the specified number of carbon atoms (i.e., C)₁-C₄Meaning 1-4 carbons). Examples of the saturated hydrocarbon group include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, cyclopropyl, cyclopropylmethyl, methylene, ethylene, and n-butylene. Unsaturated alkyl groups are groups having one or more double and/or triple bonds. Examples of the unsaturated aliphatic group include vinyl, 2-propenyl, crotyl, -2- (butadienyl), 1-propynyl and 3-propynyl.

The term "oxyaliphatic" refers to those groups having an aliphatic group attached to the remainder of the molecule through an oxygen atom.

The above terms (e.g., "alkyl", "acyl") are meant to include both substituted and unsubstituted forms of the indicated moiety, respectively.

Substituents for aliphatic groups may be various groups selected from: -OR ', -O, ═ S, ═ NR', -N-OR ', -NR' R ', -SR', -halogen, -SiR 'R "R'", -oc (O) R ', -c (O) R', -CO₂R′，-CONR′R″，-OC(O)NR′R″，-NR″C(O)R′，-NR′-C(O)NR″R*，-NR″C(O)₂R′，-NH-C(NH₂)＝NH，-NR′C(NH₂)＝NH，-NH-C(NH₂)＝NR′，-S(O)R′，-S(O)₂R′，-S(O)₂NR 'R', -CN and-NO₂The number is zero to (2m '+ 1), where m' is the total number of carbon atoms in these groups. R ', R "and R'" independently mean hydrogen and unsubstituted (C), respectively₁-C₄) An aliphatic group. From the above discussion of substituents, it will be understood by those skilled in the art that the term "alkyl" is meant to include haloalkyl (e.g., -CF)₃and-CH₂CF₃) Such groups and the like.

Unless otherwise indicated, the term "halo" or "halogen" by itself or as part of another substituent refers to a fluorine, chlorine, bromine, or iodine atom. In compounds having multiple halogen substituents, the halogens may be the same or different.

The term "pharmaceutically acceptable salt" is meant to include salts of the active compounds prepared using relatively non-toxic acids or bases, depending on the particular substituents on the compounds described herein. If the compounds of the invention contain relatively acidic functionalities, base addition salts can be obtained by adding the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts. If the compounds of the invention contain relatively basic functionalities, acid addition salts may be obtained by adding the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydriodic acid, or phosphorous acid and the like, as well as salts derived from relatively nontoxic organic acids such as acetic acid, propionic acid, isobutyric acid, oxalic acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as arginine salts and analogs, and salts and analogs of organic acids such as glucuronic acid or galacturonic acid (see, e.g., Berge, s.m., et al, "drug salts", journal of pharmaceutical sciences, 1977, 66, 1-19). Certain specific compounds of the present invention comprise both basic and acidic functionalities, enabling the compounds to be converted into base or acid addition salts.

The neutral form of these compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. For the purposes of the present invention, the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are identical to the parent form of the compound.

In addition to salt forms, the present invention provides compounds in prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Alternatively, prodrugs can be converted to the compounds of the present invention by chemical or biochemical means in an ex vivo environment. For example, prodrugs are slowly converted to the compounds of the present invention when placed in a transdermal patch with a suitable enzyme or chemical agent.

Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms (including hydrated forms). In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain of the compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses to which the invention pertains and are intended to be within the scope of the invention.

Certain compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers and individual isomers are all included within the scope of the present invention.

The compounds of the present invention may also contain an abnormal proportion of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be substituted with radioactive isotopes, such as tritium(s) ((iii))³H) Iodine-125 (¹²⁵I) Or carbon-14 (¹⁴C) And performing radioactive labeling. All isotopologues of the compounds of the inventionElemental variants, whether radioactive or not, are included within the scope of the invention.

Introduction to

To improve the safety of vaccines, manufacturers are avoiding whole cell inactivated vaccines and producing recombinant or subunit vaccines. In preparing these safer vaccines, additional bacterial or viral components are eliminated while leaving the smallest structural or antigenic epitopes that are considered necessary for protective immunity. The safety of these vaccines is improved by eliminating additional bacterial or viral components that are often proven to be toxic and pyrogenic. The same components that cause toxicity provide non-specific immune stimulation, making whole cell vaccines very effective. Recombinant and subunit vaccines containing minimal structural and antigenic epitopes are often poorly immunogenic without additional immune stimulation.

A disaccharide molecule derived from LPS from Salmonella minnesota R595, MPL * immunostimulant (Corixa Corp.) has immunostimulating properties. MPL * immunostimulant, monophosphoryl lipid a, is a structural derivative of lipid a (or LPS) and has an improved therapeutic index relative to lipid a (see the structure of monophosphoryl lipid a of U.S. patent 4,987,237; U.S. patent nos.4,436,727 and 4,436,728, which describe the preparation of monophosphoryl lipid a). Other useful immunostimulants include 3-de-O-acylated monophosphoryl lipid A (3D-MPL) described in U.S. Pat. No.4,912,094. The compounds can be safely administered to humans at doses up to at least 20 μ g/kg, but some patients experience elevated temperatures, flu-like symptoms, increased heart rate and moderate blood pressure drops at dosage levels ≥ 10 μ g/kg. Cell culture and animal evaluation demonstrated that MPL * immunostimulant still retained some of the immunostimulatory activity of the maternal LPS, as the pyrogenicity and ability to induce pro-inflammatory cytokines such as TNF and IL-8 were retained, despite higher dose levels. Thus, the need for effective vaccine adjuvants is well established. Ideally, these adjuvants will boost the protective immune response without inducing undesirable toxicity and pyrogenicity.

To obtain an immunostimulant with low pyrogenicity, a synthetic molecule structurally similar to MPL * immunostimulant has been prepared. These new molecules, commonly known as Aminoalkyl Glucosaminide Phosphates (AGPs), consist of an acylated glucose moiety bonded to an acylated aminoalkyl group (Johnson et al, bioorg. Med. chem. Lett.9: 2273-2278(1999) and PCT/WO98/50399 and references therein). Each molecule has 6 fatty acid tails, which is considered to be the best number for peak adjuvant activity. Substitutions of different chemical moieties within the aminoalkyl structure are designed into AGPs to optimize stability and solubility properties. AGPs can therefore be broadly divided into several families according to the structure of their aminoalkyl groups. After an initial biological evaluation, it was clearly found that aminoalkyl motifs can significantly affect the pyrogenicity of AGP (see U.S. patent application No.09/074,720 and US patent nos.6,113,918 and 6,303,347 filed 5, 7 of 1998). Rabbit pyrogenicity data was determined as part of the initial screening process for synthetic adjuvant compounds. It can be seen that several compounds did not elicit a fever response when administered intravenously at a dose of 10. mu.g/kg. Generally, these same compounds are unable to induce detectable levels of the inflammatory cytokines TNF- α or IL-1 β in an ex vivo cytokine induction assay on human peripheral blood mononuclear cells. Here we report on studies of adjuvant performance of a class of AGPs that induce minimal activity in both rabbit pyrogen assays and ex vivo cytokine assays.

Compounds and compositions

The present invention provides compounds generally represented by structural formula (I):

and pharmaceutically acceptable salts thereof, wherein X is-O-or-NH-and Y is-O-or-S-; r¹，R²And R³Are each independently (C)₉-C₁₄) Acyl groups, including saturated, unsaturated and branched acyl groups; r⁴is-H or-PO₃R⁷R⁸Wherein R is⁷And R⁸Are each independently H or (C)₁-C₄) An aliphatic group; r⁵is-H, -CH₃or-PO₃R⁹R¹⁰Wherein R is⁹And R¹⁰Are each independently selected from-H and (C)₁-C₄) An aliphatic group; r⁶Independently selected from H, OH, (C)₁-C₄) Oxyaliphatic radical, -PO₃R¹¹R¹²，-OPO₃R¹¹R¹²，-SO₃R¹¹，-OSO₃R¹¹，-NR¹¹R¹²，-SR¹¹，-CN，-NO₂，-CHO，-CO₂R¹¹and-CONR¹¹R¹²Wherein R is¹¹And R¹²Are each independently selected from H and (C)₁-C₄) An aliphatic group; provided that R is⁴And R⁵One is a phosphorus-containing group and if R is⁴is-PO₃R⁷R⁸，R⁵Is not-PO₃R⁹R¹⁰Wherein^*1-3"and^**"denotes a chiral center;

wherein n, m, p and q are each independently integers of 0 to 6, provided that the sum of p and m is 0 to 6.

Although the hexopyranoside of structural formula I is given in the glucose configuration, other glycosides are within the scope of the invention. For example, glucopyranosides, including other hexopyranosides (e.g., allose glycoside, altrose glycoside, mannoside, gulose glycoside, idoside, galactosides, talose glycoside), are within the scope of the invention.

In the above general formula, the symbol^*1″，″^*2"and³"the configuration of the 3' -stereocenters (Stereogenic centers) to which the normal fatty acyl residues are attached is R or S, but R is preferred. To which R is directly or indirectly attached⁶And the carbon atom of the cyclic aglycone unit of the glucosamine unit (marked ″)^**") may be R or S. In the above formula, Y may be in a flat or upright position, but is preferably in a flat or upright positionFlat and flat. All stereoisomers, enantiomers, diastereomers and mixtures thereof are contemplated as being within the scope of the invention.

In a preferred embodiment of the invention, X and Y are-O-, R⁴Is phosphono, R⁵And R⁶Is H, and n, m, p, and q are integers from 0 to 3, and more preferably from 0 to 2. Most preferably, the integer n is 1, the integer m is 2, and the integers p and q are 0. In this preferred embodiment, the compounds of the invention are 2-pyrrolidinylmethyl β -D-glucosaminide 4-phosphate having the general formula (V):

in a preferred embodiment of the invention, the 3' -stereocenters (") to which they are attached^*1-3") has the configuration R, Y in equatorial position, and a pyrrolidine stereogenic center (")^**") is S.

A particularly preferred embodiment is N- [ (R) -3-tetradecanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-tetradecanoyloxytetradecanoylamino ] -3-O- [ (R) -3-tetradecanoyloxytetradecanoyl ] -beta-D-glucopyranoside, and pharmaceutically acceptable salts thereof, depicted in structural formula (II):

(N- [ (R) -3-dodecanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-dodecanoyloxytetradecanoylamino ] -3-O- [ (R) -3-dodecanoyloxytetradecanoyl ] -beta-D-glucopyranoside and pharmaceutically acceptable salts thereof, structural formula (III),

and (N- [ (R) -3-decanoyloxytetradecanoyl ] - (S) -2-pyrrolidinomethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-decanoyloxytetradecanoylamino ] -3-O- [ (R) -3-decanoyloxytetradecanoyl ] -beta-D-glucopyranoside and pharmaceutically acceptable salts thereof, structural formula (IV),

preparation of the Compounds

The compounds of the invention can be used as described in Johnson et al, bioorg.med.chem.lett.9: 2273-2278(1999) and PCT/WO98/50399 and the references cited therein. In general, the synthetic methods described in the above references are widely applicable to the preparation of compounds having different acyl groups and substitutions. It will be appreciated by those skilled in the art that the various methods described therein may be varied to employ alternative acylating agents, or may be initiated with commercially available materials having attached suitable acyl groups.

Evaluation of Compounds

The compounds provided herein can be evaluated in various assay formats to select compounds with appropriate pharmacophore distributions. For example, U.S. patent No.6,013,640 describes an animal model suitable for assessing the cardioprotective effects of the compounds described herein. The following examples also provide assays for assessing the pyrogenicity of a subject compound, and other assays for assessing the pro-inflammatory effects of the compound.

The present invention further provides pharmaceutical compositions comprising a compound provided herein in admixture with one or more pharmaceutically acceptable carriers. Suitable carriers depend on the condition to be treated and on the route of administration. Thus, a discussion of the vector is provided below along with methods of use.

Pharmaceutical composition and use thereof

In one embodiment, the present invention provides a pharmaceutical composition comprising a compound of the present invention and a pharmaceutically acceptable carrier. The compound is present in a therapeutically effective amount required for the compound to achieve the desired effect in treating a disease, disorder, or biological event. The pharmaceutical composition may be used as an adjuvant when co-administered with an antigen.

The compositions of the invention include compositions that are combined with a vaccine or other active agent, or separately formulated to deliver the active compound directly to a patient without dilution, as well as more concentrated compositions of the compound that can be formulated for later dilution to avoid shipping and/or storage of large amounts of diluent (e.g., water, saline, or aqueous materials). In general, a pharmaceutical composition of the invention for direct or immediate administration (i.e., undiluted) to a subject will comprise one or more therapeutically effective amounts of the compound. This amount will vary depending on the particular therapeutic compound and the desired therapeutic effect. More concentrated compositions comprise one or more compounds of the invention in an amount suitable for these compositions.

For the preparation of pharmaceutical compositions, the pharmaceutically acceptable carrier may be solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

In powders, the carrier is a finely divided solid which is in admixture with the finely divided active component. In tablets, the active ingredient is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

Compositions in solid form may also be prepared by spray drying an aqueous formulation of the active adjuvant (e.g. in the form of a salt) or by lyophilization and trituration with excipients.

Suitable carriers for use in the solid compositions of the present invention include, for example, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term "formulation" is meant to encompass a formulation of an active compound that provides encapsulation with the encapsulating material as a carrier, with or without the active ingredient of the other carrier being surrounded by a carrier, and thus in association with, the two. Similarly cachets and sugar lakes are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

To prepare suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active ingredient is dispersed homogeneously therein, for example by stirring. The molten homogeneous mixture is then poured into suitably sized moulds and cooled, thus solidifying.

Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, the liquid preparation may be formulated as a solution in an aqueous polyethylene glycol solution. In certain embodiments, the pharmaceutical composition is formulated as a stable emulsion formulation (e.g., a water-in-oil emulsion or an oil-in-water emulsion) or preferably an aqueous formulation comprising one or more surfactants. Suitable surfactants well known to those skilled in the art may be used in these emulsions. In one embodiment, the composition is in the form of a micellar dispersion comprising at least one suitable surfactant. Surfactants that may be used in these micellar dispersions include phospholipids. Examples of phospholipids include: diacylphosphatidylglycerols, such as: dimyristoyl phosphatidyl glycerol (DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), and distearoyl phosphatidyl glycerol (DSPG); diacyl phosphatidyl cholines, such as: dimyristoylphosphatidylcholine (DPMC), Dipalmitoylphosphatidylcholine (DPPC), and Distearoylphosphatidylcholine (DSPC); diacylphosphatidic acids, such as: dimyristoyl phosphatidic acid (DPPA), dipalmitoyl phosphatidic acid (DPPA), and distearoyl phosphatidic acid (DSPA); and diacylphosphatidylethanolamines such as: dimyristoyl Phosphatidylethanolamine (DPME), dipalmitoyl phosphatidylethanolamine (DPPE), and distearoyl phosphatidylethanolamine (DSPE). Other examples include, but are not limited to, derivatives of ethanolamine (e.g., phosphatidylethanolamine, as described above, or cephalin), serine (e.g., phosphatidylserine), and 3 '-O-lysyl glycerol (e.g., 3' -O-lysyl-phosphatidylglycerol).

Aqueous solutions suitable for oral administration can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavors, stabilizing agents, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water together with viscous materials, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

Also included are solid form preparations which are converted shortly before use to liquid form preparations for oral administration. These liquid forms include solutions, suspensions, and emulsions. In addition to the active ingredient, these formulations may contain coloring agents, perfumes, stabilizing agents, buffering agents, artificial and natural sweeteners, dispersing agents, thickening agents, solubilizing agents, and the like.

The pharmaceutical preparation is preferably in unit dosage form. In these forms, the formulations are subdivided into unit doses containing appropriate amounts of the active ingredient. The unit dosage form may be a packaged preparation, a package containing discrete quantities of the preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Alternatively, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

Thus, the adjuvant system of the present invention is particularly advantageous for the manufacture and use of vaccines and other immunostimulant compositions for the treatment or prevention of disease, for example to induce autoimmunity against antigens in mammals, preferably humans. Vaccine formulations are a well-developed technology, and general guidance for the preparation and formulation of vaccines is readily available from any of a variety of sources. An example of this is "New trends and developments in vaccines" (edited by Voller et al, University park Press, Baltimore, Md., U.S. A.1978).

In an illustrative embodiment, the antigen in the vaccine composition of the invention is a peptide, polypeptide, or immunogenic portion thereof. As used herein, an "immunogenic portion" is a portion of a protein that is recognized by (i.e., specifically binds to) a B-cell and/or T-cell surface antigen receptor. These immunogenic portions generally comprise at least 5 amino acid residues, more preferably at least 10, and even more preferably at least 20 amino acid residues of the antigenic protein or a variant thereof.

Immunogenic portions of antigenic polypeptides are generally identified using well-known techniques, such as those summarized in Paul, basic immunology, third edition, 243-247(Raven Press, 1993) and the references cited therein. These techniques include screening polypeptides for their ability to react with antigen-specific antibodies, anti-serum and/or T-cell lines or clones. Antisera and antibodies as used herein are "antigen-specific" if they bind specifically to an antigen (i.e., they react with the protein in an ELISA or other immunoassay, and do not react detectably with unrelated proteins). These antisera and antibodies can be made as described herein, and using well-known techniques. The immunogenic portion of the protein is that portion which reacts with these antisera and/or T-cells at a level which is not significantly less than the reactivity of the full-length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). These immunogenic portions can be reacted within these assays at a level similar to or greater than the reactivity of the full-length polypeptide. Such screens can generally be performed using methods well known to those of ordinary skill in the art, such as those described in Harlow and Lane, antibodies: a laboratory manual, Cold spring harbor laboratory, 1988. For example, the polypeptide may be immobilized on a solid support and contacted with patient serum such that antibodies in the serum bind to the immobilized polypeptide. Unbound serum may then be removed and used, for example,¹²⁵i-labeled protein A bound antibody was detected.

Peptide and polypeptide antigens are made using any of a variety of well-known techniques. Recombinant polypeptides encoded by the DNA sequences can be readily prepared from the isolated DNA sequences using any of a variety of expression vectors known to those of ordinary skill in the art. Expression may be effected in any suitable host cell which has been transformed or transfected with an expression vector comprising a DNA molecule encoding a recombinant polypeptide. Suitable host cells include prokaryotes, yeast, and higher eukaryotic cells, such as mammalian cells and plant cells. Preferably, the host cell used is E.coli, yeast or a mammalian cell line such as COS or CHO.

Portions and other variants of protein antigens having less than about 100 amino acids, and generally less than about 50 amino acids, can also be produced by synthetic means using techniques well known to those of ordinary skill in the art. For example, these polypeptides can be synthesized using any commercially available solid phase technique, such as the Merrifield solid phase synthesis method, in which amino acids are added sequentially to the growing amino acid chain. See Merrifield, j.am.chem.soc.85: 2149-2146, 1963. Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer/Applied biosystem division (Foster City, CA) and may be operated according to the manufacturer's instructions.

In certain particular embodiments, the polypeptide antigen used in the vaccine compositions of the invention may be a fusion protein comprising two or more different polypeptides. The fusion partner may, for example, contribute to the provision of T helper epitopes (immunological fusion partners), preferably T helper epitopes recognized by humans, or may contribute to the expression of the protein (expression enhancer) in higher yield than the original recombinant protein. Certain preferred fusion participants are both immunological and expression enhancing fusion participants. Other fusion partners may be selected to increase the solubility of the protein or to enable the protein to be targeted to a desired compartment. Other fusion partners include affinity tags that aid in the purification of the protein.

Fusion proteins can generally be made using standard techniques, including chemical conjugation. Preferably, the fusion protein is expressed as a recombinant protein such that an increased level relative to the non-fusion protein is produced in the expression system. Briefly, DNA sequences encoding the polypeptide components may be assembled separately and ligated into a suitable expression vector. The 3 'end of the DNA sequence encoding one polypeptide component is linked to the 5' end of the DNA sequence encoding the second polypeptide component, with or without a peptide linker, such that the reading frames of the sequences are in phase. This translates into a single fusion protein that retains the biological activity of the two component polypeptides.

Peptide linker sequences may be used to separate the first and second polypeptide components by a sufficient distance so as to ensure that each polypeptide folds into its secondary and tertiary structures. Such peptide linker sequences are introduced into the fusion protein using standard techniques well known in the art. Suitable peptide linker sequences may be selected based on the following factors: (1) its ability to assume a flexible extended conformation; (2) its ability to not exhibit secondary structure that can interact with functional epitopes on the first and second polypeptides; and (3) lack hydrophobic or charged residues reactive with functional epitopes of polypeptides. Preferred peptide linker sequences comprise Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala, can also be used for the linker sequence. Amino acid sequences that may be advantageously used as linkers include those disclosed in Maratea et al, Gene 40: 39-46, 1985; murphy et al, proc.natl.acad.sci.usa 83: 8258-8262, 1986; those of U.S. patent No.4,935,233 and U.S. patent No.4,751,180. The linker sequence may generally have a length of 1 to about 50 amino acids. If the first and second polypeptides have an optional N-terminal amino acid region that can be used to separate the functional domains and prevent steric interference, a linker sequence is not required.

In a preferred embodiment, the immunological fusion participant is derived from protein D, the surface protein of the gram-negative bacterium Haemophilus influenzae B (WO 91/18926). Preferably, the protein D derivative comprises about the first third of the protein (e.g., the first 100-110 amino acids of the N-terminus), and the protein D derivative may be lipidated. In certain preferred embodiments, the first 109 residues of the lipoprotein D fusion participant are included on the N-terminus to provide polypeptides with other exogenous T-cell epitopes and to increase expression levels in E.coli (and thus serve as expression enhancers). The lipid tail ensures optimal presentation of the antigen to the antigen presenting cells. Other fusion partners include the non-structural protein from influenza virus, NS1 (hemagglutinin). Typically, the N-terminal 81 amino acids are used, but different fragments including T-helper epitopes can be used.

In another embodiment, the immunological fusion partner is a protein known as LYTA, or a portion thereof (preferably the C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine amidase called the amidase LYTA (encoded by the LytA Gene; Gene 43: 265-292, 1986). LYTA is an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal region of the LYTA protein is responsible for its affinity for choline or for some choline analogues such as DEAE. This property has been exploited to develop E.coli C-LYTA expressing plasmids to express fusion proteins. Purification of hybrid proteins containing a fragment of C-LYTA at the amino terminus has been described (see biotechnologies 10: 795-798, 1992). In a preferred embodiment, the repeat portion of LYTA may be introduced into the fusion protein. The repeat portion is present in the C-terminal region starting at residue 178. Particularly preferred repeat portions comprise residues 188-305.

In another embodiment of the invention, the adjuvant system described herein is used to prepare a DNA-based vaccine composition. An illustrative vaccine of this type comprises DNA encoding one or more polypeptide antigens such that the antigens are produced in situ. The DNA may be present in any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Many gene delivery technologies are well known in the art, such as, for example, Rolland, crit. rev. tlaverp. pharmaceutical carrier system 15: 143, 198, 1998, and those described in the references cited therein. Suitable nucleic acid expression systems comprise DNA sequences (e.g., suitable promoter regions and termination signals) required for expression in a patient. Bacterial delivery systems include the provision of immunogenic portions of the polypeptide expressed on its cell surface or bacteria that secrete such epitopes (e.g., Bacillus-Calmette-Guerrin). In a preferred embodiment, the DNA is introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), typically involving the use of a nonpathogenic (defective), replication competent virus. Illustrative systems are disclosed, for example, in Fisher-Hoch et al, proc.natl.acad.sci.usa 86: 317 and 321, 1989; flexner et al, ann.n.y.acad.sci.569: 86-103, 1989; flexner et al, vaccine 8: 17-21, 1990; U.S. patent nos.4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. patent nos.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; berkner, biotechnology 6: 616-627, 1988; rosenfeld et al, science 252: 431-434, 1991; kolls et al, proc.natl.acad.sci.usa 91: 215-; Kass-Eisler et al, proc.natl.acad.sci.usa 90: 11498. 11502, 1993; guzman et al, cycle 88: 2838 2848, 1993; and Guzman et al, cir.res.73: 1202-1207, 1993. Techniques for introducing DNA into these expression systems are well known to those of ordinary skill in the art.

In addition, DNA may be "naked," for example, by Ulmer et al, science 259: 1745. cndot. 1749, 1993 and published by Cohen, science 259: 1691 1692, 1993. Uptake of naked DNA can be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells. Obviously, the vaccine may comprise both polynucleotide and polypeptide components as required.

In addition, the vaccine may obviously comprise pharmaceutically acceptable salts of the desired polynucleotide, polypeptide and/or carbohydrate antigen. For example, such salts can be prepared from pharmaceutically acceptable non-toxic bases including organic bases (e.g., salts of primary, secondary, and tertiary amines and basic amino acids) and inorganic bases (e.g., sodium, potassium, lithium, ammonium, calcium, and magnesium salts).

The adjuvant system of the present invention has a strong adjuvant effect when administered under a wide dosage range and ratio.

The amount of antigen in each vaccine dose is generally selected to induce an immune protective response in a typical vaccine without significant adverse side effects. These amounts depend on which particular immunogen is used and how it is presented. In general, each dose is expected to contain about 1 to 1000. mu.g of protein, most usually about 2 to 100. mu.g, preferably about 5 to 50. mu.g. Of course, the dose given may depend on age, weight, type of concurrent treatment (if any) and nature of the antigen administered.

The immunogenic activity of a given amount of a vaccine composition of the invention can be readily determined, for example, by monitoring the increase in antibody titer to the antigen used in the vaccine composition (Dalsgaard, K.A. ActaVeteria Scandinavica 69: 1-40 (1978)). Another common method involves intradermal injection of CD-1 mice with various amounts of vaccine composition, followed by collection of serum from the mice and testing for anti-immunogen antibodies by, for example, ELISA. These and other similar methods will be apparent to the skilled artisan.

The antigen can be from and/or isolated from essentially any desired source, depending on the infectious disease, autoimmune disease, disorder, cancer, pathogen, or disease to be treated with a given vaccine composition. For example, the antigen may be derived from a viral source, such as influenza virus, feline leukemia virus, feline immunodeficiency virus, human HIV-1, HIV-2, herpes simplex type 2 virus, human cytomegalovirus, hepatitis A, B, C or E, respiratory syncytial virus, human papilloma virus, rabies, measles, or foot and mouth disease virus. Illustrative antigens may also be derived from bacterial sources such as anthrax, pertussis, Lyme disease, malaria, tuberculosis, leishmaniasis, t. Antigens are typically composed of natural or synthetic amino acids, e.g., in the form of peptides, polypeptides, or proteins, may be composed of polysaccharides, or may be mixtures thereof. Illustrative antigens can be isolated from natural sources, synthesized using solid phase synthesis, or can be obtained using recombinant DNA techniques.

In another embodiment, tumor antigens are used in the vaccine composition of the invention to prevent and/or treat cancer. Cancer cells typically have specific antigens on their surface, such as truncated epidermal growth factor, folate binding proteins, epithelial mucins, melanoferrin, carcinoembryonic antigen, prostate specific membrane antigen, HER2-neu, which are candidates for therapeutic cancer vaccines. Because tumor antigens are normal or associated with normal components of the body, the immune system is generally unable to generate an effective immune response against those antigens to repel tumor cells. To achieve this response, the adjuvant systems described herein may be used. As a result, the exogenous protein can enter a pathway for processing the endogenous antigen, resulting in the production of cytolytic or cytotoxic T Cells (CTLs). This adjuvant effect contributes to the generation of antigen-specific CTLs that seek out and destroy those tumor cells that carry tumor antigens on their surface for immunization. Illustrative types of cancer in which this approach can be used include prostate, colon, breast, ovarian, pancreatic, brain, head and neck, melanoma, leukemia, lymphoma, and the like.

In another embodiment of the invention, the adjuvant system of the invention may be administered alone, i.e., without co-administered antigen, thus boosting the immune system to treat chronic infectious diseases, especially in immunocompromised patients. Illustrative examples of infectious diseases that can be treated or prevented using this approach can be found in U.S. Pat. No.5,508,310. Such a boosting effect on the immune system may also be used as a prophylactic measure to limit the risk of nosocomial and/or postoperative infections.

In another embodiment, the antigen present in the vaccine composition is not a foreign antigen, but is an autoantigen, e.g., the vaccine composition is directed against autoimmune diseases such as type 1 diabetes, general organ-specific autoimmune diseases, neurological diseases, rheumatic diseases, psoriasis, connective tissue diseases, autoimmune cytopenia, and other autoimmune diseases. These conventional organ-specific autoimmunities may include thyroiditis (Graves + Hashimoto's), gastritis, adrenalitis (Addison's), oophoritis, primary biliary cirrhosis, myasthenia gravis, gonadal failure (gonadal failure), hypoparathyroidism, alopecia, malabsorption syndrome, pernicious anemia, hepatitis, anti-receptor antibody disease and vitiligo. These neurological disorders may include schizophrenia, Alzheimer's disease, depression, hypopituitarism, diabetes insipidus, sjogren's syndrome and multiple sclerosis. These rheumatic/connective tissue diseases may include rheumatoid arthritis, Systemic Lupus Erythematosus (SLE) or lupus, scleroderma, polymyositis, inflammatory bowel disease, dermatomyositis, ulcerative colitis, Crohn's disease, vasculitis, psoriatic arthritis, exfoliative psoriatic dermatitis, pemphigus vulgaris, Sjogren's syndrome. Other autoimmune-related diseases can include autoimmune uvoretinitis, glomerulonephritis, myocardial posterior wall infarction, cardiotomy syndrome, pulmonary hemosiderosis, amyloidosis, sarcoidosis, aphthous stomatitis, and other immune-related diseases, such as those given herein and known in the relevant arts.

Although any suitable carrier known to those of ordinary skill in the art may be used in the vaccine compositions of the present invention, the type of carrier will generally vary depending on the desired mode of administration. The compositions of the present invention may be formulated for any suitable mode of administration, including, for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, intradermal, subcutaneous, or intramuscular administration. For parenteral administration, such as subcutaneous injection, the carrier will usually comprise water, saline, alcohol, fat, wax or buffer. For oral administration, the above carriers are generally used, or solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate may also be employed. Biodegradable microspheres (e.g., polylactic polyglycolic acid) may also be used as carriers for the compositions of the present invention. Suitable biodegradable microspheres are disclosed, for example, in U.S. patent nos.4,897,268; 5,075,109; 5,928,647; 5,811,128, respectively; 5,820,883, respectively; 5,853,763, respectively; 5,814,344 and 5,942,252, the disclosures of which are hereby incorporated by reference in their entirety. Modified hepatitis B core protein carrier systems are also suitable, such as those described in WO/9940934, and the references cited therein, all of which are incorporated herein by reference. Carriers comprising particle-protein complexes, such as those described in U.S. patent No.5,928,647, the disclosure of which is incorporated herein by reference in its entirety, can also be employed, which are capable of inducing a class I-restricted cytotoxic T lymphocyte response in a subject.

In an illustrative embodiment, the vaccine formulation is administered to the mucosa, especially the oral cavity, and preferably to the sublingual space, for eliciting an immune response. Oral administration is in many cases preferred over traditional parenteral delivery due to the ease and convenience of non-invasive administration techniques. In addition, the regimen further provides a means for eliciting mucosal immunity, which is difficult to achieve with conventional parenteral delivery, and which can provide protection against airborne pathogens and/or allergens. Other advantages of buccal administration are that patient compliance can be improved using sublingual vaccine delivery, particularly for pediatric applications, or for applications where multiple injections over an extended period of time are often required, such as for allergic desensitization therapy.

The vaccine composition may also contain buffering agents (e.g., neutral buffered saline, phosphate buffered saline or phosphate buffered w/o saline), carbohydrates (e.g., glucose, mannose, sucrose or dextran), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of the recipient, suspending agents, thickening agents and/or preservatives. Additionally, the compositions of the present invention may also be formulated as a lyophilized body. The compositions may also be encapsulated within liposomes using well known techniques.

Thus, in one embodiment, the vaccine composition is an aqueous formulation comprising an effective amount of one or more surfactants. For example, the composition may be in the form of a micellar dispersion comprising at least one suitable surfactant, e.g., a phospholipid surfactant. Illustrative examples of phospholipids include diacylphosphatidylglycerols, such as Dimyristoylphosphatidylglycerol (DPMG), Dipalmitoylphosphatidylglycerol (DPPG), and Distearoylphosphatidylglycerol (DSPG), diacylphosphatidylcholines, such as Dimyristoylphosphatidylcholine (DPMC), Dipalmitoylphosphatidylcholine (DPPC), and Distearoylphosphatidylcholine (DSPC); diacylphosphatidic acids, such as dimyristoyl phosphatidic acid (DPMA), dipalmitoyl phosphatidic acid (DPPA), and distearoyl phosphatidic acid (DSPA); and diacylphosphatidylethanolamines such as Dimyristoylphosphatidylethanolamine (DPME), Dipalmitoylphosphatidylethanolamine (DPPE), and Distearoylphosphatidylethanolamine (DSPE). Typically, the surfactant in the aqueous formulation: the adjuvant molar ratio is from about 10: 1 to about 1: 10, more typically from about 5: 1 to about 1: 5, although any effective amount of surfactant may be used in the aqueous formulation to best suit the particular target of interest.

In another embodiment, the composition is an emulsion, such as a water-in-oil emulsion or an oil-in-water emulsion. These emulsions are generally well known to those skilled in the art.

The adjuvant system of the present invention may be used as the sole adjuvant system or, alternatively, may be administered with other adjuvants or immune effectors. For example, these adjuvants may include oil-based adjuvants (e.g., freund's complete and incomplete adjuvants), liposomes, mineral salts (e.g., AlK (SO)₄)₂，AlNa(SO₄)₂，AlNH₄(SO₄) Silica, alum, Al (OH)₃，Ca₃(PO₄)₂Kaolin, and carbon), polynucleotides (e.g., poly IC and poly AU acids), polymers (e.g., nonionic block polymers, polyphosphazenes, cyanoacrylates, polymerase- (DL-lactide-co-glycosides), and others, as well as certain natural substances (e.g., lipid a and its derivatives, wax D from mycobacterium tuberculosis, and substances found in corynebacterium parvum, bordetella pertussis, and bacteria of the genus brunella), bovine serum albumin, diphtheria toxoid, tetanus toxoid, ephedrine, keyhole limpet hemocyanin, pseudotoxin a, cholera toxin, pertussis toxin, viral proteins, and eukaryotic proteins such as interferons, interleukins, or tumor necrosis factors. These proteins may be obtained from natural or recombinant sources according to methods well known to those skilled in the art. If obtained from a recombinant source, the adjuvant may comprise a protein fragment that includes at least the immunostimulatory portion of the molecule. Other known immunostimulatory macromolecules useful in the invention include, but are not limited to, polysaccharides, tRNA's, non-metabolic synthetic polymers such as polyvinylamine, polymethacrylic acid, polyvinylpyrrolidone, mixed polycondensates of 4 ', 4-diaminodiphenylmethane-3, 3 ' -dicarboxylic acid and 4-nitro-2-aminobenzoic acidRelatively high molecular weight) (see Sela, m., science 166: 1365-1374(1969)) or glycolipids, lipids or carbohydrates.

In one embodiment, the adjuvant system is preferably designed to induce a predominantly Th 1-type immune response. High levels of Th 1-type cytokines (e.g., IFN-. gamma., TNF. alpha., IL-2 and IL-12) tend to be beneficial in inducing cell-mediated immune responses to administered antigens. In contrast, high levels of Th 2-type cytokines (e.g., IL-4, IL-5, IL-6, and IL-10) tend to be beneficial in inducing humoral immune responses. Following application of the vaccines provided herein, the patients will support immune responses, including Th 1-and Th 2-type responses. In a preferred embodiment, wherein the response is predominantly a Th 1-type, the level of Th 1-type cytokine is increased to a greater extent than the level of Th 2-type cytokine. The levels of these cytokines can be readily assessed using standard assays. For a review of cytokine classes, see Mosmann and Coffman, ann.rev.immunol 1.7: 145-173, 1989.

For example, other adjuvants for eliciting predominantly Th 1-type responses include, for example, monophosphoryl lipid A, such as 3-de-O-acylated monophosphoryl lipid A (3D-MPL), in combination with an aluminum salt. MPL adjuvant WAs obtained from Corixa corporation (Seattle, WA; see U.S. Pat. Nos.4,436,727; 4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides, in which the CpG dinucleotide is unmethylated, also induce a predominantly Th1 response. These oligonucleotides are well known and described, for example, in WO 96/02555, WO99/33488, and U.S. patent nos.6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, in Sato et al, science 273: 352,1996. Other illustrative adjuvants that may be included in the vaccine composition include Montanide ISA 720(Seppic, France), SAF (Chiron, California, usa), iscoms (csl), MF-59(Chiron), Detox^TMAdjuvant (Corixa, Hamilton, MT).

The compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule, sponge or gel (e.g., composed of polysaccharides) that provides sustained release of the compound after administration). These formulations are generally prepared using well-known techniques (see, e.g., Coombes et al, vaccine 14: 1429-. Sustained release formulations may comprise a polypeptide, polynucleotide or antibody dispersed in a carrier matrix and/or contained within a reservoir surrounding a rate controlling membrane. The carriers used in these formulations are biocompatible and may also be biodegradable; preferably the formulation provides a relatively constant level of active ingredient release. These carriers include microparticles of poly (lactide-co-glycolide), polyacrylates, latex, starch, cellulose, dextran and the like. Other delayed release carriers include supramolecular biovoectors comprising a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an outer layer comprising amphiphilic compounds, such as phospholipids (see, e.g., U.S. patent No.5,151,254 and PCT applications WO 94/20078, WO/94/23701 and WO 96/06638). The amount of active compound included in the sustained release formulation will vary depending on the site of implantation, the rate and expected duration of release, and the nature of the condition to be treated or prevented.

Any of a variety of known delivery vehicles can be used in pharmaceutical compositions and vaccines to facilitate the generation of cell-targeted antigen-specific immune responses. Delivery vehicles include Antigen Presenting Cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that can be engineered to be effective APCs. These cells may, but need not, be genetically engineered to increase the capacity for presenting antigens, enhance the activation and/or maintenance of T cell responses, have an anti-target effect per se and/or are immunologically compatible with the recipient (i.e., the matched HLA haplotype). APCs can generally be isolated from any of a variety of biological fluids and organs, including tumors and peritumoral tissues, and can be autologous, allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the invention use dendritic cells or prototypes thereof as antigen presenting cells. Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392: 245-. In general, dendritic cells can be defined by their typical shape (star-shaped in situ, visible in vitro distinct cytoplasmic processes (dendrites)), their absorptive capacity, their ability to efficiently process and present antigen and their ability to activate naive T cell responses.

Dendritic cells can of course be designed to express specific cell surface receptors or ligands that are not normally found on dendritic cells in vivo or in vitro, and these modified dendritic cells are contemplated by the present invention. As an alternative to dendritic cells, dendritic cells loaded with secretory vesicle antigens (called exosomes) can be used in vaccines (see Zitvogel et al, Nature Med.4: 594-600, 1998).

Dendritic cells and precursors can be obtained from peripheral blood, bone marrow, tumor-infiltrating cells, peri-tumor tissue-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood, or any other suitable tissue or fluid. For example, dendritic cells can be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, IL-4, IL-13, and/or TNF α to a culture of monocytes obtained from peripheral blood. Alternatively, CD34 positive cells obtained from peripheral blood, umbilical cord blood or bone marrow may be differentiated into dendritic cells by adding to the culture medium a combination of GM-CSF, IL-3, TNF α, CD40 ligand, LPS, f1t3 ligand and/or other compounds that induce differentiation, maturation and proliferation of dendritic cells.

Dendritic cells are conveniently classified as "immature" and "mature" cells, which allow for the discrimination of two well-characterized phenotypes in a simple manner. But this nomenclature should not be understood to exclude all possible intermediate differentiation stages. Immature dendritic cells are characterized by high capacity of APC for antigen uptake and processing, which is associated with high expression of Fcr receptor and mannose receptor. The mature phenotype is generally characterized by low expression of these markers, but high expression of cell surface molecules associated with T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-1 BB).

The APCs can generally be transfected with the polynucleotide encoding the antigenic polypeptide (or portion or other variant thereof) such that the antigenic polypeptide, or immunogenic portion thereof, is expressed on the surface of the cell. These transfections occur ex vivo, and compositions or vaccines comprising these transfected cells, and adjuvants described herein, can then be used for therapeutic purposes.

Alternatively, a gene delivery vehicle targeting dendritic cells or other antigen presenting cells can be administered to a patient, resulting in transfection occurring in vivo. In vivo and in vitro transfection of dendritic cells, for example, can generally be accomplished using any method known in the art, such as those described in WO 97/24447, or described in Mahvi et al, immunology and cell biology 75: 456-460, 1997. Antigen loading of dendritic cells can be accomplished by contacting the dendritic cells or progenitor cells with antigenic polypeptides, DNA (naked or in a plasmid vector) or RNA; or by breeding with antigen-expressing recombinant bacteria or viruses (e.g., vaccinia, avipox, adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be covalently linked to an immunological participant (e.g., a carrier molecule) that provides T cell assistance. In addition, dendritic cells can be pulsed with unconjugated immunological participants, either alone or in the presence of a polypeptide.

Treatment of nitric oxide related disorders

In one aspect, the invention provides methods for treating diseases or conditions mediated by nitric oxide, particularly ischemia and reperfusion injury. The method comprises administering to a subject in need of such treatment an effective amount of a compound of the invention. It is generally accepted that inducers of iNOS gene transcription and protein synthesis are pro-inflammatory and therefore somewhat "toxic" or not readily tolerated in animals and humans. Endotoxin (LPS) and proinflammatory cytokines such as IL-1, TNF- α and IFN- γ are known iNOS inducers. They are both toxic in nature and when administered to animals are capable of inducing systemic inflammatory responses, adult respiratory distress syndrome, multi-organ failure and cardiovascular rupture.

Studies of the cardioprotective activity of MPL * immunostimulant indicate that Induction of Nitric Oxide Synthase (iNOS) is important in the delayed cardioprotective action of this compound. In addition, Nitric Oxide (NO) signaling through the constitutive pool of NOS is estimated to be important in the acute cardioprotective action of this compound. Given the residual endotoxoid activity of MPL * immunostimulant, it is not surprising that this compound is capable of inducing nitric oxide signaling. In addition, nitric oxide signaling has been identified as a possible method by which ischemic preconditioning leads to cardioprotection. Considering that the nitric oxide donor is cardioprotective, this observation provides further support that the NOS/NO pathway is a route for the MPL * immunostimulant cardioprotection.

The compounds of the present invention are useful in methods of treating diseases or conditions that are modulated or ameliorated by nitric oxide, particularly ischemia and reperfusion injury (see, U.S. patent application serial No.: 09/808669, filed 3/14/2001, which describes the cardioprotective properties of aminoalkyl glucosaminide phosphates and methods for analyzing the cardioprotective properties).

Examples

The following examples are intended to illustrate, but not limit, the claimed invention.

Example 1 preparation of N- [ (R) -3-tetradecanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-tetradecanoyloxytetradecanoylamino ] -3-O- [ (R) -3-tetradecanoyloxytetradecanoyl ] -beta-D-glucopyranoside triethylammonium salt: triethylammonium salt of a compound of formula (II)

(1a) To 2-deoxy-4-O-diphenylphosphino-3-O- [ (R) -3-tetradecanoyloxytetradecanoyl group]A solution of-6-O- (2, 2, 2-trichloro-1, 1-dimethylethoxycarbonyl) -2- (2, 2, 2-trichloroethoxycarbonylamino) - β -D-glucopyranosyl bromide (1.05g, 0.81mmol) in dry 1, 2-dichloroethane (10mL) was added 4 * molecular sieves (0.5g), dry CaSO₄(2.2g, 16mmol), and N- [ (R) -3-tetradecanoyloxy-tetradecanoylAcyl radical]- (S) -2-pyrrolidinemethanol (0.40g, 0.75 mmol). The resulting mixture was stirred at room temperature for 1h with Hg (CN)₂(1.02g, 4.05mmol) and heated to reflux in the dark for 16 h. The cooled reaction mixture is treated with CH₂Cl₂Diluted and filtered. The filtrate was washed with 1N aq KI and dried (Na)₂SO₄) And concentrating. Flash chromatography on silica gel (gradient elution, 15 → 20% EtOAc/hexanes) afforded 0.605g (43%) of N- [ (R) -3-tetradecanoyloxytetradecanoyl]- (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-diphenylphosphino-3-O- [ (R) -3-tetradecanoyloxytetradecanoyl]-6-O- (2, 2, 2-trichloro-1, 1-dimethylethoxycarbonyl) -2- (2, 2, 2-trichloroethoxycarbonylamino) - β -D-glucopyranoside, an amorphous solid.

(1b) A solution of the compound prepared in (1a) above (0.50g, 0.29mmol) in AcOH (10mL) was treated with zinc powder (0.98g, 15mmol) in three equal portions over 1-h at 60 ℃. The cooled reaction mixture was sonicated, filtered through a pad of Celite, and concentrated. The residue obtained is in CH₂Cl₂And saturated aq NaHCO₃Divided among them and layered. The organic layer was dried (Na)₂SO₄) And concentrating. The resulting crude amino alcohol and (R) -3-tetradecanoyloxytetradecanoic acid (0.155g, 0.34mmol) in CH₂Cl₂(3.5mL) was stirred with powdered 4 * molecular sieve (0.25g) for 0.5h and then treated with 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (0.11g, 0.44 mmol). The resulting mixture was stirred at room temperature for 8h, filtered through Celite, and concentrated. Flash chromatography on silica gel with 50% EtOAc/hexanes provided 0.355g (68%) of N- [ (R) -3-tetradecanoyloxytetradecanoyl]- (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-diphenylphosphino-2- [ (R) -3-tetradecanoyloxytetradecanoylamino]-3-O- [ (R) -3-tetradecanoyloxytetradecanoyl group]- β -D-glucopyranoside, a colorless syrup.

(1c) A solution of the compound prepared in (1b) above (0.300g, 0.166mmol) in a mixture of AcOH (1mL) and tetrahydrofuran (9mL) in PtO₂(0.15g) was hydrogenated in the presence of hydrogen at room temperature and 70psig for 18 h. Reaction ofThe mixture was diluted with 2: 1CHCl₃MeOH (50mL) dilution and simple sonication. The catalyst was collected and used in 2: 1CHCl₃MeOH wash, then concentrate the combined filtrate and wash. Use of CHCl on silica gel₃-MeOH-H₂O-Et₃Flash chromatography on N (90: 10: 0.5) gave the partially purified product, which was then dissolved in ice-cold 2: 1CHCl₃MeOH (30mL) and ice-cold 0.1N aq HCl (12 mL). The organic phase was filtered and extracted from 2% aq Et₃Freeze-drying in N (5mL, pyrogen-free) gave 0.228g (79%) of N- [ (R) -3-tetradecanoyloxytetradecanoyl group]- (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-phosphono-2- [ (R) -3-tetradecanoyloxytetradecanoylamino]-3-O- [ (R) -3-tetradecanoyloxytetradecanoyl group]- β -D-glucopyranoside triethylammonium salt, a colorless powder:

mp 67-70 ℃; IR 3306, 2955, 2923, 2853, 1736, 1732, 1644, 1548, 1466, 1378, 1245, 1177, 1110, 1053, 844 cm^-1；¹H NMR(CDCl₃-CD₃OD)δ0.88(m，18H)，1.0-1.2.05(mH)，2.20-2.70(m，12H)，3.06(q，6H，J＝7.2Hz)，3.3-325(mH)，4.52(d，1H，J＝8Hz)，5.05-5.28(m，4H)，7.44(d，1H，J＝9Hz)；¹³C NMR(CDCl₃)δ173.3，173.0，170.3，169.6，168.6，101.8，100.4，75.8，72.5，72.4，70.9，70.8，70.3，70.2，69.9，69.3，67.9，66.6，56.5，56.3，54.5，47.4，45.8，44.6，41.4，41.0，39.7，39.2，39.0，34.5，34.3，34.1，32.0，29.7，29.4，28.1，27.3，25.7，25.3，25.2，25.1，24.0，22.7，21.6，14.1，8.6.

Theoretical calculation value C₁₀₁H₁₉₄N₃O₁₇P·H₂O：C，68.47；H，11.15；N，2.37；P，

1.75. Found C, 68.79; h, 11.00; n, 2.24; p, 1.97.

Example 2: preparation of N- [ (R) -3-dodecanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-dodecanoyloxytetradecanoylamino ] -3-O- [ (R) -3-dodecanoyloxytetradecanoyl ] - β -D-glucopyranoside triethylammonium salt); triethylammonium salt of compound (III).

(2a) To 2-deoxy-4-O-diphenylphosphino-3-O- [ (R) -3-dodecanoyloxytetradecanoyl]A solution of (E) -6-O- (2, 2, 2-trichloro-1, 1-dimethylethoxycarbonyl) -2- (2, 2, 2-trichloroethoxycarbonylamino) -alpha- (D-glucopyranosyl bromide (1.60g, 1.27mmol) in dry 1, 2-dichloroethane (3.2mL) was added 4 * molecular sieve (0.6g), dry CaSO₄(1.0g, 7.3mmol), and N- [ (R) -3-dodecanoyloxytetradecanoyl]- (S) -2-pyrrolidinemethanol (0.58g, 1.14 mmol). The resulting mixture was stirred at room temperature for 1h with Hg (CN)₂(0.58g, 2.3mmol) and heated to reflux in the dark for 6 h. The cooled reaction mixture is treated with CH₂Cl₂Dilute and filter through a celite bed. The filtrate was washed with 1N aq KI and dried (Na)₂SO₄) And concentrating. Flash chromatography on silica gel (gradient elution, 25 → 35% EtOAc/hexane) afforded 1.72g (82%) of N- [ (R) -3-dodecanoyloxytetradecanoyl]- (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-diphenylphosphino-3-O- [ (R) -3-dodecanoyloxytetradecanoyl]-6-O- (2, 2, 2-trichloro-1, 1-dimethylethoxycarbonyl) -2- (2, 2, 2-trichloroethoxycarbonylamino) - β -D-glucopyranoside, a colorless oil.

(2b) A solution of the compound prepared in (2a) above (1.58g, 0.806mmol) in AcOH (40mL) was treated with zinc powder (2.6g, 40mmol) in three equal portions at 60 ℃ over 1 hour. The cooled reaction mixture was sonicated, filtered through a pad of Celite, and concentrated. The resulting residue was purified in EtOAc and saturated aq NaHCO₃Divided and layered in between. The organic layer was washed with brine and dried (Na)₂SO₄) And concentrated to give 1.3g of a white solid. The resulting crude amino alcohol and (R) -3-dodecanoyloxytetradecanoic acid (0.45g, 1.05mmol) in CH₂Cl₂(20mL) was treated with 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (0.30g, 1.21 mmol). The resulting mixture was stirred at room temperature for 18h and concentrated. Flash chromatography on silica gel with 40 → 50% EtOAc/hexanes afforded 0.89g(56%) N- [ (R) -3-dodecanoyloxytetradecanoyl]- (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-diphenylphosphino-2- [ (R) -3-dodecanoyloxytetradecanoylamino]-3-O- [ (R) -3-dodecanoyloxytetradecanoyl]-beta-D-glucopyranoside, a white foam material.

(2c) A solution of the compound prepared in (2b) above (0.75g, 0.44mmol) in a mixture of AcOH (4.5mL) and tetrahydrofuran (45mL) in PtO₂(0.45g) was hydrogenated in the presence of hydrogen at room temperature and 70psig for 18 h. The reaction mixture was diluted with 2: 1CHCl₃MeOH (35mL) dilution and simple sonication. The catalyst was collected and used in 2: 1CHCl₃MeOH wash, then concentrate the combined filtrate and wash. On silica gel with CHCl₃-MeOH-H₂O-Et₃N (gradient elution; 96: 4: 0.3 → 90: 10: 0.5) was subjected to flash chromatography to give a partially purified product (0.51g), which was dissolved in ice-cold 2: 1CHCl₃MeOH (50mL) and washed with ice cold 0.1Naq HCl (20 mL). The organic phase was filtered and concentrated. The resulting white wax was washed from 2% aqEt₃Freeze-drying in N (70mL, pyrogen-free) gave 0.54g (78%) of N- [ (R) -3-dodecanoyloxytetradecanoyl]- (S) -2-pyrrolidinomethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-dodecanoyloxy tetradecanoylamino]-3-O- [ (R) -3-dodecanoyloxytetradecanoyl]- β -D-glucopyranoside triethylammonium salt, a white powder:

mp 146-; IR (film) 3292, 3100, 2958, 2922, 2852, 1739, 1731, 1659, 1651, 1644, 1562, 1555, 1468, 1455, 1433, 1377, 1339, 1310, 1253, 1238, 1183, 1160, 1107, 1080, 1047, 960, 856, 722cm^-1；¹H NMR(CDCl₃-CD₃OD)δ 0.88(m，18H)，1.0-2.10(mH)，2.20-2.75(m，12H)，3.04(q，6 H，J＝7.2Hz)，3.3-4.3(mH)，4.45(d，1H，J＝8.5Hz)，5.0-5.28(m，4H)；¹³C NMR(CDCl₃)δ173.9，173.4，173.2，170.6，170.1，169.2，101.4，75.5，74.0，70.8，70.7，70.2，68.5，60.5，56.6，53.6，47.4，45.6，40.9，39.6，38.8，34.5，34.3，34.2，34.1，31.9，29.7，29.6，29.5，29.4，29.4，29.3，29.2，27.3，25.2，25.0，23.6，22.7，21.6，14.0，8.3.

MALDI-MS calculated value [ M + Na ]]⁺1590.1900, found 1590.1866; theoretical calculation value

C₉₅H₁₈₂N₃O₁₇P·3H₂O: c, 66.20; h, 10.99; n, 2.44, found C, 66.36; h, 10.69; and N, 2.15.

Example 3: preparation of N- [ (R) -3-decanoyloxytetradecanoyl ] - (S) -2-pyrrolidinylmethyl 2-deoxy-4-O-phosphono-2- [ (R) -3-decanoyloxytetradecanoylamino ] -3-O- [ (R) -3-decanoyloxytetradecanoyl ] - β -D-glucopyranoside triethylammonium salt); triethylammonium salt of compound (IV).

(3a) To 2-deoxy-4-O-diphenylphosphino-3-O- [ (R) -3-decanoyloxytetradecanoyl group]A solution of-6-O- (2, 2, 2-trichloro-1, 1-dimethylethoxycarbonyl) -2- (2, 2, 2-trichloroethoxycarbonylamino) -alpha-D-glucopyranosyl bromide (1.70g, 1.38mmol) in dry 1, 2-dichloroethane (3.5mL) was added 4 * molecular sieves (0.6g), dry CaSO₄(1.2g, 8.8mmol), and N- [ (R) -3-decanoyloxytetradecanoyl group]- (S) -2-pyrrolidinemethanol (0.60g, 1.24 mmol). The resulting mixture was stirred at room temperature for 1h with Hg (CN)₂(0.63g, 2.5mmol) and heated to reflux in the dark for 6 h. The cooled reaction mixture is treated with CH₂Cl₂Dilute and filter through a celite bed. The filtrate was washed with 1N aq KI and dried (Na)₂SO₄) And concentrating. Flash chromatography on silica gel (gradient elution, 25 → 40% EtOAc/hexanes) afforded 1.82g (80%) of N- [ (R) -3-decanoyloxytetradecanoyl]- (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-diphenylphosphino-3-O- [ (R) -3-decanoyloxytetradecanoyl]-6-O- (2, 2, 2-trichloro-1, 1-dimethylethoxycarbonyl) -2- (2, 2, 2-triaminoethoxycarbonylamino) - β -D-glucopyranoside, a colorless oil.

(3b) A solution of the compound prepared in (3a) above (1.67g, 1.02mmol) in AcOH (50mL) was partitioned with zinc powder (3.33g, 51mmol) at 60CThree equal portions were treated in 1 h. The cooled reaction mixture was sonicated, filtered through a pad of Celite, and concentrated. The resulting residue was purified in EtOAc and saturated aq NaHCO₃Divided and layered in between. The organic layer was washed with brine and dried (Na)₂SO₄) And concentrated to give 1.25g of a white solid. The resulting crude amino alcohol and (R) -3-decanoyloxytetradecanoic acid (0.53g, 1.33mmol) were added to CH₂Cl₂(20mL) was treated with 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline (0.38g, 1.53 mmol). The resulting mixture was stirred at room temperature for 18h and concentrated. Flash chromatography on silica gel with 40 → 50% EtOAc/hexanes afforded 1.23g (74%) of N- [ (R) -3-decanoyloxytetradecanoyl]- (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-diphenylphosphino-2- [ (R) -3-decanoyloxytetradecanoylamino]-3-O- [ (R) -3-decanoyloxytetradecanoyl group]-beta-D-glucopyranoside, a white foam material.

(3c) A solution of the compound prepared in (3b) above (1.07g, 0.654mmol) in a mixture of AcOH (6.5mL) and tetrahydrofuran (65mL) in PtO₂(0.66g) was hydrogenated in the presence of hydrogen at room temperature and 70psig for 18 h. The reaction mixture was diluted with 2: 1CHCl₃MeOH (50mL) dilution and simple sonication. The catalyst was collected and used in 2: 1CHCl₃MeOH wash, then concentrate the combined filtrate and wash. The resulting waxy solid was lyophilized from 2% aq triethylamine to give about 1g of crude triethylammonium salt as a white powder. On silica gel with CHCl₃-MeOH-H₂O-Et₃Flash chromatography of N (gradient elution; 96: 4: 0.3 → 88: 12: 1: 0.6) afforded the partially purified product (0.84g), which was dissolved in ice-cold 2: 1CHCl₃MeOH (168mL) and ice-cold 0.1N aq HCl (67 mL). The organic phase was filtered and concentrated. The resulting white wax (about 0.7g) was removed from 2% aq Et₃Freeze-drying in N (70mL, pyrogen-free) gave 0.79g (79%) of N- [ (R) -3-decanoyloxytetradecanoyl group]- (S) -2-pyrrolidinylmethyl-2-deoxy-4-O-phosphono-2- [ (R) -3-decanoyloxytetradecanoylamino]-3-O- [ (R) -3-decanoyloxytetradecanoyl group]- β -D-glucopyranoside triethylammonium salt, a white powder: mp 121-122C; IR (film)

3287，3093，2961，2913，2850，1745，1738，1732，1716，1666，1660，1651，1644，1635，1565，1556，1538，1470，1455，1434，1416，1378，1337，1311，1248，1184，1104，1081，1021，964，721cm^-1；¹H NMR(CDCl₃-CD₃OD)δ0.88(m，18H)，1.0-2.05(mH)，2.20-2.75(m，12H)，3.04(q，6H，J＝7.2Hz)，3.3-4.3(mH)，4.45(d，1H，J＝8.5Hz)，5.0-5.28(m，4H)；¹³C NMR(CDCl₃)δ173.7，173.4，173.2，170.5，170.1，169.1，101.4，75.6，74.0，70.8，70.2，68.7，60.4，56.6，53.8，47.4，45.6，41.0，39.6，38.9，34.5，34.3，34.2，34.1，31.9，29.7，29.6，29.5，29.4，29.4，29.3，29.2，27.3，25.3，25.0，23.7，22.7，21.6，14.1，8.4.

MALDI-MS calculated value [ M + Na ]]⁺1506.0961, found 1506.1008; theoretical calculation value

C₈₉H₁₇₀N₃O₁₇P: c, 67.43; h, 10.81; n, 2.65, found C, 67.26; h, 10.85; and N, 2.47.

Examples 4 to 8

The primary objective of examples 4-8 was to determine whether the compound of formula (II) prepared in example 1 (as the triethylamine salt) (hereinafter "compound II") is capable of stimulating minimal pyrogenicity and mediating adjuvant activity when formulated with vaccine antigens.

Example 4 adjuvant Activity against HBsAg (hepatitis B surface antigen)

Several groups of 6-8 week old BALB/c mice (Jackson Laboratories Barharbor, Maine) were injected s.c. with 2 μ g HBsAg (laboratory Pablo Cassara). + -. 20 μ g adjuvant (MPL * immunostimulant or Compound II) on days 0 and 21. The vaccine was made by mixing an adjuvanted TEoA (triethanolamine) formulation with recombinant HBsAg. Titers to HBsAg were determined by ELISA from pooled sera (5 mice/group) collected 21 days after the second vaccination (table 1). Non-immune controls were not vaccinated.

The serum titers of mice receiving compound II were significantly higher than the anti-HBsAg response of control sera receiving antigen alone (table 1). Of particular interest is the increase in potency values of the IgG2a and IgG2b isotypes. These titers were comparable to those exhibited by the control group receiving MPL * immunostimulant.

TABLE 1 comparison of Low pyrogen adjuvants for HBsAg

Serum titer

Group heating property^* IgG IgG1 IgG2a IgG2b

Nonimmune- -100 < 100

TEoA vector N.T. 51,200102,40025,6001600

MPL*-TEoA 2-3 409,600 204,800 204,800 51,200

Cpd.II-TEoA 0.3 409,600 204,800 409,600 51,200

a. The pyrogenicity data represent the total increase in temperature (degrees centigrade) after i.v. administration of a 10. mu.g/Kg dose to 3 rabbits. In pyrogen analysis, the compound was dissolved at 100. mu.g/ml in 10% EtOH/WFI (USP water for injection) and subsequently diluted with 5% aqueous glucose. N.t. means that the compound was not tested.

Example 5 adjuvant Activity on hemagglutinin proteins in Fluzone influenza vaccine

A group of BALB/c mice at 6-8 weeks (Jackson Laboratories Bar Harbor, Maine) was injected subcutaneously on days 0 and 14 with 0.2. mu.g hemagglutinin protein. + -. 20. mu.g adjuvant (MPL * immunostimulant or Compound II) in a Fluzone influenza vaccine (Connaught Laboratories, Swiftwater, Pa.). Titers to FluZone were determined by FluZone ELISA from pooled sera of 5 mice collected at day 14 after the second inoculation (table 2). Non-immune controls were not vaccinated. The initial dilution for serum in the test group was 1: 1600.

The results are similar to the previous examples. Also, compound II had significantly higher titers than control sera receiving antigen alone (table 2). The increase in titer was also reflected in enhanced IgG2a and IgG2b responses. These titers were comparable to those exhibited by the control group receiving MPL * immunostimulant.

TABLE 2 comparison of Low pyrogen adjuvants used with influenza vaccines

Serum titer

Group heating property^* IgG IgG1 IgG2a IgG2b

Nonimmune- -100 < 100

TEoA carrier N.T. 12,80051,2001600 < 1600

MPL*-TEoA 2-3 102,400 102,400 25,600 12,800

Cpd.II-TEoA 0.3 51,200 102,400 25,600 6400

a. The pyrogenicity data represent the total increase in temperature (degrees centigrade) after i.v. administration of a 10. mu.g/Kg dose to 3 rabbits. In pyrogen analysis, the compound was dissolved at 100. mu.g/ml in 10% EtOH/WFI (USP water for injection) and subsequently diluted with 5% aqueous glucose. N.t. means that the compound was not tested.

Example 6 adjuvant Activity against HBsAg

BALB/c groups were injected subcutaneously with 2.0 μ g HBsAg (Rhein American, & Rhein Biotech). + -. 25 μ g adjuvant (MPL * immunostimulant or Compound II) on days 0 and 21. Isotypes of IgG1 and IgG2a for HBsAg were determined by ELISA from pooled sera collected at day 21 after the second vaccination (table 3). Non-immune controls were not vaccinated. In this experiment, compound II mediated an increased titer compared to the control group that received the antigen in PBS. The potency of RC-553 stimulation was equal to that of the positive control, MPL * immunostimulant (Table 3).

TABLE 3 comparison of the hypopyrogen adjuvant used with HBsAg

Serum titer

Group heating property^* IgG1 IgG2a

Nonimmune- - < 100

PBS control N.T. 64,0004000

MPL*-TEoA 2-3 128,000 1,024,000

Cpd.II-TEoA 0.3 32,000 2,048,000

a. The pyrogenicity data represent the total increase in temperature (degrees centigrade) after i.v. administration of a 10. mu.g/Kg dose to 3 rabbits. In pyrogen analysis, the compound was dissolved at 100. mu.g/ml in 10% EtOH/WFI (USP water for injection) and subsequently diluted with 5% aqueous glucose. N.t. means that the compound was not tested.

Example 7 Compound II increases CTL Activity against HBsAg-immunized mice

Some mice from each group of example 4 were additionally used as spleen cell donors to evaluate CTL activity. Specific lysis directed against HBsAg in four hours of standard⁵¹Evaluation in Cr-release analysis (Moore et al (1988) Cell 55: 777-785). Single cell suspensions were made from spleens of mice on day 9 after vaccination. Spleen cells were treated with tris-buffered NH₄Cl treatment to remove red blood cells and at a concentration of 7.5X 10⁶Resuspended at/ml in RPMI/10% FCS supplemented with 5mM HEPES, 4mM L-glutamine, 0.05mM 2-mercaptoethanol and antibiotics. Will represent the known MHCI class, L^d-synthetic peptide of restricted CTL epitope (IPQSLDSWWTSL) was added to cells at a final concentration of 75 nM. After four days incubation, cells were removed and CTL activity was assessed. Specific killing of expression L^dRestricted epitopes of antigens⁵¹Cr-labeled transfected P815S cells. The target cell is expressing L^d-a transfected P815 cell line with restricted CTL epitopes (P815S). Nonspecific lysis was less than 10% for P815 target at E: T50: 1 (Table 4). Unlike the antibody response, RC-553 stimulated significantly elevated levels of CTL activity compared to the antigen-only control (Table 4).

TABLE 4 comparison of Low pyrogen adjuvants used with HbsAg

Percent specific killing (Effector: target ratio)

Group heating property^* 50∶1 25∶1 12.3∶1 6.25∶1

Non-immunological- -6310

PBS N.T. 29 20 11 7

MPL*-TEoA 2-3 80 71 47 32

Cpd.II-TEoA 0.3 85 77 53 37

a. The pyrogenicity data represent the total increase in temperature (degrees centigrade) after i.v. administration of a 10. mu.g/Kg dose to 3 rabbits. In pyrogen analysis, the compound was dissolved at 100. mu.g/ml in 10% EtOH/WFI (USP water for injection) and subsequently diluted with 5% aqueous glucose. N.t. means that the compound was not tested.

Example 8 Ex vivo cytokine Induction of Compound II

The effect of compound II on TNF- α and IL-1 β production was assayed ex vivo for human peripheral blood mononuclear cells. MPL * immunostimulant and Compound II were formulated in 0.2% TEoA/WFI in water.

Human whole blood was used to assess the Ability of Glycolipids (AGPs) to induce proinflammatory cytokines. Human whole blood was collected into heparinized tubes and 0.45ml of whole blood was mixed with 0.05ml of phosphate buffered saline (PBS, ph7.4) containing glycolipids (i.e., test compounds). Tubes were incubated at 37 degrees celsius for 4hr in a shaker apparatus. The samples were then diluted with 1.5ml sterile PBS and centrifuged. The supernatant was removed and analyzed for cell-associated TNF-. alpha.and IL-1. beta. by sandwich ELISA using R & D Systems' Quantinkin immunoassay kit for human TNF-. alpha.and IL-1. beta.

Compound I I did not produce TNF-. alpha.levels detectable under the assay conditions in the 1, 5, and 10. mu.g/ml assays. In contrast, the positive control LPS was an effective stimulator of TNF-. alpha.secretion from cells at 1 ng/mL. MPL *) immunostimulant efficiently induced TNF- α in the concentration range of 100-10,000 ng/mL.

Similarly, compound II (at 1, 5 and 10. mu.g/ml) produced no detectable levels of IL-1. beta. To compare the effects of this compound, the level of IL-1 β induced by the MPL * immunostimulant was assigned the value 1 and the relative inducibility of Compound II was ≦ 0.05.

Discussion of examples 4 to 8

Data from these studies indicate that compound II is able to increase immunity to vaccine antigens. The compound increases serum titers to two different vaccine antigens (influenza and hepatitis surface antigens). Like MPL * immunostimulant, it mediates a shift in antibody distribution from reactions dominated by the IgG1 isotype to those with high levels of IgG2a antibody. In addition to increasing the antibody response, the compounds are good adjuvants for inducing CTL activity.

One significant feature of the results of this study is that compound II appears to affect the response without inducing measurable levels of the inflammatory cytokines TNF- α or IL-1 β. These cytokines are produced by cells in the innate immune system in response to bacterial cell wall products including lipid a. Because of the structural similarity between the compound and lipid A, it is expected that it will also stimulate TNF- α or IL-1 β, and indeed many AGP molecules.

As inflammatory cytokines, TNF- α and IL-1 β stimulate the release of a series of other cytokine mediators that are used to activate phagocytic cells and mobilize specific immunity. IL-1 is initially referred to as an endogenous pyrogen because it induces a febrile response. Thus, the detectable lack of IL-1 following administration of Compound II is consistent with the apparent lack of fever in the rabbit pyrogen assay.

It is still possible that in these studies the compound actually stimulated TNF-. alpha.and IL-1. beta. secretion at levels high enough to mediate the activation of specific immunity, but too low to be detected in an in vitro cytokine assay. Alternatively, the compound stimulates cytokine mediators other than TNF- α and IL-1 β that result in specific immune responses to the co-administered vaccine antigens. It appears that IFN γ is produced. This cytokine is thought to be useful for inducing isotype-switched antibodies to the IgG2a subclass and as a promoter of TH-1 driven CTL responses. Thus the increased IgG2a titer and active CTL population both reflected IFN γ production.

Example 9 stimulation of Inducible Nitric Oxide Synthase (iNOS) by Compound II

This example illustrates the effect of various glycolipids on iNOS induction in J774 murine macrophages. Murine macrophage line J774 can be very sensitive to IFN- γ in vitro sensitization and to LPS stimulation following iNOS upregulation, as determined by standard Greiss reagent ELISA assay procedures. The assay was performed at 1X 10 using 30 mL/flask⁶J774 cells seeded at/mL and IFN- γ added to 100 units/mL for 16-24 hrs. The cells were then collected and washed at 2X 10⁵Resuspend and adhere to the wall in 96-well plates under well. For the experimental groups, glycolipid compounds were serially diluted into wells and the resulting cultures were incubated for an additional 36-40 hours before culture supernatants were collected in the analysis of the Greiss reagent for nitrite release. Nitrite content is closely related to iNOS function.

Potency was determined as the concentration (ng/mL) of glycolipid half way capable of inducing maximal induction of nitrite in culture (ED)₅₀)。ED₅₀The lower the number, the greater the potency of iNOS induction. ED (electronic device)₅₀According to Johnson et al, (1999) j.med.chem.42: 4640-4649.

MPL * immunostimulant was found to have ED₅₀About 2ng/mL, resulted in high levels of nitrite production, while Compound II had a nominal ED₅₀About.gtoreq.3000 (ng/ml).

The very low maximal iNOS activity observed in this compound indicates that it is essentially ineffective for iNOS induction in this system.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for any purpose.

Claims (24)

1. A compound of the formula:

and pharmaceutically acceptable salts thereof, wherein X is selected from the group consisting of-O-and-NH-;

y is selected from-O-and-S-; r¹、R²And R³Are each independently selected from (C)₉-C₁₃) An acyl group;

R⁴selected from-H and-PO₃R⁷R⁸Wherein R is⁷And R⁸Are each independently selected from-H and (C)₁-C₄) An aliphatic group;

R⁵is selected from-H, -CH₃and-PO₃R⁹R¹⁰Wherein R is⁹And R¹⁰Are each independently selected from-H and (C)₁-C₄) An aliphatic group;

R⁶is selected from H, OH, (C)₁-C₄) Oxyaliphatic radical, -PO₃R¹¹R¹²，-OPO₃R¹¹R¹²，-SO₃R¹¹，-OSO₃R¹¹，-NR¹¹R¹²，-SR¹¹，-CN，-NO₂，-CHO，-CO₂R¹¹and-CONR¹¹R¹²Wherein R is¹¹And R¹²Are each independently selected from H and (C)₁-C₄) An aliphatic radical, provided that R⁴And R⁵One is a phosphorus-containing group and when R is⁴is-PO₃R⁷R⁸When R is⁵Is not-PO₃R⁹R¹⁰；

Wherein^*1″，″^*2″，″^*3"and^**"denotes a chiral center;

wherein n, m, p and q are each independently integers of 0 to 6, provided that the sum of p and m is 0 to 6.

2. The compound of claim 1, wherein X and Y are-O-, R⁴Is PO₃R⁷R⁸，R⁵And R⁶Is H, and n, m, p, and q are integers from 0 to 2.

3. The compound of claim 2, wherein R⁷And R⁸is-H.

4. The compound of claim 3, wherein n is 1, m is 2, and p and q are 0.

5. The compound of claim 1, wherein R¹，R²(ii) a And R³Are respectively C₁₀-C₁₂An acyl group.

6. The compound of claim 4, wherein R¹，R²And R³Are decanoyl residues, respectively.

7. The compound of claim 4, wherein R¹，R²And R³Are each a dodecanoyl residue.

8. The compound of claim 4, wherein^*1，^*2And are and^*3is in the R configuration.

9. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound according to any one of claims 1 to 8.

10. A pharmaceutical composition according to claim 9, which comprises a therapeutically effective amount of a compound according to any one of claims 1 to 8.

11. The pharmaceutical composition of claim 9 or 10, wherein the pharmaceutical composition further comprises at least one antigen.

12. The pharmaceutical composition of claim 11, wherein the antigen is from herpes simplex virus type 1, herpes simplex virus type 2, human cytomegalovirus, HIV, hepatitis a, hepatitis b, hepatitis c or hepatitis e, respiratory syncytial virus, human papilloma virus, influenza virus, tuberculosis, leishmaniasis, t.

13. The pharmaceutical composition of claim 11, wherein the antigen is a human tumor antigen.

14. The pharmaceutical composition of claim 13, wherein the tumor antigen is from prostate cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, brain cancer, head and neck cancer, melanoma, leukemia or lymphoma cancer.

15. The pharmaceutical composition of claim 13, wherein the antigen is an autoantigen.

16. The pharmaceutical composition of claim 15, wherein the autoantigen is an antigen associated with an autoimmune disease.

17. The pharmaceutical composition of claim 16, wherein the autoimmune disease is type 1 diabetes, multiple sclerosis, myasthenia gravis, rheumatoid arthritis, or psoriasis.

18. A pharmaceutical composition according to any one of claims 10 to 17 in the form of an aqueous formulation.

19. The pharmaceutical composition of claim 18, wherein the aqueous formulation comprises one or more surfactants.

20. The pharmaceutical composition according to any one of claims 10 to 17 in the form of an emulsion formulation.

21. The pharmaceutical composition according to any one of claims 10 to 17 in the form of a solid formulation.

22. A method of inducing an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1-8 or a composition of any one of claims 9-21.

23. A method of treating a mammal suffering from or susceptible to a pathogenic infection, cancer or autoimmune disease, comprising administering to the mammal a therapeutically effective amount of a compound according to any one of claims 1 to 8 or a composition according to any one of claims 9 to 21.

24. A method for treating a disease or disorder ameliorated by the production of nitric oxide in a subject, comprising contacting the subject with an effective amount of a compound according to any one of claims 1 to 8 or a composition according to any one of claims 9 to 21.